Land and Food Systems, Faculty of
DSpace
UBCV
Ma, Yuexi
Master of Science - MSc
University of British Columbia
Dietary factors, like polyphenols naturally occurring in plants, exert influences on human health via epigenetic mechanisms that alter gene expression without changing DNA sequence. We have previously performed genome-wide DNA methylation and expression analyses in human mammary epithelial cells and healthy rats’ livers, respectively, which were exposed to berry polyphenols with estrogenic activity (i.e., phytoestrogens). Using these datasets, we identified hypermethylated and/or downregulated genes enriched with functional categories that disrupt homeostasis and health maintenance, and promote aging, e.g., by limiting DNA break repair (RNF169) or promoting ribosomal biogenesis (rDNA). Existing evidence suggests that DNA methylation may be governed by chromatin regulatory proteins such as SIRT1 deacetylase. SIRT1 may interact with DNA methylating enzymes (e.g., DNMT3B) to repress transcription. Interestingly, SIRT1 was reported as a possible target of phytoestrogens. However, it remains unknown whether SIRT1 and its interaction with DNMT3B play a mechanistic role in phytoestrogen-mediated epigenetic effects in normal mammary epithelial cells. Thus, we test a hypothesis that phytoestrogens suppress genes related to homeostatic imbalance and aging, via SIRT1-mediated effects on DNA methylation. Human MCF10A mammary epithelial cells were treated with phytoestrogens, pterostilbene (PTS) and genistein (GEN). PTS and GEN decreased MCF10A cell growth with 50% inhibition and less than 10% dead cells at 11µM and 22µM concentrations respectively, as compared with vehicle-treated cells (control). SIRT1 occupancy at the selected hypermethylated phytoestrogen-target genes, RNF169 and rDNA, was accompanied by consistent promoter hypermethylation and gene downregulation in response to GEN, but not PTS. GEN-mediated DNA hypermethylation and SIRT1 occupancy were linked to a robust DNMT3B binding at both RNF169 and rDNA promoters. This was not observed in cells exposed to PTS, suggesting a distinct mechanism of action. Although both SIRT1 and DNMT3B bind to RNF169 and rDNA promoters upon GEN, the two proteins do not co-occupy the regions, as confirmed by double-ChIP. Through siRNA SIRT1 depletion, GEN does not decrease expression of rDNA anymore, suggesting that SIRT1 is required for epigenetic suppression of rDNA upon GEN. In conclusion, these findings further our understanding of SIRT1 role in epigenetic mechanisms mediating the impact of the environment-diet on cell biology.
Investigating the Role of SIRT1 in Epigenetic Effects of Dietary Phytoestrogens in Human Mammary Epithelial Cells by Yuexi Ma B.S., The University of British Columbia, 2022 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Human Nutrition) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2024 © Yuexi Ma, 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: Investigating the Role of SIRT1 in Epigenetic Effects of Dietary Phytoestrogens in Human Mammary Epithelial Cells submitted by Yuexi Ma in partial fulfilment of the requirements for the degree of Master of Science in Human Nutrition Examining Committee: Dr. Barbara Stefanska, Associate Professor, Human Nutrition, UBC Supervisor Dr. Rajavel Elango, Associate Professor, Human Nutrition, UBC Supervisory Committee Member Dr. Tianxi Yang, Assistant Professor, Food Science, UBC Supervisory Committee Member Dr. David Kitts, Associate Dean, Food Science, UBC Additional Examiner iii Abstract Dietary factors, like polyphenols naturally occurring in plants, exert influences on human health via epigenetic mechanisms that alter gene expression without changing DNA sequence. We have previously performed genome-wide DNA methylation and expression analyses in human mammary epithelial cells and healthy rats’ livers, respectively, which were exposed to berry polyphenols with estrogenic activity (i.e., phytoestrogens). Using these datasets, we identified hypermethylated and/or downregulated genes enriched with functional categories that disrupt homeostasis and health maintenance, and promote aging, e.g., by limiting DNA break repair (RNF169) or promoting ribosomal biogenesis (rDNA). Existing evidence suggests that DNA methylation may be governed by chromatin regulatory proteins such as SIRT1 deacetylase. SIRT1 may interact with DNA methylating enzymes (e.g., DNMT3B) to repress transcription. Interestingly, SIRT1 was reported as a possible target of phytoestrogens. However, it remains unknown whether SIRT1 and its interaction with DNMT3B play a mechanistic role in phytoestrogen-mediated epigenetic effects in normal mammary epithelial cells. Thus, we test a hypothesis that phytoestrogens suppress genes related to homeostatic imbalance and aging, via SIRT1-mediated effects on DNA methylation. Human MCF10A mammary epithelial