Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
DSpace
UBCV
Ling, Yeou Mei
Master of Science - MSc
University of British Columbia
Methyl-CpG-binding protein 2 (MECP2) plays a key role in brain homeostasis. MECP2 function is sensitive to changes in its concentration, as both overexpression and reduction of MECP2 result in neurodevelopmental disorders in humans and mice. MEPC2 was originally discovered based on its ability to bind methylated-CpG DNA to repress gene expression. Later research suggested that MECP2 may also interact with unmethylated DNA. While there have been in vitro studies supporting this idea, there were few in vivo studies. In this thesis, we demonstrate that expression of the human MECP2 in an organism lacking DNA methylation, the budding yeast Saccharomyces cerevisiae, disrupts normal yeast growth, by inhibiting passage through S phase. Further investigation revealed that MECP2-expressing yeast had defects in their cell cycle, which may be the cause of the growth defect. We found that the phenotype of yeast expressing MECP2 with disease-associated mutations reflects disease severity in humans. Using single-cell techniques such as flow cytometry and fluorescence microscopy, we confirmed the expression of MECP2 in yeast and its nuclear localization. Disease-associated mutations that reversed the growth defect either impair nuclear localization or, as previously reported, disrupt MECP2 binding to DNA. In addition, we found differences between the two MECP2 isoforms in terms of protein stability, and impact on the cell cycle and viability. This suggests that the isoforms have different functions and potentially, differing interacting partners within cells. Collectively, our findings confirmed the feasibility of using yeast as a model organism to study MECP2 function in vivo. Moreover, using this system, we are able to rapidly screen for phenotypes of MECP2-disease-associated mutations that reflect disease severity in humans.
INVESTIGATING THE EFFECTS OF EXPRESSING MECP2 IN SACCHAROMYCES CEREVISIAE by Yeou Mei Ling B.Sc., The University of British Columbia, 2020 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2023 © Yeou Mei Ling, 2023 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: INVESTIGATING THE EFFECTS OF EXPRESSING MECP2 IN SACCHAROMYCES CEREVISIAE submitted by Yeou Mei Ling in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry and Molecular Biology Examining Committee: Dr. LeAnn Howe, Professor, Biochemistry and Molecular Biology, UBC Supervisor Dr. Annie Vogel Ciernia, Assistant Professor, Biochemistry and Molecular Biology, UBC Supervisory Committee Member Dr. Ivan Sadowski, Professor, Biochemistry and Molecular Biology, UBC Supervisory Committee Member Dr. Louis Lefebvre, Associate Professor, Medical Genetics, UBC Additional Examiner iii Abstract Methyl-CpG-binding protein 2 (MECP2) plays a key role in brain homeostasis. MECP2 function is sensitive to changes in its concentration, as both overexpression and reduction of MECP2 result in neurodevelopmental disorders in humans and mice. MEPC2 was originally discovered based on its ability to bind methylated-CpG DNA to repress gene expression. Later research suggested that MECP2 may also interact with unmethylated DNA. While there have been in vitro studies supporting this idea, there were few in vivo studies. In this thesis, we demonstrate that expression of the human MECP2 in an organism lacking DNA methylation, the budding yeast Saccharomyces cerevisiae, disrupts normal yeast growth, by inhibiting passage through S phase. Further investigation revealed that MECP2-expressing yeast had defects in their cell cycle, which may be the cause of the growth defect. We found that the phenotype of yeast expressing MECP2 with disease-associated mutations reflects disease severity in humans. Using single-cell techniques such as flow cytometry and fluorescence microscopy, we confirmed the expression of MECP2 in yeast and its nuclear localization. Disease-associated mutations that reversed the growth defect either impair nuclear localization or, as previously reported, disrupt MECP2 binding to DNA. In addition, we found differences between the two MECP2 isoforms in terms of protein stability, and impact on the cell cycle and viability. This suggests that the isoforms have different functions and potentially, differing interacting partners within cells. Collectively, our findings confirmed the feasibility of using yeast as a model organism to study MECP2 function in vivo. Moreover, using this system, we are able to rapidly screen for phenotypes of MECP2-disease-associated mutations that reflect disease severity in humans. iv Lay Summary Methyl-CpG-binding protein 2 (MECP2) is a ubiquitous protein in neurons and is important for brain function. DNA in mammalian cells is often marked by tags called methylation. It was originally thought that MECP2 requires methylation to bind DNA in cells. Some research also suggests that the mark is not essential for MECP2 to interact with DNA, but there is no direct evidence showing that this is occurring within cells. In this thesis, we demonstrate that in cells without DNA methylation, such as yeast, expression of MECP2 can inhibit growth, suggesting it interacts with DNA and disrupts cell function. We also found that MECP2 mutations associated with disease impact the growth phenotypes observed in yeast. Our research provides a better understanding of MECP2 in unmethylated DNA conditions and suggests that yeast is a suitable model to rapidly study and screen for the effects of MECP2 and disease-associated mutations in cells. v Preface Unless otherwise stated, all experiments, strain generation and data analysis in this thesis were carried out by me. The initial project conceptualization was by Dr. LeAnn Howe and Dr. Juan Ausió (University of Victoria). The project was carried out under the guidance of Dr. LeAnn Howe with some feedback from Dr. Juan Ausió. The galactose-inducible MECP2-E2 plasmid was generated by Barry Young in Dr. Christopher Loewen’s lab (UBC). Plasmids expressing ZF4-ER-VP16, the untagged estradiol-inducible MECP2 and the MECP2-E2 mutants were generated by Dr. LeAnn Howe. vi Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ................................................................................................................................ iv Preface ............................................................................................................................................ v Table of Contents .......................................................................................................................... vi List of Figures ............................................................................................................................... ix List of Tables ................................................................................................................................. xi List of Symbols ............................................................................................................................. xii List of Abbreviations .................................................................................................................. xiii Acknowledgements ..................................................................................................................... xvi Dedication ................................................................................................................................... xvii Chapter 1: Introduction ................................................................................................................ 1 1.1 Chromatin ........................................................................................................................ 1 1.1.1 Histones ................................................................................................................... 1 1.1.2 Nucleosomes ............................................................................................................ 3 1.1.3 Histone post-translational modifications ................................................................. 4 1.1.4 Higher-order structure ............................................................................................. 7 1.2 The Yeast Cell Cycle ....................................................................................................... 8 1.3 DNA methylation ............................................................................................................ 9 1.3.1 CpG methylation ..................................................................................................... 9 1.3.2 CpH methylation ................................................................................................... 11 1.3.3 Hydroxymethylation .............................................................................................. 12 vii 1.4 Methyl-CpG-binding protein 2 ...................................................................................... 12 1.4.1 Structure ................................................................................................................ 13 1.4.2 Binding of MECP2 to methylated CpGs ............................................................... 16 1.4.3 Binding of MECP2 to methylated CpHs ............................................................... 17 1.4.4 Binding of MECP2 to unmethylated regions ........................................................ 18 1.4.5 MECP2 and the linker histone H1 ......................................................................... 19 1.4.6 MECP2 and co-repressors ..................................................................................... 21 1.4.7 Beyond repression ................................................................................................. 24 1.4.8 MECP2 and chromatin .......................................................................................... 25 1.4.9 MECP2 levels in cells ........................................................................................... 27 1.4.10 Disease-associated MECP2 mutations .................................................................. 29 1.4.10.1 Point mutations .............................................................................................. 29 1.4.10.2 Truncation mutations ..................................................................................... 31 1.5 Summary of research hypothesis ................................................................................... 33 Chapter 2: Materials and Methods ............................................................................................ 35 2.1 Strain generation ............................................................................................................ 35 2.2 Inducing protein expression .......................................................................................... 38 2.2.1 Galactose induction ............................................................................................... 38 2.2.2 β-estradiol induction .............................................................................................. 39 2.3 Western blots ................................................................................................................. 39 2.4 Growth assays ................................................................................................................ 41 2.5 Flow cytometry for quantifying protein expression levels ............................................ 41 2.6 Viability assay ............................................................................................................... 42 viii 2.7 Cell cycle assay ............................................................................................................. 42 2.8 Fluorescence microscopy .............................................................................................. 43 Chapter 3: Results ....................................................................................................................... 51 3.1 Testing protein expression systems ............................................................................... 51 3.1.1 Inducing MECP2 expression in S. cerevisiae with galactose ................................ 51 3.1.2 Inducing MECP2 expression in S. cerevisiae with beta-estradiol ......................... 52 3.2 Exploring the causes of growth defects for MECP2-expressing cells .......................... 63 3.3 MECP2 disease-associated mutants .............................................................................. 72 3.3.1 Disease-associated mutations and growth ............................................................. 72 3.3.2 Variability in yeast strains and protein levels ........................................................ 73 3.3.3 The impact of disease-associated mutations on protein localization ..................... 75 Chapter 4: Discussion .................................................................................................................. 91 References .................................................................................................................................... 97 ix List of Figures Figure 1. MECP2 isoforms and common RTT-associated mutations ........................................... 15 Figure 2. Expression of N-terminal tagged MECP2-E1 in S. cerevisiae results in defects in growth ............................................................................................................................................ 56 Figure 3. The addition of a tag to the N-terminus of MECP2-E1 alters protein levels when expressed in S. cerevisiae .............................................................................................................. 57 Figure 4. Maximum MECP2 induction in S. cerevisiae is reached with 1 μM estradiol .............. 58 Figure 5. Maximum MECP2 induction in S. cerevisiae is achieved with 1 μM estradiol (part 2) 59 Figure 6. Expression of MECP2 is titratable by varying the estradiol concentrations .................. 60 Figure 7. Maximum MECP2 expression in S. cerevisiae is achieved at 4 hours post-induction .. 61 Figure 8. Maximum MECP2 levels in S. cerevisiae is achieved at 4 hours post-induction (part 2) ....................................................................................................................................................... 62 Figure 9. GFP-tagged, but not untagged, MECP2-E1 inhibits the growth of S. cerevisiae .......... 67 Figure 10. Yeast strains with MECP2-induced defects in growth have altered cell cycles .......... 68 Figure 11. Yeast strains with MECP2-induced defects in growth have altered cell cycles (part 2) ....................................................................................................................................................... 69 Figure 12. The expression of GFP-MECP2-E1 in S. cerevisiae induces cell death ...................... 70 Figure 13. The expression of GFP-MECP2-E1 in S. cerevisiae induces cell death (part 2) ......... 71 Figure 14. Mutations R168X and T158M revert the growth defect induced by MECP2-E2 expression ...................................................................................................................................... 78 Figure 15. Phenotypes are variable between biological replicates of yeast expressing GFP-MECP2-E1 mutants ....................................................................................................................... 79 x Figure 16. Phenotypes were variable between biological replicates of yeast expressing GFP-MECP2-E2 mutants ....................................................................................................................... 80 Figure 17. Variability in MECP2 expression between independently derived yeast strains ......... 81 Figure 18. Variability in MECP2 expression between independently derived replicates (part 2) 82 Figure 19. Low variability in MECP2 expression between replicates generated from a single colony ............................................................................................................................................ 83 Figure 20. Low variability in MECP2 expression between replicates generated from a single colony (part 2) ............................................................................................................................... 84 Figure 21. GFP-MECP2-E1 and mutants are variably expressed in yeast .................................... 85 Figure 22. GFP-MECP2-E1 and mutants are variably expressed in yeast (part 2) ....................... 86 Figure 23. GFP-MECP2-E2 and mutants are variably expressed in yeast .................................... 87 Figure 24. GFP-MECP2-E2 and mutants are variably expressed in yeast (part 2) ....................... 88 Figure 25. MECP2-E1 wild-type and the T158M mutant, but not the R168X mutant, are nuclear-localized when expressed in yeast ................................................................................................. 89 Figure 26. MECP2-E2 wild-type and the T158M mutant, but not the R168X mutant, are nuclear-localized when expressed in yeast ................................................................................................. 90 xi List of Tables Table 1. List of plasmids used to generate the yeast strains in this study ..................................... 44 Table 2. Sequences of MECP2 isoforms and protein tags ............................................................ 45 Table 3. Primers used for cloning .................................................................................................. 48 Table 4. Genotypes of tagged and untagged MECP2 strains ........................................................ 49 Table 5. Genotypes of MECP2-E2 mutant strains ........................................................................ 49 Table 6. Genotypes of GFP-tagged MECP2-E1 mutant strains .................................................... 50 Table 7. Genotypes of GFP-tagged MECP2-E2 mutant strains .................................................... 