Medicine, Faculty of
Medical Genetics, Department of
DSpace
UBCV
Ashworth, Nolan
Master of Science - MSc
University of British Columbia
Imprinting, an epigenetic phenomenon where only one allele of a gene is expressed depending on its parental origin, plays a significant role in mammalian development. The sub-proximal MMU6 imprinting domain, particularly, includes the paternally expressed gene Mest and the maternally expressed genes Copg2 and Klf14. In addition to the mature mRNA, Mest has a much longer isoform produced via alternative polyadenylation, termed MestXL. Mest is a candidate gene for the growth restriction Silver-Russell syndrome (SRS), since maternal uniparental disomy for the chromosomal region containing MEST (mUPD7) has been identified in 5-10% of SRS patients and because Mest knockout mice exhibit a growth restriction phenotype. The tissue-specific switch from Mest to MestXL formation seems to be linked to differential expression of the downstream gene Copg2. Copg2, which is adjacent and antisense to Mest/MestXL, is normally biallelically expressed. However, in tissues where MestXL is produced, such as the developing central nervous system, Copg2 shows preferential maternal expression, suggesting a potential regulatory role for MestXL in this process since it is exclusively expressed form the paternal allele. This thesis explores the expression and regulation of MestXL, which induces secondary imprinting of Copg2 within the central nervous system (CNS). Although it is known that MestXL is expressed in the CNS, the specific cell types, mechanisms behind its production and effect on paternal Copg2 expression remain unclear. I leveraged RNA-seq data and established an ESC-derived cortical neuron differentiation protocol to investigate MestXL expression. To study the sufficiency of the Mest mRNA polyA signal (PAS) in MestXL production in cortical neurons, I also developed a double-fluorescent Mest primary PAS transgenic ESC line. Finally, I constructed an endogenous MestXL reporter-truncation vector, for eventual integration at Copg2 and forward genetics studies of factors involved in MestXL expression and Copg2 imprinting in F1 ESCs. These tools and methodologies provide a foundation for understanding the regulation and function of the Mest locus, with broader implications for APA regulation and secondary imprinting.
ALTERNATIVE POLYADENYLATION AT MEST by Nolan Ashworth B.Sc., University of Alberta, 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 (Medical Genetics) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) November 2024 © Nolan Ashworth, 2024 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Alternative Polyadenylation at Mest submitted by Nolan Ashworth in partial fulfilment of the requirements for the degree of Master of Science in Medical Genetics Examining Committee: Louis Lefebvre, Associate Professor, Medical Genetics, UBC Supervisor Carolyn Brown, Professor, Medical Genetics, UBC Supervisory Committee Member Michael Underhill, Professor, Cellular & Physiological Sciences and School of Biomedical Engineering, UBC Supervisory Committee Member Catherine Van Raamsdonk, Associate Professor, Medical Genetics, UBC Additional Examiner iii Abstract Imprinting, an epigenetic phenomenon where only one allele of a gene is expressed depending on its parental origin, plays a significant role in mammalian development. The sub-proximal MMU6 imprinting domain, particularly, includes the paternally expressed gene Mest and the maternally expressed genes Copg2 and Klf14. In addition to the mature mRNA, Mest has a much longer isoform produced via alternative polyadenylation, termed MestXL. Mest is a candidate gene for the growth restriction Silver-Russell syndrome (SRS), since maternal uniparental disomy for the chromosomal region containing MEST (mUPD7) has been identified in 5-10% of SRS patients and because Mest knockout mice exhibit a growth restriction phenotype. The tissue-specific switch from Mest to MestXL formation seems to be linked to differential expression of the downstream gene Copg2. Copg2, which is adjacent and antisense to Mest/MestXL, is normally biallelically expressed. However, in tissues where MestXL is produced, such as the developing central nervous system, Copg2 shows preferential maternal expression, suggesting a potential regulatory role for MestXL in this process since it is exclusively expressed form the paternal allele. This thesis explores the expression and regulation of MestXL, which induces secondary imprinting of Copg2 within the central nervous system (CNS). Although it is known that MestXL is expressed in the CNS, the specific cell types, mechanisms behind its production and effect on paternal Copg2 expression remain unclear. I leveraged RNA-seq data and established an ESC-derived cortical neuron differentiation protocol to investigate MestXL expression. To study the sufficiency of the Mest mRNA polyA signal (PAS) in MestXL production in cortical neurons, I also developed a double-fluorescent iv Mest primary PAS transgenic ESC line. Finally, I constructed an endogenous MestXL reporter-truncation vector, for eventual integration at Copg2 and forward genetics studies of factors involved in MestXL expression and Copg2 imprinting in F1 ESCs. These tools and methodologies provide a foundation for understanding the regulation and function of the Mest locus, with broader implications for APA regulation and secondary imprinting. v Lay Summary Imprinting is a genetic process where only one copy of a gene is used, depending on whether it came from the mother or the father. The gene Mest is only turned on in the father’s copy. Mest has a longer version called MestXL. Copg2, which is located next to Mest/MestXL, usually gets expressed from both the mother's and father's copies. In tissues where MestXL is active, Copg2 is preferentially turned on from the mother's copy. I investigated how MestXL is made and how it affects Copg2 in the brain and nervous system. To explore this, I made MestXL-expressing brain cells out of embryonic stem cells. I also created a cell line to see if the sequence that ends Mest mRNA is enough to produce MestXL in brain cells. Additionally, I used a genetic editing tool to try to create a line that turns green when MestXL is expressed. vi Preface N. Ashworth performed all experiments, except preliminary brain organoid qRT-PCR and qRT-PCR analysis, which was performed by undergraduate honours student Abdullah Khawaja. The work described in this thesis was approved by the UBC Biosafety Committee and covered under protocol number B22-009. ChatGPT was used in the drafting of this thesis to suggest alternative word choice (as a thesaurus) and to provide suggestions on how to separate sub-chapters. The original plasmids used for this study were obtained from L. Lefebvre and A. Bogutz. The initial project conceptualization and design was done by L. Lefebvre and carried out under the guidance of L. Lefebvre and A. Bogutz. vii Table of Contents Abstract ........................................................................................................................ iii Lay Summary ................................................................................................................. v Preface ......................................................................................................................... vi Table of Contents ......................................................................................................... vii List of Tables .................................................................................................................. x List of Figures ............................................................................................................... xi Acknowledgements ..................................................................................................... xii Dedication .................................................................................................................. xiii Chapter 1: Introduction ................................................................................................ 1 1.1 Overview of Genomic Imprinting ........................................................................ 1 1.1.1 History ........................................................................................................ 1 1.1.2 Evolutionary Theories of Genomic Imprinting ............................................. 2 1.1.3 DNA Methylation in Genomic Imprinting ..................................................... 3 1.1.4 Regulation of Imprinting by gDMRs and ICRs .............................................. 5 1.1.5 Tissue-specific Imprinting ........................................................................... 9 1.2 Sub-proximal MMU6 Imprinted Domain ............................................................10 1.2.1 Mest ...........................................................................................................12 1.2.2 Mest Knockout Models ...............................................................................13 1.2.3 miR-335 ......................................................................................................14 1.2.4 Klf14 ...........................................................................................................15 1.2.5 Copg2 .........................................................................................................15 1.2.6 Human Homology and Disease ..................................................................16 1.4 Alternative Polyadenylation and 3’ UTR choice .................................................18 1.4.1 Overview ....................................................................................................18 1.4.2 Functions of APA/3’UTR choice ..................................................................19 1.4.3 Regulation of 3’UTR APA .............................................................................20 1.4.3.1 Cis- acting factors regulating APA .............................................................20 viii 1.4.3.2 Specific Trans-Acting Proteins and APA .....................................................22 1.4.3.3 Influence of Transcriptional and Translational Status on 3'UTR Choice ....23 1.4.4 Tissue-Specific APA/3’UTR Choice .............................................................25 1.4.5 Human Homology and Disease ..................................................................27 1.5 Thesis Theme and Objectives ............................................................................28 Chapter 2: Materials and Methods ...............................................................................30 2.1 Cortical Neuron Differentiation ..........................................................................30 2.2 Gibson Assembly ................................................................................................30 2.3 Bacterial Transformation ....................................................................................31 2.4 Polymerase Chain Reaction ................................................................................31 2.5 RNA Extraction ...................................................................................................32 2.6 Reverse Transcription .........................................................................................32 2.7 qRT-PCR ..............................................................................................................32 2.8 Lipofection In-suspension ..................................................................................33 2.9 Immunohistochemistry ......................................................................................34 2.10 Plasmid Construction .......................................................................................34 2.10.1 Endogenous Reporter Construct ................................................................34 2.10.2 Exogenous Mest PAS Construct with Neo Selectable Marker .....................35 2.10.3 Floxed pCTPIE for RMCE .............................................................................35 2.11 ESC Line Generation Attempts .........................................................................35 2.11.1 C2 Mest PAS Double Fluorescent Reporter.................................................35 2.11.2 pCTPIE RMCE Transfection .........................................................................36 2.11.3 Endogenous Reporter Construct ................................................................36 Chapter 3: Results .......................................................................................................42 3.1 Readthrough of Exogenous Mest PAS ..................................................................42 3.1.1 Background/Rationale .................................................................................42 3.1.2 Plasmid Construction and ESC Transfections ..............................................43 3.1.3 Cortical Neuron Differentiation ....................................................................49 3.2 Trapping and Truncating Endogenous MestXL expression ...................................50 ix 3.2.1 Background/Rationale .................................................................................50 3.2.2 Plasmid Construction and ESC Transfection ................................................51 3.3 Cortical Neuron Differentiation in Brain Organoids ............................................56 3.3.1 Background/Rationale .................................................................................56 3.3.2 Cell-Type and Protocol Choice .....................................................................57 3.3.3 Troubleshooting and Protocol Establishment ..............................................59 Chapter 4: Discussion .................................................................................................67 4.1 Mest Primary PAS Readthrough ..........................................................................67 4.2 Endogenous MestXL Reporter .............................................................................70 4.3 Conclusion .........................................................................................................74 References ..................................................................................................................75 Appendix: Supplemental Figures .................................................................................91 x List of Tables TABLE 2.1 LIST OF OLIGONUCLEOTIDE PRIMERS USED ......................................................................... 37 xi List of Figures FIGURE 1. 1 MECHANISMS OF IMPRINTING REGULATION ....................................................................... 9 FIGURE 1.2 MEST SUB-PROXIMAL MMU6 IMPRINTED DOMAIN .............................................................. 