cells were treated with phytoestrogens, pterostilbene (PTS) and genistein (GEN). PTS and GEN decreased MCF10A cell growth with 50% inhibition and less than 10% dead cells at 11µM and 22µM concentrations respectively, as compared with vehicle-treated cells (control). SIRT1 occupancy at the selected hypermethylated phytoestrogen-target genes, RNF169 and rDNA, was accompanied by consistent promoter hypermethylation and gene downregulation in response to GEN, but not PTS. GEN-mediated DNA hypermethylation and iv SIRT1 occupancy were linked to a robust DNMT3B binding at both RNF169 and rDNA promoters. This was not observed in cells exposed to PTS, suggesting a distinct mechanism of action. Although both SIRT1 and DNMT3B bind to RNF169 and rDNA promoters upon GEN, the two proteins do not co-occupy the regions, as confirmed by double-ChIP. Through siRNA SIRT1 depletion, GEN does not decrease expression of rDNA anymore, suggesting that SIRT1 is required for epigenetic suppression of rDNA upon GEN. In conclusion, these findings further our understanding of SIRT1 role in epigenetic mechanisms mediating the impact of the environment-diet on cell biology. v Lay Summary Pterostilbene and genistein are natural plant-based compounds found in food like blueberries and soybeans, which are commonly accessible in our daily diets. These compounds offer various health benefits, including potential disease-fighting properties and supporting our overall health. While past studies mainly focused on how these compounds affect breast cancer cells, I aim to explore their effects in healthy breast cells, which are crucial for women's health. My research fills this gap in knowledge by investigating how these compounds can promote health maintenance of breast cells. Additionally, I delved into how they interact with proteins in human body to achieve their specific roles and functions. This research sheds light on the importance of our dietary choices in contributing to health promotion of breast tissue and overall well-being. vi Preface This research project was identified by my supervisor, Dr. Barbara Stefanska, and jointly designed by both of us. I conducted the entire research and analyzed the research data. vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ................................................................................................................................ xi List of Figures .............................................................................................................................. xii List of Abbreviations ................................................................................................................. xiii Acknowledgements .................................................................................................................. xviii Dedication .....................................................................................................................................xx Chapter 1: Introduction ................................................................................................................1 1.1 Breast Health ................................................................................................................... 1 1.2 Epigenetics ...................................................................................................................... 3 1.2.1 DNA methylation ........................................................................................................ 4 1.2.1.1 Definition and importance .................................................................................. 4 1.2.1.2 DNA methyltransferases (DNMTs) .................................................................... 4 1.2.1.3 DNA demethylation ............................................................................................ 5 1.2.1.4 DNA methylation and gene expression .............................................................. 7 1.2.1.5 Crosstalk between DNA methylation and histone modifying enzymes ............. 9 1.3 SIRT1 ............................................................................................................................ 10 1.3.1 SIRT1 in epigenetic regulation ................................................................................. 10 1.3.1.1 Deacetylation-dependent action of SIRT1 ........................................................ 11 viii 1.3.1.1.1 SIRT1 and histone targets ........................................................................... 11 1.3.1.1.2 SIRT1 and non-histones (i.e., histone modifier) ......................................... 11 1.3.1.1.3 SIRT1 and non-histones (i.e., transcription factors and coregulators) ........ 12 1.3.1.2 Deacetylation-independent action of SIRT1 ..................................................... 13 1.3.2 SIRT1 activation by natural phytochemicals ............................................................ 