50 xii List of Symbols α Alpha, anti β Beta ° Degree D Delta, deletion δ Delta, deletion g Gram l Liter μ Micro - Minus M Molar concentration m Meter n Nano % Percent, percentage + Plus X Stop codon x Times xiii List of Abbreviations BSA Bovine serum albumin ChEC Chromatin endogenous cleavage ChIP Chromatin immunoprecipitation ConA Concanavalin A CTD C-terminal domain DNMT DNA methyltransferase ECDF Empirical cumulative distribution function EDTA Ethylenediaminetetraacetic acid ESC Embryonic stem cells EtOH Ethanol GFP Green fluorescent protein GST Glutathione S-transferase HAT Histone acetyltransferase HDAC Histone deacetylase HDM Histone demethylase HMT Histone methyltransferase ID Intervening domain KO Knockout LAD Lamina-associated domain LiAc Lithium acetate MBD Methyl-CpG-binding domain xiv MECP1 Methyl-CpG-binding protein 1 MECP2 Methyl-CpG-binding protein 2 MEF Mouse embryonic fibroblast MNase Micrococcal nuclease NCoR Nuclear receptor co-repressor SMRT Silencing mediator of retinoid and thyroid hormone receptor NDR Nucleosome depleted region NID NCoR/SMRT interaction domain NLS Nuclear localization signal NRL Nucleosome repeat length NTD N-terminal domain NuA4 Nucleosome acetyltransferase of histone H4 PBS Phosphate-buffered saline PBST Phosphate-buffered saline with 0.1% Tween-20 PCR Polymerase chain reaction PEG Polyethylene glycol PGK1 Phosphoglycerate kinase 1 PI Propidium iodide qPCR Quantitative polymerase chain reaction Rpm Revolutions per minute RTT Rett syndrome SAGA Spt-Ada-Gcn5-acetyltransferase sdH2O Sterile deionized water xv seq Sequencing SDS Sodium dodecyl sulphate TAD Topologically associating domain TBL1 Transducin β-like protein 1 TBLR1 TBL1-related protein TRD Transcriptional repression domain TSS Transcription start site WT Wild-type YP Yeast extract-peptone YPD Yeast extract-peptone-dextrose xvi Acknowledgements First, I would like to thank my supervisor, Dr. LeAnn Howe, for her guidance and valuable insight throughout my studies. I learned a great deal under her mentorship. I want to thank all the members of the lab: Ellia, Jonah, and Josh, for their support and suggestions on experiments, and to the past members: Nadiyah, Liam, and Julia for supplying media and clean glassware. A special thanks to Dr. Juan Ausió at the University of Victoria, without whom this research would never have happened. I would also like to thank my committee members: Dr. Annie Vogel Ciernia for her expertise on the brain and MECP2, and Dr. Ivan Sadowski for his knowledge of everything related to yeast research. Also, to my cat Chloe, thanks for always being cute and annoying. You never fail to make my day better. And lastly, I am grateful to my parents for their unwavering support. xvii Dedication To my cat Chloe, for all those times you sat on my keyboard while I was writing the thesis 1 Chapter 1: Introduction 1.1 Chromatin 1.1.1 Histones In eukaryotes, DNA is packaged by histones into nucleosomes. Histones form octamers composed of two H2A-H2B dimers and an H3-H4 tetramer, which are also referred to as core histones (1). Multiple nucleosomes make up chromatin. Chromatin packing is subdivided into euchromatic and heterochromatic regions. Chromatin packing dictates the accessibility of promoters by transcription factors that bind to and initiate transcription. Heterochromatin, which contains densely packaged DNA, are regions that associate with low transcription. Conversely, euchromatin is loosely packaged DNA that is highly transcribed. Thus, the structure of chromatin regulates gene expression. While the above is consistent within eukaryotes, there are several notable differences in chromatin structure between yeast (Saccharomyces cerevisiae) and mammalian cells, and these are discussed below. There are two types of histones, canonical and variant histones. In humans, there are around eight variants of H2A and six variants of histone H3 (2). The only histone variants found in yeast are H2A.Z and a centromeric H3 (CenH3) called Cse4 (3–5). Humans have a centromeric H3, called CENP-A, and three variants of H2A.Z (6, 7). In yeast, each of the core histones is encoded by two genes, while the variants H2A.Z and CenH3 are encoded by one gene (8). In contrast, the human core histones are encoded by over ten genes, but like yeast, the histone variants are encoded by one or two genes (2). Mammals contain the histone variants, H3.1 and H3.2, both of which are part of the canonical histones, and the variant H3.3 (2). H3.1 and H3.2 differ by one amino acid, and H3.2 and H3.3 differ by only four amino acids (9). However, histone H3.3 is the variant that 2 is the most similar to yeast H3, and it associates with active transcription (10, 11). Humanized yeast expressing the human core histones, hH2A, hH2B, hH3, and hH4 can still grow, suggesting highly conserved functions (12). Interestingly, despite the similarity between yeast H3 and human H3.3, yeast expressing hH3.3 had reduced growth compared to yeast expressing human H3.1, suggesting that human H3.3 is not as similar to yeast H3 as previously thought (11, 12). The linker histone H1 is not considered a core histone as it doesn’t directly form the nucleosome core particle. It instead binds the DNA entering and exiting the nucleosomes and thus assists in compacting chromatin into higher-order structures (13). Histone H1 has 11 variants in humans and mice, seven of which are expressed in all tissues, three testis-specific and one specific to the oocyte, and each variant is encoded by one gene (13). Histone H1 has been shown to inhibit the acetylation of histone H3 by sterically hindering the access of histone acetyltransferases (14). Mice with a single or double knockout of the genes encoding H1 variants were phenotypically similar to wild-type mice, suggesting that the absence of one or two linker histone variants could be compensated by other variants (15–17). Mouse embryonic stem cells (ESC) with triple knockouts (KO) of H1 genes that resulted in a 50% reduction in H1 levels had a 15 bp decrease in the average distance between nucleosomes, called the nucleosome repeat length (NRL) (189 to 174 bp), and a less compact chromatin (18). Despite H1 depletion, however, less than 1% of genes had changes in expression and little change was detected in the global DNA methylation pattern (18). This suggests that H1 may not be essential for gene regulation. The closest to the human linker histone H1 in yeast is encoded by the HHO1 gene which is about 52% similar to the human H1 and localizes to the nucleus (19, 20). Although Hho1 can compact chromatin, deletion of HHO1 did not cause a growth defect or change gene expression levels in yeast, suggesting that it is not essential for proper gene regulation (19–21). The other protein with an H1-like function in yeast 3 is Hmo1, which has also been shown to compact chromatin via its C-terminal tail (22). Deletion of HMO1 increased the sensitivity of chromatin to micrococcal nuclease (MNase) digestion and resulted in poor growth (22, 23). Moreover, the expression of a human H1 variant in the HMO1 deletion strain restored the MNase resistance of WT yeast and also alleviated the growth defect (22, 23). Thus, aside from Hho1, Hmo1 may act as a linker histone in yeast. 1.1.2 Nucleosomes The nucleosome consists of ~146 bp of DNA wrapped ~1.67 times around the core histones (1). Nucleosomes are joined together by linker DNA. Linker DNA regions are histone free and are ~25 times more easily digested than the DNA in nucleosomes (24). The NRL depends on several factors such as the ratio of linker histones to core histones, or the interactions between nucleosomes (25). Actively transcribed regions tend to have a shorter NRL, while a longer NRL is located in heterochromatin (26, 27). There is a difference in the NRL between yeast and across organisms. The average NRL in eukaryotic cells is around 190 bp, although the NRL is much shorter in mouse and rat neurons. In neurons, the NRL is approximately 165 bp, which is similar to the NRL of S. cerevisiae (28, 29). Expression of the humanized core histones in yeast did not change its NRL, suggesting that NRLs are not dictated by histones alone (12). However, NRLs affect histone H1 binding, as a shorter NRL sterically hinders H1 and H1 can bind to regions with longer NRLs (30). Yeast chromatin is more diffused and less structured than that of mammalian cells, mostly existing as an open state with no higher-order compaction (31–33). About 85% of the yeast genome is actively transcribed, which may explain why yeast chromatin is in an open state (34). However, having an open chromatin does not mean that it is homogenous. Although yeast chromatin does not form large heterochromatic regions like mammalian chromatin, it can still form small 4 nucleosome clusters (31). The few heterochromatic regions in yeast are hypoacetylated and are located at telomeres and the mating loci, HML and HMR (35, 36). During mitosis, yeast chromosomes do not form condensed structures and there is no increase in the local nucleosome concentration (31). In contrast, human chromosomes form compact structures during mitosis (37). The rules governing nucleosome position in yeast have been shown to be a good predictor of nucleosome positioning in flies and humans, so we will focus our discussion on yeast nucleosome occupancy (38). Nucleosomes occupy about 81% of the total yeast genome and most of the nucleosomes are in transcribed regions (39). There is a depletion of nucleosomes in the region immediately upstream of the transcription start site (TSS), called the nucleosome depleted region (NDR), which averages about 150 bp in length (39, 40). A NDR is found at the majority of yeast gene promoters and includes transcription factor binding sites and poly(dA-dT) sequences (40, 41). Poly(dA-dT) sequences destabilize histone-DNA interactions which reduce nucleosome formation in vitro, and function to exclude nucleosomes at the NDR (38, 41, 42). Poly-A stretches in human chromatin are also depleted of nucleosomes (43). Unlike yeast promoters, human promoter regions are high in GC sequence and low in poly (dA-dT) (44). In humans, NDRs at the TSS are only found at expressed genes or unexpressed genes with preinitiation complexes (PIC) at the promoters (43). Unexpressed genes without a PIC do not have a detectable NDR (43). 1.1.3 Histone post-translational modifications The disordered N-terminal tails of histones can be post-translationally modified to regulate gene expression. Acetylation on lysine neutralizes the positive charge of histones, decreasing their affinity to the negatively charged DNA backbone and correlates with actively transcribed regions (45). This is facilitated by histone acetyltransferases (HATs) that transfer the acetyl group from 5 acetyl-coenzyme A (CoA) to lysine (K) (46, 47). Conversely, histone deacetylase complexes (HDACs) reverse the process and remove the acetyl groups from lysines (47). Histones can also be methylated on lysines (K) or arginines (R) by histone methyltransferases (HMTs) and this modification can be removed by histone demethylases (HDMs) (46). Methylation marks are associated with active or inactive gene expression depending on the site (46). Moreover, there can be multiple methylations such as mono, di, tri-methylation on lysine and mono or di-methylation on arginine (46). We will focus on some of the key histone acetylation and methylation marks in the section below. In yeast, H3 or H4 acetylation mostly occurs at nucleosomes downstream of the promoters of active genes (48). The two major HATs in yeast are the complexes Nucleosome acetyltransferase of histone H4 (NuA4) and Spt-Ada-Gcn5-Acetyltransferase (SAGA). Esa1, part of the NuA4 complex, acetylates lysines on H4 and is essential for yeast growth as depletion of Esa1 inhibits progression through G2/M (49–51). Gcn5 is part of the SAGA complex and mostly acetylates H3 and H2B (52–54). In S. cerevisiae, histone methylation is added by Set1, Set2 and Dot1 (55–57). Set1 is enriched at the 5’end of genes and is recruited by serine 5-phosphorylated-RNA polymerase II (55). Only H3K4me3 correlates with Set1 localization at the 5’ end of active genes, while H3K4me2 and H3K4me do not correlate with Set1 localization as H3K4me2 is located throughout the coding region, and H3K4me at the end of genes (48, 55). Due to the association between Set1 and RNAPII, H3K4 methylation marks are associated with active transcription (55). H3K36 methylation is targeted by the interaction between Set2 and phosphorylated RNAPII and is associated with transcription elongation (56, 58, 59). H3K36me3 is depleted at the 5’ ends of genes and is instead found in regions with elongating RNAPII, while H3K79me3 is uniformly spread across gene bodies (48). The methylation marks, H3K9me3, 6 H3K27me3, and H4K20me3 are all located in heterochromatin, and are associated with repression in mammalian cells, but are absent in S. cerevisiae (60–62). H3K9 methylation is associated with constitutive heterochromatic regions and depletion of this mark results in a loss of heterochromatin organization in mouse embryonic fibroblasts (MEF) (63). Both H3K9me3 and H4K20me3 localize to the pericentric heterochromatin while H3K27me3 is located on facultative heterochromatin (60, 64). The lack of heterochromatic marks in yeast is unsurprising given that chromatin in yeast mostly exists as an open chromatin state (31, 32). Overall, most histone PTMs in yeast are associated with active gene expression and S. cerevisiae lacks the common heterochromatic marks in mammalian cells. The yeast HDAC, Rpd3, forms a complex with Sin3 and plays an important role in transcription repression (65). Rpd3 in yeast is homologous to the human HDACs 1-3 and 8, with HDAC2 having the highest similarity (82%) to Rpd3 (66–68). Expression of human HDAC1 in place of Rpd3 in yeast showed that it can interact with Sin3 and weakly represses transcription (65, 69). This suggests that HDAC1 and Rpd3 have functional similarities. The Sin3-Rpd3 complex has also been shown to play a role in replication, as disruption of the complex results in early activation of usually late-replicating origins (70). The human homologs of yeast Sin3 are the mammalian corepressors mSin3A and mSin3B, which are one of the many repressors that associate with the Mad-Max complex (71). mSin3A associates with HDAC1 and HDAC2, and the deacetylase activity of these proteins is essential for repression by Mad (71–74). The nuclear receptor co-repressor (NCoR/SMRT) complex is also involved in transcription repression and consists of the proteins NCoR1 or SMRT plus HDAC3, GPS2, TBL1 and TBLR1 (75–77). Both NCoR1 and SMRT bind to HDAC3 and are required to activate its deacetylase activity (75, 78–80). The NCoR/SMRT complex interacts with the mSin3A-HDAC1/2 complex (74, 81, 82). The 7 Set3 corepressor complex in yeast is homologous to the mammalian NCoR/SMRT corepressor complex (83). The Set3 complex is made up of two histone deacetylases, Hos2 and Hst1, and the proteins Set3, Snt1, YIL112w, Sif2 and Cpr1 (83). Both Snt1 and NCoR1 have SANT domains, Sif2 and TBL1 have WD40 repeats, and Hos2 is the closest homolog to HDAC3 (80, 83, 84). Set3 contains a PHD finger that recognizes H3K4me2, targeting the HDAC activity of Hos2 to transcriptionally active genes, leading to the deacetylation of histones H3 and H4 (85). Consistent with this, deletion of SET1 results in increased histone acetylation at the 5’ end of genes, likely due to disruption in the recruitment of the Set3 complex to the region (85). 1.1.4 Higher-order structure Aside from euchromatin and heterochromatin, the chromatin of eukaryotes is organized into distinct, megabase-sized, regions called topologically associating domains (TADs) (86). TADs likely function to promote interactions between distal regions while insulating regulatory elements away from genes in other TADs (86). In humans and mice, TADs are further divided into sub-TADs and these are kilobases in size (87–89). Chromatin regions that are associated with the nuclear lamina in the nucleus are called lamina-associated domains (LADs) (90). Lamina-associated domains were characterized in humans, flies (Drosophila melanogaster), and worms (Caenorhabditis elegans), but yeast do not have lamins and thus do not have LADs (90–94). LADs are associated with transcription repression and enriched with repressive marks H3K27me3 and H3K9me2 (91). Like TADs, LADs are megabases in size (0.1-10 megabases) (86, 91). Some borders of TADs and LADs are bound by the CTCF protein, which is important for TAD formation (86, 87, 89, 91). Despite a smaller size, yeast chromatin can arrange into similar, although not as complex organizations as the chromatin in the mammalian nucleus because yeast lacks CTCF (95). 8 TADs are also found in yeast and are about 200 kb in size (96). TADs in yeast help organize early and late replication timing, but there is no difference in the transcription levels or histone PTMs between different TADs (96). In addition to LADs and TADs, the genomes of multicellular organisms appear to be divided into two compartments, designated A and B (97). Regions in the A compartment tend to be more highly transcribed, are associated with active chromatin marks like H3K36me3, H3K79me2, H3K27ac and H3K4me3 and tend to replicate early, while regions in the B compartment replicate later and are enriched in H3K27me3 and H3K9me3 (87, 97, 98). Although the driving force that generates these compartments is not clear, it has been proposed that liquid-liquid phase separation (LLPS) plays a role (99, 100). This process involves the clustering of high concentrations of proteins, RNA or DNA into distinct hubs, called condensates (100). Proteins or complexes that can phase separate usually have intrinsically disordered regions and are able to participate in multivalent interactions, as is the case of chromatin arrays (100, 101). Disruption of condensate formation in the nucleus perturbs the boundary formation of 20% of TADs, usually at actively transcribed regions (102). Moreover, LLPS plays a role in clustering transcription factors at super-enhancers, the formation of CTCF-independent chromatin loops, and can be regulated by histone PTMs (103–106). 1.2 The Yeast Cell Cycle The yeast cell cycle involves four stages: G1, S, G2 and Mitosis (M) phase (107). The G1 and G2 phases involve cell growth, and the S phase is where DNA replication occurs. Cytokinesis occurs during Mitosis (M phase) and following this phase, the cycle restarts. The cell can also enter a quiescent stage (G0) when exposed to harsh environmental conditions (108). It is possible to visually track the stage of the cell cycle based on yeast morphology. In G1, cells are round but 9 at S phase, a small bud forms, and this bud progressively grows larger throughout the cell cycle until G2/M, where the nucleus migrates to the bud junction (107). During cytokinesis, the nucleus splits into two, one in the parent and one in the daughter cell, each with its copy of DNA (107). Cell cycle checkpoints are controlled by the presence or absence of cell division cycle proteins (CDC) to regulate various steps of the cell cycle such as ensuring that replication is only initiated once, or to prevent replication in the presence of DNA damage (109, 110). 1.3 DNA methylation 1.3.1 CpG methylation DNA methylation plays a crucial role in a wide range of functions from embryonic development, X chromosome inactivation, to the maintenance of gene expression. DNA methyltransferases (DNMTs) transfer the methyl group from S-adenosyl-L-methionine (SAM) onto DNA bases (111). Methylation primarily occurs on the 5’ carbon of cytosine resulting in 5-methylcytosine (5mC) on CG dinucleotides (CpG), and ~60-80% of CpGs in the mammalian genome are methylated (112, 113). Proteins with methyl CpG-binding domains (MBDs), such as methyl-CpG-binding protein 2 (MECP2), bind preferentially to these mCpG regions to regulate chromatin structure (114, 115). Note that there are also regions of densely clustered CpGs, called CpG islands, that are located at transcription start sites (TSS), and these are usually not methylated (116). There are five DNMTs in humans, DNMT1, DNMT2, DNMT3A, DNMT3B and DNMT3L (117). DNMT1 is responsible for ensuring that methylation marks are passed on during replication. DNMT3A and DNMT3B are de novo methyltransferases that add new methylation marks to DNA during development (118). While DNMT3A and 3B have some redundant 10 functions, they also methylate different sites. For example, during embryo development, DNMT3A has a preference for methylating promoters and gene bodies of developmental genes, but DNMT3B mostly targets methylation on the X chromosome (119). In contrast, DNMT2 and DNMT3L do not contain catalytic activity toward DNA (120, 121). DNMT2 (TRDMT1) methylates tRNAs (120). DNMT3L mostly assists DNMT3A in enhancing CpG methylation (121). The C-terminal domain of DNMT3L forms a tetramer with the DNMT3A catalytic domain (two DNMT3L-DNMT3A dimers) to stabilize the active site and enhance binding to CpGs (122). Moreover, DNMT3L potentially interacts with unmethylated H3K4, preventing DNA methylation in H3K4-methylated regions (123). There is no DNA methylation in yeast, but several studies have expressed the mouse DNA methyltransferases in S. cerevisiae (124–126). Expression of DNMT3B or DNMT3A and DNMT3L resulted in methylation of less than 1% of the whole genome (124, 125). However, the expression of the four DNMTs, DNMT1, DNMT3A, DNMT3B and DNMT3L, was able to increase this to 2% methylation, and up to 5% when the induction time was extended to over three days (126). DNMTs expressed in yeast preferentially methylate linker DNA regions (125, 126). Moreover, DNA methylation was depleted at H3K4-trimethylated regions and enriched in regions with H3K36 methylation (124–126). DNA methylation at H3K36 is likely added by DNMT3A, which has specificity for H3K36me2 and H3K36me3 through its PWWP domain (127, 128). A yeast strain with a SET1 deletion, which reduces H3K4 methylation showed higher DNA methylation levels (124, 125). This is consistent with the fact that H3K4 methylation is associated with active regions and the fact that the PHD finger of DNMT3L has specificity for unmethylated H3K4 (55, 123). Yeast expressing DNMTs have narrower NDRs, fuzzier nucleosomes, and condensed centromeres, likely due to increased rigidity of methylated DNA (126). 