10 FIGURE 1.3 MEST-COPG2 REGULATORY RELATIONSHIP ........................................................................ 12 FIGURE 1.4 MEST PRIMARY PAS REGION ............................................................................................... 21 FIGURE 1.5 MECHANISMS OF APA ......................................................................................................... 24 FIGURE 3.1 EXOGENOUS MEST PAS CONSTRUCT .................................................................................. 46 FIGURE 3.2 C2 MEST PAS INTEGRANTS .................................................................................................. 48 FIGURE 3.3 ENDOGENOUS REPORTER CONSTRUCT ............................................................................. 55 FIGURE 3.4 MEST LOCUS RNA-SEQ & ENDOGENOUS REPORTER TARGET .............................................. 58 FIGURE 3.5 HEALTHY BRAIN ORGANOID MORPHOLOGY PROGRESSION ............................................... 63 FIGURE 3.6 CONFOCAL MICROSCOPY OF D21 BC11 CORTICAL NEURON ORGANOIDS ......................... 64 FIGURE 3.7 D21 CN ORGANOID QRT-PCR .............................................................................................. 65 FIGURE 4.1 MEST PAS FOLLOW-UP EXPERIMENTAL WORKFLOW ........................................................... 70 FIGURE S1 MESTXL SINGLE CELL RNA SEQUENCING ANALYSIS ............................................................ 91 FIGURE S2 MESTXL SECONDARY STRUCTURE ....................................................................................... 93 xii Acknowledgements Of critical importance to my completion of this body of work were firstly the members of the Lefebvre lab. Louis for hiring me and his tireless support in designing, progressing and troubleshooting my project. Sherry Fang has been an incredible friend and provided invaluable second opinions and support throughout my time here. Amanda Ha was an outstanding mentor and friend during our overlapping time in the lab. Finally, Aaron Bogutz has been and continues to be a phenomenal mentor, instrumental in progression of my project and unendingly patient in answering my plethora of daily questions. Additionally, the Teves, Lorincz and Penninger labs were invaluable in both advice, providing reagents and protocol optimization. Lastly, Jeremy Wong, Uyseok Lee and Mahara Mtawali (pictured below) kept me sane and grounded and have been three of the best teammates and friends I could ever ask for. xiii Dedication To my family, for being themselves. Chapter 1: Introduction 1.1 Overview of Genomic Imprinting 1.1.1 History Most mammalian genes are expressed biparentally, and identification of an excepting phenomenon did not occur until the 1980s. The first studies in mammals on what came to be known as “imprinting” were performed by generating and observing the phenotypic differences between androgenetic and gynogenetic mouse embryos1–3. Neither of these experimentally derived uniparental embryos survived to term: they had phenotypic differences that suggested that both the maternal and paternal genomes were not only necessary, but also unique in their contributions. Haploid eggs with the male pronuclei removed and replaced with a second maternal genome (gynogenetic embryos) developed up to the 25-somite stage but had very poorly developed trophoblast tissue. Conversely, those embryos comprised of two paternal genomes (androgenetic embryos) had very short-lived development (6-8 somite stage) but significant trophoblast tissue development3. From these observations it was determined that although the parental genomes can be identical in sequence, they are functionally inequivalent, likely because of a germline-specific epigenetic “imprint” created during gametogenesis. Following the observation and terming of this imprinting phenomena, the first imprinted genes - genes preferentially expressed from either the maternal or paternal allele as the result of epigenetic marks acquired during gametogenesis - were identified, all of which showed essential roles in growth phenotypes4–6. Insulin-like growth factor type-2 (Igf2) and Igf2 receptor (Igf2r) were two of the first discovered imprinted genes 2 and their parental expressions are inverse to one another. Igf2 was shown to be paternally expressed, with paternal and homozygous mutants showing identical phenotypes5. Contrarily, Igf2r was identified as maternally expressed, as was the coevally discovered imprinted gene H194,6. Since then, approximately 200 imprinted genes have been identified, 63 of which exhibit homology in the human genome7. Many of these imprinted genes have essential developmental functions and are highly expressed in developmental tissues7, facts that have led to hypotheses as to their evolutionary value. 1.1.2 Evolutionary Theories of Genomic Imprinting As with any biological occurrence outside the norm, the evolutionary value of imprinting has been heavily theorized. One theory as to the origin of imprinting is the parental conflict hypothesis8. This hypothesis proposes that the inequivalence observed between the maternal and paternal genomes favours propagation of the respective parent’s genetic information. Because many imprinted genes have essential roles in embryonic and extraembryonic tissues it was proposed that imprinting arose in therian mammals, where embryos receive direct nourishment from maternal tissues, to regulate nutrient transfer. Summarily, this theory proposes that maternally expressed genes reduce nutrient transfer, favouring maternal genetic information by enabling multiple healthy pregnancies, whereas paternally expressed genes enhance nutrient transfer to conceptuses, increasing the likelihood that the paternal genetic information of an individual is passed on compared to intra- or inter- pregnancy competing paternal information8. Some popular alternative hypotheses as to the evolutionary origin of imprinting include the host defense model and the ovarian time bomb hypothesis9,10. 3 The host defense model proposes that genomic imprinting evolved to protect the organism from invasion by foreign DNA through epigenetic silencing9. In support of the host defense model there have been at least 10 retrotransposed imprinted genes identified thus far11,12. The ovarian time bomb hypothesis however, proposes that certain genes expressed solely from the paternal allele are vital for the development of extraembryonic tissues and that this requirement is crucial as it shields against the spontaneous activation of eggs that might otherwise result in the invasive growth of extraembryonic-like tissue, potentially leading to cancer10. While these theories differ in many regards, they agree on the idea that imprinting arose as an important mechanism of regulating gene dosage in mammals. 1.1.3 DNA Methylation in Genomic Imprinting As the parent-of-origin differences observed in imprinted genes could not be due to genetic sequence, imprinting was thought to be accomplished by epigenetic mechanisms. Since then, this has been confirmed, and much has been learned as to the specific epigenetic mechanisms responsible for imprinting. After the identification of the first imprinted genes, paternal-specific DNA methylation coinciding with closed-chromatin conformation was identified at the H19 promoter region, suggesting the possible involvement of DNA methylation (DNAme) in the regulation of imprinting13,14. Consistent with this finding, the DNA methyltransferase (DNMT) DNMT1 was shortly thereafter found to be necessary in maintaining imprinted expression of Igf2, Igf2r and H1915. The establishment and maintenance of proper gamete-specific imprinted DNAme is contingent upon a multi-step cycle in mouse development. After fertilization, there is a 4 global erasure of DNAme, except for specific regions called imprinted gametic differentially methylated regions (gDMRs), which retain their methylation patterns acquired from one gamete and persisting throughout development. Following implantation, DNAme is newly established (de novo) in the epiblast. During primordial germ cell development (gamete-precursors in the developing embryo), another wave of demethylation occurs, this time including imprinted gDMRs. This process allows for the resetting of epigenetic marks prior to the establishment of new germline DNAme patterns in a gamete-specific manner16. DNA methyltransferases are enzymes essential to imprinting that catalyze the addition of a methyl group to the 5th position carbon of cytosine residues in 5’-cytosine-guanine-3’ (CpG) dinucleotides. In the germline, DNAme is established by the action of the de novo DNA methyltransferase 3A (DNMT3A) and the non-catalytic cofactor DNMT3L, along with DNMT3B and DNMT3C, which function similarly to DNMT3A but with differing specificity17–22. While the process of selecting specific sequences for allele-specific DNAme in the germline is still not fully understood, some research suggests that transcription across ICR sequences serves as a crucial instructive step for DNA methyltransferases23,24. After fertilization, maintaining the parental-specific imprints is essential despite the extensive genome reprogramming and DNA demethylation that occur during preimplantation development16. The maintenance DNA methyltransferase DNMT1 and its cofactor necessary for recruitment, UHRF125, recognize hemi-methylated DNA and methylates the newly synthesized DNA strand. Additionally, the recognition of unique cis-acting sequences by trans-acting factors offers protection against post-fertilization reprogramming. For instance, the zinc finger protein homolog 5 57 (ZFP57) specifically recognizes the methylated allele of most gDMRs at imprinted genes and protects them from demethylation26. 1.1.4 Regulation of Imprinting by gDMRs and ICRs Imprinted genes are often found in clusters throughout the genome and within these clusters are regulated by cis-elements, termed imprinting control regions (ICRs) 27. An ICR is any cis-acting element responsible for the regulation of 2 or more imprinted genes and is necessary for their imprinted expression. These elements are usually gDMRs in CpG-rich promoters, and their gamete-specific differential DNAme results in the regulation of other genes within the cluster through several different mechanisms28. Apart from differential allele-specific DNAme patterns, imprinting control regions (ICRs) also display allele-specific histone modifications at histone H3 linked to both active and suppressed chromatin conditions. Active alleles are associated with trimethylation of lysine 4 (H3K4me3) whereas suppressed alleles show trimethylation at lysines 9 and/or 20 (H3K9me3 and H3K20me3)29,30. While there are currently only 25 confirmed ICRs in the human genome31, recent bisulphite sequencing analyses have identified 322 putative ICRs with gamete-specific DNAme32, suggesting that imprinting control regions may be a much more pervasive method of gene-cluster regulation than previously thought. Imprinted gene expression occurs via three main mechanisms: direct promoter DNAme, insulator interactions, and long non-coding RNA-mediated silencing33. The most well-known mechanism for regulating imprinted genes is allele-specific direct promoter DNAme, involving 5-methyl-cytosine at CpGs, resulting in gDMRs (Fig. 1.1A). In this process, the allele with a hypermethylated promoter remains inactive, while the 6 allele with a hypomethylated promoter is transcribed. This is perfectly exemplified at the Mest locus, where the promoter-gDMR is methylated and therefore not transcribed in oocytes but remains unmethylated and active in sperm, both of which are maintained following fertilization34,35. Imprinted genes dependent on a gDMR at their promoter will be termed to be “primary imprinted genes” in this thesis as their imprinting is not dependent on distal regions or indirect mechanisms. Most imprinted clusters contain at least one long non-coding RNA (lncRNA)36: a mechanism by which gDMRs can result in secondary (less direct than promoter DNAme) imprinting of genes surrounding an ICR. In this case, the promoter of a lncRNA is located within the ICR. On the paternal allele the ICR is unmethylated, thus allowing expression of the lncRNA, which in turn silences several genes in the domain in cis. This occurs by either attracting machinery that lay down repressive chromatin marks37 or by preventing RNA polymerase II recruitment at promoters (transcriptional interference)38. An illustration is seen in the Kcnq1 imprinting domain on mouse chromosome 7, where the lncRNA Kcnq1ot1 is regulated by maternal-specific methylation over the IC2 gDMR, situated within intron 10 of the Kcnq1 gene (Fig. 1.1C). IC2 encompasses the promoter of Kcnq1ot1, permitting only paternal expression of this lncRNA, which subsequently suppresses the paternal expression of Cdkn1c, Kcnq1, and other adjacent genes upstream and downstream of IC2, resulting in maternal allele expression of those protein-coding genes39–41. The third mechanism by which imprinted expression can be regulated is via methylation-sensitive insulator function (Fig. 1.1B). Regulation of the Igf2/H19 locus is illustrative of the function of this mechanism42–44. In this instance, on the maternal allele, 7 the imprinting control region (ICR) upstream of H19 remains unmethylated and interacts with the insulator protein CCCTC binding factor (CTCF). This interaction inhibits the downstream enhancers, which are shared by both H19 and Igf2, from interacting with the Igf2 promoter. However, it allows these enhancers to access H19 and stimulate its expression. Conversely, on the paternal allele the ICR is methylated, preventing CTCF binding and insulator formation, allowing Igf2 activation. 8 9 Figure 1. 1 Mechanisms of Imprinting Regulation (A) Direct promoter methylation occurs via allele-specific DNAme of CpG island promoters, creating a gDMR. The allele with the hypermethylated promoter remains silent, while the allele with the hypomethylated promoter is transcribed and expressed. (B) Methylation-sensitive insulator function is exemplified using the H19/Igf2 domain. IC1, an intergenic DMR between Igf2 and H19, binds the CTCF protein on the unmethylated maternal allele but is methylated on the paternal allele, allowing or precluding access of the enhancer to Igf2, respectively. (C) LncRNA-mediated epigenetic silencing, illustrated using the IC2 domain. Transcription of the Kcnq1ot1 lncRNA from IC2 is linked to bi-directional silencing of genes in cis on the paternal chromosome, resulting in the expression of only the maternal alleles of adjacent genes, such as Cdkn1c and Kcnq1. Grey gene regions indicate lack of transcription 1.1.5 Tissue-specific Imprinting Many imprinted genes exhibit differential tissue-specific expression or lack-thereof. The majority of these exhibit their preferential imprinting in the placenta, yolk sack or embryonic tissues45, as would be expected based on the proposed evolutionary value of imprinted genes (discussed earlier). However, a significant percentage of tissue-specific imprinted genes exhibit their specificity in neural tissues45,46. These neural-imprinted regions appear to be relatively abundant46 (on the scale of imprinted genes) and have 10 proposed significance in molecular pathways critical to neural development47. The keystone imprinted locus of this thesis, the Mest/Copg2 locus, exhibits such tissue-specificity (discussed later). 