14 1.4 Polyphenols ................................................................................................................... 16 1.4.1 Phytoestrogens .......................................................................................................... 16 1.4.1.1 Characteristics ................................................................................................... 16 1.4.1.2 Classification and chemical structures .............................................................. 17 1.4.1.3 Estrogenic signaling pathway ........................................................................... 18 1.5 Pterostilbene .................................................................................................................. 20 1.5.1 Food sources ............................................................................................................. 20 1.5.2 Metabolism and bioavailability................................................................................. 21 1.5.3 Toxicity ..................................................................................................................... 22 1.5.4 Health related effects ................................................................................................ 22 1.5.5 Epigenetic regulation of PTS .................................................................................... 23 1.5.5.1 PTS and DNA methylation ............................................................................... 23 1.5.5.2 PTS and SIRT1 ................................................................................................. 24 1.6 Genistein ....................................................................................................................... 24 1.6.1 Food sources ............................................................................................................. 24 1.6.2 Metabolism and bioavailability................................................................................. 25 1.6.3 Toxicity ..................................................................................................................... 26 1.6.4 Health related effects ................................................................................................ 26 ix 1.6.5 Epigenetic regulation of GEN ................................................................................... 27 1.6.5.1 GEN and DNA methylation .............................................................................. 27 1.6.5.2 GEN and SIRT1 ................................................................................................ 27 1.7 Research question ......................................................................................................... 28 1.7.1 Knowledge gaps ........................................................................................................ 28 1.7.2 Hypothesis................................................................................................................. 29 1.7.3 Objectives ................................................................................................................. 29 Chapter 2: Materials and Methods ............................................................................................31 2.1 Cell Culture and Treatment with Pterostilbene and Genistein ...................................... 31 2.2 Trypan Blue Exclusion Test.......................................................................................... 32 2.3 RNA Isolation and Real-Time qPCR ............................................................................ 32 2.4 Whole-cell Protein Extraction....................................................................................... 33 2.5 Western Blot Analysis .................................................................................................. 33 2.6 DNA Extraction and Pyrosequencing ........................................................................... 34 2.7 Chromatin Immunoprecipitation and Quantitative ChIP (qChIP) ................................ 35 2.8 Double Chromatin Immunoprecipitation Assay (Double-ChIP) .................................. 36 2.9 Small-interfering RNA (siRNA) Transfection.............................................................. 36 2.10 Data Analysis ................................................................................................................ 37 2.11 Statistical Analysis ........................................................................................................ 37 Chapter 3: Results........................................................................................................................40 3.1 Putative Phytoestrogen-target Genes were selected from Existing Datasets ................ 