11 1.3.2 CpH methylation Methylation can also occur at non-CpG sites (mCpH; H= A, C, or T) and is more prevalent in stem cells and neurons than in other somatic cells (129–131). In pluripotent stem cells, the levels of CpA methylation correlate with that of CpG methylation (131). In neurons, mCpH is depleted at actively transcribed regions and enriched at regions with low CpG methylation, while in ESCs mCpH is enriched at highly expressed genes (112, 130, 132). DNMT3A has been shown to play an important role in adding methylation to CpA in neurons (130, 132, 133). Knockout of Dnmt3a results in significant depletion of methylated CpA and CpT, but not CpC or CpG in the mice cerebellum (133). Knockdown of Dnmt3b in human ESCs is also able to reduce CpA methylation but has minimal effects on the level of CpG methylation, suggesting overlapping activities between DNMT3A and DNMT3B (131). However, in mouse brains, DNMT3A levels are higher than DNMT3B, suggesting that DNMT3A plays a larger role in methylating CpH in the brain (132). Analysis of CpH methylation across species revealed that CpH methylation in the brain is specific to vertebrates (134). During mice and human brain development, CpH methylation increases and accumulates in neurons but not in the glia (130). In mature neurons, CpH methylation, specifically CpA, is the dominant form of methylation, and regions with CAC sequences have the highest methylation level (130, 135). In mice neurons, the genes in TAD compartments with high mCpA levels correlate with genes that are repressed by MECP2, a crucial neuron protein, suggesting that changes in mCpA levels during neuron maturation influence global gene expression (136). CpH methylation also changes during stem cell differentiation, CpH starts out highly methylated but methylation decreases as cell differentiate. Overall, the methylation pattern of mCpH changes during neuron maturation and stem cell differentiation, suggesting that it is crucial in regulating gene expression during development. 12 1.3.3 Hydroxymethylation 5-methylcytosine (5mC) is oxidized into 5-hydroxymethylcytosine (5hmC) by Tet 1, 2 and 3 (137, 138). Knockdown of Tet1/2 reduces 5hmC levels while the triple knockout Dnmt1, Dnmt3a, and Dnmt3b eliminates 5hmC in mESC (139). This suggests that 5mC is required for oxidation into 5hmC (139). Unlike 5mCs, 5hmCs are enriched at CpG islands and promoters and are associated with active transcription in mouse ESCs (139). In the brain, the bodies of genes that are highly transcribed are enriched in 5hmC and depleted of 5mC (130, 140). Similar to mCpH, 5hmC levels also decrease as ESCs differentiate (141). Moreover, 5hmCs are depleted at regions containing the repressive marks, H3K27me3 and H3K9me3 in the brain and during mouse embryo development (142, 143). 5hmC is crucial for regulating pluripotent genes in embryonic stem cells and localizes to euchromatin and CpG islands (139). In the brain, 5hmC is localized at active gene bodies and depleted around the TSS (144). Like mCpH, 5hmC is enriched in mature, but not immature neurons in the mouse cerebellum and hippocampus (144). Genes that gained 5hmC during aging are associated with neurodegenerative disorders (145). In addition, the important neuron protein, MECP2 has a specificity for 5hmC that is not found in other methyl-CpG-binding domains, suggesting that 5hmC may help target MECP2 to distinct regions of the genome in neurons (140). 1.4 Methyl-CpG-binding protein 2 Methyl-CpG-binding protein 2 is ubiquitously expressed in all cell types but is the most abundant in neurons, making it a crucial brain protein (115, 146). Mutations that result in loss of MECP2 function are associated with the neurodevelopmental disorder, Rett syndrome (RTT) (147). The MECP2 gene is located on the X chromosome, making RTT a predominantly female 13 disorder. Rett syndrome involves a delayed onset of symptoms involving a progressive loss of speech and motor skills in children 6-18 months after birth (147). On the other hand, overexpression of MECP2 due to duplication of the MECP2 gene on the same X chromosome, called MECP2-duplication syndrome also results in RTT-like phenotypes (148). Unlike RTT, duplication syndrome is commonly found in males with female mothers being the carrier (148). Within, neurons, MECP2 is so abundant that its amount is close to the level of histone H3 (115). While abundant, deviation in its amounts can be detrimental to brain homeostasis as mice with a 50% reduction in MECP2 display RTT-like phenotypes, such as disruptions in breathing, social behaviour and movement (149). Likewise, mild over-expression of MECP2 in mice results in seizures and in some cases, death (150). These results suggest that small changes in MECP2 levels have a drastic effect on brain homeostasis. 1.4.1 Structure MECP2 consists of five major domains, the N-terminal-domain (NTD), methyl CpG-binding domain (MBD), intervening domain (ID), transcriptional repression domain (TRD), and the C-terminal domain (CTD) (Figure 1) (114, 151–154). About 60% of MECP2 is disordered, with the MBD being mostly structured (152). The MBD was one of the few MECP2 domains to be crystallized and studied structurally (155). Structural analysis of the MBD showed that the domain not only recognizes and binds to methylated CpG regions, but can also bind to methylated and unmethylated CA (114, 155). The CTD is composed of two parts, CTD-α and CTD-β (154). The CTD-β region can interact with nucleosome arrays, but not naked DNA in vitro, suggesting that it can interact with histones (152). The TRD is highly disordered, and deletion of the TRD abrogates the transcriptional repressive function of MECP2 (151, 154). The NCoR/SMRT 14 interaction domain (NID) (amino acids 285-309) is located at the C-terminal region of the TRD and interacts with the co-repressor complex NCoR/SMRT (156, 157). The NID is the other part of MECP2 that was studied structurally (157). Crystallization of part of the NID (amino acids 297-307) bound to the paralog of TBL1, TBLR1, a part of the NCoR/SMRT complex revealed that the four important amino acids for the interactions were mutated in people with RTT (157). The Nuclear localization signal (NLS) is also located within the TRD at amino acids 255-271, but interestingly, deletion of the NLS does not hinder the localization of MECP2 to the nucleus (114, 158, 159). Thus, it has been suggested that the affinity of the MBD for DNA is enough for MECP2 to localize to the nucleus (158). There are three AT-hook regions, located within the ID, the TRD, and another in the CTD, which assist in the binding of MECP2 to chromatin (160–162). The ID region helps stabilize the MBD and enhances its interactions with methylated or unmethylated double-stranded DNA (152, 153). There are two isoforms of MECP2, generated via alternative splicing (163, 164). MECP2-E1 contains the 1, 3 and 4th exon, skipping exon 2, while MECP2-E2 contains exons 1-4, but translation initiates from the start codon in exon 2, so only exons 2,3 and 4 are translated (163, 164). The two isoforms are similar but E1 has a longer N-terminus compared to E2 (163, 164). The E1 isoform is more abundant and also has greater stability than E2 in neurons (164, 165). A large majority of mutations associated with RTT occur in exons 3 or 4, which is common to both E1 and E2 (166). To date, RTT-associated mutations are found on exon 1 but not exon 2 (167–170). The deletion of exon 1, but not exon 2, in mice results in RTT-like neurological defects, suggesting that the two isoforms play different roles in the brain (165, 171). 15 Figure 1. MECP2 isoforms and common RTT-associated mutations Shown are two isoforms of the methyl-CpG-binding protein 2 (MECP2), called E1 and E2. The isoforms are identical aside from the beginning of the N-terminal region. The sequences unique to each isoform are labelled. NTD: N-terminal domain, MBD: methyl-CpG-binding domain, ID: intervening domain, TRD: transcription repression domain, NLS: nuclear localization signal, NID: NCoR/SMRT-interaction domain, CTD: C-terminal domain. The NLS and NID are located within the TRD and thus are delineated with dashed lines. The X denotes a stop codon. RTT-associated mutations are numbered according to the amino acid (aa) position on MECP2-E2 (172). Note that the diagram is not drawn to scale. 16 1.4.2 Binding of MECP2 to methylated CpGs MECP2 was initially discovered to bind to methylated CpGs (mCpGs), hence the name methyl-CpG-binding-protein 2 (114, 173). The first study that identified MECP2 from rat nuclear extracts found that, unlike MECP1, MECP2 can bind to DNA probes containing only a single mCpG, but not to unmethylated probes (162). Moreover, they found that MECP2 localizes to, and forms puncta, at pericentric heterochromatic regions in rat liver fibroblasts and mouse fibroblasts (114, 162). This region is rich in methylated CpGs (~40% of mCpG in the mouse genome), supporting the results of the in vitro DNA probe binding assay (162). MECP2 can also interact with methylated DNA in vitro to form phase-separated condensates (174, 175). An in vitro transcription assay using purified MECP2 from rat brain and HeLa nuclear extract showed that MECP2 reduced expression of a methylated, but not an unmethylated template (151). Likewise, using a fusion MECP2-GST construct, they found that the level of transcription repression depends on the density of CpG methylation (151). Expression of exogenous rat MECP2 in WT or mutated mouse ESCs with reduced mCpG (5% mCpG compared to WT) found that decreased methylation also disrupted the localization of MECP2 in the nucleus (114). Several deletions of MECP2 were tested to explore which part of MECP2 is crucial for binding methylated CpGs. A partial deletion of the MBD of rat MECP2 (amino acids 99-126) disrupted binding to methylated CpGs in vitro, and the mutated protein was largely dispersed throughout nuclei instead of forming puncta at heterochromatin, despite having an intact NLS (114). Moreover, ChIP-seq or ChIP-PCR analysis of MECP2 showed that its occupancy pattern matches that of methylated CpG density in the mouse brain (115). These studies confirmed that the MBD of MECP2 binds mCpG regions to and is involved in suppressing transcription. 17 1.4.3 Binding of MECP2 to methylated CpHs Although studies have shown that MECP2 binds mCpG regions, there were some discrepancies in the results. For example, although MECP2 distribution matches that of mCpG globally, misregulated genes in Mecp2-null or Mecp2-overexpressing adult mice brains do not correlate with mCpG regions (176). In addition, while the D99-126 deletion disrupted binding of the MBD to mCpGs and dispersed the majority of MECP2 throughout nuclei, around 20% of MECP2 still localized weakly to heterochromatin (114, 151). These results suggest that MECP2 may target regions aside from mCpGs through domains aside from the MBD. There are two other regions that MECP2 may target, either non-CpG methylation (mCpH, H= A, C, or T), or unmethylated regions. In the mouse brain, at regions where mCpG is low, MECP2 localizes to areas with high mCpH (176). In addition, higher levels of MECP2 are found at gene bodies with higher mCpH, and many genes in these areas are misregulated in the brains of Mecp2-null or Mecp2-overexpressing adult mice (176). The same pattern was observed at long genes (>100kb), where ChIP-seq analysis revealed that there is a high correlation between high mCpA density and MECP2 localization (133). It is important to note that long genes are more highly expressed in the brain compared to other tissues in mice and humans, and long genes upregulated in Mecp2 knockout (KO) or mutant mice are often associated with neuronal functions (133). The importance of MECP2 and mCpH in the brain is supported by the fact that ChIP-qPCR showed that MECP2 levels are enriched at mCpH sites, but not mCpG, on the Bdnf locus as mice mature (176). This suggests that an increase in MECP2 localization at mCpH regions as the brain matures may explain the delayed symptoms associated with RTT. 18 In vitro, the MBD of MECP2 binds not just to methylated CpGs but also to methylated CA (mCpAs) and hydroxymethylated CA (hmCpAs) (133). However, it does not interact with hmCpG, hm/m CC or hm/mCT (133). Thus, we will focus the rest of the discussion on methylated CpA. MECP2 interacts more strongly with CpA repeats that are five repeats or longer than a single CpA (177). At CpA repeats, MECP2 has a preference for hmCpA over mCpA or unmethylated CpA (177). MECP2 can distinguish hmCpA from other modifications through arginine 133 (177). Further, at long genes with high levels of mCpA in the brain, Mecp2-KO mice have an increased amount of RNA polymerase II at transcription start sites compared to WT (178). No change in RNAPII at gene bodies was detected between WT and Mecp2-KO mice, suggesting that at long genes with high mCpA, MECP2 affects RNAPII initiation but not elongation (178). A recent study generated a chimeric MECP2 with the MBD swapped with its ortholog, MBD2 that can bind mCpG, but not mCpA both in vitro and in vivo (179). All other parts of MECP2 were retained, but mice expressing the MECP2-MBD2 protein (MM2) had increased severity in RTT-like phenotypes, and decreased lifespan and weight, although not as severe as Mecp2-null mice (179). The MM2 mice also had motor defects and hindlimb clasping that are reminiscent of RTT-like phenotypes in mice (179). Collectively, these results demonstrates that the unique specificity of MECP2 for methylated CpA sites may be crucial for its function in the brain. 1.4.4 Binding of MECP2 to unmethylated regions Aside from methylated CpG or CpA regions, MECP2 also interacts with unmethylated regions. Global analysis using ChIP-chip for MECP2 distribution in human SH-SY5Y neuronal cells showed that there was no correlation between highly methylated sites and MECP2 distribution on the genome, and only 6% of MECP2 bound sites are CpG methylated (180). These 19 results suggest that MECP2 is not limited to methylated regions. This is supported in vitro, where rat MECP2 also binds unmethylated DNA probes and can inhibit the transcription of unmethylated templates albeit less than methylated ones (181). In mouse olfactory epithelium (OE) cells, MECP2 associates with H3K27me3 at hypomethylated regions, suggesting that DNA methylation does not dictate MECP2 distribution across the genome (182). Therefore, methylation alone cannot fully explain how MECP2 interacts with chromatin. In vitro, MECP2 cannot form phase-separated droplets on its own but can when it interacts with unmethylated DNA and droplet size increase if the DNA is methylated (174). Thus, it is likely that methylation merely enhances the interaction, but is not essential for MECP2 to bind DNA. Importantly, MECP2 can bind and compact unmethylated nucleosome arrays into secondary structures in vitro, demonstrating that regulation of chromatin structure does not rely on DNA methylation (183). In addition, overexpression of human MECP2 in Drosophila melanogaster, which lacks methylated DNA, results in defects in flight capabilities and dendritic development (184). These phenotypes mirror MECP2-duplication syndrome and suggest that even in the absence of DNA methylation, overexpression of MECP2 can still cause neurological defects. Collectively, these results suggested that DNA methylation is not essential for MECP2 to interact with chromatin. 1.4.5 MECP2 and the linker histone H1 MECP2 localizes to linker regions on chromatin. The 5hmC sites that MECP2 preferentially binds to are easily digested with low MNase concentrations in cerebellar nuclei, meaning that the sites are highly accessible to nucleases, such as linker regions (140). This is supported via in vitro binding studies, which showed human MECP2 binds with similar affinity to nucleosomes with either one or two linkers, but not to the nucleosome core particle without a linker 20 (185). An increase in H1 levels in a Mecp2-null compared to WT was also observed in neurons derived from mouse ESCs (186). Fluorescence microscopy demonstrated that H1 and MECP2 both co-localize to the pericentric heterochromatin in the nucleus of mouse fibroblast cells (187). Likewise, the distribution of MECP2 on the genome matches that of H1 in mouse OE cells, as was demonstrated through ChIP-seq, MNase-seq and ChIP-qPCR (182). While it may seem like MECP2 is simply an H1 substitute, several properties distinguish these two proteins. Salt extractions of MECP2 from rat brain nuclei demonstrated that MECP2 is eluted easier from nuclei than H1 which requires a higher salt concentration, suggesting that MECP2 and H1 interact with chromatin differently (181). And a recent study showed that MECP2 and H1 form distinct phase-separated condensates that are close but do not overlap in hippocampal neurons or NIH 3T3 cells (175). Therefore, MECP2 localizes to similar regions as H1, but the two proteins may regulate chromatin differently. MECP2 not only localizes to the same region as H1 but has also been suggested to compete with H1 for the same binding sites. Quantitative Western Blot analysis of neuronal, but not glial nuclei, demonstrated that depletion of MECP2 levels increases the abundance of histone H1 (115). An in vitro competition assay has shown that there is an increasing amount of H1 displaced from methylated nucleosomes with increasing concentrations of MECP2 added to the mixture, and vice versa (187). It has also been shown that MECP2 competes better with the mobile (readily exchangeable) fractions of H1 rather than H1 tightly bound to the chromatin (187). This agrees with the initial study that observed that MECP2 cannot displace all H1, and mostly displaces H1 from methylated, but not unmethylated, minichromosomes in Xenopus oocytes (151). However, MECP2 is better at displacing H1 from nucleosomes than H1 is at displacing MECP2 from nucleosomes in vitro (187). This may explain why the overexpression of H1 did not alter MECP2 21 distribution in mouse forebrain excitatory neurons (188). If MECP2 competes with H1, it may explain the reason why the loss of MECP2 results in a global increase in H1 levels (115). However, ChIP-seq analysis of H1 in mouse neurons disputed this result, as H1 occupancy and distribution did not change when MECP2 was depleted (188). A likely explanation is that the increase in H1 levels observed by Western blot encompasses both free and bound H1, and thus increases in H1 levels in nuclear extracts does not mean that the distribution of H1 on chromatin is altered, as demonstrated with H1 ChIP-seq (115, 188). The other point is that although the in vitro competition assay detects loss of H1 from chromatin, it does not prove that MECP2 occupies the same region freed by H1 (187). There are also several differences between the experiments that may explain the conflicting results. First, in the H1 ChIP experiment, they used transgenic mice that overexpress flag-tagged H1 such that it was 1.5 times greater than normal (188). Thus, the displacement of H1 by MECP2 may be masked by the increased abundance of H1. Second, it is important to mention that the H1 ChIP-seq study used unsorted cortex tissues, while Skene et al. noted that they only observed changes in H1 levels between WT and Mecp2-null mice in sorted neuronal nuclei (115, 188). Therefore, the results may vary due to the cells used in the experiment. The other contradictory finding was that MECP2 overexpression does not change H1 levels in mouse neuronal nuclei even though MECP2 depletion increases the amount of H1, (115, 188). If MECP2 does compete with H1, then more MECP2 should displace more H1. Thus, a direct competition between MECP2 and H1 cannot fully explain their relationship. 