1.2 Sub-proximal MMU6 Imprinted Domain Two imprinted domains are situated at the proximal end of mouse Chr6 (MMU6). Maternal UPD of this region leads to slowed embryonic growth, while paternal UPD results in overgrowth48,49. The sub-proximal region of this chromosome includes the paternally expressed gene Mest and the maternally expressed genes Copg2 and Klf14 (Fig. 1.2) 50–52. Remarkably, transcription from the Mest promoter also yields the paternally expressed microRNA miR-335, encoded within intron 2 of Mest53–55. Other imprinted transcripts have been described at the Mest-Copg2 locus, such as Copg2AS and Mit1/Lb952, which are now believed to be part of MestXL, as discussed below56. This thesis focuses on the regulation of Mest, MestXL, and Copg2 within this context. Figure 1.2 Mest Sub-proximal MMU6 Imprinted Domain UCSC browser view of the Mest Sub-proximal MMU6 imprinted domain. From left to right: Mest oocyte specific TSS, Mest TSS, Mir335 microRNA, Copg2 and Klf14. 11 Despite all three genes in this Mest-Copg2-Klf14 locus showing various levels of imprinting, the only confirmed gDMR in the region overlaps with the Mest promoter CpG island. This gDMR acquires and maintains maternal DNAme and is thought to function as an ICR in addition to its direct effect on Mest expression34,57. The maternal DNAme present at the Mest gDMR (and therefore the Mest promoter) not only inhibits expression at the Mest promoter but also inhibits binding of CTCF, further precluding transcription of the maternal allele58. In addition to directly regulating Mest expression, this gDMR is putatively responsible for secondary imprinting effects on Copg256 and Klf14. Mest and Copg2 genes are adjacent and antisense to each other, with their 3’UTR regions overlapping by less than 1 kb in most tissues (Fig. 1.3A). However, within derivatives of the central nervous system (CNS), an extended form of the Mest transcript, termed MestXL, emerges via alternative polyadenylation (APA)56. The generation of this elongated MestXL isoform somehow disrupts Copg2 expression from the paternal allele, potentially through transcriptional interference or as a lncRNA. Consequently, this leads to a preference for maternal Copg2 expression within these cells56 (Fig. 1.3B). In a Mest paternal knockout model previously generated by this laboratory, MestXL undergoes truncation due to the insertion of a PAS (PAS), facilitating biallelic expression of Copg256. While Klf14 expression was unaffected in this model, subsequent unpublished work in our lab by Amanda Ha has elucidated a possible mechanism for Mest gDMR imprinting regulation of Klf14 via long-range chromatin looping of the unmethylated allele. 12 Figure 1.3 Mest-Copg2 Regulatory Relationship (A) Mest-Copg2 locus in non-CNS tissues. Maternal DNAme precludes transcription of Mest from the maternal allele, while Copg2 is transcribed from both alleles, antisense and adjacent to Mest. (B) Mest-Copg2 locus in CNS-tissues. The primary Mest PAS is bypassed in favour of one much further downstream, extending the new MestXL transcript ~40kb into the Copg2 locus. This results in reduced paternal Copg2 expression in these cell types, we propose by some lncRNA-like or transcriptional interference-related mechanisms. 1.2.1 Mest Mest (aka Peg1) encodes a putative enzyme belonging to the α/β-hydrolase fold superfamily, which is highly conserved and primarily localized to the endoplasmic reticulum50,59,60. Despite being identified as a putative epoxide hydrolase, MEST has yet 13 to demonstrate enzymatic activity, leaving its substrate unidentified. Mest was initially discovered as a paternally expressed imprinted gene through a systematic screening process involving subtraction hybridization between cDNAs sourced from normal and parthenogenetic mouse embryos50. Structurally, Mest contains 12 coding exons, with its promoter overlapping a CpG island spanning exon 157. Notably, this CpG island undergoes specific maternal DNAme during oogenesis, persisting throughout embryonic development34,57. This imprinting pattern holds true across all marsupials and placental mammals studied thus far48,50,61,62. In mice, Mest exhibits robust expression in embryonic and extraembryonic mesodermal lineages, particularly during early embryonic stages and in several tissues in the placenta35,63. Additionally, Mest plays a crucial role in embryonic neuronal development, particularly in the cortex and midbrain regions64,65. Mest knockout mice display embryonic growth restriction, in line with the parental conflict hypothesis of imprinted genes35. 1.2.2 Mest Knockout Models The Mesttm1Masu (MGI: 2181803) mouse model (mentioned previously in the MestXL-Copg2 interaction), referred to as Mest KO, was created using a targeted promoter trap technique35,57. This involved replacing the predicted catalytic regions spanning exons 3-8 and part of 9 with a splice acceptor-IRES-βgeo-PAS cassette. The inserted PAS stops transcription upstream of exon 9, truncating Mest and MestXL transcripts. Through this allele, Mest imprinted expression can be observed by X-gal staining of reciprocal mKO and pKO embryos, revealing a signal only upon paternal transmission of the KO allele. Importantly, the sequences essential for miR-335 production within Mest intron 2 remain unaffected in this KO allele. Consequently, miR-335 maintains paternal expression, 14 although it is reduced compared to WT56. Paternal inheritance of the Mest KO allele results in slowed embryonic growth and Mest KO mothers exhibit abnormal maternal behavior, characterized by a lack of maternal care35. This (and other) Mest KO models have also been utilized to explore Mest's role in neurogenesis, where its loss is associated with aberrant neuronal development and migration, and even implicated in adult neuron function in Alzheimer’s disease, possibly due to a putative role in WNT-signalling64,66,67. Additionally, Mest is implicated in muscle regeneration independently of miR-335 and has been shown to influence lipid metabolism, as its upregulation correlates with adipocyte enlargement and adipose tissue expansion in various obesity models55,68–71. The precise mechanism linking Mest function to these phenotypes remains unknown. 1.2.3 miR-335 MiR-335 is a micro-RNA originating from intron 2 of Mest and displaying paternal expression and is expressed concomitantly with Mest from the same promoter55,72,73. Most pertinently for mine and previous projects focusing on Mest and MestXL, parsing out whether observations stem from these transcripts or from miR-335 is very important, as all three products arise from the same promoter. For instance, the discussed Mest KO allele preserved the crucial sequences for miR-335 expression and processing35,56. Consequently, Mest KO mice experience a loss of Mest expression, whereas miR-335 is merely downregulated. In this scenario, the growth inhibition phenotype observed in Mest KO mice could result from the dysregulation of either Mest or miR-335. However, a separate deletion of miR-335 showed no slowing of growth, suggesting that Mest is 15 probably the primary gene responsible for this abnormal growth phenotype seen in the KO mice55. 1.2.4 Klf14 Klf14, a maternally expressed gene located approximately 60 kb downstream from the Copg2 promoter and transcribed from the negative strand, codes for a Krüppel-like transcription factor. This gene has been demonstrated to exhibit expression and imprinting across all embryonic and extra-embryonic tissues12. The KLF14 protein belongs to the Sp/KLF zinc finger family of transcription factors, bearing the closest resemblance to KLF1612. These transcription factors, known for their diverse roles in development, are implicated in the regulation of cell proliferation, differentiation, and metabolism74. In humans, KLF14 was identified as a regulatory protein of the Transforming Growth Factor β pathway and is suggested to serve as a key regulator of gene expression in adipose tissue75,76. 1.2.5 Copg2 Copg2 encodes a subunit of the coatomer subcomplex crucial for Golgi-intracellular transport and exhibits cell-type specific maternal expression77,78. Observed expression of Copg2 has shown variability across strains and cell types, ranging from strict maternal expression to biallelic expression in certain crosses52,77,79–81. The variation in imprinting across different hybrid strains may be caused by genetic differences in the Mest PAS or 3’UTR, as suggested by my predecessor56. Overall, Copg2 maternal expression seems less strict and primarily confined to neuronal lineages, with biallelic expression in other tissues. 16 1.2.6 Human Homology and Disease The sub-proximal MMU6 domain, along with the Mest gDMR, exhibits strong conservation in humans on 7q32.282,83. Most genes within this domain share similar imprinting patterns, except for COPG2, where the imprinting status is debated, ranging from paternal expression to biallelic expression with no clear imprinting12,48,83,84. Humans also possess two major isoforms of MEST: isoform 1, originating from exon 1c, shows preferential paternal expression, while isoform 2, originating from exon 1a, displays more variable and tissue-specific imprinting85. In fetal placenta and kidney, MEST isoform 2 is paternally expressed, but it exhibits biallelic expression in other fetal tissues such as the brain and heart (Nakabayashi et al., 2002). Additionally, the locus includes a paternally expressed antisense lncRNA, MESTIT1, located in the first intron of isoform 2, possibly involved in MEST gene regulation86. A longer 3’UTR was also identified for human MEST, and an antisense COPG2 transcript, COPG2IT1, was found in intron 20, analogous to mouse Copg2as and Mit1 (MestXL), respectively84.6 In addition to previously mentioned putative roles in Alzheimer's disease, cancerous pathways and relevance in obesity models, dysfunction within the MEST imprinting domain is linked to growth restriction phenotypes in Silver-Russell syndrome and other adult-onset diseases. As all four main transcripts from this region exhibit some level of regulation by the MEST gDMR, dysregulation of this gDMR can lead to changes in expression and disease phenotypes resulting from any of these transcripts. For instance, neural tube defects have been associated with MEST upregulation and reduced DNAme over the gDMR87. Conversely, MEST downregulation and gDMR hypermethylation have been linked to different cancers and adverse pregnancy 17 outcomes72,88–90 .Since the intronic MIR-335 is conserved in humans, the relative roles of this miRNA and the MEST protein in these phenotypes is still unknown. Silver-Russell syndrome (SRS), initially described by Silver and Russell, is characterized by intrauterine and postnatal slowed growth, macrocephaly, body asymmetry, triangular facial features, fifth finger clinodactyly, and associated with congenital malformations including renal and cardiac defects, with gestational and adult growth restriction phenotypes91,92. SRS cases are made up by two primary genetic subtypes, each presenting distinct growth features and implicating different imprinted gene clusters. Hypomethylation at the H19 ICR on chromosome 11p15.5 is responsible for a significant portion of cases, while maternal uniparental disomy (mUPD) for chromosome 7 accounts for a smaller percentage93,94. SRS mUPD7 cases typically exhibit more severe growth and neurocognitive problems compared to those with H19 ICR hypomethylation95,96.While the specific genomic region responsible for SRS due to mUPD7 remains unidentified, maternal duplications 97,98 and paternal microdeletions 99 specifically at 7q32.2, encompassing the MEST imprinting domain, have been reported in some SRS cases. Recently, a paternally inherited microdeletion encompassing the imprinted genes MEST, MESTIT1, MIR-335, and 3’ of COPG2 was observed in a patient with SRS-like symptoms, indicating the potential involvement of these genes in SRS pathogenesis100. Despite such implications of MEST in mUPD7 SRS, no definitive link between the gene itself and the phenotype has been established. However, since the slowed growth phenotype observed in Mest knockout mice resembles mUPD7 SRS, MEST is considered a candidate gene35. The mechanism by which SRS and similar 18 cases arise in mUPD7 remains largely unknown, highlighting the need for further research in this complex region. 1.4 Alternative Polyadenylation and 3’ UTR choice 1.4.1 Overview 3' untranslated regions (3' UTRs) are sections of mRNA transcripts located in between the translation stop codon and the poly(A) tail (poly(A)), playing key regulatory roles from transcript abundance and localisation to cellular phenotypes101. Processing of mRNA, which includes 5’ end capping, splicing, and cleavage and polyadenylation (CPA) at the 3' end102,103, is crucial for mRNA maturation. 3’ end formation involves endonucleolytic cleavage followed by poly(A) tail synthesis by poly(A) polymerase104. This process, essential for transcription termination, is guided by the CPA machinery, including factors like CPSF, CFI, and CSTF that recognize specific sequence motifs in the mRNA105. Alternative polyadenylation (APA) is a widespread RNA-processing mechanism that generates distinct 3' termini on mRNAs, thus producing isoforms with 3' UTRs of different lengths106–108. This can occur either in the terminal exon (3' UTR APA) or in upstream introns (intronic APA)101, the former producing mRNA isoforms encoding the same protein but with variable regulatory regions, and the latter often generating truncated proteins. 3’ UTR choice is gene- and cell-type-specific106,108,109 and significantly influences gene regulation by determining 3' UTR length and regulatory potential through PAS choice. 19 1.4.2 Functions of APA/3’UTR choice Three prime untranslated regions (3' UTRs) play crucial roles in gene regulation without directly affecting the protein-coding sequence. They contain numerous regulatory sequences and are often targets for microRNAs (miRNAs)110, RNA-binding proteins (RNABPs), mRNA destabilizing elements, and other cellular regulatory mechanisms. These sequences enable 3' UTRs to regulate protein output, mRNA export, stability, translation rates, and mRNA localization within the cell111–117. Generally, these effectors result in genes with longer 3’ UTRs having lower expression, by virtue of them containing more RBP and miRNA binding sites and destabilizing elements. However, there are a not insignificant number of cases where longer UTRs actually lead to the same or increased gene product, with changes in cellular localization or protein aggregation only. One key function of 3' UTRs is the regulation of subcellular localization. For example, in neurons, the brain-derived neurotrophic factor mRNA (BDNF) has shorter isoforms restricted to the cell body, while longer isoforms migrate to dendrites for translation111. Localization effects are not constrained to neural cell types however; for example, 3' UTRs can direct mRNA to the endoplasmic reticulum (ER) for membrane protein synthesis115,118. Sequences within 3' UTRs can also affect protein localization independently of mRNA localization. For instance, the 3' UTR of CD47 acts as a scaffold for protein complexes, resulting in different localization and functions depending on the UTR used112. 