40 3.2 Growth Rate of MCF10A Human Mammary Epithelial Cells in Response to Pterostilbene or Genistein. ........................................................................................................ 46 x 3.3 SIRT1 Level is Upregulated at IC50 concentration of Pterostilbene or Genistein in MCF10A Human Mammary Epithelial Cells, Despite Unchanged mRNA Levels. ................ 49 3.4 SIRT1 Occupancy is Consistently Enriched at All Target Genes in Response to Genistein, but not Pterostilbene, in MCF10A Human Mammary Epithelial Cells. ................. 51 3.5 Expression of RNF169 and rDNA, but Not RNF114, is Downregulated in Response to Pterostilbene and Genistein....................................................................................................... 53 3.6 Differential DNA Methylation within Promoters of the Target Genes in Response to Pterostilbene or Genistein in MCF10A Human Mammary Epithelial Cells. ........................... 55 3.7 DNMT3B Occupancy is Enriched at RNF169 and rDNA Promoters in Response to Genistein in MCF10A Human Mammary Epithelial Cells....................................................... 57 3.8 SIRT1 and DNMT3B do Not Co-occupy RNF169 and rDNA Promoter Regions in Response to IC50 Genistein in MCF10A Human Mammary Epithelial Cells. ........................ 59 3.9 SIRT1 is Required for Genistein-mediated Downregulation of rDNA, but Not RNF169, in MCF10A Human Mammary Epithelial Cells. ...................................................................... 61 Chapter 4: Discussion & Conclusions ........................................................................................63 Bibliography .................................................................................................................................74 xi List of Tables Table 1.1 The Concentration of Pterostilbene (PTS) in Various Types of Foods ........................ 21 Table 1.2 The Concentration of Genistein (GEN) in Various Types of Foods ............................ 25 Table 2.1 Pyrosequencing, qRT-PCR and Quantitative ChIP (qChIP) Primer Sequences for the Selected Gene Targets................................................................................................................... 38 Table 3.1 Bioinformatics Data for Selected Top Genes from our Existing Databases ................. 45 xii List of Figures Figure 1.1 DNA methylation and demethylation. ........................................................................... 7 Figure 1.2 Chemical structure of 17β-estradiol. ........................................................................... 17 Figure 1.3 Four subclasses of phytoestrogens. ............................................................................. 18 Figure 1.4 Diagram illustrating research objectives. .................................................................... 30 Figure 3.1 Putative phytoestrogen-target genes that are differentially methylated or expressed in healthy experimental models. ....................................................................................................... 44 Figure 3.2 MCF10A cell growth in response to PTS or GEN. ..................................................... 48 Figure 3.3 Impact of PTS or GEN on SIRT1 expression in MCF10A cells. ................................ 50 Figure 3.4 SIRT1 binding at RNF114, RNF169 and rDNA promoters upon 9-day exposure to PTS or GEN in MCF10A cells. .................................................................................................... 52 Figure 3.5 Expression of RNF114, RNF169 and rDNA at mRNA levels in response to 9-day exposure to PTS or GEN in MCF10A cells. ................................................................................. 54 Figure 3.6 PTS- and GEN-mediated changes in DNA methylation of CpG loci within RNF169 and rDNA promoters in MCF10A cells. ....................................................................................... 56 Figure 3.7 DNMT3B binding at RNF169 and rDNA promoters upon 9-day exposure to PTS or GEN in MCF10A cells.................................................................................................................. 58 Figure 3.8 Evaluation of a co-occupancy of SIRT1 and DNMT3B at RNF169 and rDNA promoter regions upon 9-day exposure to 22 μM GEN in MCF10A cells as measured by double ChIP. ............................................................................................................................................. 60 Figure 3.9 Expression of SIRT1, RNF169 and rDNA upon exposure to 22 μM GEN in MCF10A cells with SIRT1 knockdown. ....................................................................................................... 