1.4.6 MECP2 and co-repressors Expression of only the N-terminus and the MBD results in weak transcription inhibition in vitro, suggesting that binding to methylated CpG alone is insufficient for the repressive function 22 of MECP2 (151). MECP2 was found to associate with several co-repressors, such as mammalian Sin3A (mSin3A) and NCoR/SMRT (156, 189, 190). Immobilized GST-MECP2 fusion protein was able to pull down the mSin3A-HDAC complex (mSin3A and HDAC1 and 2) from HeLa nuclear extracts (72, 189). The same interaction was observed when using an anti-MECP2 antibody that isolated mSin3A from rat brains (189, 191). Several deletions of the GST-MECP2 fusion construct were used to show that the intervening domain and a part of the TRD on MECP2 interact with the N-terminus of the HID domain on mSin3A and also with the C-term of mSin3A (189). Repression by GAL4-TRD in vivo was not completely abolished with trichostatin A (TSA) (an HDAC inhibitor) in mouse fibroblasts, suggesting that repression is not completely dependent on the interaction between MECP2 and an HDAC (189). Moreover, size exclusion chromatography of rat brain extracts was unable to detect MECP2 bound to mSin3A and vice versa but was able to detect an HDAC1/2 association with mSin3A (191). Therefore, it is likely that the interaction between MECP2 and mSin3A-HDAC is unstable or transient. The interaction between NCoR/SMRT and MECP2 has been extensively studied. MECP2 interacts with NCoR/SMRT via its NID which is located on the C-terminal region of the TRD (156). Immunoprecipitation from mouse brain extracts identified mSin3A, and the NCoR/SMRT complex as interaction partners of MECP2 (156). Deletions of various MECP2 segments in HeLa cells revealed that the removal of the amino acids 285-309 (the NID of MECP2) abolished the binding of MECP2 to NCoR/SMRT but did not affect binding to mSin3A (156). Mice expressing MECP2 with only the MBD and NID region co-immunoprecipitated with the components of the NCoR/SMRT complex—NCoR, HDAC3 and TBLR1 (192). Thus, MECP2 interacts with NCoR/SMRT through the NID and this region does not bind mSin3A-HDAC. Like with the mSin3A-HDAC complex, MECP2 does not directly bind to HDAC3 and instead interacts with the 23 complex at the N-terminal region of NCoR1 and the C-terminal region TBR1 of the NCoR/SMRT complex (156, 157). Several RTT-associated mutations occur on the NID of MECP2: P302R, K304E, K305R, and R306C (156). These amino acids play important roles in the interaction between MECP2 and the NCoR/SMRT complex as demonstrated with in vitro structural studies and in vivo pull-down assays from mouse brain extracts (156, 157). MECP2 carrying these mutations failed to co-immunoprecipitate TBLR1 and HDAC3, and treatment of mouse embryonic fibroblast cells expressing WT MECP2 with TSA decreased the repression to the same level as the mutants (156, 157). Moreover, RNA-seq of the forebrain of mature mice with an R306C mutation showed that genes were upregulated in the mutant compared to WT mice, especially at sites with high mCpA and long genes (178). The same mutation also increased RNAPII stalling at the TSSs of long genes with dense mCpA regions (178). Collectively, these results demonstrate that RTT-associated mutations that disrupt the interaction of MECP2 with NCoR/SMRT lead to misregulation of transcription at MECP2 target regions. Male mice expressing only a minimal MECP2 protein with all regions deleted except the MBD and the NID regions (DNIC) had no significant difference in neurological phenotypes compared to mice expressing the full-length MECP2 (192). Importantly, injecting Mecp2-null mature mice with human DNIC protein packaged in a viral vector was able to alleviate the neurological defects compared to those treated with an empty vector (192). That this minimal protein is sufficient to rescue RTT-like phenotypes, highlights the importance of the MBD and NID regions for MECP2 function. Indeed, mice with the T308X truncation mutation that removed four amino acids from the NID have delayed RTT-like symptoms such as motor impairments, hypoactivity and seizures after six weeks of age (193). Yet staining of mice brain tissues for MECP2 with the T308X truncation showed that it still localized to heterochromatic regions like 24 WT, demonstrating that the mutation affects MECP2 function, not the localization (193). A subsequent study using MECP2 T308X-expressing mice demonstrated that less HDAC3 was pulled down with the mutant compared to WT MECP2, confirming that the mutation disrupts the interaction between MECP2 and the NCoR/SMRT complex (156). Note that mice with DNIC lack amino acids 174-271, meaning that a large portion of the AT-hook 2 is deleted (amino acids 264-273) (160, 192). This suggests that AT-hook 2 is not crucial for MECP2 function. This is also supported by initial studies of localization of rat MECP2 truncations which found that deletion of either the AT-hook 1 or 2 regions (amino acids 184-195 and 264-273 respectively) did not compromise puncta formation at heterochromatin, suggesting that MECP2 can still interact with methylated regions despite the absence of the AT-hook domain (114, 160). Collectively, these results demonstrated that the MBD and the NID region of MECP2 are essential for it to function. 1.4.7 Beyond repression Although MECP2 is well known for its association with repressors, it has been shown to play a multi-faceted role in gene regulation. Large-scale analysis of MECP2 binding sites in neurons revealed that 63% of promoters bound by MECP2 are active promoters (180). Moreover, microarray analysis of mouse hypothalamus tissues showed the majority (85%) of the genes regulated by MECP2 were upregulated when MECP2 is overexpressed and downregulated in null mice (194). While it is also likely that the upregulation could be indirect, the study did not find any repressor-encoding genes that are repressed by MECP2 (194). MECP2 also binds to transcription activators such as CREB1 in the mouse hypothalamus and also upregulates the expression of Creb1 by binding to its promoter regions (194). Given that MECP2 levels differ between different parts of the brain and that small changes in MECP2 levels affect its function, 25 studies using the hypothalamus may not be directly comparable to other regions (146, 149, 150, 195). However, when the same group repeated their experiment with cerebellum tissues, they found similar results. The majority of the genes (75%) in the cerebellum were upregulated following MECP2 overexpression and downregulated in Mecp2-null mice (196). Interestingly, they found more overlap between genes activated in the cerebellum and hypothalamus than those that were repressed (196). However, whether MECP2 also interacts with CREB1 in the cerebellum was not explored in this study. Collectively, these data demonstrated that MECP2 is not merely a repressor, but potentially an activator and has a broad role in gene regulation. 1.4.8 MECP2 and chromatin MECP2 binds to DNA and nucleosomes to regulate chromatin. Solubilization of MECP2 from rat brain nuclei requires digestion with MNase, demonstrating that MECP2 binds directly to chromatin (181). MECP2 was found to bind throughout entire genes, has a preference for long genes, and can repress transcription initiation through three-dimensional TSS-gene body contacts (115, 133, 178). This suggests that it is unlikely for MECP2 to control transcription at specific sites, but instead, regulate expression by altering chromatin structure. MECP2 regulates nucleosome occupancy at CpA repeats by binding to these sites and creating nucleosome-free regions on chromatin (177). Analysis of hippocampal neurons revealed that loss of MECP2 alters heterochromatic organization (197). In addition, MECP2 can condense chromatin in vitro (183). Collectively, these results suggest that MECP2 can regulate chromatin structure. Aside from the fact that MECP2 may substitute for linker histones in neurons, MECP2 also plays a role in regulating histone PTMs. This is not surprising given that MECP2 binds to or close to H3 on the nucleosome in vitro (185). In vivo, MECP2 co-immunoprecipitated with H3 and 26 weaker with H4 from mouse OE nuclear extracts treated with nuclease (182). MECP2 seems to regulate histone acetylation levels as mice depleted of MECP2 had 1.5 times more H3 acetylation in unsorted brain nuclei, and 2.6 times more H3 acetylation in neuronal nuclei compared to WT (115). Immunostaining of human and mouse cerebral cortex confirmed that H3K9ac levels change with MECP2 during development and ChIP-seq showed that H3K9ac distribution matches MECP2 occupancy around TSSs in mouse OE cells (182, 198). During early development, H3K9ac levels are high and MECP2 levels low in mouse neurons but as the levels of MECP2 increase during maturation H3K9ac levels decrease (198). In contrast, H3K9ac levels do not decrease in Mecp2-null mice, suggesting that misregulation of histone acetylation during development may be associated with RTT (198). MECP2 may regulate H3 acetylation through the recruitment of the NCoR/SMRT complex, which includes HDAC3 (156). This has been supported by the finding that there is less HDAC3 pulled down with the MECP2 T308X mutant, which deletes part of the NID, compared to the WT protein (156). Like Mecp2-null mice, T308X mice also have elevated levels of histone H3 acetylation (115, 193). Moreover, the change in acetylation is most drastic in tissues that have the most MECP2 such as the cerebellum, cerebral cortex and spleen, but not in the liver where MECP2 levels are low (193). Interestingly, male mice expressing MECP2 T308X survive longer than Mecp2-null mice (156, 193). Since the T308X truncation reduces but does not abolish the interaction of MECP2 with the NCoR/SMRT, likely, it can still recruit HDAC3 to some sites and regulate deacetylation (156, 193). It should be noted that histone H4 acetylation was unchanged in T308X (193). This is likely because MECP2 only associates weakly with H4 in vivo (182). Collectively, these findings demonstrate that MECP2 is crucial in regulating histone acetylation on chromatin, through its interaction with the NCoR/SMRT complex. 27 MECP2 not only affects histone acetylation but also histone methylation. ChIP-seq analysis found that the distribution of H3K27me3 matches MECP2 in mouse olfactory epithelium (OE) cells (182). The MBD of MECP2 has a greater preference for binding H3K27me3 compared to non-modified H3 in vitro (182). However, high-resolution imaging of hippocampal pyramidal neurons showed contradictory results, as MECP2 and H3K27me3 do not co-localize on heterochromatin (197). In WT hippocampal neurons, H4K20me3 localizes adjacent to MECP2 and heterochromatic regions, but in Mecp2-null mice, H4K20me3 location is altered and moved into the pericentric chromatin (197). This observation is cell-type specific, as there was no change in the distribution of H4K20me3 in cerebellar granule cells between WT and Mecp2-null mice (197). Aside from changes in the location of histone PTMs, there ~10% increase in the level of H4K20me3 and H3K9me3 throughout the cell in Mecp2-null compared to WT hippocampal neurons (197). Contradictory results have been observed in mouse cerebral cortex, where there is no change in H3K9me3 distribution, but only a decrease in H3K9me3 levels in mice without MECP2 (198). The different results could be due to the use of different cells and methods. Due to contradictory findings, the effects of MECP2 on histone methylation are more ambiguous than the effect on acetylation. Despite this, it is possible that MECP2 can alter chromatin structure by changing the distribution of acetylation and methylation on chromatin. 1.4.9 MECP2 levels in cells Given the high conservation of MECP2, it is no surprise that a lot of MECP2 studies were done in mice and rats (192). However, MECP2 levels vary depending on the cell, tissue, and age of animals used in the study. Studies of mouse and rat extracts found that MECP2 levels are the highest in the brain, followed by the kidney, spleen, and liver, and lowest in the testis (181). 28 MECP2 was also detected in HeLa cells but is very low in mouse ESCs and the gene for a MECP2 homolog is absent in Drosophila (181). Initial microscopy images have shown that the distribution of MECP2 in rat and mouse fibroblast cells is different (162). In mice, MECP2 is concentrated in heterochromatic regions with high DNA methylation while in rats, it is uniformly distributed throughout the genome (162). The distribution of MECP2 is the same between rat cells, monkey COS7 and human HeLa cells (151). Thus, these results suggest that MECP2 location deviates between mice and other organisms. Studies of mouse CNS brain development showed that MECP2 is expressed throughout the brain but only in mature neurons and not the glial cells (146, 195). Moreover, MECP2 levels do not stabilize or reach maximum abundance until mice reach five weeks of age (115). The change in MECP2 abundance likely impacts the distribution of MECP2 in the nucleus as the mouse matures (115, 146). Staining of brain tissues showed that MECP2 forms distinct puncta at heterochromatic regions in mature mice neurons, but is uniform in embryos or newborn mice (146). In addition, in neurons derived from stem cells, they found that the percentage of cells expressing MECP2 increases as neurons mature, from ~20% of cells expressing MECP2 at day 3 to 80% by day 7 (146). Mecp2-null mice have less complex and smaller neurons, and Mecp2-null mouse ESC-derived neurons also have smaller nuclei compared to WT, suggesting that neuron maturation is disrupted (146, 186). Likewise, neurons that express less MECP2 than WT also have smaller nuclei than WT, although the difference was not as drastic as Mecp2-null (186). Importantly, expression of MECP2 in these Mecp2-null neurons is able to partially rescue the phenotype and these neurons have larger nuclei than those without MECP2 (186). The timing of size difference in neuronal nuclei matches with the difference in mRNA levels between the genotypes. During the first three days of differentiation, the total RNA levels were the same between Mecp2-null and WT neurons, but starting at 8 days, the Mecp2-null had 29 significantly lower total RNA amounts than WT (186). The changes in transcript levels may explain the importance of MECP2 in neuron maturation (146, 186, 195). Overall, the variability of MECP2 across different cells and ages, and the fact that MECP2 is sensitive to small changes in its levels (149, 150) may explain contradictory studies regarding its function. 1.4.10 Disease-associated MECP2 mutations Common MECP2 mutations fall into two major categories, missense mutations, and truncation mutations. Figure 1 shows the location of common RTT-associated mutations on MECP2. Genetic analysis of RTT patients found that about 97% of these patients had mutations in MECP2 and the severity of the symptoms varied between the mutations (166). The most common mutations were R255X (13%), T158M (12.2%), and R168X (11.9%) (166). The X denotes mutation to a stop codon which results in a shorter MECP2. Large deletions, such as R168X, result in the most severe phenotypes while smaller deletions like R294X or C-terminal truncations result in milder phenotypes (166). RTT-related mutations are commonly studied in vitro and in vivo as aberrations in mutant MECP2 inform on protein function. In the sections below, we will discuss some of the RTT-related mutations. 1.4.10.1 Point mutations Missense RTT mutations cluster in two major groups, either in the MDB or the NID (156). The three most common missense mutations are T158M and R133C in the MBD, and R306C in the NID (199). Arginine 133 of MECP2 in the MBD plays a key role in helping MECP2 distinguish the 5hmC (177). Although the R133C mutation disrupts the ability of MECP2 to recognize and bind 5hmC sites, patients with this mutation have milder symptoms compared to other mutants 30 (140, 166, 177). While the binding of R133C to 5mC was decreased, the difference was not as drastic as its interaction with 5hmC (140). Thus, it is likely that the specificity of R133 for 5hmC is not essential for MECP2 function, as the protein can still interact with mCpA, mCpG, or unmethylated DNA (115, 133, 177, 183). This has also been supported in vitro, where the R133C mutation does not disrupt the ability of MECP2 to compact chromatin and does not alter the regions that MECP2 binds to on chromatin (183, 185). This may explain why the R133C mutation results in the least severe phenotype out of the three common point mutations (166, 199). As discussed previously, arginine 306 is located in the NID of MECP2 that interacts with the NCoR/SMRT (156). Mice with the R306 mutation have reduced brain weight and premature death (156). Binding interaction and structural analyses revealed that the R306C mutation completely abolishes the interaction between MECP2 and the C-terminus of TBLR1, due to the loss of salt bridges and hydrogen bonding which destabilizes the interaction (157). Fluorescence microscopy images of neurons generated from mouse ESCs showed that the R306C mutation does not disrupt the localization of MECP2, as it localized like WT to heterochromatin regions rich in 5mC (156). However, MECP2 R306C failed to shuttle TBL1, which lacks an NLS, into the nucleus, proving that its interaction with NCoR/SMRT is disrupted (156). Out of the three point mutations, T158M is the most common and results in the most severe phenotype (166, 199). Structural analysis revealed that T158M disrupts a hydrogen bond in the C-terminal region of the MBD, destabilizing the structure and disrupting the binding of MECP2 to methylated DNA in vitro (200). The instability of the T158M has been shown in vivo, where the level of T158M-GFP is 70% less than WT in mature mice (173, 199). Moreover, unlike R306C, T158M does not show concentrated foci and is diffused throughout the nucleus (156, 199). T158M also disrupts binding to mCpG or mCpA oligonucleotides (199). These results demonstrate that T158M is unable to associate with DNA. 31 Interestingly, overexpression of MECP2 T158M partially alleviates the RTT-like phenotype in male and female mice (173). This suggests that protein levels are key in MECP2 function. This has been supported in vitro, where MECP2 can form phase-separated condensates, and this is sensitive to protein concentrations (174, 175). Both the T158M and R133C mutations drastically reduced droplet size compared to WT MECP2, and this reduction was less pronounced in the R306C mutant, indicating that mutations in the MBD disrupt transient protein interactions (174). Collectively, these results show that point mutations that destabilize the protein or disrupt the interactions between MECP2, DNA and co-repressors alter function, leading to RTT. 1.4.10.2 Truncation mutations Common RTT-associated truncation mutations cut off the C-terminal region after the MBD of MECP2 (166, 174). Both R168X and R255X proteins could not be detected through Western blot or immunofluorescence from mouse brains, suggesting that these proteins are unstable or rapidly degraded (201–203). The instability of these two truncated proteins agrees with their inability to form phase-separated condensates even at high concentrations in vitro (174). This makes it challenging to study the effect of these mutations on MECP2 function in mice on a molecular level. However, R168X has been studied in vitro (183). The R168X truncation, which only has the NTD-MBD region, can still interact with nucleosome arrays, but cannot condense them into secondary structures (183). This suggests that the MBD is responsible for DNA binding and that the C-terminal region is required for higher-order chromatin regulation. Both R270X and G273X truncations have defects in repression compared to WT, yet localize normally to heterochromatin, suggesting that the TRD function is disrupted (160). R270X and G273X interact with chromatin differently. The G273X truncation is slightly better at oligomerizing methylated 32 nucleosome arrays than R270X, and G273X, but not R270X can bind DNA probes in vitro (160). Interestingly, the lifespan of MECP2 R270X-expressing mice is much shorter than G273X (160). The premature death could be explained by the lower brain weight of R270X during development which is similar to Mecp2-KO mice (160). The only difference between these two is the extra three amino acids that coincide with the location of AT-hook 2 (160). It should be noted that although tremors and motor deficits of G273X are delayed compared to R270X or KO mice, these phenotypes eventually develop (160). Thus, the AT-hook domain may be important, but not crucial to MECP2 function. Mice with the R294X mutation have milder phenotypes and survive longer than R255X mice (202). Unlike R255X, the R294X protein is more stable and can be detected by immunoblot of mouse brain lysates (202, 203). The R294X truncation localizes to heterochromatin like WT MECP2, but purification of R294X from mouse brain nuclei requires higher salt concentration than WT MECP2, suggesting that it may interact with chromatin differently (202). In vitro, R294X can still bind to nucleosome arrays and generate a similar MNase digestion pattern as WT MECP2, but it is unable to compact chromatin, suggesting that the C-terminal region is crucial for broad range or multiple interactions with chromatin (185). However, studies of mouse cortical neurons transfected with MECP2-R294X-GFP did not affect dendritic growth compared to WT in the three days that were tested, which may explain why the mutation is milder than the shorter truncations (166, 204). The P389X truncation (also called CTD2) can localize to heterochromatin, bind NCoR/SMRT and mSin3A, and shuttle TBL1 to the nucleus, suggesting that this mutation does not disrupt the function of MBD or NID (205). Mouse brains and cultured neuron cells from MECP2 P389X-expressing mice had similar protein and mRNA levels as WT MECP2 and these mice have similar phenotypes as WT mice (205). Coinciding with its mild phenotype, the condensate formation of P389X was slightly smaller than WT MECP2 (174). 