20 The specific 3' UTRs utilized can determine the half-life and subcellular localization of the mRNAs and proteins as well as facilitating protein complex assembly113,114,119–121. Long 3' UTRs can lead to mRNA degradation through mechanisms such as nonsense-mediated decay, with Upf1 recognizing the 3' UTR length114. Additionally, 3' UTRs are common targets for mRNA destabilization elements like AU-rich elements (AREs), GU-rich elements (GREs), and PUF protein-binding elements113. Alternative polyadenylation (APA) can also allow mRNA isoforms with specific 3' UTRs to be translated at membranes or within biomolecular condensates, such as TIS granules, facilitating protein complex assembly119–121. This precise regulation by alternative 3' UTRs ensures proper protein production and localization, essential for various cellular processes. 1.4.3 Regulation of 3’UTR APA Regulation of 3' UTR alternative polyadenylation (APA) is influenced by a multitude of factors operating co- and post-transcriptionally (Fig. 1.5). These factors include cis-regulatory sequence elements, trans-acting cellular elements like RNA-binding proteins (RBPs), RNA polymerase II processivity, and various extracellular signals (Fig. 1.5C). Gene-specific sequence elements in promoters and enhancers have also recently been shown to play a role in determining 3' UTR isoform choice122–124. 1.4.3.1 Cis- acting factors regulating APA Cleavage and polyadenylation (CPA) are dictated by key cis-acting sequence motifs (Fig. 1.5B) including the PAS consensus hexamer (AAUAAA or variants), recognized by the CPSF complex, UGUA motifs bound by cleavage factor I (CFI), and downstream U-21 rich or GU-rich regions bound by cleavage stimulation factor (CSTF)101,103,122,125. These elements are essential because, despite the frequent occurrence of the PAS hexamer in pre-mRNAs, only specific sites are utilized for CPA, which ensures accurate mRNA processing and gene expression regulation106. These sequences work together to determine the relative strength and utilization of PASs126. Variants in the core hexamer and the absence of upstream and downstream elements often result in "weaker" PASs that are less likely to be utilized127. Another unique example is that recently, enhancer-mediated recruitment of factors to promoters has also been shown to regulate APA in human cells in some cases122. Figure 1.4 Mest primary PAS region Mest region surrounding the primary PAS site with candidate AAUAAA consensus hexamers highlighted in grey. This provides context as to the frequency of this hexamer as well as the necessity of up and downstream contributing sequence elements in PAS choice, to differentiate between the common consensus sequences. 22 1.4.3.2 Specific Trans-Acting Proteins and APA Trans-acting factors, including RBPs, transcription elongation and termination factors, and CPA machinery components, influence APA and 3' UTR choice (Fig. 1.5C). For instance, CSTF2 upregulation in B cells increases usage of weaker intronic PASs128. Changes in nuclear export receptor protein levels also affect 3' UTR isoform ratios, as longer 3' UTRs rely more on these receptors129–131. Enhancer-mediated recruitment of factors to promoters can also regulate APA in human cells. Several RBPs, such as NOVA2, ELAV, and Hu proteins, have been implicated in APA regulation132–134. NOVA2 can inhibit or enhance PAS usage depending on its binding position. ELAV suppresses proximal PAS choice in D. melanogaster neurons, while its mammalian homolog, Hu proteins, inhibit polyadenylation by targeting U-rich elements. A remarkably balanced example is mouse SRSF3 and SRSF7. These RBPs influence 3' UTR length in mouse cells, with SRSF3 lengthening and SRSF7 shortening the 3' UTRs131. Extracellular signals, including cold exposure, nutrient availability, immune pathway activation, and developmental cues, can regulate levels of RBPs and transcription factors (TFs) and by extension APA108,122,135–140. For example, BMAL1, a transcription factor, influences 3' UTR choice in concert with circadian rhythms, causing widespread alternative UTR (aUTR) choice for a multitude of specific genes138. 23 1.4.3.3 Influence of Transcriptional and Translational Status on 3'UTR Choice Three prime UTR choice is influenced by the transcriptional and translational status of the gene. RNA polymerase II pausing or slowing its processivity downstream of a functional PAS site enhances CPA machinery recruitment to that site, promoting effective polyadenylation141,142 (Fig. 1.5C). Additionally, APA can be regulated post-transcriptionally through sequential CPA, nuclear RNA degradation, regulation of mRNA export, and control of mRNA stability. One recent and noteworthy discovery is that transcription start site (TSS) choice can heavily influence 3’UTR choice in many genes (Fig. 1.5B), often linking “strong” TSSs (strong in this context meaning often used and the seemingly preferred choice) with strong PASs143. Larger-scale genetic states such as open vs closed status of chromatin have also been linked to APA influence144. 24 Figure 1.5 Mechanisms of APA (A) Possible PAS locations and their effect. Upstream or intronic PAS exist in some genes (the proximal PAS in this figure) and due to their locale can often result in truncations (in this case between exons 2 and 3). 3’UTR PAS, while not affecting protein-coding sequence of the gene, results in different length 3’UTRs being expressed, with varying levels of miRNA and RBP binding sites and therefore different regulatory potentials. (B) Cis-acting regulatory elements in PAS choice. A zoomed-in look at the PAS along with upstream TSSs. Transcription start sites (and alternatives, labelled ATSS1 and 2), along with upstream UGUA and U-rich elements, the consensus hexamer and downstream GU/U rich elements are important regulators in PAS choice. Presence or 25 absence of, in addition to variations of these, can result in the PAS being stronger or weaker and heavily influences recruitment of CPA machinery and use of the PAS. (C) Trans-acting regulatory factors in PAS choice. Some of the primary trans-acting factors in PAS choice are exampled here. Relative levels of CPA factors in the cell directly relate to levels of assembled CPA machinery, recruitment of which is essential for PAS utilization. Additionally, enhancer-promoter interactions along with RNA Pol II processivity exhibit relevance in PAS choice as the levels and speed at which transcription occurs through the PAS is affects utilization. In accordance with the above, as transcription factors play roles in not only transcriptional status of the gene, but also levels of other gene products in the cell (such as RBPs or CPA factors), and RBPs affect levels of TFs and CPA factors, these also fall under important trans-acting factors in PAS choice and subsequent 3’UTR length. Unlabelled grey boxes represent exons, pink circles represent PASs, green triangles and orange stars represent miRNA and RBP binding sites respectively, the purple triangle represents a looping enhancer as in figure 1.1. 1.4.4 Tissue-Specific APA/3’UTR Choice Multi-3’ UTR genes can be expressed ubiquitously, but their 3' UTR isoform selection varies by cell type, providing tissue-specific regulation106,108. 3’UTR profiles differ significantly between cell types, largely due to the abundance of APA regulators such as CPSF5 and CPSF6107,145–147. This regulation is relevant for both cell proliferation and differentiation102,148,149 and while pervasive in many cell types, in mice and humans it 26 occurs most often in neuronal lineages110. Neuronal lineages trend towards use of much longer distal 3’UTRs, most in the range of 500 to 2000 base pairs but in rarer but not an insignificant number of cases can be upwards of 10kb110. Early analyses combined with more recent deep sequencing have revealed numerable detailed tissue-specific APA profiles106,109,150,151. Some tissues globally favor certain APA isoform types150: neurons as well as bone marrow, pancreatic islet, and uterine tissues typically have much longer UTRs110,150, whereas testis tissues, retina, placenta, blood and ovary tissues tend to have shorter UTRs144,150,152. Comparative analyses across the tissues of five different mammals indicate that APA profiles are well conserved across species, with ubiquitous genes using APA to achieve tissue-specific expression109. In neurons, correct local protein synthesis is often dependent on specific 3' UTRs, which contain elements necessary for differential mRNA and protein localization111,116,153–160. This is of particular import in neurons due to the extended axonic morphology of many neuronal cell types. Their large size and the need for mRNA and protein products far from the nucleus necessitates robust mRNA localization in the distal portions of these cells. For example, brain-derived neurotrophic factor (BDNF) 3' UTR-APA isoforms show distinct localization patterns, with the long isoform localizing to dendrites to support local protein synthesis111. Similarly, the 3' UTR of the gene encoding the mouse chromatin remodeler HMGN5 directs its translation to neuronal growth cones, where locally synthesized HMGN5 is phosphorylated and able to mediate transcription activation after being trafficked to the nucleus157. Putative mechanisms promoting longer 3' UTRs in neurons include the inhibition of ELAV (which typically 27 favors shorter 3' UTRs) and the suppression of AU-rich element-mediated decay123,133,161. Environmental signals can also affect particular sets of neurons 162 by differentially expressing surface receptors or through the alternative 3' UTRs of transcription factors and other regulatory proteins154,156–158. This selective expression enables neurons to respond specifically to these signals, establishing a selective response through APA isoform expression. 1.4.5 Human Homology and Disease Alternative polyadenylation (APA) profiles vary across tissues but are generally conserved among species109, indicating their crucial role in gene regulation. While polyadenylation factors are largely conserved in mammals, some differences are observed in yeast and plants163. APA and 3' UTR choices have significant implications for human diseases. Research has shown that 5-19% of APA quantitative trait loci (QTLs)—mutations affecting APA—are linked to the heritability of human traits or diseases164–166. These mutations often alter polyadenylation sites (PASs) or RNA-binding protein binding sites within 3' UTRs167. For example, in systemic lupus erythematosus (SLE), a single nucleotide polymorphism that reduces the use of the proximal PAS leads to the production of longer IRF5 isoforms, resulting in lower IRF5 levels168. This genetic variation defines risk and protective haplotypes for SLE168. In cancer, APA is frequently dysregulated, with a notable trend towards 3' UTR shortening in solid tumors145,169,170. A key APA regulator involved in this process is 28 NUDT21 (CPSF5, CFIm25)145,146,171,172: reducing NUDT21 levels through small hairpin RNAs results in the 3' UTR shortening of nearly 1,500 genes172. Low NUDT21 expression is associated with poor prognosis in several cancers, making it a potential biomarker and therapeutic target145,173–176. 1.5 Thesis Theme and Objectives The sub-proximal MMU6 imprinting domain includes the paternally expressed gene Mest and the maternally expressed genes Copg2 and Klf14. Mest is considered a candidate gene for Silver-Russell syndrome (SRS) due to the growth phenotype observed in knockout mice, although the exact function of the MEST protein remains unknown. In tissues expressing MestXL, such as the developing embryo and adult brain, Copg2 is reported to be preferentially maternally expressed, suggesting that the production of the larger MestXL may play a role in regulating Copg2, by down-regulating its expression in cis. This fact, combined with the inordinate size of the MestXL 3’ UTR, its proximity and extension into the Copg2 locus and the presence of numerous stem-loop structures (key factors in lnc-like silencing) in the highly repetitive region downstream of the primary Mest PAS (Fig. S2) have led to our model that MestXL evolved to suppress Copg2 paternal expression in neuronal cell lineages by acting similarly to a lncRNA, inhibiting transcription of Copg2 from the paternal allele by recruiting silencing marks, through transcriptional interference, or via enhancer occlusion. 29 While aspects of this model, the regulation of Mest57 and Copg256 imprinting are well characterized, the mechanism by which the primary Mest PAS is bypassed in favour of the distal MestXL PAS, and exactly how this leads to secondary imprinting of Copg2 are still unknown. In this thesis I attempt to elucidate this mechanism of PAS bypass at Mest and gain information on how the MestXL transcript interferes with Copg2 expression. Briefly, the aims pursuant to this are: 1. To determine if the primary Mest PAS is sufficient for readthrough in cell types expressing MestXL. This hypothesis will be tested by generating randomly integrated transgenes in mouse embryonic stem cells (mESCs) allowing expression of different fluorescent reporters located on either side of small sequences encompassing the Mest PAS and differentiating these transgenic mESCs into cortical neuron (CN) cell types to assess readthrough. 2. In the event that the Mest PAS is not sufficient, I also attempted to assay the necessity of full-length MestXL in inhibiting paternal Copg2 expression, as well as probing for candidate genes necessary for Mest primary PAS readthrough. This will be accomplished by creating and differentiating a MestXL-reporter mESC line that truncates MestXL at the endogenous locus, allowing simultaneous readout of MestXL formation and assay of Copg2 allelic usage with this abrogated MestXL to gain information on the mechanism of transcriptional interference. 