62 xiii List of Abbreviations 5caC………………5-carboxylcytosine 5fC………………5-formylcytosine 5hmC………………5-hydroxymethylcytosine 5mC………………5-methylcytosine ACSS3………………Acyl-CoA Synthetase Short Chain Family Member 3 ACVR1C………………Activin A Receptor Type 1C AEBP2………………AE Binding Protein 2 AGTPBP1………………ATP/GTP Binding Carboxypeptidase 1 ALKBH5………………AlkB Homolog 5, RNA Demethylase AMPK………………AMP-activated Protein Kinase AP-1………………Activating Protein 1 AROS………………Active Regulator of SIRT1 ATP2B1………………ATPase Plasma Membrane Ca2+ Transporting 1 BRCA1………………BRCA1 DNA Repair Associated BTG3………………BTG Anti-Proliferation Factor 3 CA8………………Carbonic Anhydrase 8 cAMP………………Cyclic Adenosine Monophosphate c-fos………………Encoded by Fos Proto-Oncogene Cmax………………The Peak Plasma Concentration CpG………………5’-C-phosphate-G-3’ CRISPR-Cas9………………Clustered Regularly Interspaced Short Palindromic Repeats xiv CXCR4………………C-X-C Motif Chemokine Receptor 4 DNMTs………………DNA Methyltransferases DPP4………………Dipeptidyl Peptidase 4 DVL1………………Dishevelled Segment Polarity Protein 1 E2………………Estradiol E2F6………………E2F Transcription Factor 6 EBF3………………EBF Transcription Factor 3 EGFR………………Epidermal Growth Factor Receptor EHMT1………………Euchromatic Histone Lysine Methyltransferase 1 EHMT2………………Euchromatic Histone Lysine Methyltransferase 2 ENO………………Enolase 1 eNoSC………………Energy-dependent Nucleolar Silencing Compelx ER………………Estrogen Receptor ERCs………………Extrachromosomal rDNA circles EREs………………Estrogen Response Elements ERK………………Extracellular Signal-regulated Kinases ETAA1………………ETAA1 Activator Of ATR Kinase EZH2………………Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit FE………………Iron FOXO………………Forkhead Box O G9a………………= ENMT2 (Euchromatic Histone-lysine N-methyltransferase 2) GAP43………………Growth Associated Protein 43 Gcg………………Prolucagon xv GEN………………Genistein GLI2………………GLI Family Zinc Finger 2 GLP1………………Glucagon-Like Peptide 1 GPER………………G Protein-Coupled Estrogen Receptor GULP1………………GULP PTB Domain Containing Engulfment Adaptor 1 H2AX………………H2A.X Variant Histone HATs………………Histone Acetyltransferases HDACs………………Histone Deacetylases HMTs………………Histone Methyltransferases HNF1A………………HNF1 Homeobox A HOXA5………………Homeobox A5 IGF1………………Insulin Like Growth Factor 1 KCNK9………………Potassium Two Pore Domain Channel Subfamily K Member 9 LDL………………Low Density Lipoprotein m6A………………N6- Methyladenosine MBD………………Methyl-CpG Binding Domain Mcm10………………Minichromosome Maintenance 10 Replication Initiation Factor NAD………………Nicotinamide Adenosine Dinucleotide NF-κB………………Nuclear Factor Kappa B NOX5………………NADPH Oxidase 5 NRF2/Nrf2………………Nuclear Factor Erythroid 2-related Factor 2 OPA1………………OPA1 Mitochondrial Dynamin Like GTPase OXR1………………Oxidation Resistance 1 xvi PAX6………………Paired Box 6 Pc3………………Proprotein Convertase 3 PCDHB4………………Protocadherin Beta 4 PDCD6IP………………Programmed Cell Death 6 Interacting Protein PDE10A………………Phosphodiesterase 10A PGC-1α………………Peroxisome proliferator-activated receptor-gamma coactivator PIAS1………………Protein Inhibitor of Activated STAT 1 PIN………………Pinostilbene PRC2………………Polycomb Repressive Complex 2 PRC4………………Polycomb Repressive Complex 4 PRKCA………………Protein Kinase C Alpha PTPRD………………Protein Tyrosine Phosphatase Receptor Type D PTS………………Pterostilbene RAD50………………RAD50 Double Strand Break Repair Protein Rad51………………RAD51 Recombinase RASSF1A………………Ras Association Domain Family Member 1 RB1………………RB Transcriptional Corepressor 1 rDNA………………Ribosomal DNA RNF114………………Ring Finger Protein 114 RNF169………………Ring Finger Protein 169 ROS………………Reactive Oxygen Species RSV………………Resveratrol RUNX3………………RUNX Family Transcription Factor 3 xvii SAM………………S-adenosyl-L-methionine SEMA3A………………Semaphorin 3A SFTA2………………Surfactant Associated 2 SIPA1………………Signal-Induced Proliferation-Associated 1 SIRT………………Sirtuin SLC7A5………………Solute Carrier Family 7 Member 5 SUV39H1………………SUV39H1 Histone Lysine Methyltransferase TAGAP………………T Cell Activation RhoGTPase Activating Protein TBK1………………TANK Binding Kinase 1 TERT………………Telomerase Reverse Transcriptase TET………………Ten-Eleven Translocation TGF-β………………Transforming Growth Factor-beta TMS1………………Target of Methylation-induced Gene Silencing TNNI3………………Troponin I3, Cardiac Type TNXB………………Tenascin XB UHRF2………………Ubiquitin Like With PHD And Ring Finger Domains 2 WNT4………………Wnt Family Member 4 α-SMA………………Alpha-Smooth Muscle Actin Expression xviii Acknowledgements I would like to express my heartfelt gratitude to my supervisor, Dr. Barbara Stefanska, for her invaluable guidance, understanding, unwavering support and caring demeanor throughout my master's program. I will never forget her warm welcome and acceptance when I wanted to join her research group (as international student with limited funding resources). I am deeply grateful to my committee supervisors, Dr. Rajavel Elango and Dr. Tianxi Yang, for their responsible mentorship and approachable nature. Their insightful suggestions on my course plan and thoughtful questions have fostered my growth. I appreciate their genuine interest in my research work and their willingness to address my queries. Working with them has truly been a rewarding experience during my master's journey. Special