33 However, when the human MECP2 is expressed in mouse and human cultured neurons, the P389X mutation destabilizes MECP2, resulting in less mRNA than WT (205). Overall, larger deletions result in more severe phenotypes and premature death which could be caused by a decrease in MECP2 abundance due to unstable protein or disruptions in MECP2 function due to deletions of critical domains. 1.5 Summary of research hypothesis Due to associations between MECP2 mutations and neurodevelopmental disorders, a large majority of in vivo studies have focused on studying MECP2 in human/mouse tissues or cell lines. DNA methylation plays an important role in development (118). Thus, knocking out DNMTs may result in undesired side effects, making it difficult to distinguish whether the effects were directly associated with MECP2 function. Although it has been shown that MECP2 regulates unmethylated chromatin in vitro, and overexpression of MECP2 in flies that lack DNA methylation results in motor defects (183, 184), it remains unclear whether MECP2 can regulate unmethylated DNA in vivo. We hypothesized that MECP2 interacts with unmethylated chromatin in vivo. To test our hypothesis, we expressed human MECP2 in Saccharomyces cerevisiae. Like Drosophila, there is no DNA methylation in yeast. Moreover, the nucleosome repeat length of yeast and neurons are similar (both ~165 base pairs), while most mammalian cells have a NRL of 190 base pairs (28, 29). This allowed us to partially recapitulate the neuron chromatin conditions in yeast. Note because we are expressing human MECP2, throughout the thesis, we will refer to the gene as MECP2 and the protein as MECP2, which coincides with the conventions for human genes and proteins. We first confirmed that we could induce the expression of human MECP2 in yeast in section 3.1. Next, we analyzed the consequences of expressing MECP2 in yeast by analyzing 34 changes in viability and cell cycle in section 3.2. Finally, we tested various disease-associated mutations to see whether they disrupt the function of MECP2 in section 3.3. Our results demonstrated the advantage of using yeast as a model organism to study and screen for MECP2 mutants in vivo. Furthermore, we were able to recapitulate the effects of MECP2-disease-associated mutations in yeast. 35 Chapter 2: Materials and Methods 2.1 Strain generation All plasmids generated in this study were created with NEBuilder. A list of the plasmids used in this study and their sources are in Table 1. Unless otherwise stated, all enzymes and buffers used for cloning were from New England Biolabs (NEB). All plasmid restriction digestions were carried out by incubating the plasmid and restriction enzyme mix at 37°C for 15 minutes. The human MECP2-E1 gene in a pMA plasmid was custom ordered from Invitrogen. The sequence was codon optimized for yeast and listed in Table 2. All primers were designed with NEBuilder Assembly Tool (NEB). The primers contained sequences homologous to the integration site on the vector. The primers used to generate the plasmids made in this study is listed in Table 3. The MECP2-E1 gene was amplified with polymerase chain reaction (PCR) using the primers listed in Table 3. The PCR enzyme used in the study was KAPA HiFi HotStart ReadyMix (Kapa Biosystems, KM2605). The size of the amplicon was confirmed via gel-electrophoresis using a 1% agarose gel with SYBR safe. The DNA was purified from the PCR reaction with the E.Z.N.A Cycle Pure Kit (Omega Bio-Tek). The galactose-inducible plasmid that contained an ampicillin marker for bacteria selection, pGAL1pr-CYC1t-416 was generated by Dr. LeAnn Howe. This plasmid was digested with BamHI. The amplified MECP2-E1 gene was incubated with the linearized plasmid at a 1:2 ratio (vector:insert) in 2 µL and topped to 10 µL with sdH2O. Next, 5 µL of this mixture was mixed with 5 µL of NEBuilder HiFi DNA Assembly Master Mix (NEB, M5520A) and incubated at 50°C for 15 minutes. Following incubation, 5 µL of the ligated product was mixed with 50 µL of DH5-α competent cells and incubated on ice for 30 minutes. The cells 36 were heat shocked at 42°C for 90 seconds, incubated on ice for 1 minute and then plated on LB-ampicillin and incubated at 37°C overnight to select for cells containing the plasmid. Individual colonies were picked from the plate and grown in liquid LB-ampicillin cultures at 37°C overnight. The next day, cells were pelleted at 4000 rpm for 3 minutes. The plasmids were isolated from bacteria cells using the E.Z.N.A Plasmid DNA Mini Kit (Omega Bio-Tek). The plasmid sequences were confirmed with Sanger Sequencing. Superfolding GFP (sfGFP) or Glutathione S-transferase (GST) were conjugated to MECP2 via cloning. The sequences for sfGFP and GST are listed in Table 2. To clone sfGFP or GST to the N-terminus of MECP2, primers were designed to remove the stop codon at the end of sfGFP or GST. The sequences for the stop codons are highlighted in red in Table 3. The pGAL1pr-MECP2-E1-CYC1t-416 plasmid was linearized with SpeI. Cloning sfGFP or GST to the C-terminus of MECP2 required two PCR amplifications, one to amplify MECP2 to remove its stop codon, and one to amplify the protein tag. The pGAL1pr-CYC1t-416 plasmid was digested with BamHI and joined with the two amplicons. The three fragments were combined at a ratio of 1:2:2 (vector:insert1:insert2). The plasmids for the estradiol-inducible MECP2 and the MECP2-E2 mutants were generated by Dr. LeAnn Howe. The sequence for the MECP2-E2 isoform was derived from MECP2-E1 using the plasmid pGAL1pr-MECP2-E1-CYC1t-416 as a template with primers containing sequences unique to E2. The sequences unique to each isoform are highlighted in red in Table 2. All the GFP-tagged MECP2-E1 and E2 mutants were generated using the same primer pair, but different vectors. First, each of the six MECP2-E2 mutants was digested with HindIII and gel purified with the E.Z.N.A Cycle Pure Kit (Omega Bio-Tek) to isolate the 6000 bp vector fragment. The primers amplified the sfGFP and the N-terminus of MECP2 on pZFpro-sfGFP-MECP2-E1-CYC1t-406 or pZFpro-sfGFP-MECP2-E2-CYC1t-406. The sfGFP-MECP2-E1 amplicon was joined to the linearized vectors containing the mutations to 37 make the sfGFP-MECP2-E1 mutants. Likewise, the sfGFP-MECP2-E2 fragment was combined with the mutant vectors to make sfGFP-MECP2-E2 mutants. All yeast strains were derived from FY602, a “wild-type” strain that is isogenic to S288C. Strains used in the experiments are listed in Tables 4-7. To generate the galactose-inducible MECP2 strains, pGAL1-MECP2-E1-CYC1t-416 was transformed into FY602, and cells with the plasmid were selected for on uracil-depleted media. The galactose-inducible plasmids were not integrated into the genome. Unlike the galactose-inducible strains, the estradiol-inducible strains were generated by integrating multiple plasmids into the yeast genome. First, a plasmid containing the sequence for ZF4-ER-VP16 and a LEU2 selection marker was transformed into the yeast genome. This was carried out by linearizing pZF4-ER-VP16-405 by cutting at LEU2 with EcoRV. Once the linearized plasmid was transformed into yeast, it integrated into the genome via homologous recombination at the leu2Δ1 locus. This generated the ZF4-ER-VP16-expressing strain. Next, in a similar manner, plasmids expressing either tagged or untagged MECP2-E1 or MECP2-E2, or the MECP2 mutants and URA3 selection markers were linearized at URA3 using NcoI and transformed to the ZF4-ER-VP16 strain for integration into the ura3-52 locus. The transformation protocol used in the above experiments is as follows. The corresponding starter strain was grown to saturation overnight at 30°C with a shaking speed of 225 rpm. The next day, the optical density (OD600) was measured, and the culture was diluted to an OD of 0.10 and grown to mid-log phase (OD ~0.4-0.5). The cells were pelleted at 4000 rpm for 3 minutes, and washed with 5 mL sdH2O and resuspended in 1 mL sdH2O. Following resuspension, the cells were pelleted again at 7000 rpm for 1 minute. Then, the pellet was resuspended in 240 µL of 50% polyethylene glycol (PEG), 36 µL 1M LiAc, 50 µL 2 mg/mL denatured carrier (calf thymus) DNA, and 34 µL of linearized plasmid at 30°C for 30 minutes. Note that in the generation 38 of the galactose-inducible strains, the plasmids were not linearized because it contains a yeast origin of replication and thus could be maintained without genome integration. After incubation, the suspension was heat shocked at 42°C for 20 minutes, and the cells were pelleted at 7000 rpm for 1 minute and resuspended in 300 µL sdH2O. The cells were plated on either -uracil for the galactose-inducible strains, or on -leucine media for the ZF4-ER-VP16-expressing strain, or on -leucine-uracil for the estradiol-inducible strain containing the sequence encoding MECP2 and ZF4-ER-VP16. All media were supplemented with 2% dextrose. The plates were incubated at 30°C for 3 days and three colonies were randomly selected and frozen in the same media with 10% DMSO at -80°C. Fresh cells were streaked out from freezer stocks onto the appropriate media and grown for three days at 30°C before using them for downstream experiments. Fresh cells were streaked out every week from freezer stocks. Plates older than a week were discarded and not used for experiments. 2.2 Inducing protein expression 2.2.1 Galactose induction Cultures were grown to saturation in yeast extract-peptone (YP) with 2% dextrose (YPD) overnight at 30°C with a shaking speed of 225 rpm. The next day, the optical density (OD) of each sample was measured and the cultures were diluted in YP with 2% raffinose to an OD of 0.10 and grown until mid-log phase (OD ~0.4-0.5). Once cultures reached mid-log phase, galactose (final concentration: 2%) was added to the cultures and the cultures were incubated for 1-3 hours at 30°C and 225 rpm. 39 2.2.2 β-estradiol induction Unless overwise stated, cultures were grown to saturation in -uracil-leucine with 2% dextrose overnight at 30°C, with shaking at 225 rpm. The next day, the OD of each sample was measured, and the cultures were diluted in -uracil-leucine with 2% dextrose to an OD of 0.003 and grown until mid-log phase. Once cells reached mid-log, they were diluted to OD 0.4. A mixture of 2 µM β-estradiol (Sigma-Aldrich, SKU: E8875-250MG; resuspended in ethanol) in -uracil-leucine with 2% dextrose was added to the cultures at a 1:1 ratio (final OD 0.2 with 1 µM β-estradiol). The mixture was incubated for 1 hour at 30°C, 225 rpm to induce expression of the target protein. For 4-hour induction times, mid-log cultures were diluted to OD 0.3 (final OD 0.15 after adding estradiol) to prevent them from reaching saturation during estradiol induction. For the experiment that tested various concentrations of estradiol, 0-100 µM β-estradiol was spiked directly into a mid-log culture that was diluted to OD 0.2, and the culture was incubated for 1 hour to induce protein expression. For the experiment that tested different induction times, a mid-log culture was diluted to OD 0.1 and 5 mL was aliquoted into another flask. To the aliquot, 1 µM β-estradiol was spiked in and the culture was induced for 7 hours at 30°C, 225 rpm. The culture without estradiol (original culture) was also incubated at 30°C 225 rpm. At 1-hour intervals, 5 mL was aliquoted from the original culture and 1 µM β-estradiol was spiked into the aliquot. After 7 hours, eight flasks (T = 0, 1, 2, 3, 4, 5, 6, 7 hours) for each strain were removed from the incubator and the cells were pelleted for subsequent experiments. 2.3 Western blots After protein induction, cultures were pelleted at 7000 rpm for 1 minute to remove the supernatant. The pellets were resuspended in sdH2O and normalized to an optical density unit 40 (ODU) of 1 (OD 1 per 1 mL). Following normalization, the cells were incubated with 2.4 µL of 2M NaOH for 15 minutes at room temperature. Next, 14 µL of 3x SDS solvent (18 mM tris-HCl pH 6.8, 9% SDS, 27% glycerol, 10% β-mercaptoethanol, and 0.03% bromophenol blue) and 8 µL 5x running buffer (959 mM glycine, 125 mM tris-base, and 17.34 mM sodium dodecyl sulphate (SDS)) were added to the lysates and boiled for 5 minutes at 100°C to denature the proteins. The lysates were loaded onto 10% SDS-polyacrylamide gels and the proteins separated at 150V for 1 hour. The gel and nitrocellulose membrane were then equilibrated in transfer buffer (16.65 mM glycine, 0.00128% ethanolamine, and 0.2% methanol) for 10 minutes at room temperature. Following equilibration, the proteins were transferred from the gel to the membrane with 100V for 1 hour at 4°C. Phosphate-buffered saline (PBS) was made in-house and was used at 1x concentration (136.89 mM NaCl, 2.68 mM KCl, 10.14 mM Na2HPO4, and 1.76 mM KH2PO4 at pH 7.4 unless otherwise specified). To make PBST, Tween-20 was diluted in 1x PBS to 0.1%. Once the proteins were transferred onto the membrane, the blot was blocked with 1% bovine serum albumin (BSA) (VWR, CAS: 9048-46-8, diluted in 1x PBST) at room temperature for 1 hour. After blocking, the blots were incubated with the primary rabbit-anti-MECP2 antibody (Sigma-Aldrich, SKU: M9317-200UL; diluted 1:4000 in 1x PBST) overnight on a rotating platform at 4°C. The next day, blots were washed once with 1x PBST for 15 minutes followed by two 5-minute washes. The blots were incubated with a secondary antibody, IRDye 680RD goat anti-rabbit IgG (Li-Cor, P/N: 926-68071; diluted 1:25000 in 1x PBST) for 45 minutes at room temperature. Next, the blots were washed once with 1x PBST for 15 minutes followed by two 5-minute washes before imaging with a Chemidoc (Biorad). For loading control analysis, the same blots were further washed once with 1x PBST for 15 minutes followed by two 5-minute washes and then probed with a mouse anti-PGK1 monoclonal antibody (Invitrogen, Catalog # 459250; 41 diluted 1:4000 in 1x PBST) overnight on a rotating platform at 4°C. The next day, blots were washed once with 1x PBST for 15 minutes followed by two 5-minute washes and incubated with a secondary antibody, IRDye 800CW goat anti-mouse IgG (Li-Cor, P/N: 926-32210; diluted 1:50000 in 1x PBST) for 45 minutes at room temperature and washed once with 1x PBST for 15 minutes followed by two 5-minute washes before imaging. 2.4 Growth assays Colonies were picked from freshly streaked plates and resuspended in 200 µL of sdH2O. Approximately equal-sized colonies were chosen to ensure the suspensions were similar in concentration. The suspensions were serially diluted 10-fold five times from 1x to 10000x in sdH2O and 5 µL of each dilution was spotted onto YPD, -uracil/dextrose, or -uracil/galactose plates for the galactose-inducible strains, or onto -leucine-uracil/dextrose with 0 µM β-estradiol or -leucine-uracil/dextrose with 1 µM β-estradiol for the estradiol-inducible strains. The plates were incubated at 30°C and imaged on days 2 and 3 to monitor changes in growth. 2.5 Flow cytometry for quantifying protein expression levels After the strains were induced with estradiol to express GFP-MECP2, 1 mL of cultures were pelleted at 7000 rpm for 1 minute, washed once with 1x PBS, and resuspended in 1 mL 1x PBS. The samples were vortexed, and the fluorescence signal was measured with the Guava easyCyte System (Millipore Corporation) using the built-in InCyte software. The FSC files were plotted in R Studio using a combination of the following flow-cytometry-analysis packages from Bioconductor: flowCore, openCyto, flowClust, flowViz and ggcyto. 42 2.6 Viability assay The MECP2-expressing cells were harvested by pelleting 1 mL of the culture at 4000 rpm for 2 minutes. The supernatant was removed and 50 µL of 1x PBS was added to resuspend the cells. Next, 10 µg/mL of acridine orange (Sigma Aldrich, SKU A6014-10G; solubilized in sdH2O) and 5 µg/mL of propidium iodide (Invitrogen, Cat no. P12493; solubilized in sdH2O) were incubated with the cells for 15 minutes at room temperature in the dark. Following incubation, 2 µL of the samples were loaded onto glass slides and imaged with an Optika B-380 microscope at 40x magnification using the green and red laser for fluorescence microscopy. To quantify the percentage of dead cells in each sample, equal-sized boxes were drawn close to the centre of each microscopy image. Live and dead cells within the box were manually counted three times and averaged. Three different areas of the glass slide were imaged and counted to generate three data points per sample. A minimum of 20 cells were counted for each data point. If the cells within the box contain less than 20 cells, all the cells in the images were counted. The number of dead cells was divided by the total number of cells to calculate the percentage of dead cells. The buds on the yeast were not counted as individual cells. The results were plotted as dot plots using the ggplot2 package, and the two-way ANOVA (significance level: p<0.05) was calculated using the aov function in R. 2.7 Cell cycle assay Following treatment with estradiol, 1 mL of cultures were pelleted at 13 000 rpm for 1.5 minutes and washed once with 70% ethanol (EtOH). The pellets were fixed with 70% EtOH at 4°C in the dark overnight. After fixation, the samples were pelleted at 13 000 rpm for 1.5 minutes and washed three times with 1 mL 1x PBS. The pellets were rehydrated for 1 hour at room 43 temperature in 1ml 1x PBS. The resuspensions were then pelleted at 13 000 rpm for 1.5 minutes and incubated with 100 µL of RNase buffer (0.2M Tris-Cl pH 7.5, 0.02M EDTA, 0.1 mg/mL RNAse A (ThermoFisher, Catalog # EN0531)) at 37°C for 3 hours to remove RNA from the samples. Next, the supernatants were removed, and the cells were stained with 100 µL of propidium iodide staining solution (propidium iodide diluted to 5 µg/mL in 1x PBS) for 1 hour at room temperature in the dark. After staining, 800 µL 1x PBS was added to the samples. The samples were kept on ice and vortexed before they were measured with a CytoFLEX LX flow cytometer (Beckman Coulter). Data collection and analysis were carried out using the CytExpert software for CytoFLEX. 2.8 Fluorescence microscopy Glass slides were washed with 70% ethanol and dried before they were coated with 5 µL of concanavalin A (ConA) (Sigma Aldrich, SKU: C2010-25MG; solubilized in sdH2O to 0.1 mg/mL). For sample preparation, 1 mL of culture was pelleted at 7000 rpm for 1 minute and washed once with 1 mL 1x PBS. The cells were resuspended in 50 µL of 1x PBS and 5 µL of the suspension was loaded onto glass slides. The samples were imaged with an Optika B-380 microscope at 100x magnification with brightfield or fluorescence microscopy. For fluorescence microscopy, the green laser was used to detect GFP in cells. The fluorescent and brightfield images were overlaid in ImageJ. 