30 Chapter 2: Materials and Methods 2.1 Cortical Neuron Differentiation To generate cortical neurons, ESCs were differentiated in 3D brain organoids in accordance with Le Digarcher et al 2022, with some modifications177. Briefly, after being detached using Accutase, approximately 3000 cells were plated per well of a 96-well ultra-low attachment plate in 100uL corticogenesis medium 1(CM1): DMEM/F-12/GlutaMAX (Life Technologies, 31331028) supplemented with 10% KSR (Life Technologies, 10828010) 0.1 mM of non-essential amino acids 1 mM of sodium pyruvate 0.1 mM of 2-mercaptoethanol 100 U/mL penicillin/streptomycin 1 μM DMH1-HCl (BMP inhibitor, Stemcell Technologies, 73632 73634), and 240 nM IWP-2 (Wnt inhibitor, Stemcell Technologies, 72122 72124). After 4 days an additional 100uL of CM1 was added to each well. After 7 days the developing organoids were moved using a cut 1ml pipette tip to 6 well plates on orbital shakers within the incubator (set at 100 rpm), 8-10 healthy organoids (smooth edges) per well in 2mL of corticogenesis medium 2 (CM2): DMEM/F-12/GlutaMAX supplemented with N2 and B27 (without vitamin A) supplements, 500 μg/mL of BSA 0.1 mM of non-essential amino acids 1 mM of sodium pyruvate 0.1 mM of 2-mercaptoethanol 50 U/mL penicillin/streptomycin. On day 14 of differentiation, an additional 2mL of CM2 was added. Organoids were collected on day 21 of differentiation, for immunofluorescence and qRT-PCR analysis. 2.2 Gibson Assembly PCR primers were generated to amplify pertinent plasmid fragments and add 20-30 nt specific overhangs between segments. Following PCR amplification, the vector was 31 linearized appropriately and Gibson assembly proceeded with NEB’s NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621) as per their protocol. Briefly, vector and insert fragments were combined in molar ratios of 1:2 (vector to insert), with total DNA adding up to 0.03-0.2 pmoles. To this, 10 μl of NEBuilder HiFi DNA Assembly Master Mix was added and total volume was topped up to 20 μl with dH2O. This mixture was incubated at 50°C for 15 minutes and stored at -20°C. Controls without inserts and without master mix were created in parallel and 10μl of each were transformed into DH5α competent E. coli cells. 2.3 Bacterial Transformation 10 uL of plasmid DNA was combined with 100 ul DH5α competent E. coli cells. This mixture was then put on ice for 30 minutes. Following icing the mixture was heat shocked for 90s at 42°C, then topped up to 1 ml with SOC buffer. This was incubated at 37°C for 1 hour following which 100-200 ul was spread onto LB+Ampicillin plates which were placed at 37°C for 16 hours before picking colonies. 2.4 Polymerase Chain Reaction General PCR cycling conditions are as follows: Pause at 95°C, followed by 35 cycles of 30s at 95°C, 30s at 58°C, 1 min at 72°C, and a final 5 min final elongation at 72°C. Touchdown PCR cycling conditions are as follows: Pause at 95°C, followed by 8 cycles of 30s at 95°C, 30s at 62°C decreasing to 56°C over the course of the 8 cycles, and 60s (varying depending on target length) at 72°C, these 8 cycles were subsequently followed by 32 cycles of 30s at 95°C, 30s at 56°C and 60s at 72°C, followed by a final 5 minutes at 72°C. 32 PCR products were visualized by agarose gel electrophoresis. 2.5 RNA Extraction Samples were snap frozen on dry ice and stored at -80°C. Total RNA was extracted using Trizol (Invitrogen Life Technologies, CA) according to the manufacturer’s directions and resuspended in 20-40 μl DEPC dH2O. 10 μl of RNA was treated with 1 μl RQ1 DNaseI (Promega) in 2.5 μl of 10x RQl buffer (Promega), 1 μl of RNase inhibitor (Promega), and 10.5 μl of dH2O for 1-1.5 hours at 37°C. DNaseI was inactivated by incubation at 65°C for 15 min. 2.6 Reverse Transcription Complementary DNA (cDNA) was generated using M-MLV reverse transcriptase (Invitrogen) and random pentadecamers (N15). 10 μl of DNase-treated RNA was added to 9 μl of the master mix (2 μl of N15, 5 μl dH2O, 2 μl 10uM dNTPs) and incubated at 65°C for 10 minutes. 19 μl of the second master mix (8 μl 5X first strand synthesis buffer (Invitrogen), 4 μl 0.1M DTT (Invitrogen), 0.5 μl RNase inhibitor, 6.5 μl dH2O) was added.19 μl of the final mix was aliquoted into separate tubes as RT- controls. 1 μl of MMLV reverse transcriptase was added to the positively labelled tubes. All tubes were incubated at 42°C for 1 hour, and inactivated at 72°C for 15 minutes. 2.7 qRT-PCR Quantitative RT-PCR was conducted using a 96-Well QuantStudio Real-Time PCR System (Applied Biosystems) with the QuantStudio Design & Analysis Software (Version 2.7.0), with Eva Green dye (Biotium). For the reactions, either 1 μl or 2 μl of the synthesized cDNA was used in a total volume of 20 μl. The amplification conditions 33 were 40 cycles at 95°C for 30 seconds, an annealing temperature of 58–62°C for 30 seconds depending on the primer pair, and extension at 72°C for 30 seconds. This was followed by a melt curve analysis. Ct values of technical triplicates were averaged and used to calculate relative expression using the 2-ΔΔCT method, normalized against the reference housekeeping gene Ppia (Mamo et al., 2007). All primers used were either pre-existing or designed using Primer3, aligned against MGI transcripts to preclude their presence in introns and confirm appropriate product size for qPCR, and Tms were tested via temperature gradient PCR prior to use in qRT-PCR. All RNA extraction, RT and qRT-PCR troubleshooting and early analyses were performed by the undergraduate honours student Abdullah Khawaja including but not limited to: UCSC checks for size and locations of primers, Tm determination, qPCR troubleshooting on WT mouse brains, and analyses of CN organoids generated before protocol optimizations and mycoplasma cleaning. 2.8 Lipofection In-suspension ESC transfections were performed in suspension using Lipofectamine 3000 Transfection Reagents (Thermofisher L3000001). For each transfection, 3.75 uL of L3000 and 125 uL DMEM were combined with 1 ug of each DNA sample to be transfected, 10 uL P3000 and 125 uL DMEM then incubated for 15 minutes. ESCs were grown to confluence on 10 cm dishes and 1:4 passaged onto 10 cm dishes in combination with the incubated liposome mixture. No-DNA controls were plated similarly, and both were allowed to grow overnight before addition of antibiotic selection media. 34 2.9 Immunohistochemistry For immunodetection of various proteins, day 21 organoids were fixed in 4% PFA in PBS overnight at 4°C. The following day they were washed twice in PBS and blocked in 10% goat serum, 3% BSA, 0.3% Triton-X100 in PBS for 1 hour at room temperature. Following this, organoids were incubated with 1° antibodies in 1% serum, 3% BSA and 0.1% Triton-X100 in PBS overnight at 4°C. Primary antibody concentrations were as follows: PAX6 1:1000, TUBB3 1:250, NESTIN 1:1000, TBR1 1:250. Organoids were washed 3X in PBS then incubated with 2° antibodies in 1% serum, 3% BSA and 0.1% Triton-X100 in PBS overnight at 4°C. Secondary antibody concentrations were as follows: goat-α-mouse Alexafluor660 1:250, goat-α-rabbit488 1:250, DAPI 1:1000. Organoids were then washed 3X with PBS and stored at 4°C until imaging. Organoids were imaged via confocal Microscopy on a Leica Stellaris 5 confocal microscope. 2.10 Plasmid Construction 2.10.1 Endogenous Reporter Construct Half of the eventual complete endogenous reporter plasmid construct was assembled using restriction enzyme cloning. This was accomplished by digesting our existing PGKneolox2DTA plasmid (Lefebvre lab) with NheI and SalI, digesting our PCR amplified Copg2 3’ arm of homology with the same, and ligating them together before transforming this mixture into DH5α competent E. coli cells. This construct was confirmed by whole plasmid sequencing (Plasmidsaurus). Assembly of the completed endogenous reporter construct was accomplished by Gibson assembly. PGKneolox2DTA was linearized, and PCR primers with tails 35 overlapping with their insertion sites were used to amplify the IRES-EGFP fragment of our existing pCX-IRES-EGFP plasmid (Lefebvre lab) and our Copg2 5’ arm of homology. These components were assembled via Gibson assembly as previously described (see 2.2) to produce the final plasmid (pCopg2HA5-IRES-EGFP-PGKneolox2-Copg2HA3-DTA). 2.10.2 Exogenous Mest PAS Construct with Neo Selectable Marker This plasmid was entirely assembled by Gibson assembly. Gibson tailed PCR primers were generated for our PGKneolox2DTA plasmid and our existing pCTPIE plasmid (Lefebvre lab) was linearized. These components were assembled as previously described (see 2.2) to produce the final plasmid (pCTPIE-pLoxpNeo) 2.10.3 Floxed pCTPIE for RMCE This plasmid was entirely assembled by Gibson assembly. An existing pBlueKSL1-MGFhGFP-1L plasmid built for the purpose of RMCE (contains a floxed MFGhGFP which can be easily replaced) was amplified with designed Gibson PCR primers excluding the MFGhGFP segment, with overhangs matching to the linearized pCTPIE construct. These components were assembled as previously described (see 2.2) 2.11 ESC Line Generation Attempts 2.11.1 C2 Mest PAS Double Fluorescent Reporter C2 ESCs were grown to ~80% confluence on a 10 cm dish, then passaged 1:4 and plated with the lipofection mixture described above (see 2.8) with 1 µg of our newly generated pCTPIE-pLoxpNeo construct. Cells were allowed to recover for 24 hours and 36 were then subjected to 7 days of 250 ug/ml G418 selection (concentration determined via kill curves), following which surviving clones were allowed to recover on normal ESM and were subsequently picked and expanded. 2.11.2 pCTPIE RMCE Transfection HA36 ESCs (Lorincz lab, RMCE line) were grown to ~80% confluence on a 10 cm dish, then passaged 1:4 and plated with the lipofection mixture described above (see 2.8) with a total of 1 µg of plasmid DNA (attempts were made with molar ratios of 7:1, 1:1 and 1:7) of floxed-pCTPIE and our Cre-PGK-puro plasmid (cre expressing vector with puromycin selectable marker) (Lefebvre lab). Attempts were also made with 1 ug of each of these co-transfected plasmids. Cells were allowed to recover for 24 hours and were then subjected to 72 hours of 2 ug/ml puromycin treatment, followed by 48 hours of normal ESM (to allow cells to lose all HSV TK, integrated in HA36 cells, if RMCE was successful), and 7 days of 7.5 uM gancyclovir treatment. No clones survived gancyclovir treatment. 2.11.3 Endogenous Reporter Construct F1 ESCs (BC1.1 and BC1.3, generated by Amanda Ha in a previous project) were grown to confluence on a 10 cm dish, then passaged 1:4 and plated with the lipofection mixture described above (see 2.8) with 1 ug each of pCopg2HA5-IRES-EGFP-PGKneolox2-Copg2HA3-DTA and our CRISPR-Cas9 plasmid with our Copg2 intron22 guide RNA inserted. Cells were allowed to recover for 24 hours following which they were treated with 2 µg/ml puromycin for 48 hours. Cells were allowed to recover for 48 hours to allow for counting of surviving colonies (to estimate transfection efficiency) and 37 were then treated with 500 µg/ml G418 (concentration determined via kill curve). This was followed by recovery on ESM and surviving colonies were picked and expanded. Expanded clones were tested for integration using PCR primers made to assay presence of the 5’ and 3’ ends of the construct, and a genomic control reaction spanning the insertion site (Table 2.1). Table 2.1 List of Oligonucleotide Primers Used Primer Name Sequence Utility Reference SLIC-F-Cop1 ATCTTGTGGAAAGGACGAAACACCGCCTTCCTCACTAATGTGGTCGTTTTAGAG Cogp2 guide 1 This project SLIC-R-Cop1 CCTTATTTTAACTTGCTATTTCTAGCTCAAACGACCACATTAGTGAGGAAGG Copg2 guide 1 This project SLIC-F-Cop2 ATCTTGTGGAAAGGACGAAACACCGCTAGGACCTATAGCTAGCATGTTTTAGAG Copg2 guide 2 This project SLIC-R-Cop2 CCTTATTTTAACTTGCTATTTCTAGCTCAAACATGCTAGCTATAGGTCCTAG Copg2 guide 2 This project Copg2 3’ HA F1 CCCAATGCTAGCTATAGGTCCT Copg2 3’ homology arm This project Copg2 3’ HA R1 GGCCGTCGACTCCCACCAAAATCTCTCCCC Copg2 3’ homology arm This project Copg2 5’ HA F1 GGCCGTCGACAGTGAGAGTAGCAACAGCATCT Copg2 5’ homology arm This project Copg2 5’ HA R1 CCGGCCGCGGCTCACCCAGACCACATTA Copg2 5’ homology arm This project 38 Gibson SA-IRES-EGFP pA TAAGGGTTCCGGATCCACTAGTTCTAGAGCCTTCCGGCTCGTATGTTGTG Endogenous reporter Gibson assembly segment This project Gibson SA-IRES-EGFP int TAATGTGGTCTGGGTGAGCCTGACCGGCGGCTCTAGAG Endogenous reporter Gibson assembly segment This project Gibson Copg2 5’ HA R1 CTCTAGAGCCGCCGGTCAGGCTCACCCAGACCACATTA Endogenous reporter 5’ HA Gibson assembly This project Gibson Copg2 5’ HA F1 TAGGGCGAATTGGAGCTCCACCGCGGTGGCAGTGAGAGTAGCAACAGCATCT Endogenous reporter 5’ HA Gibson assembly This project Pctpie rmce F1 AATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTGATCCGGCCGGCCCCATAAC Pctpie RMCE Gibson assembly This project Pctpie rmce R1 ATTGATTACTATTTATAACTAGTCAATAATCAATGAGCAAAGCTTATCGAATAAC Pctpie RMCE Gibson assembly This project Pctpie ploxpneo Gibson F ATTTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCAGGAACCGTAAAAAGGCC PCTPIE-pLoxpNeo Gibson assembly This project Pctpie ploxpneo Gibson R ATTGATTACTATTATTAATAACTAGTCAATAATCAATGTGGCGGCCGCTCTAGAACTA PCTPIE-pLoxpNeo Gibson assembly This project Fabp7 F1 TCCAGCTGGGAGAAGAGTTT qRT-PCR CN diff Fabp7 adult CNS marker 178 Fabp7 R1 CCAACCHAACCACAHACYYA qRT-PCR CN diff Fabp7 adult CNS marker 178 39 Tbr1 F1 GGTCAAGCGGTCCATGTC qRT-PCR CN diff Tbr1 cortical neuron marker 179 Tbr1 R1 CTGTCACCGCTTACCAGAAC qRT-PCR CN diff Tbr1 cortical neuron marker 179 Pax6 F1 CAAACAACCTGCCTATGCAA qRT-PCR CN diff Pax6 dorsal-NPC marker 180 Pax6 R1 GGCAGCATGCACCAGTATGA qRT-PCR CN diff Pax6 dorsal-NPC marker 180 Tubb3 F1 CCAGTGCGGCAACCAGATAGG qRT-PCR CN diff β-tubulin adult neuronal marker 181 Tubb3 R1 AAAGGCGCCAGACCGAACACT qRT-PCR CN diff β-tubulin adult neuronal marker 181 Nanog F1 GCCTCTCCTCGCCCTTCCTCT qRT-PCR CN diff ESC marker 181 Nanog R1 CCACCGCTTGCACTTCATCCTT qRT-PCR CN diff ESC marker 181 Mest F1 CAACAATGACGGCAACCTGGT qRT-PCR Mest reaction 182 Mest R1 TCTGAATTTCTTCCTTTGATTAATGTACTGTA qRT-PCR Mest reaction 182 Mit1F6 F1 GTGTGGGTCCCCTGAGTTATCTA qRT-PCR MestXL reaction 56 Mit1F6 R1 CTTTCATTGCTTCAGAATTCTTCCA qRT-PCR MestXL reaction 56 Copg2_in22_qF1 CCCACCATGTCCTAAGAGCT qRT-PCR MestXL proximal reaction This project 40 Copg2_in22_qR1 GGGGCCTGTCTTCATCATCT qRT-PCR MestXL proximal reaction This project Copg2IE5’HA flank F1 GTCAGTGACACTGCCTGGTG Endogenous reporter integration 5’ test PCR This project Copg2IE5’HA flank R1 CAATAACCAGCACGTTGCCC Endogenous reporter integration 5’ test PCR This project Copg2IE3’HA flank F1 GGGGCTCGAATCAAGCTGAT Endogenous reporter integration 3’ test PCR This project Copg2IE3’HA flank R1 ATAGCCTTTTTGACCTACATGACAAA Endogenous reporter integration 3’ test PCR This project Copg2IEcontrol F1 CCATGCAAAGGGCCTGAGGG Endogenous reporter genomic control PCR This project Copg2IEcontrol R1 AGTCATCCCTGCCGAGATCT Endogenous reporter genomic control PCR This project Ppia F CGCGTCTCCTTCGAGCTGTTTG qRT-PCR housekeeping gene control 183 Ppia R TGTAAAGTCACCACCCTGGCACAT qRT-PCR housekeeping gene control 183 MCGpF11 ACACCATGGGAG(C/T)TGGTAAT Mycoplasma test reaction 184 MCGpR1 CTTC(A/T)TCGACTT(C/T)CAGACCCAAGGCAT Mycoplasma test reaction 184 Cop E17 F2 CATTGGAGCCTTCAGAAAAACC qRT-PCR Copg2 exon 17-19 56 Cop E19 R2 GCTTCACACAGCGAACGAAAT qRT-PCR Copg2 exon 17-19 56 Cop E5 F2 TCCTTGATGGTGAGGTAGCA qRT-PCR Copg2 exon 1-5 56 41 Cop E1 R1 TCCAATCCTTTCCAGCATTT qRT-PCR Copg2 exon 1-5 56 42 Chapter 3: Results 3.1 Readthrough of Exogenous Mest PAS 3.1.1 Background/Rationale Mest and Copg2 are adjacent genes transcribed in opposite