thanks go to Dr. David Kitts for generously serving as my external examiner. I want to express my sincere thanks to Dr. Carolyn Brown, Dr. Louis Lefebvre, and Dr. Matthew Lorincz for their guidance over the past two years in the course MEDG 545, through which I’ve grown significantly, improving my knowledge, paper-reading skills, and presentation abilities. I also thank to my lab collogues, especially Cayla Boycott, who helps me with my understanding of research-related and experimental knowledge. xix I appreciate the technical support from Wayne Tamagi and Patrick Leung, as well as the lab support from Peter Hoffman Furthermore, I am indebted to my parents (Hao Ma & Qi Zhang), who enable me to study abroad with a broad vision and financially support me throughout my 6-year education at UBC. I also extend my thank to my husband Mr. Mengwen Zhu, for his continuous support of my mental well-being. Although these words cannot fully capture my gratitude, I want to express my sincere appreciation to all those who have supported and guided me. Thank you! xx Dedication Dedicated to my husband, Mengwen Zhu, who selflessly supported me throughout my academic pursuit at UBC and encouraged me to be my true self. 1 Chapter 1: Introduction 1.1 Breast Health The mammary gland consists of two main tissue compartments: the epithelium, which includes ducts and milk-producing alveolar cells, and the stroma. Throughout a lifetime, it undergoes distinct developmental stages, beginning from embryo to puberty, pregnancy, lactation, and involution (1). Additionally, there is a dynamic change in cellular homeostasis during each phase of the menstrual cycle, primarily due to the influence of ovarian hormones (i.e., estrogen and progesterone) (2). In the luteal phase, where progesterone dominates over estrogen, the mammary epithelial cells will reach a peak in proliferation rate and milk producing alveolar cells will continue to mature to become specialized in milk production (3). A study identified 255 genes showing significant differential expression in response to hormonal fluctuations during the menstrual cycle, based on the analysis of 20 samples of normal human breast epithelium. The majority of these differentially expressed genes exhibited the highest changes during the luteal phase (4). Altered gene expression in response to hormonal signals suggests that epigenetic mechanisms play a recognized role in orchestrating normal breast development and regulating functions of specific cell types. Compared to pre-menopausal individuals, there is a decline in ovarian estrogen and progesterone production in post-menopausal stage, which is regarded as a hallmark of ovarian aging (5). This decline is associated with reduced proliferation of mammary epithelial cells, increased deposition of breast adipocytes, and alterations in stromal tissues (6). These shifts in cellular homeostasis are potential contributors to the development of breast diseases and even cancer (6, 7). 2 Benign breast diseases refer to a group of non-cancerous conditions that affect the breast tissue. Some common examples are breast cysts, fibroadenomas, mastitis, ductal ectasia (8). It is common to observe these benign changes in women of childbearing age, with the prevalence peaking between the ages of 30 and 50. Over a median follow-up period of 7.6 years, women diagnosed with benign breast diseases were found to have a 1.77 times greater risk of developing breast cancer compared to those without any breast conditions (9). Breast cancer significantly impact the morbidity and mortality in women, with an annual incidence surpassing two million new cases globally (10). Predominantly affecting post-menopausal women, it stands as a leading cause of death in this demographic group (11, 12). The development and progression of breast cancer are shaped by a multitude of biological and societal risk factors, including age, ethnicity, pregnancy status, dietary habits, and genomic changes, in which advancing age is the foremost contributor (13). Aging is an intricate and inevitable process marked by a variety of biological changes within the body, which can be influenced by genetics, environmental factors, and lifestyle (14). Research has demonstrated that aging significantly impairs the functions of the immune system, which serves as a notable risk factor for breast cancer (15). The reason is because the immune microenvironment plays a crucial role in regulating tissue homeostasis and resistance to tumor development (16). Specifically, chronic inflammation linked to aging can lead to the accumulation of reactive oxygen species (ROS), further contributing to DNA damage and gene mutations (17). The resultant tissue damage and fibrosis caused by chronic inflammation create a microenvironment conducive to the growth and spread of cancer (18). Indeed, aging triggers epigenetic alterations that