44 Table 1. List of plasmids used to generate the yeast strains in this study Plasmids Sources pRS405 Addgene pRS406 Addgene pRS416 Addgene pMA-MECP2-E1 Invitrogen pGAL1pr-CYC1t-416 LeAnn Howe pGAL1pr-MECP2-E1-CYC1t-416 This study pGAL1pr-sfGFP-MECP2-E1-CYC1t-416 This study pGAL1pr-GST-MECP2-E1-CYC1t-416 This study pGAL1pr-MECP2-E1-sfGFP-CYC1t-416 This study pGAL1pr-MECP2-E1-GST-CYC1t-416 This study pZF4-ER-VP16-405 LeAnn Howe pZFpro-406 LeAnn Howe pZFpro-MECP2-E1-CYC1t-406 LeAnn Howe pZFpro-MECP2-E2-CYC1t-406 LeAnn Howe pZFpro-sfGFP-MECP2-E1-CYC1t-406 LeAnn Howe pZFpro-sfGFP-MeCP2-E2-CYC1t-406 LeAnn Howe pZFpro-MECP2-E2-R168X-CYC1t-406 LeAnn Howe pZFpro-MECP2-E2-R255X-CYC1t-406 LeAnn Howe pZFpro-MECP2-E2-R270X-CYC1t-406 LeAnn Howe pZFpro-MECP2-E2-R294X-CYC1t-406 LeAnn Howe pZFpro-MECP2-E2-P389X-CYC1t-406 LeAnn Howe pZFpro-MECP2-E2-T158M-CYC1t-406 LeAnn Howe pZFpro-sfGFP-MECP2-E1-R168X-CYC1t-406 This study pZFpro-sfGFP-MECP2-E1-R255X-CYC1t-406 This study pZFpro-sfGFP-MECP2-E1-R270X-CYC1t-406 This study pZFpro-sfGFP-MECP2-E1-R294X-CYC1t-406 This study pZFpro-sfGFP-MECP2-E1-P389X-CYC1t-406 This study pZFpro-sfGFP-MECP2-E1-T158M-CYC1t-406 This study pZFpro-sfGFP-MECP2-E2-R168X-CYC1t-406 This study pZFpro-sfGFP-MECP2-E2-R255X-CYC1t-406 This study pZFpro-sfGFP-MECP2-E2-R270X-CYC1t-406 This study pZFpro-sfGFP-MECP2-E2-R294X-CYC1t-406 This study pZFpro-sfGFP-MECP2-E2-P389X-CYC1t-406 This study pZFpro-sfGFP-MECP2-E2-T158M-CYC1t-406 This study 45 Table 2. Sequences of MECP2 isoforms and protein tags Genes Sequences MECP2-E1 ATGGCTGCTGCAGCTGCTGCTGCACCATCTGGTGGTGGTGGCGGTGGTGAAGAAGAAAGATTGGAAGAAAAATCCGAAGATCAAGACTTGCAAGGTTTGAAGGATAAGCCATTGAAGTTCAAGAAGGTTAAGAAGGACAAGAAAGAAGAGAAAGAGGGTAAACACGAACCAGTTCAACCATCTGCTCATCATTCTGCTGAACCAGCTGAAGCTGGTAAAGCTGAAACTTCTGAAGGTTCTGGTTCTGCTCCAGCTGTTCCTGAAGCTTCTGCTTCTCCAAAACAAAGAAGATCCATCATCAGAGATAGAGGTCCAATGTATGATGATCCAACTTTGCCAGAAGGTTGGACTAGAAAATTGAAGCAAAGAAAGTCTGGTAGATCCGCTGGTAAATACGATGTTTACTTGATTAACCCACAAGGTAAGGCCTTCAGATCTAAGGTTGAATTGATTGCCTACTTCGAAAAGGTTGGTGATACTTCTTTGGACCCAAACGATTTCGATTTCACTGTTACTGGTAGAGGTTCTCCATCTAGAAGAGAACAAAAACCACCAAAGAAGCCAAAATCTCCAAAAGCTCCAGGTACTGGAAGAGGTAGAGGTAGACCAAAAGGTTCAGGTACAACTAGACCTAAAGCTGCTACATCAGAAGGTGTTCAAGTTAAGAGAGTCTTGGAAAAATCACCAGGTAAGTTGTTGGTTAAGATGCCATTTCAAACTTCTCCAGGTGGTAAGGCTGAAGGTGGCGGAGCTACTACTTCTACTCAAGTTATGGTTATCAAAAGGCCAGGTAGAAAGAGAAAAGCTGAAGCAGATCCACAAGCCATTCCAAAAAAGAGAGGTAGAAAACCAGGTTCTGTTGTTGCTGCTGCCGCAGCCGAAGCTAAAAAGAAAGCTGTAAAAGAATCCTCCATCAGGTCTGTTCAAGAAACTGTTTTGCCAATCAAGAAGCGTAAGACTAGAGAAACCGTTTCCATCGAAGTAAAAGAAGTTGTCAAGCCCTTGTTGGTTTCTACTTTGGGTGAAAAATCTGGTAAGGGTTTGAAAACCTGTAAGTCTCCTGGTAGGAAGTCGAAAGAATCATCTCCTAAAGGTAGATCTTCCTCTGCTTCATCTCCACCAAAAAAAGAACATCACCATCACCACCATCATTCCGAATCACCAAAAGCACCTGTTCCATTATTGCCACCATTGCCTCCACCACCTCCAGAACCTGAATCATCTGAAGATCCTACTTCTCCACCAGAGCCTCAAGATTTGTCATCTTCTGTATGCAAAGAAGAAAAAATGCCAAGAGGTGGTTCCTTGGAATCTGATGGTTGTCCAAAAGAACCAGCTAAAACTCAACCAGCTGTTGCTACTGCTGCTACAGCAGCTGAAAAGTACAAACATAGAGGTGAAGGTGAGAGGAAGGATATCGTTTCTTCATCTATGCCAAGACCAAACAGAGAAGAACCAGTTGATTCTAGAACTCCAGTTACCGAAAGAGTCTCTTGA 46 Genes Sequences MECP2-E2 ATGGTTGCTGGTATGCTTGGTTTAAGAGAAGAAAAATCCGAAGATCAAGACTTGCAAGGTTTGAAGGATAAGCCATTGAAGTTCAAGAAGGTTAAGAAGGACAAGAAAGAAGAGAAAGAGGGTAAACACGAACCAGTTCAACCATCTGCTCATCATTCTGCTGAACCAGCTGAAGCTGGTAAAGCTGAAACTTCTGAAGGTTCTGGTTCTGCTCCAGCTGTTCCTGAAGCTTCTGCTTCTCCAAAACAAAGAAGATCCATCATCAGAGATAGAGGTCCAATGTATGATGATCCAACTTTGCCAGAAGGTTGGACTAGAAAATTGAAGCAAAGAAAGTCTGGTAGATCCGCTGGTAAATACGATGTTTACTTGATTAACCCACAAGGTAAGGCCTTCAGATCTAAGGTTGAATTGATTGCCTACTTCGAAAAGGTTGGTGATACTTCTTTGGACCCAAACGATTTCGATTTCACTGTTACTGGTAGAGGTTCTCCATCTAGAAGAGAACAAAAACCACCAAAGAAGCCAAAATCTCCAAAAGCTCCAGGTACTGGAAGAGGTAGAGGTAGACCAAAAGGTTCAGGTACAACTAGACCTAAAGCTGCTACATCAGAAGGTGTTCAAGTTAAGAGAGTCTTGGAAAAATCACCAGGTAAGTTGTTGGTTAAGATGCCATTTCAAACTTCTCCAGGTGGTAAGGCTGAAGGTGGCGGAGCTACTACTTCTACTCAAGTTATGGTTATCAAAAGGCCAGGTAGAAAGAGAAAAGCTGAAGCAGATCCACAAGCCATTCCAAAAAAGAGAGGTAGAAAACCAGGTTCTGTTGTTGCTGCTGCCGCAGCCGAAGCTAAAAAGAAAGCTGTAAAAGAATCCTCCATCAGGTCTGTTCAAGAAACTGTTTTGCCAATCAAGAAGCGTAAGACTAGAGAAACCGTTTCCATCGAAGTAAAAGAAGTTGTCAAGCCCTTGTTGGTTTCTACTTTGGGTGAAAAATCTGGTAAGGGTTTGAAAACCTGTAAGTCTCCTGGTAGGAAGTCGAAAGAATCATCTCCTAAAGGTAGATCTTCCTCTGCTTCATCTCCACCAAAAAAAGAACATCACCATCACCACCATCATTCCGAATCACCAAAAGCACCTGTTCCATTATTGCCACCATTGCCTCCACCACCTCCAGAACCTGAATCATCTGAAGATCCTACTTCTCCACCAGAGCCTCAAGATTTGTCATCTTCTGTATGCAAAGAAGAAAAAATGCCAAGAGGTGGTTCCTTGGAATCTGATGGTTGTCCAAAAGAACCAGCTAAAACTCAACCAGCTGTTGCTACTGCTGCTACAGCAGCTGAAAAGTACAAACATAGAGGTGAAGGTGAGAGGAAGGATATCGTTTCTTCATCTATGCCAAGACCAAACAGAGAAGAACCAGTTGATTCTAGAACTCCAGTTACCGAAAGAGTCTCTTGA 47 Genes Sequences sfGFP ATGTCCAAGGGTGAAGAGCTATTTACTGGGGTTGTACCCATTTTGGTAGAACTGGACGGAGATGTAAACGGACATAAATTCTCTGTTAGAGGTGAGGGCGAAGGCGATGCCACCAATGGTAAATTGACTCTGAAGTTTATATGCACTACGGGTAAATTACCTGTTCCTTGGCCAACCCTAGTAACAACTTTGACATATGGTGTTCAATGTTTCTCAAGATACCCAGACCATATGAAAAGGCATGATTTCTTTAAAAGTGCTATGCCAGAAGGCTACGTGCAAGAGAGAACTATCTCCTTTAAGGATGACGGTACGTATAAAACACGAGCAGAAGTGAAATTCGAAGGGGATACACTAGTTAATCGCATCGAATTAAAGGGTATAGACTTTAAGGAAGATGGTAATATTCTCGGCCATAAACTTGAGTATAATTTCAACTCGCATAATGTGTACATTACAGCTGACAAACAAAAGAACGGAATTAAAGCGAATTTTAAAATCAGGCACAACGTCGAAGATGGGTCTGTTCAACTTGCCGATCATTATCAGCAAAACACCCCTATTGGTGATGGTCCAGTCTTGTTACCCGATAATCACTACTTAAGCACACAGTCTAGATTGTCAAAAGATCCGAATGAAAAGCGTGATCACATGGTTTTATTGGAATTTGTCACCGCTGCAGGAATAACTCACGGAATGGACGAGCTTTATAAGGGATCCTAA GST ATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCTTGTGCAACCCACTCGACTTCTTTTGGAATATCTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGGTGATAAATGGCGAAACAAAAAGTTTGAATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTTAAATTAACACAGTCTATGGCCATCATACGTTATATAGCTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCATAAAACATATTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCATCCTCCAAAATGA 48 Table 3. Primers used for cloning Plasmids Forward primers (5’-3’) Reverse primers (5’-3’) pGAL1pr-MECP2-E1-CYC1t-416 AAACCCCGGATTCTAGAACTAGTATGGCTGCTGCAGCTGCT TCGAATTCCTGCAGCCCGGGTCAAGAGACTCTTTCGGTAACTGGAG pGAL1pr-sfGFP-MECP2-E1-CYC1t-416 (N-terminal tag) AAAAACCCCGGATTCTAGAAATGTCCAAGGGTGAAGAG CAGCAGCTGCAGCAGCCATACCAGCACCAGCACCTGCTCCCTTATAAAGCTCGT CCATTC pGAL1pr-MECP2-E1-sfGFP-CYC1t-416 (C-terminal tag) CCCGGATTCTAGAACTAGTGATGGCTGCTGCAGCTGCT (MECP2) CAGTTACCGAAAGAGTCTCTGGAGCAGGTGCTGGTGCTGGTATGTCCAAGGGTG AAGAG (sfGFP) AGAGACTCTTTCGGTAACTGGAGTTC (MECP2) CGAATTCCTGCAGCCCGGGGTTAGGATCCCTTATAAAGCTC (sfGFP) pGAL1pr-GST-MECP2-E1-CYC1t-416 (N-terminal tag) AAAAACCCCGGATTCTAGAAATGTCCCCTATACTAGGTTATTG CAGCAGCTGCAGCAGCCATACCAGCACCAGCACCTGCTCCTTTTGGAGGATGGT CGCC pGAL1pr-MECP2-E1-GST-CYC1t-416 (C-terminal tag) CCCGGATTCTAGAACTAGTGATGGCTGCTGCAGCTGCT (MECP2) GGAGCAGGTGCTGGTGCTGGTATGTCCCCTATACTAGGTTATTG (GST) ACCAGCACCAGCACCTGCTCCAGAGACTCTTTCGGTAACTGGAGTTC (MECP2) CGAATTCCTGCAGCCCGGGGTCATTTTGGAGGATGGTCGCC (GST) pZFpro-sfGFP-MECP2 mutants TGGAGCTCGATGAATTGAAAAGCTTTATGGACCCAGATCTTTATATTGAATTTTC CTTTGTTTTGGAGAAGCAGAAGCTTCAGGAACAGCTGGAGCAG 49 Table 4. Genotypes of tagged and untagged MECP2 strains Strain Genotype FY602 (Wild-type) his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 ZF4-ER-VP16 his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 Vector only his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr MECP2-E1 his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr MECP2 E1 MECP2-E2 his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr MECP2 E2 GFP-MECP2-E1 his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr sfGFP-MECP2 E1 GFP-MECP2-E2 his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr sfGFP-MECP2 E2 Table 5. Genotypes of MECP2-E2 mutant strains Strain Genotype MECP2-E2-R168X his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr MECP2 E2 R168X MECP2-E2-R255X his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr MECP2 E2 R255X MECP2-E2-R270X his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr MECP2 E2 R270X MECP2-E2-R294X his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr MECP2 E2 R294X MECP2-E2-P389X his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr MECP2 E2 P389X MECP2-E2-T158M his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr MECP2 E2 T158M 50 Table 6. Genotypes of GFP-tagged MECP2-E1 mutant strains Strain Genotype GFP-MECP2-E1-R168X his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr sfGFP-MECP2 E1 R168X GFP-MECP2-E1-R255X his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr sfGFP-MECP2 E1 R255X GFP-MECP2-E1-R270X his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr sfGFP-MECP2 E1 R270X GFP-MECP2-E1-R294X his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr sfGFP-MECP2 E1 R294X GFP-MECP2-E1-P389X his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr sfGFP-MECP2 E1 P389X GFP-MECP2-E1-T158M his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr sfGFP-MECP2 E1 T158M Table 7. Genotypes of GFP-tagged MECP2-E2 mutant strains Strain Genotype GFP-MECP2-E2-R168X his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr sfGFP-MECP2 E2 R168X GFP-MECP2-E2-R255X his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr sfGFP-MECP2 E2 R255X GFP-MECP2-E2-R270X his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr sfGFP-MECP2 E2 R270X GFP-MECP2-E2-R294X his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr sfGFP-MECP2 E2 R294X GFP-MECP2-E2-P389X his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr sfGFP-MECP2 E2 P389X GFP-MECP2-E2-T158M his3Δ200 leu2Δ1 lys2-128δ ura3-52 trp1Δ63 LEU2::ZF4-ER-VP16 URA3::ZFpr sfGFP-MECP2 E2 T158M 51 Chapter 3: Results 3.1 Testing protein expression systems 3.1.1 Inducing MECP2 expression in S. cerevisiae with galactose To express the human protein, methyl-CpG-binding protein 2 (MECP2), in Saccharomyces cerevisiae, we cloned the MECP2 gene downstream of the GAL1 promoter. The MECP2 gene was synthesized and codon-optimized for yeast expression by Thermofisher. The GAL1 promoter allowed us to induce yeast to express MECP2 by adding galactose to the media. The MECP2-expressing plasmid contained its own centromere (CEN) and origin of replication (ARS) and thus could replicate and segregate without integration into the chromosome. Aside from a CEN/ARS, it also carried the URA3 gene, a selection marker that allowed the transformed yeast to grow in uracil depleted media (-uracil). We also generated strains expressing MECP2 with a green fluorescent protein (GFP) tagged to the N or C terminus. Adding the GFP tag allowed us to track MECP2 in vivo in downstream experiments such as flow cytometry or fluorescence microscopy. The plasmid was transformed into yeast and selected for on -uracil plates to pick out successfully transformed yeast. If MECP2 can indeed interact and bind with unmethylated DNA, it will potentially disrupt normal transcription or replication and result in changes in growth. To test this, we performed a growth assay by spotting yeast onto YPD, -uracil/dextrose, or -uracil/galactose plates. The samples were serially diluted from 1-10000x to ensure we could visualize single colonies. We spotted WT, vector-alone, and MECP2-expressing strains onto the plates. The vector-alone strain contained the backbone plasmid with the GAL1 promoter and URA3, while the WT strain lacked the plasmid and should not grow on -uracil. We found that expression of an N-terminal, but not C-terminal 52 tagged MECP2-E1 severely inhibited growth (Figure 2A). Yeast expressing untagged MECP2-E1 had no growth defect and grew like vector-alone. The effect of the tag was not unique to GFP alone, as if we added a similar sized Glutathione-S-transferase (GST) tag to MECP2 (the GFP tag is 28kDa, while the GST tag is 26kDa), again, we saw that only N-terminal tagged MECP2 affected growth (Figure 2B). We also obtained a galactose-inducible MECP2-E2-expressing yeast strain from the Loewen lab at UBC and tested this in parallel with MECP2-E1. Unlike MECP2-E1, yeast expressing MECP2-E2 showed only a slight reduction in growth compared to vector-alone (Figure 2A). We then explored whether the difference in the growth of cells expressing the tagged versus untagged MECP2 was due to changes in protein levels. To test this, we added 2% galactose to cultures grown in -uracil/raffinose media, harvested the cells after 1 hour, and analyzed the lysates via Western blot. Consistent with the impacts on yeast growth, the N-terminal GFP and GST-tagged MECP2-E1 were more abundant than the untagged MECP2-E1 (Figure 3A). Surprisingly, although the expression of untagged E2, but not untagged E1, impacted yeast growth, the amount of untagged MECP2-E1 appeared marginally more than MECP2-E2 levels. The C-terminal GFP/GST tagged MECP2-E1 was not detected through Western blot and was still absent even after we extended the induction time from 1 to 3 hours (Figures 3A and B). These results show that the expression of MECP2 E1 and E2 inhibits yeast growth, and growth inhibition by E1 is only observed when carrying an amino-terminal tag, which greatly increases the level of MECP2. 3.1.2 Inducing MECP2 expression in S. cerevisiae with beta-estradiol Following our initial findings on the growth inhibition due to expressing MECP2 in S. cerevisiae, we decided to switch from galactose to a β-estradiol inducible system (206). This would 53 give us the ability to control protein expression by varying the concentration of estradiol or the induction time, or both. Moreover, unlike galactose-induction which required changing the media from glucose to raffinose before induction, this system enabled us to add estradiol directly to cells that were grown in glucose. We expanded on the constructs outlined in McIssac et al. (2013) to generate a yeast strain expressing the artificial transcription factor (ATF): the fusion construct Zinc finger (Z4)-VP16-estrogen receptor. We then cloned N-terminally tagged GFP-MECP2-E1 or GFP-MECP2-E2 downstream of an artificial promoter with binding sites for the ATF. The vector-alone strain expressed the ATF and contained the artificial promoter but had no MECP2 gene. First, we tested the concentration of estradiol required for maximum protein expression by detecting the changes in GFP intensity with flow cytometry. We opted for flow cytometry due to its ability to quantify protein levels in single cells. The cultures were grown to mid-log phase and 0-100 μM of β-estradiol was added to induce protein expression for 1 hour. The results indicated that we reached a maximum protein level with β-estradiol concentrations between 0.1 to 1 μM (Figures 4 and 5). The GFP signal remained consistent beyond 1 μM suggesting that this level of estradiol triggered maximal induction. The trend was similar between GFP-MECP2-E1 and GFP-MECP2-E2. It should be noted that yeast exhibit autofluorescence, thus cells without estradiol treatment will have some background fluorescence signal detected by flow cytometry. To distinguish autofluorescence from GFP signal, we only looked for shifts in GFP intensities between cells treated with or without estradiol to identify whether the cells are expressing GFP. When we added β-estradiol to vector-alone strains, there were no increases in the GFP signal compared to untreated cells, indicating no GFP expression (Figures 4 and 5, left panel). We then confirmed the flow cytometry results with Western blot. We again found that a maximum protein level was reached when we added between 0.1 to 1 μM estradiol to the cultures (Figure 6). 54 Consistent with Figure 3, the GFP-tagged proteins were more abundant than the untagged ones. Before, we observed that the expression of the untagged E1 was slightly more than E2 when the proteins were expressed from a galactose-inducible promoter (Figure 3). Similar to the untagged proteins, the GFP-tagged E1 isoform was also more abundant than the GFP-tagged E2 isoform (Figure 6C). We were unable to detect the untagged MECP2-E2 when expressed from the estradiol-inducible promoter, but untagged MECP2-E2 inhibited cell growth. This is not unexpected since the band corresponding to MECP2-E1 in Figure 6A was very faint, and expression of MECP2-E2 was lower than MECP2-E1 when expressed with the galactose-inducible promoter. However, because the untagged MECP2-E2 inhibits cell growth, it is likely that the MEPC2-E2 protein was present but too low to be detected (Figures 6A-B). Next, we tested the minimum time required to express maximal levels of MECP2. Instead of varying the concentration of estradiol, we varied the induction time from 0-7 hours in 1 μM estradiol and measured changes in GFP levels with flow cytometry. While we found that 1 hour is enough time for proteins to be induced, 4-5 hours with estradiol is required for yeast to reach maximum protein abundance (Figures 7 and 8). Similar results were observed between GFP-MECP2-E1 and GFP-MECP2-E2. We noticed that there were two peaks in the GFP histograms of the GFP-MECP2-expressing cells (Figure 7). We suspect this was because some populations of cells were expressing different amounts of protein. However, without additional experiments, at this time, we do not know the cause of the bimodal distribution. Despite differences in estradiol uptake, the histogram peaks did not shift position beyond 4 hours, indicating that 4 hours was the shortest time required for maximum protein expression with the estradiol-inducible system in our strains. Overall, our results confirmed that we can use the β-estradiol inducible system to induce 55 expression of MECP2 in S. cerevisiae and that the conditions required for maximum MECP2 expression are 1 μM β-estradiol and an induction time of 4 hours. 56 Figure 2. Expression of N-terminal tagged MECP2-E1 in S. cerevisiae results in defects in growth Cells were diluted 1 to 10000-fold before plating on YPD, -uracil/dextrose, or -uracil/galactose. The strains contained a plasmid with the GAL1 promoter sequence upstream of the MECP2 gene and an URA3 selection marker. A GFP or GST tag was added to the N or C terminus of MECP2 as indicated. Vector-alone contained a GAL1 promoter and the URA3 marker, but without MECP2. Wild-type (WT) strains did not contain any plasmid and thus should not grow on -uracil plates. YPD and -uracil/dextrose plates were grown at 30°C for two days, and the -uracil/galactose plates were grown for three days before imaging. 57 Figure 3. The addition of a tag to the N-terminus of MECP2-E1 alters protein levels when expressed in S. cerevisiae A, Cells were grown to mid-log phase in -uracil/raffinose before adding galactose to induce MECP2 expression. Samples were collected after 1 hour incubation with galactose; extracts were prepared, and proteins were separated on a 10% SDS-PAGE gel and immunoblotted with the indicated antibodies. The effects of expressing the proteins on cell growth are summarized below the blot, with +++ indicating the most severe growth defect and – indicating no growth defect compared to vector only. B, Similar to A but samples were collected 0-3hrs after induction. The expected sizes for MECP2 are 75kDa, sfGFP-MECP2 102kDa, and GST-MECP2 101kDa. Phosphoglycerate kinase 1 (Pgk1) was blotted as a loading control.58 Figure 4. Maximum MECP2 induction in S. cerevisiae is reached with 1 μM estradiol GFP intensities of yeast expressing either vector-only, GFP-MECP2-E1 or GFP-MECP2-E2 were quantified with flow cytometry. Yeast cultures were grown to mid-log phase and 0-100 μM β-estradiol was added to induce expression for 1 hour before quantification. The mean GFP intensities of 5000 cells are displayed as histograms. 59 Figure 5. Maximum MECP2 induction in S. cerevisiae is achieved with 1 μM estradiol (part 2) The same as Figure 4, but the results are displayed as empirical cumulative distribution function (ECDF) plots to show the relative distribution of the GFP intensity. The relative frequency is plotted on the y-axis and the GFP intensity is plotted in log-scale on the x-axis. Vector GFP−MECP2−E1 GFP−MECP2−E21e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+050.000.250.500.751.00GFP intensityECDFβ−estradiol (µM)1001010.10.01060 Figure 6. Expression of MECP2 is titratable by varying the estradiol concentrations Yeast cultures were grown to mid-log and 0-1 μM β-estradiol was added to induce expression of MECP2 for 1 hour before cell lysates were collected and analyzed by Western blot. A and B, Wild-type yeast containing the indicated plasmids. C, Same samples as A and B, but with GFP-MECP2-E1 and GFP-MECP2-E2 loaded on the same gel. GFP was added to the N-terminus of MECP2. The expected sizes for MECP2 are ~75kDa and GFP-MECP2 ~100kDa. 61 Figure 7. Maximum MECP2 expression in S. cerevisiae is achieved at 4 hours post-induction GFP intensities of yeast containing vector-alone and yeast expressing GFP-MECP2-E1 or GFP-MECP2-E2 were quantified with flow cytometry. Cultures were grown to mid-log and 1 μM β-estradiol was added to induce expression for 0-7 hours before harvesting and quantifying with flow cytometry. The mean GFP intensities of 5000 cells are displayed as histograms. 