directions. Based on UCSC genome build mm10, the typical predicted overlap between these transcripts is approximately 900bp in the majority of tissues. There is however the possibility of alternative transcripts of differing lengths and endpoints depending on the assayed cell types (Fig. 3.4). In the CNS, longer transcripts of Mest, known as MestXL, are formed through alternative polyadenylation56. This process involves skipping the primary PAS, resulting in a larger transcript that ends much further downstream, approximately 40 kb into the Copg2 transcribed region. In tissues expressing MestXL, Copg2 shows preferential maternal expression instead of its typical biparental expression, inverse to the paternal expression of Mest. Previous work in our lab has shown that this secondary imprinting interaction likely occurs because of MestXL: in a Mest KO allele where a PAS is introduced at exon 9, both Mestxl expression and the imprinted expression of Copg2 are lost. Although production of MestXL and its effect on Copg2 paternal expression have been well documented, the factors allowing for the readthrough of the upstream primary Mest PAS and utilization of the distal MestXL PAS have yet to be elucidated. As outlined previously in this thesis, there are many factors involved in PAS choice in transcripts, among the most integral of which are the cis-acting elements making up the PAS. We hypothesize that in cell types expressing MestXL, the primary Mest PAS is sufficient for 43 readthrough. To address this hypothesis, I attempt to transfect our pCTPIE-pLoxpNeo plasmid (Fig. 3.1) into an ESC line and differentiate this line into cortical neurons. This plasmid strategy hinges on utilizing two fluorescent reporters (tdTomato and IRES-EGFP) separated by the Mest PAS to assess readthrough in MestXL-expressing cells. The key experimental readout would be that cells showing PAS readthrough should show both red and green fluorescence, while those integrants without PAS readthrough would show red fluorescence only. The results we expected, contingent with our hypothesis, would be that, once differentiated, a subset of transfected cells will show both fluorescence and that these cells would be the only ones expressing MestXL. These results would indicate that our integrated construct is mimicking endogenous Mest PAS readthrough, and that the PAS provided is sufficient for readthrough in cortical neuron cells. Observing differing results would also be valuable as it would suggest that there is a unique cis-acting mechanism such as chromatin looping or unique RNA polymerase processivity occurring allowing readthrough specifically at the endogenous locus but not exogenous mimics. 3.1.2 Plasmid Construction and ESC Transfections Initial attempts in this aim used a previously generated plasmid, named pCTPIE. This plasmid contains a CMV immediate early enhancer and chicken β-actin promoter (CAG) upstream of a tdTomato fluorescent reporter, followed by the primary Mest PAS which is subsequently followed by an internal ribosome entry site (IRES) and a green fluorescent reporter. This plasmid was co-transfected into F1 ESCs with a PGK-puro construct for transient selection. Over several transfection attempts, many colonies survived puromycin selection, but no positive clones were generated. These results informed our 44 development of two alternative strategies focussed on providing a built-in selection for the construct: i) adding a PGK-neo cassette flanked by loxP sites (floxed) to allow for direct selection of integrants (plasmid termed “pCTPIE-pLoxpNeo”); and ii) floxing the pCTPIE construct with inverted loxP sites to allow for use in transfecting a previously generated RMCE docking site cell line. At the outset of plasmid construction, my strategies were built around restriction-digest cloning. While sound in principle, these attempts were largely unsuccessful due to factors including incompatibility of plasmid fragments necessitating lower efficiency blunt-end ligations, and the expected low efficiency of restriction digest cloning. These unsuccessful trials, combined with those in aim 2 (chapter 3.2) detailed later, lead to our eventual decision to abandon restriction-digest cloning in favour of Gibson assembly. Primers with appropriate tails were designed for our fragments of interest and these plasmids were successfully generated via Gibson assembly (primers available in Table 2.1), with integrity being confirmed using whole-plasmid sequencing (Plasmidsaurus) and amino acid sequence confirmation. Although the floxed pCTPIE construct was successfully generated for RMCE transfection, over several attempts to co-transfect it with a PGK-puro-Cre construct into a previously generated RMCE cell line, no positive clones were generated. This result, combined with similar outcomes from another student using the cell line led to our suspicion that this line may no longer be RMCE-competent. Despite our RMCE approach failing to generate integrants, I had much more success with pCTPIE-pLoxpNeo trials performed in parallel. 45 46 Figure 3.1 Exogenous Mest PAS Construct (A) Complete plasmid map with regions to scale. Clockwise from the CMV-IE: A strong constitutive human CMV-IE enhancer (pale green)+ chicken β-actin promoter (purple), the pCAGGS promoter, driving expression of a tdTomato red fluorescent reporter (red); followed by the primary Mest PAS (bright pink) (sequence shown in Fig. 1.4); an internal ribosome entry site (IRES) (soft pink); an EGFP green fluorescent reporter (green); a PAS (bright pink); the two combined plasmid backbones containing a ColE1 origin of replication, an ampicillin resistance gene (two side-by-side as result of the Gibson assembly of multiple plasmid fragments) and an F1 origin of replication (grey and sky blue respectively); two loxP sites (yellow) flanking a PGK-neo-PAS cassette. All polyAs (except Mest) are constitutive. (B) Linear representation of the “core” plasmid construct I randomly integrated into ESCs. Left to right: pCAGGS strong constitutive promoter (light green and purple, for CMV-IE and β-actin promoter respectively), tdTomato (red), Mest PAS (bright pink), IRES (soft pink), EGFP (green), and the last PAS (bright pink). The entire linearized (PVU1) construct (A) was randomly integrated into the genome, but this core is the region key for our aim. Following plasmid construction, we realized that allelic information was not actually important for this aim as we are randomly integrating the construct and not examining the endogenous locus except to confirm MestXL expression (which can be done without allelic information). Because of this and previous difficulties integrating constructs into our F1 ESC lines, we decided to switch to transfecting our very 47 integration-competent pure C57BL/6 C2 ESCs185. The new pCTPIE-pLoxpNeo construct (Fig. 3.1) allowing for direct antibiotic selection of integrated construct and single-stage antibiotic treatment, combined with new cell line choice as well as increased DNA amounts and cell numbers compared to previous trials, made huge improvements to efficacy of this transfection. In my first attempt I picked 4 red fluorescent colonies, compared to 0 over multiple trials before these changes. Due to the nature of a random integration transfection, allowing the possibility of multiple insertions, position effects by adjacent promoter/enhancer regions, as well as other possibly unforeseen effects, additional clones were generated in subsequent transfections. Clones were deemed positive and picked on the basis of observed red fluorescence by microscopy which while dim, was demonstrably higher than autofluorescence observed in negative controls (Fig. 3.2). 48 Figure 3.2 C2 Mest PAS Integrants C2 pCTPIE-pLoxpNeo integrants 1-3 (the first three generated) pictures taken on 100x zoom. From left to right the observations states are as follows: brightfield, L5 (green fluorescence), tx2 (red fluorescence), and tx2 at high exposure. The high exposure column allows visualization of the fact that while for clones 1 and 3 fluorescence is dim, it is still much higher than WT C2 ESCs. Also note that all EGFP readouts are blank, this is expected as these are undifferentiated clones. We don’t expect EGFP expression until they have been differentiated into cortical neurons. 49 3.1.3 Cortical Neuron Differentiation The eventual goal for both aims of this thesis was to differentiate the generated ESC clones into MestXL-expressing cell types, in our case being cortical neurons. As outlined in chapter 3.3, initial attempts to differentiate Mest-PAS ESC clones fell short due to presence of mycoplasma contamination and lack of subsequent protocol optimizations. After a 2-week plasmocin treatment, I have performed a single differentiation trial on one of these clones up to day 21. Morphology of aggregates throughout the protocol was consistent with successful and healthy differentiation: smooth edges, intact aggregates and increasing size up to day 21. While red fluorescence was consistently observable up to and including d21, EGFP signal observation by fluorescent microscopy was questionable, with dim signal if any being present, which was confounded further by the presence of autofluorescence due to the cell-density of the organoids. On day 21, I bulk-prepared these organoids for fluorescence activated cell sorting (FACS). Disaggregation into a single-cell suspension was accomplished via a Trypsin-based approach, counterstaining for dead cells with PI. Although such a suspension was eventually achieved, FACS results indicated that most cells died in this process (upwards of 90%). The remaining cells showed red fluorescence but not convincing presence of EGFP signal. 50 3.2 Trapping and Truncating Endogenous MestXL expression 3.2.1 Background/Rationale While the sequence components of an individual PAS are integral in PAS 3’UTR choice, many other cis and trans factors (detailed earlier) can often be at play. In the event that the primary Mest PAS is not sufficient for readthrough in appropriate cell types, our second aim allows us to elucidate other factors involved including candidate RBPs. Additionally, while the previous aim addresses the regulation of MestXL production and provides a proxy for MestXL, it does not allow for sorting cells based directly on MestXL expression, a function that is necessary for identifying involved factors and one that this second aim attempts to provide. As previously explained, large-scale 3’UTR lengthening is a pervasive phenomenon in nervous tissue. Generic 3’UTR lengthening in nerve cells is often related to the need for differing localization or regulation of gene products. While it is entirely possible that the transition from Mest to MestXL simply falls under this umbrella, a possibility that has informed our identification of candidate RBPs, the MestXL 3’UTR exhibits some oddities that have led to our model of its unique function. Due to its apparent necessity for paternal down-regulation of the Copg2 transcript, proximity to and extension into the Copg2 transcribed region, along with its massively inordinate size (upwards of 37kb 3’UTR) compared to the average of ~1-2kb lengthened 3’UTRs observed in neural tissues (Miura et al., 2013), we believe that MestXL evolved as a 51 long RNA acting on Copg2 as a mechanism of allele-specific gene dose regulation in nervous tissue. In this aim, I attempt to address these questions by creating and introducing into ESCs a fluorescent construct trapping and truncating MestXL approximately 1.8kb after the primary Mest polyA, in intron 22 of Copg2, truncating MestXL by ~35kb. The core of this construct is composed of a splice acceptor and an internal ribosome entry site (IRES) followed by an EGFP and PAS to terminate transcription. When properly integrated via CRISPR-Cas9, this construct should report and truncate MestXL, allowing purification and analysis of expressing cells. Following successful integration in F1 ESCs, I planned to differentiate positive clones into cortical neurons (a neuronal cell type expressing MestXL) to allow sorting of construct-expressing cells and assay of Copg2 and MestXL expression as well as probing for candidate factors. In this aim we hypothesize that we would see 100% correlation between MestXL-expressing cells and the fluorescent and that there will be candidate factors for involvement in MestXL transcription that we can identify via analysis of these sorted fluorescent cells. Regarding the MestXL truncation, we hypothesize that in these sorted fluorescent cells, Copg2 will be expressed biallelically. 3.2.2 Plasmid Construction and ESC Transfection The plasmid construct upon which this aim hinges is composed of: a splice acceptor-IRES-EGFP-PAS segment to trap and report expression of MestXL; adjacent to a floxed PGK-neo selectable marker to allow for selection of integrants following transfection; both of which are flanked by 5’ and 3’ homology arms (HA) mapping to intron 22 of Copg2; adjacent to which is a PGK-driven diphtheria toxin fragment A (DTA), a suicide 52 gene resulting in cell death if anything beyond the core construct (in between the homology arms) is randomly integrated (Fig. 3.3). The splice acceptor site was added in case some unknown splicing event takes place 3' of the primary Mest PAS in the formation of MestXL. As this approach is CRISPR-Cas9-based, I began by designing, PCR amplifying, and SLIC-cloning two alternate CRISPR guides against Copg2 intron 22 in a vector expressing Cas9, followed by ESC transfection and surveyor endonuclease assay to determine efficacy of each guide. Both candidate gRNAs were similarly highly efficient, with a slight advantage for guide 2. Initial attempts to derive the donor plasmid in this CRISPR approach involved a multi-step restriction-digest cloning plan. This involved digesting and ligating the designed and amplified 3’ arm of homology into an existing PGK-neo-lox2-DTA construct (ploxPneo); digesting and ligating the 5’ homology arm into an existing pCX-IRES-EGFP plasmid (PCIE); then blunt-end ligating these two plasmid products together. Extensive troubleshooting was necessary to only successfully insert the 3’ homology arm into our PGK-neo-lox2-DTA construct, with the remaining steps uncompleted via restriction digest cloning after multiple strategy adjustments including but not limited to: selection of new enzymes, dephosphorylating cut 5’ and 3’ ends and generation and use of new cloning bacteria. Following our abandonment of restriction digest cloning, as in aim 1 I moved on to Gibson assembly. With one component of the final plasmid already generated, the Gibson assembly design entailed linearizing our pCX-IRES-EGFP construct, then designing overlapping tailed-oligos for each of the 5’ arm of homology and the 3’HA-53 ploxPneo to be PCR amplified and assembled into PCIE. This approach was much more effective than restriction digest cloning and I successfully generated the final plasmid in one trial. Following generation, I confirmed the structure of the plasmid with whole-plasmid sequencing (Plasmidsaurus) and amino acid sequence analysis to ensure any mismatches didn’t affect protein sequences. 