contribute to 3 cancer initiation (6). On the one hand, hypermethylation and silencing of tumour suppressor genes is observed in breast cancer, for example HOXA5, TMS1, p16, RASSF1A, and BRCA1 (19). On the other hand, genes associated with oncogenic functions and signal transduction are aberrantly hypomethylated and activated in breast cancer, including SIPA1 promoting metastasis, KCNK9 inhibiting apoptosis, GLI2 from the Hedgehog pathway, and WNT4 from the Wnt signaling (20-23). Interestingly, the Wnt pathway plays a crucial role in the healthy development of the mammary gland and maintenance of mammary epithelium, however it exhibits aberrantly upregulated activity in 40-60% of breast cancer cases (24). Genome-wide investigations of the DNA methylation landscapes bring further insights on cis-specific methylation at numerous promoters and distal elements which correlate with transcriptional alterations in known tumor suppressors and oncogenes (25). Epigenetic studies are predominantly conducted on breast cancer and tumorigenesis rather than healthy and benign breast conditions (26). Further research is needed to elucidate the specific epigenetic alterations linked to pre-malignant conditions, as well as their potential implications for diagnosis, prognosis, and treatment. 1.2 Epigenetics The term 'epigenetics' was introduced by Conrad Waddington in 1942. Epigenetics refers to the mechanisms of regulating gene expression in a heritable way without changes in the underlying DNA sequence (i.e., a change in the phenotype without a change in the genotype) (27). On the contrary, genetics focuses on heritable changes resulting from direct alterations to the DNA sequence, such as gene mutations or deletions, among others (28). Epigenetic mechanisms include 4 DNA methylation, histone covalent modifications, chromatin remodeling complexes, and regulation by non-coding RNA mechanisms (29). Aberrations in these mechanisms are prevalent contributors to the development of human diseases, influenced by a range of factors, both internal and external. For example, poor diet, aging, stress, smoking, environmental pollutants and lack of physical activity can pose health threats via disruptions in regulation of epigenetic modifications; thus changing gene expression and susceptibility to disease (30). 1.2.1 DNA methylation 1.2.1.1 Definition and importance DNA methylation refers to a transfer of a methyl group from S-adenosyl-L-methionine (SAM), the ubiquitous methyl donor, to the C5 position of the cytosine ring, resulting in the formation of 5-methylcytosine (5mC). It is vital for establishing cellular identity during development through de novo methylation or demethylation. In addition, it serves as a relatively stable component of the epigenome, persistently maintaining specific methylation patterns across multiple cycles of cell divisions, thus regulating the tissue-specific gene expression (28, 30). 1.2.1.2 DNA methyltransferases (DNMTs) There are three classes of epigenetic regulatory enzymes closely associated with DNA methylation, which are known as writers, erasers, and readers (31). Writers facilitate the addition of methyl groups to cytosine residues. Erasers are responsible for modifying and eliminating the methyl groups. Readers, in turn, identify and attach to methyl groups, eventually influencing gene expression. 5 DNA methylation is catalyzed by a family of DNA methyltransferases (DNMTs), which are known as writers. The DNMTs consist of an N-terminal domain containing various protein and DNA interaction sites, and a C-terminal methyltransferase domain responsible for its catalytic activity (32). DNMTs are a conserved family of cytosine methylases – DNMT1, DNMT3A and DNMT3B - with a central role in epigenetic regulation and development of an embryo (33). DNMT1 is a maintenance DNMT responsible for preserving DNA methylation patterns from parental strand during cell replication (34). DNMT3A and DNMT3B are both regarded as de novo DNMTs that establish specific methylation patterns onto unmodified DNA in specific tissue types (35). Another two members of the DNMT family, DNMT2 and DNMT3L, by contrast, demonstrate sequence homology with canonical DNMTs (mentioned above) but lacks catalytic DNMT activity. The primary function of DNMT2 is in the methylation of tRNA, while DNMT3L has been shown to cooperate with DNMT3A and DNMT3B and regulated their function to carry out de novo methylation (36-38). Overall, de novo DNA methylation occurs at CpG sites, starts with DNA methyltransferase (DNMT) 3A/3B, and is maintained by DNMT1. 