62 Figure 8. Maximum MECP2 levels in S. cerevisiae is achieved at 4 hours post-induction (part 2) The same as Figure 7, but the results are displayed as empirical cumulative distribution function (ECDF) plots to show the relative distribution of the GFP intensity. The relative frequency is plotted on the y-axis and the GFP intensity is plotted in log-scale on the x-axis. Vector GFP−MECP2−E1 GFP−MECP2−E21e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+050.000.250.500.751.00GFP intensityECDFTime (hrs)7654321063 3.2 Exploring the causes of growth defects for MECP2-expressing cells After confirming the feasibility of using the estradiol-induction system, we carried out a growth assay on the cells expressing untagged and N-terminal GFP-tagged MECP2-E1 and E2. Similar to galactose-induced expression, plasmid constructs driving estradiol-dependent expression of MECP2 also used URA3 as a selection. In addition to -uracil selection, the estradiol-induction system also employed the gene for the ATF construct, which was selected using LEU2. Thus, strains that grew in -leucine-uracil contained both the ATF required for the estradiol induction system to function and the MECP2 gene for the ATF to induce. We spotted the cells onto -leucine-uracil dextrose with 0 or 1 μM β-estradiol. The vector-alone strain was included as a control which contained the selection markers LEU2 and URA3 and the gene for ATF but lacked the MECP2 gene. We found that adding a GFP tag to MECP2-E1 reduced its growth compared to the yeast with the untagged MECP2-E1, which grew similarly to vector alone (Figure 9). However, contrary to untagged MECP2-E2 induced using the GAL1 promoter, which caused a modest growth defect, yeast expressing MECP2-E2 had a strong growth defect and adding a GFP-tag to it did not alter this. These results were consistent across three independently derived biological replicates. We noticed that the results from the growth assay deviated between galactose and estradiol-induced expression (Figures 2 and 9). MECP2-E2 had smaller colonies when expressed with the estradiol-inducible system compared to galactose-induction. Moreover, there was no growth of the GFP-MECP2-E1 expressing strains when induced with galactose, but colonies were visible when induced with estradiol. The different results were likely due to different expression systems. Aside from different expression systems, the galactose-induced MECP2 was expressed off an ARS/CEN vector, but the genes encoding the ATF and MECP2 for the estradiol system were integrated into the yeast genome via homologous recombination at the LEU2 or URA3 sites. 64 Yeast can carry multiple ARS/CEN vectors or could potentially have multiple integrations. Thus, it is a possibility that the variability in the growth assay is due to varying copies of the MECP2 gene between the two-expression systems. Next, we delved deeper to explore the possible causes of the growth defects that we observed in the growth assays. We tested whether GFP-MECP2-E1, GFP-MECP2-E2, MECP2-E1, MECP2-E2-expressing strains, and the vector-alone strain had changes in their cell cycles. From our previous data, we concluded that 1 μM β-estradiol for 4 hours was enough to reach maximum protein abundance. Thus, we used this condition for our experiment. The cells were harvested and fixed with ethanol and stained with propidium iodide (PI) to stain the DNA, and the PI signal was measured with flow cytometry. Cells in G1 will have 1N DNA content while those in G2 will have 2N DNA content due to duplication of DNA from replication. S phase cells are any signal between the G1 and G2 peaks. A sample with vector-alone treated with 5 μM alpha factor for 2 hours was included as a positive control for cell cycle arrest. The vector-alone strain treated with 5 μM alpha factor had a single peak that corresponded with G1 cells, which confirmed cell cycle arrest (Figure 10). Intriguingly, only the strains that had poor growth from the growth assay, namely those expressing MECP2-E2, GFP-MECP2-E1, and GFP-MECP2-E2 had defects in their cell cycles. Instead of two peaks, they had one broad single peak at the S phase junction region. However, we were unable to conclude whether they were indeed S phase arrested as the whole peak seemed to shift toward the right, including the smaller shoulder peak beside the larger one, which we suspected to be cells arrested in G2. Yeast expressing MECP2-E1, and all the uninduced strains (0 μM β-estradiol) had similar cell cycle peaks as the vector-alone strain. We also tested the same strains and conditions after reducing the induction time to 1 hour instead of 4 hours. At 1 hour post-induction, the MECP2-E2, GFP-MECP2-E1, and GFP-MECP2-E2-65 expressing strains did not have significant changes in their cell cycle compared to the controls (Figure 11). It is likely that 1 hour is not enough time or not enough protein to cause an effect on the cell cycle. Since the cells were fixed for the cell cycle assay, we were not able to discern whether the cells were dead or alive. Therefore, we followed up on our experiment with a viability assay. Cell cycle arrest could be due to the activation of a checkpoint or death. To explore whether death is the cause of altered cell cycle in our strains, we induced expression of GFP-MECP2-E1 and GFP-MECP2-E2 with 1 μM β-estradiol for 4 hours and stained with propidium iodide, but on live instead of fixed cells. Without fixation, propidium iodide stains dead, but not live cells. Acridine orange was added as a counterstain to stain all cells. Dead cells will stain red while other cells will be green. We included a vector-alone sample treated with a high concentration of alpha factor (10 μM) for 2 hours as a positive control as we have previously found this to result in a high incidence of cell death. Vector-alone treated with 10 μM alpha factor had some red cells, indicating death, which confirmed our viability assay can identify dead cells. At 4 hours post-induction, yeast expressing GFP-MECP2-E1, but not GFP-MECP2-E2 contained dead cells (Figure 12A). We then increased our expression time from 4 to 18 hours to see whether prolonged induction affects GFP-MECP2-E2-expressing cells. After 18 hours, yeast expressing GFP-MECP2-E2 indeed had some dead cells, but a similar number of dead cells was also observed in the unexpressed sample (0 μM β-estradiol) (Figure 12B). We then quantified the percentage of dead cells in the microscopy images for each strain (Figure 13). At 4 hours post-induction, GFP-MECP2-E1-expressing cells, but not GFP-MECP2-E2-expressing cells had a significantly greater percentage of dead cells compared to the unexpressed sample (Figure 13A). However, after 18 hours, the percentage of dead cells was not significantly different between cells treated with or without estradiol (Figure 66 13B). Therefore, after 18 hours, we were unable to discern whether the death was due to the presence of GFP-MECP2-E2 or natural cell death from multiple doublings. We noticed that strains expressing GFP-MECP2-E1 had more death at 4 hours versus 18 hours post-induction. It was likely that expressing GFP-MECP2-E1 killed off a portion of cells early, but the ones that survived continued to proliferate and became the majority of the population at 18 hours. Overall, strains expressing GFP-MECP2-E2 had a cell cycle arrest but were viable despite growth inhibition and this likely indicates that these strains had checkpoint arrest. Unlike E2, GFP-MECP2-E1 also had cell cycle arrest, but growth inhibition correlates with a significant loss of viability, suggesting that the cell cyle arrest in this case is detrimental to these strains. 67 Figure 9. GFP-tagged, but not untagged, MECP2-E1 inhibits the growth of S. cerevisiae Comparison of yeast strains containing the MECP2-E1, MECP2-E2-expressing vectors, or the isoforms with a GFP tag at the N-terminus. Cells were serially diluted 10-fold from (1x-10000x) and spotted onto -leucine-uracil drop-out plates containing 0 or 1 μM β-estradiol and grown for 2 days at 30°C. A vector-only strain was included as a control. Three independently generated replicates were tested for each strain.68 Figure 10. Yeast strains with MECP2-induced defects in growth have altered cell cycles Cells were grown to log phase and treated with 0 or 1 μM estradiol for 4 hours before staining with propidium iodide (PI) and analyzed with flow cytometry. One sample was arrested at G1 with 5 μM alpha factor for 2 hours, with 2.5 μM alpha factor spiked in every 1.5 hours to maintain arrest. The first peak represents cells in G1, the second peak cells in G2 and the valley in between the peaks are cells in S phase. A no PI control was included to show the background fluorescence. 69 Figure 11. Yeast strains with MECP2-induced defects in growth have altered cell cycles (part 2) Cells were grown to log phase and treated with 0 or 1 μM estradiol for 1 hour before staining with propidium iodide (PI) and analyzed with flow cytometry. One sample was arrested at G1 with 5 μM alpha factor for 2 hours, with 2.5 μM alpha factor spiked in every 1.5 hours to maintain arrest. The first peak represents cells in G1, the second peak cells in G2 and the valley in between the peaks are cells in S phase. A no PI control was included to show the background fluorescence. 70 Figure 12. The expression of GFP-MECP2-E1 in S. cerevisiae induces cell death Cells were kept at log phase and 0 or 1 μM β-estradiol was added to the cultures to induce expression of GFP-MECP2-E1 or GFP-MECP2-E2. A, cells induced for 4 hours, B, cells induced for 18hrs. A vector-only strain was used as a control. A sample of the vector-only strain was also treated with 10 μM alpha factor which arrests cells and causes death. Dead cells stain red, and all cells stain green due to staining with acridine orange or expression of GFP-tagged MECP2. Scale bars represent 10 μm. Vector GFP-MECP2-E1 GFP-MECP2-E2 Alpha factor 10μM0μM1μMβ-Estradiol A4 hrsBVector GFP-MECP2-E1 GFP-MECP2-E2 Alpha factor 10μMβ-Estradiol 0μM1μM 18 hrs71 Figure 13. The expression of GFP-MECP2-E1 in S. cerevisiae induces cell death (part 2) Dead and live cells were manually counted from three different sections of the glass slide for each strain. These are the same samples in Figure 12. The number of dead cells was divided by the total number of cells counted to obtain the percentage of dead cells. A minimum of 20 cells were counted for each data point. The black dots represent the mean and the vertical line error bars. The samples were compared using the two-way ANOVA (significance level: p<0.05). The * indicates the level of significance while n.s. means not significant. Treatment: 0 or 1 μM β-estradiol, or 10 μM alpha-factor. Strain: vector, GFP-MECP2-E1, or GFP-MECP2-E2. 72 3.3 MECP2 disease-associated mutants 3.3.1 Disease-associated mutations and growth We next asked whether we could use the growth-inhibiting properties of MECP2 to identify mutants that impact MECP2 function. If the mutations altered the growth levels, it is likely that the mutated region plays a role in the function of MECP2. First, we generated common disease-associated mutants, such as the truncations R168X, R255X, R270X, R294X and P389X, and the point mutation T158M on MECP2-E2. We tested strains expressing these mutants with the growth assay by spotting them on -leucine-uracil plates with 0 or 1 μM β-estradiol. The results showed that R168X and T158M mutations reverted the growth to levels similar to the growth of the vector-alone strain (Figure 14). This result was consistent between the three biological replicates. Next, we generated the same mutations on GFP-MECP2-E1 and GFP-MECP2-E2 and carried out the same experiment to see whether the mutations also affect growth. Like what we saw with the MECP2-E2 mutants, the R168X truncation and the T158M mutant on GFP-MECP2-E1 or GFP-MECP2-E2 rescued growth to vector-alone-like levels, and the pattern was consistent between the three biological replicates (Figures 15 and 16). We also noticed that GFP-MECP2-E1-R255X partially alleviated the growth defect of GFP-MECP2-E1, but this was only observed in the second and third replicates, not the first one (Figure 15). Likewise, GFP-MECP2-E2-R255X also rescued growth, but there was also variability, as we only observed this in the first and third replicates, and not the second replicate (Figure 16). Moreover, the truncated GFP-MECP2-E2-P389X grew slightly better than GFP-MECP2-E2, but this trend was only seen in the first replicate, and not the second or third (Figure 16). Overall, our results showed that a subset of disease-associated mutations in MECP2 rescued their toxicity when expressed in yeast. Interestingly, however, some of the other mutants, especially R255X, had a variegated effect on MECP2 growth-inhibiting 73 properties. 3.3.2 Variability in yeast strains and protein levels Due to the variability of phenotypes between replicates from our growth assay, we wished to discern whether these were caused by different protein abundance. For example, one replicate may show minimal growth because of higher MECP2 amounts, while another had normal growth because it had minimal levels of MECP2. To explore this, we again turned to flow cytometry. First, we selected five independently generated strains as biological replicates for yeast expressing vector-alone, GFP-MECP2-E1, and GFP-MECP2-E2. Since we just wanted to check whether the protein expression varied, we set the protein induction time at 1 hour with 1 μM β-estradiol, which was enough time to express the protein (Figures 7-8). There were no changes in the fluorescence signal of the vector-alone strain with or without estradiol, indicating no GFP expression and this was consistent between the replicates (Figures 17-18). However, the GFP levels between the replicates differed for GFP-MECP2-E1 and GFP-MECP2-E2-expressing strains. They fell into two groups, one group with higher expression and another with lower expression. For example, in GFP-MECP2-E1, we found that the GFP intensities of replicates 2 and 5 were lower than 1,3 and 4. However, we also noticed that even with no estradiol, the higher protein group (replicates 1,3 and 5) also had higher GFP signals to begin with compared to replicates 2 and 5 (Figures 17-18). We saw a similar pattern for GFP-MECP2-E2, where replicates 1-3 had higher GFP signal, but also had slightly higher signal before estradiol induction compared to replicates 4-5. We suspected that the variability in MECP2 amounts may be due to unequal copies of DNA integrating during the transformation. Note that we used the same first three replicates for both E1 and E2 in this experiment as we did in the growth assays (Figures 9, 15-16). While the protein levels differed 74 between the replicates for GFP-MECP2-E1 and GFP-MECP2-E2, these replicates all displayed similar poor growth (Figures 9, 17-18). Therefore, is likely that the minimal threshold of GFP-MECP2 abundance is can be very low and still disrupt growth in yeast. To rule out whether technical issues were causing variability between the replicates, we switched our approach and tested three replicates that were generated from a single colony. In addition to vector-alone and GFP-MECP2-E1, we selected the mutant GFP-MECP2-E1-R255X due to inconsistent phenotypes between the replicates, and another mutant GFP-MECP2-E1-T158M which had consistent phenotypes between the replicates. The GFP levels were consistent between the replicates (Figures 19-20). We did note that there was a slight difference in the GFP signal between replicate 2 with 1 and 3 for the R255X mutant. However, this difference was marginal compared with Figures 17-18. Overall, our results confirmed that differences in protein amounts were due to strain-to-strain variation and not technical inconsistencies. Next, we moved forward and tested whether the protein levels of MECP2 mutants correlated with phenotypic variations. While protein abundance was variable between the biological replicates, all GFP-MECP2-E1 mutants were expressed (Figures 21-22). The same was observed with GFP-MECP2-E2 (Figures 23-24). We found that the protein abundance correlates with growth for the R255X mutants of both MECP2 isoforms. For example, the first replicate of GFP-MECP2-E1-R255X had the worst growth out of the three and this was also the one with the highest GFP intensity (Figures 15, 21-22). Similarly, the second replicate of GFP-MECP2-E2-R255X had the greatest GFP level and the least growth out of the three replicates (Figures 16, 23-24). While the altered phenotype can be explained by altered protein amounts for the R255X mutant, the same argument cannot be applied to the other mutants. For example, replicates 2 and 3 of GFP-MECP2-E2-P389X had similar growth inhibition as wild-type MECP2 but replicates 1 75 and 3 had similar GFP intensities (Figures 16, 23-24). In this case, there was no correlation between protein level and growth. Thus, we cannot conclude that the variability in phenotypes of yeast expressing GFP-MECP2 is due exclusively to differences in MECP2 levels. 3.3.3 The impact of disease-associated mutations on protein localization Our findings suggest that the mutations R168X and T158M disrupt MECP2 function in yeast, likely by impacting its ability to bind to chromatin. Alternatively, it is also possible that these mutations abrogate the nuclear import of MECP2. To verify this, we used fluorescence microscopy to confirm the location of GFP-MECP2 within yeast. We also imaged a yeast strain containing a GFP-tagged H3 as a positive control to show the location of the nucleus. The GFP-H3 is constitutively expressed and did not need to be induced with estradiol. As before, we treated mid-log cultures with 0 or 1 μM β-estradiol before harvesting and imaging. We tested the strains containing vector-alone, strains expressing GFP-MECP2-E1, and the mutants that did not exhibit any growth defects GFP-MECP2-E1-R168X and GFP-MECP2-E1-T158M. Note that we picked the first biological replicate from the flow cytometry and growth assay experiments for our microscopy analysis. Without estradiol, there was no GFP signal. When estradiol was added to induce the expression of MECP2, we observed GFP in the cells. Both GFP-MECP2-E1 and GFP-MECP2-E1-T158M localized to the nucleus. In contrast, GFP-MECP2-E1-R168X was in the cytosol (Figure 25). We repeated the experiment with the other isoform, GFP-MECP2-E2, and again found that only the protein with the R168X mutation localized to the cytosol while the others were in the nucleus (Figure 26). This isn’t surprising since the nuclear localization signal (NLS) of MECP2 is located at amino acids 225-271, and the R168X mutation truncates the protein before the NLS (114). We noticed that the fluorescence signal of GFP-MECP2-E2-R168X was much 76 fainter than the GFP-MECP2-E1-R168X. This fainter fluorescence signal was due to variability in protein expression between the strains. If we look at the median GFP intensities of the first replicate of these strains from the ECDF plots (y-axis=0.50), the GFP-signal of GFP-MECP2-E2-R168X was approximately 500, while GFP-MECP2-E1-R168X was about 5000, almost ten times greater than E2 (Figures 22 and 24). Furthermore, GFP-MECP2-E2 and GFP-MECP2-E2-T158M both had median GFP signals of ~1000, which were greater than GFP-MECP2-E2-R168X (Figure 24). The GFP fluorescence of GFP-MECP2-E2 and GFP-MECP2-E2-T158M looked similar and were brighter than GFP-MECP2-E2-R168X under the microscope (Figure 26). Likewise, the first replicate of GFP-MECP2-E1 and GFP-MECP-E1-R168X had the strongest GFP signal from microscopy and had the highest median GFP intensity ~5000 compared to GFP-MECP2-E1-T158M, which looked less bright with microscopy and had a median GFP intensity of ~1000 (Figures 22 and 25). Therefore, variations in the GFP signal from microscopy correlate with variable protein levels from our flow cytometry quantification. We suspect that the R168X truncation likely hinders the nuclear import of MECP2, resulting in vector-alone-like growth levels from the growth assays (Figures 15-16). Although the T158M mutation did not affect nuclear localization, the point mutation in the methyl-CpG-binding domain (MBD) likely abrogated the normal function of MECP2 in yeast and thus, yeast with this mutation did not have growth defects (166, 173). Collectively, our results demonstrated that expression of MECP2 under unmethylated conditions in yeast inhibits growth by either disrupting cell cycle progression in the case of MECP2-E2, or rendering the cells inviable, as may be the case for MECP2-E1. Moreover, we found that both isoforms of GFP-MECP2 localize to the nucleus, suggesting that they may interact with unmethylated chromatin in yeast. Finally, although the mechanism of growth inhibition by 77 MECP2 is unknown, this is disrupted by the RTT-associated mutation T158M, suggesting that yeast may be a good model to study the pathology of other disease-associated mutations. 78 Figure 14. Mutations R168X and T158M revert the growth defect induced by MECP2-E2 expression The growth of yeast strains expressing MECP2-E2 were compared to strains expressing the Rett-associated truncation mutations R168X to P389X and the Rett-associated point mutant T158M. Cells were serially diluted 10-fold from (1x-10000x) and spotted onto -leucine-uracil drop-out plates containing 0 or 1 μM estradiol and grown for 2 days at 30°C. A vector-only strain was included as a control. Three independently generated replicates were tested for each strain. 