54 55 Figure 3.3 Endogenous Reporter Construct (A) Complete plasmid map with regions to scale. Clockwise from the dark orange 5’HA: Copg2 intron 22 5’ arm of homology (dark orange); splice acceptor (SA, light grey); IRES (soft pink); EGFP green fluorescent reporter (green); PAS (bright pink); two loxP sites flanking a PGK promoter (bright blue)-driven neo/kanamycin resistance gene (soft orange) and a bGH polyA; the Copg2 intron 22 3’ arm of homology (dark orange); a PGK-driven DTA to control for correct integration, followed by a bGH polyA; the two backbones from the combined plasmids, containing two origins of replication (pale blue) and two ampicillin resistance genes (grey) (two of each due to the combination of plasmid fragments via Gibson assembly). All PASs are constitutive. (B) Linear representation of the “core” plasmid construct I aimed to integrate into MestXL in F1 ESCs via CRISPR-Cas9 insertion. Left to right: Copg2 intron 22 5’ arm of homology; IRES; EGFP; PAS; loxP-flanked PGK-neo-PAS resistance cassette; Copg2 intron 22 3’ arm of homology. Following successful plasmid construction, I began trials co-transfecting the final construct with our CRISPR guide in F1 ESCs. The choice to perform this transfection in F1 cells was to allow for parsing out allelic information in downstream analyses: single nucleotide polymorphisms (SNPs) between the paternal and maternal alleles us to assay for presence or loss of secondary Copg2 imprinting in the eventual knock-in. These transfections relied on 48-hour of puromycin treatment to select for the transiently-expressed CRISPR guide vector, followed by 7 days of G418 treatment 56 selecting for integration of the endogenous reporter. Despite both plasmids having confirmed sequence, and testing of several different amount of each plasmid in the lipofection mixes, I routinely observed cells surviving the initial puro selection but no surviving colonies following G418 treatment. This was confirmed in two different cell lines and amounts of cells lipofected and indicated that although transfection efficiency was acceptable (cells were receiving the CRISPR guide plasmid), the construct was failing to integrate into the ESC genome. These results over several trials, combined with previous failed knock-in attempts in these cell lines in the lab, lead us to suspect that these particular lines were not homologous-recombination competent. Moving on from these F1 cell lines, I attempted to integrate this construct into our C2 ESCs. While not F1 cells, we wanted to check for differences in transfection/integration efficiency between these cell lines. Several clones survived both selections, a marked increase from transfection of our F1 line. However, none of these clones showed successful integration upon PCR testing with our designed reactions. These PCR reactions assay for the presence/absence of both the 5’ and 3’ ends of our endogenous reporter, in addition to a genomic control reaction spanning the insertion site. 3.3 Cortical Neuron Differentiation in Brain Organoids 3.3.1 Background/Rationale While not an aim per se, an instrumental component to both aims of this project is differentiating the generated cell lines into MestXL-expressing cells. Our exogenous reporter line requires differentiation to allow the possibility of readthrough and 57 transcription of EGFP, along with confirmation of MestXL expression. Meanwhile, the purpose of the endogenous reporter is to allow sorting of MestXL-expressing cells, obviously requiring differentiation into such cell types. Though it has been previously established that MestXL is expressed in the CNS56, specificity beyond that has yet to be delineated. In this section I outline our choice of, troubleshooting and establishment of a MestXL-expressing cell type differentiation protocol. 3.3.2 Cell-Type and Protocol Choice To identify a specific CNS cell type that would express MestXL when differentiated, we examined RNA-seq data gathered in multiple cell types186. Due to the recent filling of the sequencing gap in the Mest-Copg2 locus in the most recent mouse reference genome build (mm39), we were able to look at expression of the entire length of MestXL in this dataset, by downloading the published RNA-seq data and re-aligning it to mm39. Although we see some expression of MestXL in motor neurons in other datasets187, mESC-derived motor neurons did not show this readthrough (ESC-MN track) and expression was observed to be more consistent in cortical neurons (Fig. 3.4). These RNA-seq data, corroborated with SC-RNAseq data analysis (Fig. S1) and having an established lab-contact (Tristan Bouschet) well-versed in ESC-cortical neuron differentiation, lead to our decision to choose cortical neuron differentiation as our means of generating MestXL-expressing cells for these aims. Although strong two-dimensional culture cortical neuron differentiations exist, we decided on a 3D brain 58 organoid approach177 due to increased speed and simplicity compared to 2D, with suggestions and optimizations provided by Tristan Bouschet at the University of Montpellier. Figure 3.4 Mest Locus RNA-seq & Endogenous Reporter Target UCSC Genome Browser track hub containing RNA-seq data from published studies, aligned to mm39186,187. These datasets include sequencing from ESCs, neural progenitor cells (NPC), cortical neurons (CN), and motor neurons (MN). Readthrough past the primary Mest PAS is observable in tracks 4 and 5, however the most expression and strongest ratio of normal length to readthrough Mest (least dropoff in expression at the end of normal-length Mest) is observable in track 4 (cortical neurons). Track 1 labels the location of the endogenous reporter homology arms (within intron 22 59 of Copg2, downstream of the primary Mest PAS). Note that in all tracks only one Mest TSS is visible (one clear initial expression peak, with no clear start points up or down stream). While alternative TSSs can be a strong regulator of PAS choice143, this is not the case for Mest. Gaps in RNA-seq reads alignment within intron 20 of Copg2 correspond to repeats of low mappability (Fig. S2). 3.3.3 Troubleshooting and Protocol Establishment To measure effective production of cortical neurons and MestXL production during brain organoid differentiation we first designed qRT-PCR and immunofluorescence (IF) assays. Primers are detailed in Table 2.1 but in brief, we selected qRT-PCR primers based on precedent and recommendations from the Bouschet group: markers for undifferentiated, early dorsal neuron progenitor stages and late-stage neurons and cortical neurons. For the Mest-Copg2 locus we used previously lab-established Mest, Copg2, and MestXL (Mit1) primers56, in addition to a newly designed MestXL-proximal reaction to assay expression directly downstream of the primary Mest PAS. All of these were analyzed by ΔΔCT with a reaction for the housekeeping gene Ppia as the reference183. For IF analysis of developed brain organoids, we decided on two pairs of filamentous and nuclear (transcription factor) markers. NESTIN and TBR1 act as neuronal progenitor and advanced cortical neuron markers, respectively, whereas PAX6 and β-tubulin (TUBB3) are used as markers for dorsal progenitor identity and adult neurons, respectively. This suite of qRT-PCR and IF markers provided an excellent proxy for expression in the Mest locus as well as the full range of neuronal differentiation, showing the progression from undifferentiated ESCs, to dorsal neuronal 60 progenitors to adult cortical neurons. I began trials by differentiating what were at the time our 3 cell lines of interest – HA36 (RMCE line), BC1.1 (F1), and C2 (B6) ESCs – and collecting organoids at D14 and 21 for analysis. For all three cell lines, I observed smaller organoids with much rougher edges than expected, and RNA yields were much lower than expected resulting in questionable qRT-PCR results. For the HA36 cell line we had promising qRT-PCR results, showing upregulation of Tbr1, Fabp7, Pax6 and Tubb3, with concomitant reduction of the pluripotency marker Nanog between differentiated and undifferentiated cells. That being said, we also had less than ideal qRT-PCR curves and the scales for our downstream analysis were quite small, likely due to the unexpectedly low RNA yield. Additionally, we observed another anomaly in Mest expression being much higher in differentiated compared to undifferentiated cells. These preliminary results in the CN differentiation were promising, if somewhat unideal. The cause of these questionable results became clearer upon confocal IF analysis. Only day 21 organoids were antibody-stained due to their larger size, greater RNA yield allowing for more to be set aside for staining, and the recommendations of the Bouschet lab that we were more likely to see the markers we were assaying for at this later timepoint. Although we observed what appears to be some cell-specific fluorescence for Tbr1 and Pax6, the vast majority of these organoids showed very punctate fluorescence, exclusively so for the filamentous markers. This punctate staining was consistent with dead cells and autofluorescence, more so than our expected staining patterns. This result, combined with the unideal qRT-PCR results and poor size and morphology of our organoids made us question their quality. Suspecting large amounts of cell death, we performed a viability stain on a cellular aggregate at day 61 7 of the differentiation with Trypan Blue. This stain both on the intact aggregate and after we dissociated it, revealed that the vast majority of cells at the end of the first stage of our differentiation were in fact dead. Suspecting significant optimizations in the first stage of our CN differentiation needed to be made, I performed several trials varying media components and with increased frequency of media changes. Finding precedent for varied concentrations of KSR in similar protocols188–190 I tried 5,10,15 and 20% KSR. While differing slightly between treatments, these aggregates maintained the key problems I was trying to circumvent. I also set up trials including N2 and B27 neural supplements in the media189,190, to facilitate growth once differentiation began, again showing the same small size, large amount of cell death and altered morphology as previous attempts. Suspecting that the cells were not receiving enough nutrition and removal of wastes from the media, I performed half media changes daily, finding precedent for more frequent media changes in several similar protocols189–192. I also performed the differentiation without one or both of the cortical-lineage-guiding cytokines, substituting both for cyclopamine193 and finally simply seeded ESCs in low attachment 96 wells with ESM without leukemia inhibitory factor (LIF), to facilitate generic differentiation. All of these trials included propidium iodide as a viability stain to allow progressive tracking of cell death and disaggregating and staining organoids with Trypan Blue to allow for quantitative live/dead cell counts at day 4 and 5. The various treatments resulted in similar observations of widespread cell death by day 3; rough, broken edges (contrary to the smooth borders expected based on images from the Bouschet group) and small size. With most (if not all) of the logical, simple to fix, and practical ideas tested, we 62 proceeded to move on to the less likely and much less convenient explanation for my observations. Using Hoescht staining as well as PCR testing (primers provided in Table 2.1), I tested our cell lines for mycoplasma contamination. After two weeks of growth without antibiotics I tested our C2 parental cell line as well as several of the exogenous PAS construct clones and our BC1.1 F1 line for mycoplasma. Hoescht staining as well as an established PCR reaction for contamination confirmed the presence of mycoplasma in all of these, but not most of the other cell lines we tested. Following these results and subsequent containment and cleaning measures, we cleaned one of the exogenous PAS construct clones with 2 weeks of plasmocin treatment, then attempted the cortical neuron differentiation on this and another clean F1 line (BC11). With modifications to the protocol suggested by the Bouschet group, namely the use of Accutase instead of Trypsin to collect the initial undifferentiated ESCs; switching to stage 2 of the differentiation at day 7 instead of 8; and incubating the plates on a rotating platform within the incubator from day 7 onwards, combined with use of cell lines clean of mycoplasma, the results of the cortical neuron differentiation improved drastically. Following these optimizations, aggregate edges were much smoother in days 1-7, indicating less cell death and healthier cells (Fig. 3.5). In the second stage of differentiation (days 8-21), we observed much larger and less opaque organoids forming, with much smoother edges (Fig. 3.5), again much more in line with our expectations for this protocol and very promising for the health and differentiation status of the cells. 63 Figure 3.5 Healthy Brain Organoid Morphology Progression Light microscopy images taken at various timepoints (days 1,2,7,14 and 21) in our healthy brain organoid differentiation. Smooth edges were observable throughout, with increasing size and opacity nearer to endpoint. Beyond the morphological improvements observed, IF analysis of organoids via confocal microscopy was significantly improved and very positive, showing cell-specific staining and filamentous intercellular staining of appropriate markers (Fig. 3.6). In this round of staining, I showed that while not the majority, there is a significant portion of cells in this differentiation expressing the cortical neuron marker TBR1, indicating there are likely MestXL-expressing cell populations within these organoids. Additionally, I 64 confirmed the presence of PAX6, NESTIN and TUBB3-expressing populations, showing an array of differentiation stages including dorsal-NPCs (PAX6) and adult neurons (TUBB3) within our CN organoids. Finally, I performed RNA extraction and qRT-PCR on these day 21 organoids and confirmed the presence of MestXL (with similar levels of Mest) and Tbr1, with concomitant loss of the ESC marker Nanog (Fig. 3.7). Figure 3.6 Confocal Microscopy of D21 BC11 Cortical Neuron Organoids (A) Day 21 brain organoid antibody stained for nuclear TBR1 (cortical neuron marker) and filamentous cytoplasmic NESTIN (CNS NPC marker), counterstained with DAPI. (B) Day 21 brain organoid antibody stained for nuclear PAX6 and filamentous cytoplasmic TUBB3, counterstained with DAPI. 65 Figure 3.7 D21 CN Organoid qRT-PCR qRT-PCR of day 21 cortical neuron organoids compared to undifferentiated C2 ESCs. *Data analysis performed by undergraduate honours student Abdullah Khawaja. Error bars represent error between qPCR technical replicates. (A) Tbr1 cortical neuron marker expression, confirming presence of cortical neurons in generated organoids (B) Nanog ESC marker expression. This marker should decline as differentiation progresses, allowing tracking of movement away from pluripotency. 66 (C) Mit1/MestXL expression. Confirming presence of MestXL-expressing populations in our late differentiated cells. (D) Mest expression. We observed higher expression in undifferentiated ESCs as expected, and the expression in D21 CNs is comparable to MestXL expression in the same cells. This aligns with our expectations assuming readthrough at the Mest PAS, the Mest and MestXL reactions should detect the same RNA. These results combined with IF results confirm efficacy of our CN differentiation in producing cortical neurons and MestXL-expressing cells. Having confirmed the presence of Tbr1 and MestXL-expressing populations in these mycoplasma-free organoids via IF and qRT-PCR, we were content with the establishment of this protocol and could move on to differentiating our transgenic ESC lines. 67 Chapter 4: Discussion Although the secondary imprinting relationship between MestXL and Copg2 has been described, the mechanism of Copg2 repression, exactly how primary Mest PAS bypass occurs, and the specific cell types where this occurs in all have yet to be elucidated. In this thesis I have outlined my processes in extensive troubleshooting and establishment of a battery of tools and beginning experiments to aid in answering these questions. 4.1 Mest Primary PAS Readthrough Pursuant to our first aim, I successfully generated a double-fluorescent Mest primary PAS construct with added antibiotic selection. This plasmid was successfully integrated into C2 ESCs and we attempted to differentiate these into MestXL-expressing cortical neurons via 3D organoid differentiation. As of yet, we have observed no EGFP expression in these differentiated clones, but these observations were confounded by cell density-related autofluorescence and significant cell death during the FACS single cell suspension preparation. An additional limitation of the progress made in this aim is the absence of explicit confirmation of the ability of the pCTPIE-pLoxpNeo clones to express EGFP. Whole-plasmid sequencing and amino acid sequence analysis confirmed that all pertinent regions are correct in the construct, but confirmation of EGFP ability in these cell lines would still be relevant. Additionally, the random integration of our construct into these ESCs presents with some complicating possibilities. Most saliently, the construct could be integrated into a region of the genome that is transcriptionally silent in cortical neuron cell types, rendering any 68 differentiation of these clones pointless. This possibility is partially negated by production of multiple clones but warrants consideration in any downstream analyses. Future endeavours for this aim should be primarily focused on optimizing and completing the FACS, accompanying controls and downstream confirmation of MestXL expression. While the addition of other enzymes such as collagenase can aid in this process, there are two main approaches for disaggregation of neuronal tissues. The first is trypsin-based, the second using papain. We have already attempted this with trypsin and while there are assuredly optimizations to be made, the immense percentage of cell death that occurred strongly suggests a change in tactic is appropriate. Subsequent attempts to produce single-cell suspensions of cortical neuron organoids for FACS will focus on papain and other gentler enzymes such as Accutase, with the primary goal being maintenance of cell health and integrity in the resultant suspension. As salient downstream analysis of these sorted cells is qRT-PCR confirmation that only cells showing both red and green fluorescence are those expressing MestXL, perfect cell health is not a necessity, but we do need a significant portion of live cells before FACS to know the sort results are faithful to expression in the CN organoids. Another significant limitation of this aim is that even upon completion of this progress, we do not have the tools in hand for obtaining a positive control for PAS readthrough. As simpler approaches to induce changes in APA such as heat shock and siRNA-induced Nudt21 knockdown favour use of proximal, rather than distal PAS sites and the pervasiveness of their effects across the genome is not yet well understood194,195, we will be restricted to more high effort approaches for inducing APA at our randomly integrated construct. These approaches would likely involve construction and utilization of CRISPR-Cas9 69 technologies within one or multiple of these generated clones. This could include CRISPR-iPAS196, wherein we would use catalytically dead RNA-specific Cas13 targeted at the fluorescence-intervening Mest PAS, restricting access of CPA machinery and theoretically allowing PAS bypass in favour of the downstream PAS. Following completion of the above progressions and possible controls, downstream understanding and experiments will be contingent on the results of the FACS and qRT-PCR of differentiated clones. Should we find that there is a population of cells that show both red and green fluorescence, and confirm that these are the only cells expressing MestXL, we show that in MestXL cell types, the PAS we have provided is sufficient for readthrough. In this case, downstream experiments would centralize around finding out what specific cell types these are, likely by bulk RNA sequencing of sorted cell populations looking for cell subtype-specific markers (Fig 4.1). One interpretation of the readthrough occurring at the primary Mest PAS is that this PAS is insufficient for readthrough. This interpretation involves the necessity of other cis-acting locus-specific sequence factors that allow for this readthrough to occur. If we do not see readthrough in any of the differentiated clones, this would suggest that there is something unique occurring at the endogenous locus resulting in readthrough in the CNS and downstream experiments would prioritize finding which sequence elements are responsible, possibly including unique promoter/enhancer interactions or other cis-acting elements. These experiments would use the eventually produced endogenous MestXL reporter line (Aim 2) as a forward-genetics tool for finding key sequence elements in expression of MestXL. Forward genetics in this case means we would be able to ascertain the genetic basis of loss of green fluorescence (which should be a 70 proxy for loss of MestXL), instead of a reverse genetics approach where we would knock out genes without knowing an expected phenotype. One very interesting approach we could employ would be to use paired-guide CRISPR-Cas9 deletion mapping197, beginning with nearby promoter/enhancer regions and if need be, moving on to multiplex RNA guide random pairing in this reporter line, looking for deletions that preclude green fluorescence in brain organoids (Fig 4.1). Figure 4.1 Mest PAS Follow-up Experimental Workflow Flow chart detailing experimental progression in the cases of red+green cells observed in differentiated Mest PAS readthrough cell lines, vs if only red cells are observed. 4.2 Endogenous MestXL Reporter 71 Although I experienced difficulties obtaining targeted integration into F1 ESCs thus far, I did successfully assemble our CRISPR-Cas9 endogenous MestXL EGFP reporter construct. Once eventually integrated, this line will serve as an invaluable forward genetics tool for identifying factors integral in MestXL expression. Over the course of multiple attempts to integrate our EGFP reporter into our F1 ESCs, transfection efficiencies have been strong, we observed many colonies surviving transient puromycin selection for the CRISPR-Cas9 plasmid, and previous projects in the lab have accomplished CRISPR-Cas9 deletions in these cells. Additionally, I performed surveyor endonuclease assay on the guide we used, confirming its cutting efficiency is acceptable from the CRISPR-Cas9 plasmid we are currently using for transfections. Due to these factors, we strongly suspect that the characterized F1 ESC line we have been attempting to integrate this construct into may be at least partially incompetent for homologous recombination. As such, ongoing attempts to create this reporter line will utilize other F1 lines. We have our own F1 ESCs, as well as established lines procured from other labs, to integrate this construct into, likely resolving my previous difficulties. My single transfection attempt in C2 ESCs showed several colonies surviving both selections but no positive integrant, this is likely owing to the inherent inefficiency of CRISPR integrations and only having 9 picked clones. Upon eventual construction of our F1 ESC endogenous MestXL reporter line, downstream experiments would be contingent upon progression of these cells through our now established CN differentiation. Following successful differentiation and sorting of EGFP-expressing cells, our first step would be to check via qRT-PCR that EGFP cells are the only ones expressing MestXL. This will ensure that our reporter is acting as 72 intended and that follow up experiments would function properly. Subsequently, due to the F1 nature of this reporter line, we will be able to assay by RNA-seq for paternal Copg2 expression in these cells. Due to the very significant ablation of MestXL by this reporter, we expect biparental expression of Copg2 in this line, and assaying for this will give us significant insights into the mechanism by which MestXL produces secondary imprinting of paternal Copg2. We currently propose two models for how MestXL might suppress paternal Copg2 expression. The first model suggests that MestXL functions similarly to a long non-coding RNA, with most of its length and secondary structure (Fig. S2) being essential for repressing paternal Copg2 by recruiting silencing factors in cis. The second model posits that this secondary imprinting may function through a transcriptional interference mechanism, leading to truncated paternal Copg2 that is then degraded. In this case, any amount of readthrough transcription past the primary PAS of MestXL might be sufficient for this repression. By observing whether paternal Copg2 repression still occurs in the absence of full-length MestXL, we would aim to gain information on which model is correct. If repression persists, it will support the idea that only a minimal amount of MestXL readthrough is needed for imprinting, while a lack of repression would indicate that the complete length and structure of MestXL are necessary for this process. Repression persisting would not completely exclude our lncRNA-like mechanism idea, but it would show that the sequence downstream of our EGFP insertion is not necessary for Copg2 secondary imprinting. In addition to this experiment, we can examine NET-seq data of Copg2 in MestXL-expressing cells to determine if paternal Copg2 is being expressed at normal levels, just at lower stability. This would provide strong support to the transcriptional interference model, in that 73 paternal Copg2 primary transcript is being produced, just in an incomplete form that is getting degraded. We can also examine ChIP-seq in MestXL-expressing cell types to find the expression status of paternal Copg2 via presence/absence of repressive marks such as H3K27me3 or H3K9me3, or active markers such as H3K4me1 and Hk327ac. Assaying the chromatin environment at this locus in MestXL expressing cells would give us strong insight into whether or not lncRNA-like recruitment of repressive marks is occurring on paternal Copg2. In addition to elucidating cis-acting sequence factors involved in Mest PAS readthrough explained in section 4.1, this EGFP MestXL reporter line will allow sorting of MestXL-expressing cells to identify which specific CN cell subtypes are showing this readthrough, as well as functioning as a forward genetics tool for identifying trans-acting cellular factors necessary for MestXL expression. While we have determined that MestXL is expressed in cortical neurons, there are multiple unique cell types that fall under this umbrella, and identifying which of these cells show expression of MestXL can provide additional understanding of MestXL regulation and function. FACS sorting for EGFP-expressing cells, combined with subsequent qRT-PCR analysis assaying for cell type-specific markers will allow us to determine which exact cells are producing this longer form of Mest and what is unique about them. In addition to this analysis, work done by our lab outside this thesis analyzing RNA-seq data has identified candidate RNA binding proteins possibly necessary for MestXL expression including known regulators of APA such as NOVA2. 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J Neurodev Disord 8, 1–14 (2016). 91 Appendix: Supplemental Figures Figure S1 MestXL Single Cell RNA Sequencing Analysis SC-RNA-seq data analysis from198 performed using Loupe Browser 7 software with custom annotation to allow identification of MestXL expression. (A) Marker analysis of postnatal day 1 mice, generating identifiable clusters of different cell types. (B) MestXL expression, exhibiting the strongest expression in projection neuron cluster. (C) Mest expression, showing highest levels of expression in progenitor cell clusters, with far less expression in MestXL-expressing clusters 92 (D) Cux2, a marker of layers 2 and 3 cortical neurons199 (E) Satb2, a marker of layers 2,3,4 and 5 cortical neurons199 (F) RorB, a known marker of layers 4 and 6 cortical neurons199 (G) ETV1, a marker of layers 5 and 6 cortical neurons199 (H) Tbr1, a marker of layers 5 and 6 cortical neurons199 These expression patterns confirmed our identification of cortical neurons as a suitable cell type for MestXL expression, as several oft-used cortical neuron markers show very similar clustering to MestXL. Note* that Tbr1 appears to mark best for a more intermediate stage of cortical neuron development based on comparison to marker analysis (A). Despite this, due to its significant use in the literature as a cortical neuron marker, as well as the easily observable clustering similarity of other cortical neuron markers to MestXL expression, we maintained that cortical neurons were an appropriate proxy for MestXL expression and that Tbr1 is a suitable marker for progression of this differentiation especially as it shows almost no expression in NPC populations (H). 93 Figure S2 MestXL Secondary Structure 94 Analysis of repeats within the Copg2 intron 20 DNA sequence, filled in the mm39 build of the mouse genome. Secondary structure analysis of this region, performed by Aaron Bogutz, reveals highly repetitive nature of the MestXL 3’ UTR and the corresponding stem-loop secondary structures that may arise in this RNA as a result.
Thesis/Dissertation
eng
Medical Genetics
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Lefebvre, Louis
Alternative polyadenylation at Mest
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