79 Figure 15. Phenotypes are variable between biological replicates of yeast expressing GFP-MECP2-E1 mutants The yeast strains expressing GFP-MECP2-E1 were compared to strains expressing Rett-associated truncation mutations R168X to P389X and the RTT-associated point mutant T158M. Cells were serially diluted 10-fold from (1x-10000x) and spotted onto -leucine-uracil drop-out plates containing 0 or 1 μM estradiol and grown for 2 days at 30°C. A vector-only strain was included as a control. Three independently generated replicates were tested for each strain. 80 Figure 16. Phenotypes were variable between biological replicates of yeast expressing GFP-MECP2-E2 mutants The growth of yeast strains expressing GFP-MECP2-E2 were compared to strains expressing RTT-associated truncation mutations R168X to P389X and the Rett-associated point mutant T158M. Cells were serially diluted 10-fold from (1x-10000x) and spotted onto -leucine-uracil drop-out plates containing 0 or 1 μM estradiol and grown for 2 days at 30°C. A vector-only strain was included as a control. Three independently generated replicates were tested for each strain.81 Figure 17. Variability in MECP2 expression between independently derived yeast strains GFP intensities of yeast expressing either vector-only, GFP-MECP2-E1 or GFP-MECP2-E2 were quantified with flow cytometry. Cultures were grown to mid-log and 0 or 1 μM β-estradiol (bottom and top row, respectively) was added to induce expression for 1 hour before harvesting and quantifying with flow cytometry. The mean GFP intensities of 5000 cells are displayed as histograms. Five independently generated colonies were tested for each strain. Vector GFP−MECP2−E1 GFP−MECP2−E2101e+00 1e+01 1e+02 1e+03 1e+04 1e+00 1e+01 1e+02 1e+03 1e+04 1e+00 1e+01 1e+02 1e+03 1e+04GFP intensityReplicate54321β-estradiol (μM)82 Figure 18. Variability in MECP2 expression between independently derived replicates (part 2) The same as Figure 17, but the results are displayed as empirical cumulative distribution function (ECDF) plots to show the relative distribution of the GFP intensity. The relative frequency is plotted on the y-axis and the GFP intensity is plotted in log-scale on the x-axis. Vector GFP−MECP2−E1 GFP−MECP2−E2101e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+050.000.250.500.751.000.000.250.500.751.00GFP intensityECDFReplicate54321Vector GFP−MECP2−E1 GFP−MECP2−E2101e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+050.000.250.500.751.000.000.250.500.751.00GFP intensityECDFReplicate54321β-estradiol (μM)83 Figure 19. Low variability in MECP2 expression between replicates generated from a single colony GFP intensities of yeast expressing either vector-only, GFP-MECP2-E1, GFP-MECP2-E1-R255X, or GFP-MECP2-E1-T158M were quantified with flow cytometry. Cultures were grown to mid-log and 0 or 1 μM β-estradiol (bottom and top row, respectively) was added to induce expression for 1 hour before harvesting and quantifying with flow cytometry. The mean GFP intensities of 5000 cells are displayed as histograms. Three biological replicates were tested for each strain. The replicates were generated from a single colony. Vector GFP−MECP2−E1 GFP−MECP2−E1−R255X GFP−MECP2−E1−T158M101e+00 1e+01 1e+02 1e+03 1e+04 1e+00 1e+01 1e+02 1e+03 1e+04 1e+00 1e+01 1e+02 1e+03 1e+04 1e+00 1e+01 1e+02 1e+03 1e+04GFP intensityReplicate321β-estradiol (μM)84 Figure 20. Low variability in MECP2 expression between replicates generated from a single colony (part 2) The same as Figure 19, but the results are displayed as empirical cumulative distribution function (ECDF) plots to show the relative distribution of the GFP intensity. The relative frequency is plotted on the y-axis and the GFP intensity is plotted in log-scale on the x-axis. β-estradiol (μM)Vector GFP−MECP2−E1 GFP−MECP2−E1−R255X GFP−MECP2−E1−T158M101e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+050.000.250.500.751.000.000.250.500.751.00GFP intensityECDFReplicate321Vector GFP−MECP2−E1 GFP−MECP2−E1−R255X GFP−MECP2−E1−T158M101e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+050.000.250.500.751.000.000.250.500.751.00GFP intensityECDFReplicate32185 Figure 21. GFP-MECP2-E1 and mutants are variably expressed in yeast GFP intensities of yeast expressing either vector-only, GFP-MECP2-E1 or RTT-associated truncation mutations R168X to P389X and the point mutation T158M were quantified with flow cytometry. Cultures were grown to mid-log and 0 or 1 μM β-estradiol (bottom and top row, respectively) was added to induce expression for 1 hour before harvesting and quantifying with flow cytometry. The mean GFP intensities of 5000 cells are displayed as histograms. Three independently generated colonies were tested for each strain. β-estradiol (μM)Vector GFP−MECP2−E1 GFP−MECP2−E1−R168X GFP−MECP2−E1−R255X GFP−MECP2−E1−R270X GFP−MECP2−E1−R294X GFP−MECP2−E1−P389X GFP−MECP2−E1−T158M101e+00 1e+01 1e+02 1e+03 1e+04 1e+00 1e+01 1e+02 1e+03 1e+04 1e+00 1e+01 1e+02 1e+03 1e+04 1e+00 1e+01 1e+02 1e+03 1e+04 1e+00 1e+01 1e+02 1e+03 1e+04 1e+00 1e+01 1e+02 1e+03 1e+04 1e+00 1e+01 1e+02 1e+03 1e+04 1e+00 1e+01 1e+02 1e+03 1e+04GFP intensityReplicate32186 Figure 22. GFP-MECP2-E1 and mutants are variably expressed in yeast (part 2) The same as Figure 21, but the results are displayed as empirical cumulative distribution function (ECDF) plots to show the relative distribution of the GFP intensity. The relative frequency is plotted on the y-axis and the GFP intensity is plotted in log-scale on the x-axis.Vector GFP−MECP2−E1 GFP−MECP2−E1−R168X GFP−MECP2−E1−R255X GFP−MECP2−E1−R270X GFP−MECP2−E1−R294X GFP−MECP2−E1−P389X GFP−MECP2−E1−T158M101e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+050.000.250.500.751.000.000.250.500.751.00GFP intensityECDFReplicate321Vector GFP−MECP2−E1 GFP−MECP2−E1−R168X GFP−MECP2−E1−R255X GFP−MECP2−E1−R270X GFP−MECP2−E1−R294X GFP−MECP2−E1−P389X GFP−MECP2−E1−T158M101e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+050.000.250.500.751.000.000.250.500.751.00GFP intensityECDFReplicate321β-estradiol (μM)87 Figure 23. GFP-MECP2-E2 and mutants are variably expressed in yeast GFP intensities of yeast expressing either vector-only, GFP-MECP2-E2 or RTT-associated truncation mutations R168X to P389X and the point mutation T158M were quantified with flow cytometry. Cultures were grown to mid-log and 0 or 1 μM β-estradiol (bottom and top row, respectively) was added to induce expression for 1 hour before harvesting and quantifying with flow cytometry. The mean GFP intensities of 5000 cells are displayed as histograms. Three independently generated colonies were tested for each strain. β-estradiol (μM)Vector GFP−MECP2−E2 GFP−MECP2−E2−R168X GFP−MECP2−E2−R255X GFP−MECP2−E2−R270X GFP−MECP2−E2−R294X GFP−MECP2−E2−P389X GFP−MECP2−E2−T158M101e+00 1e+01 1e+02 1e+03 1e+04 1e+00 1e+01 1e+02 1e+03 1e+04 1e+00 1e+01 1e+02 1e+03 1e+04 1e+00 1e+01 1e+02 1e+03 1e+04 1e+00 1e+01 1e+02 1e+03 1e+04 1e+00 1e+01 1e+02 1e+03 1e+04 1e+00 1e+01 1e+02 1e+03 1e+04 1e+00 1e+01 1e+02 1e+03 1e+04GFP intensityReplicate32188 Figure 24. GFP-MECP2-E2 and mutants are variably expressed in yeast (part 2) The same as Figure 23, but the results are displayed as empirical cumulative distribution function (ECDF) plots to show the relative distribution of the GFP intensity. The relative frequency is plotted on the y-axis and the GFP intensity is plotted in log-scale on the x-axis.Vector GFP−MECP2−E1 GFP−MECP2−E1−R168X GFP−MECP2−E1−R255X GFP−MECP2−E1−R270X GFP−MECP2−E1−R294X GFP−MECP2−E1−P389X GFP−MECP2−E1−T158M101e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+050.000.250.500.751.000.000.250.500.751.00GFP intensityECDFReplicate321β-estradiol (μM)Vector GFP−MECP2−E2 GFP−MECP2−E2−R168X GFP−MECP2−E2−R255X GFP−MECP2−E2−R270X GFP−MECP2−E2−R294X GFP−MECP2−E2−P389X GFP−MECP2−E2−T158M101e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+05 1e+01 1e+03 1e+050.000.250.500.751.000.000.250.500.751.00GFP intensityECDFReplicate32189 Figure 25. MECP2-E1 wild-type and the T158M mutant, but not the R168X mutant, are nuclear-localized when expressed in yeast Cells were treated with 0 μM or 1 μM estradiol for 1 hour before they were imaged with brightfield or fluorescence microscopy. The GFP tag was added to the N-terminus of MECP2-E1, MECP2-E1-R168X, or MECP2-E1-T158M. A GFP-H3 sample was included as a positive control to show the location of histones in the nucleus. Scale bars represent 10 micrometres (μm). Bright!eldGFPMergeβ-estradiol (μM) 1Vector0 0GFP-MECP2-E11 0 1GFP-MECP2-E1-R168X0 1GFP-MECP2-E1-T158M GFP-H390 Figure 26. MECP2-E2 wild-type and the T158M mutant, but not the R168X mutant, are nuclear-localized when expressed in yeast Cells were treated with 0 μM or 1 μM estradiol for 1 hour before they were imaged with brightfield or fluorescence microscopy. The GFP tag was added to the N-terminus of MECP2-E2, MECP2-E2-R168X, or MECP2-E2-T158M. A GFP-H3 sample was included as a positive control to show the location of histones in the nucleus. Scale bars represent 10 micrometres (μm). Bright!eldGFPMergeβ-estradiol (μM) 1Vector0 0GFP-MECP2-E21 0 1GFP-MECP2-E2-R168X0 1GFP-MECP2-E2-T158M GFP-H391 Chapter 4: Discussion In this thesis, we demonstrated the power of using yeast as a model organism to study MECP2 in vivo. We found that in a system that lacks DNA methylation, MECP2 was able to alter the growth phenotype of yeast. We confirmed we can overexpress MECP2 using a galactose-inducible promoter, or fine-tune the expression using the beta-estradiol induction system. The maximum inducible MECP2 amount in yeast was achieved by treating cells with 1 µM β-estradiol for 4 hours. Using flow cytometry, we were able to detect the expression of both MECP2 isoforms, E1 and E2, as well as the disease-associated mutations, T158M, R168X, R255X, R270X, R294X and P389X in single cells in vivo. We confirmed that we could observe changes in yeast phenotypes in strains expressing MECP2 and MECP2 mutants using propidium iodide staining combined with fluorescence microscopy and flow cytometry. We noticed that adding a GFP or a GST tag to the N-terminus of MECP2, but not the C-terminus of MECP2 expressed in yeast, alters the protein abundance and cell phenotype, especially for the E1 isoform of MECP2. Since the longer N-terminus of MECP2-E1 is mostly disordered, we suspect that the presence of a structured domain at the N-terminus stabilizes the protein and reduces degradation (152, 163, 164, 207). Indeed, it has been demonstrated that in yeast, proteins with longer disordered regions at the terminal ends have a much shorter half-life than proteins with shorter disordered regions (208). A similar trend is observed in mice and humans, suggesting that rules governing protein stability are conserved across evolution (208). Moreover, the stability of MECP2-E1 is sensitive to changes in the N-terminus, as a mutation at the very end of the protein, A2V, results in faster degradation than WT MECP2 (209). Perhaps this may explain why RTT-related mutations are found on exon 1, but not on exon 2 (167–170). Since MECP2 levels are close 92 to histone octamer levels in neurons, the higher protein abundance of GFP-tagged versus untagged-MECP2 in yeast may be more reflective of the protein concentration in neurons (115). Interestingly, the majority of studies using mouse tissues or mammalian cell lines utilize MECP2 with a GFP tag on the C-terminus, but not the N-terminus (160, 176, 179, 192, 197, 199, 210). However, because we did not observe changes in the growth of yeast expressing the C-terminal tagged MECP2-E1, nor did we detect the protein on immunoblots, we did not explore the C-terminally tagged MECP2 further in our studies. We found that the RTT-associated mutation, T158M, alleviates the growth defect of MECP2-expressing yeast, but did not alter protein localization within the cell. This suggests that the MBD is important for the growth-inhibiting properties of MECP2 in yeast. In vitro, MECP2 can form phase-separated condensates in the presence of unmethylated DNA, but not on its own, and this ability is disrupted by the T158M mutation (174). Collectively, these results support the possibility that growth inhibition by MECP2 might be due to DNA-dependent phase separation. However, due to the small size of the yeast nucleus (~1 µm in radius), we were unable to determine whether LLPS occurs in the yeast nucleus, or whether MECP2 clusters at distinct regions on the yeast chromatin with our microscope (211). Given that MECP2 has been shown to bind directly to DNA, it is equally likely that the binding of MECP2 to yeast chromatin blocks the progression of RNA polymerase or DNA replication complexes (155, 183). Indeed, because the stalling of replication forks activates the G1/S DNA damage checkpoint, inhibition of DNA replication may explain the cell cycle arrest in cells expressing MECP2 (212, 213). A test for this would be fluorescently tagging the Rad52 protein in yeast and imaging with fluorescence microscopy (214). The formation of Rad52 foci in arrested, MECP2-expressing yeast would provide support for checkpoint activation. While the localization and binding of MECP2 to unmethylated chromatin 93 in vivo have not been directly tested in our study, we would like to caution that it may be challenging to study MECP2 binding using common techniques that study protein binding to chromatin such as ChIP-seq. For example, ChIP-qPCR, which requires formaldehyde crosslinking, was not able to detect differences in the distribution of MECP2-R270X and G273X on the genome, but when using native ChIP-qPCR that avoided the crosslinking step, the difference between the two mutants was evident (160). A similar issue has been observed in other studies. The interactions between transcription factors and chromatin are dynamic, and crosslinking with formaldehyde can cause artifactual exclusion of transcription factors from mitotic chromosomes (215). Given that MECP2 is a transient, dynamic protein that can phase separate, it should be no surprise that crosslinking will disturb its interactions in vivo (174, 175). Unfortunately, crosslinking is required for ChIP in yeast to maintain protein-DNA contacts during processes such as sonication or enzyme treatment. Therefore, instead of ChIP-seq, we propose using non-crosslinking alternatives like ChEC-seq to study protein localization in yeast (216). However, since MECP2-expressing yeast has defects in the cell cycle, it would be challenging to rule out any effects due to the S phase arrest. Although not tested in our study, it is possible that MECP2 interacts with proteins in yeast and disrupts their function. For example, MECP2 may interact with the yeast Sin3-Rpd3 or the Set3 complex, the homologs of mammalian Sin3A-HDAC1/2 and NCoR/SMRT, respectively. This is likely since the HDAC-interaction domain (HID) of mSin3A, one of the regions that MECP2 binds to, is also present in the yeast Sin3 (189, 217). If MECP2 binds to these deacetylase complexes, it may interfere with their normal function, resulting in cell cycle arrest and reduced viability as we observed in our research. Yeast Sin3-Rpd3 associates with the promoter of the G2/M-specific gene, CLB2, during G1 and G2 phase, but not during S phase (218). It is possible 94 that the association of MECP2 with Sin3-Rpd3 prevents the complex from reassociating with the promoter, causing defects in cell cycle progression at the S/G2 transition. Alternatively, strains with an RPD3 deletion fire replication origins earlier than WT yeast cells and this is not observed in other deacetylase mutants, and thus MECP2 could interfere with proper replication timing during S phase (70). Since the Set3 complex is involved in the repression of cryptic transcription at the 5’ end of genes, disruption of Set3 may result in aberrant transcription of genes required for cell-cycle regulation, which may also explain the cell cycle arrest and death in the MECP2-expressing yeast (219). The binding of MECP2 to Sin3-Rpd3 or the Set3 complex may also alter their repressor activities. To test this, we could use RNAPII ChIP-seq to compare WT yeast versus MECP2-expressing yeast to detect changes in gene expression. We noticed that at four hours post-induction, GFP-MECP2-E1, but not GFP-MECP2-E2-expressing yeast contained dead cells. Therefore, it is possible that the two isoforms of MECP2 interact with different proteins. Future experiments that test protein interactions may explain the different effects that expression of the two isoforms have on yeast viability. We can explore this using MECP2 immunoprecipitation followed by mass spectrometry to identify any binding partners. Another method we could use is split-GFP (220, 221). In this approach, GFP is split into two domains, and each is conjugated onto two different proteins. A GFP signal will only be detected when the two proteins are in close proximity, allowing GFP to form. We can conjugate part of the GFP domains to MECP2 and another to proteins we suspect could interact, such as Sin3 or Set3, and measure the change in fluorescence signal using flow cytometry or fluorescence microscopy to detect interacting proteins in vivo. Since MECP2 binds to linker regions and competes with the linker histone H1 in neurons, it is also likely that MECP2 may interact with the linker-histone-like proteins Hmo1 or Hho1 in 95 yeast (115, 140, 185). Considering the fact that HMO1 deletion, but not HHO1 deletion affects yeast viability, it is more likely that MECP2 interacts with Hmo1 to disrupt its function in yeast, resulting in the growth defect we observed in MECP2-expressing yeast (20, 22, 23). We can confirm this by expressing MECP2 in yeast strains with HMO1 deletion to see if they revert the growth defects of MECP2-expressing yeast. Overall, it is likely that MECP2 interacts with a combination of proteins such as Sin3-Rpd3, Set3 complex, and Hmo1 to disrupt their normal function in yeast. A major challenge we encountered in the research is the inconsistency in protein expression for our biological replicates. Multiple copies of the non-centromeric plasmids can be integrated into the yeast genome (222). Therefore, we suspect that there may be multiple integrations of the MECP2 gene near the URA3 site. It is unlikely that multiple integrations for the artificial transcription factor (ATF) were the cause of protein level inconsistency because all the biological replicates for the MECP2-expressing yeast were generated from the same ZF4-ER-VP16-expressing starter strain. Since our biological replicates are generated independently, each colony likely took up a different number of plasmids during transformation, resulting in a different number of copies of MECP2 integrated into the genome. To determine if this is the case, it is possible to design primers that specifically amplify tandem copies of the integrated vector with PCR. It is also possible to quantify the number of MECP2 genes with quantitative PCR (qPCR), which will allow us to select strains with the same number of copies of the gene to use for biological replicates for our experiments. It is also possible to avoid multiple integrations by using a low copy number plasmid containing CEN3, which usually limits the plasmid number to one copy (223). Our research validated the potential of using the budding yeast, Saccharomyces cerevisiae as a model organism to study the mammalian methyl-CpG binding protein 2 (MECP2). We were 96 able to observe phenotypes in yeast that coincide with the RTT-like phenotypes. Despite a lack of DNA methylation and large heterochromatic regions in yeast, expression of MECP2 greatly perturbed yeast viability, suggesting that it may disrupt normal replication and or transcription processes. Future research exploring protein-chromatin interactions, protein-protein interactions, and DNA damage may provide an in-depth understanding of how MECP2 functions under unmethylated chromatin conditions in vivo. 97 References 1. Luger, K. (1997) Crystal structure of the nucleosome core particle at 2.8 A˚ resolution. 389, 10 2. Buschbeck, M., and Hake, S. B. 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Thesis/Dissertation
eng
Biochemistry and Molecular Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Howe, LeAnn
Investigating the effects of expressing MECP2 in Saccharomyces cerevisiae
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