1.2.1.3 DNA demethylation DNA methylation can be reversed by DNA demethylation, which occurs through either passive or active mechanisms. Passive DNA demethylation occurs over successive rounds of DNA replication when maintenance methylation is not restored to the newly synthesized DNA strand. In this process, the DNA methyltransferase responsible for maintaining DNA methylation patterns (DNMT1) may fail to methylate the newly synthesized strand. Consequently, the newly replicated DNA strand lacks 6 methyl groups at CpG sites, resulting in a gradual loss of DNA methylation over successive cell divisions (39). Active DNA demethylation is replication independent and involves enzymatic removal of methyl groups. It is a consensus that the Ten-Eleven Translocation (TET) family of proteins facilitates the oxidation of 5mC, converting it to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and finally to 5-carboxylcytosine (5caC). These oxidized forms of 5mC can be recognized and excised by DNA repair machinery, leading to the replacement of the modified cytosine with an unmethylated cytosine (40). The active DNA demethylation can be triggered in response to certain signals/stimuli, including changes in energy metabolism, nutrients and hormonal balance. For instance, alpha-ketoglutarate, which is an important metabolic intermediate, acts as a cofactor for TETs by participating in oxidation of Fe (II) to Fe (IV) and rendering TETs inactive (41). Vitamin C, in turn, is involved in regeneration of Fe (II) restoring TETs catalytic activity (41) which resulted in inducing DNA demethylation, for example, in embryonic stem cells (42). Besides, the administration of thyroid hormones has been demonstrated to recruit TET3 and facilitate DNA demethylation at specific DNA loci (43). 7 Figure 1.1 DNA methylation and demethylation. 1.2.1.4 DNA methylation and gene expression DNA methylation is considered a major epigenetic modification of eukaryotic genomes and known to play a crucial role in the regulation of gene expression. Most of the methylation of DNA takes place on cytosines that are followed by a phosphate-linked guanine nucleotide, commonly known as CpG sites (28). CpG islands are defined as DNA regions ranging from 200 to 1000 base pairs in length and show a high frequency of CpG sites (44). Approximately 60% of human gene promoters are situated within CpG islands that typically remain unmethylated allowing for transcriptional activity (45, 46). However, methylation of these regions contributes to transcriptional silencing facilitating a formation of condensed chromatin (47). In addition to promoters, gene transcription can also be regulated by sequence elements located in distal regions. Enhancers can boost the transcription of target genes through direct physical interaction with promoters (48), whereas their repressive counterparts, silencers, decrease transcription from target promoters (49). There is another group of CpG islands that lack association with any functional or 8 gene elements (i.e., promoters), known as orphan CpG islands. Orphan CpG islands have not only been revealed to exhibit characteristics of highly active enhancers with stronger transcriptional activity compared to classical enhancers, but also to contribute to gene regulation by amplifying poised enhancers (50, 51). DNA methylation can modulate gene transcription directly or indirectly. Firstly, methylated CpG sites in gene regulatory regions can disrupt the binding of transcription factors, resulting in the decreased gene transcriptional activity (45) (47). Secondly, DNA methylation can attract and recruit proteins, that are associated with gene repression, to methylated DNA loci. These proteins are ‘readers’ of DNA methylation, for example, the methyl-CpG binding domain (MBD) proteins that help to prevent the binding of other transcription factors to silence gene activity upon their recruitment to hypermethylated regions (52). Another example is Polycomb Repressive Complex 2 (PRC2), which is involved in epigenetic gene silencing. PRC2 can localize to methylated DNA sequences with the assistance of AEBP2, suggesting a crosstalk with DNA methylation in chromatin repression (53). Overall, DNA methylation within gene regulatory regions is thought to contribute to the formation of a condensed chromatin structure (i.e., heterochromatin) and transcriptional silencing of gene-specific loci (54). Therefore, hypermethylation of DNA is often associated with suppression of transcriptional activity, whereas DNA hypomethylation is associated with activation of gene expression. Moreover, alterations of DNA methylation patterns can ultimately lead to changes in transcriptional regulation and result in various diseases: neurological disorders, immunological 9 and cardiovascular diseases, diabetes inflammatory disorders, genetic syndromes, and cancer (55, 56). 1.2.1.5 Crosstalk between DNA methylation and histone modifying enzymes DNA methylation does not operate in isolation. Instead, it engages in a crosstalk with other epigenetic mechanisms to regulate gene expression. DNA methylation is closely linked and mechanistically cooperates with histone covalent modifications, which generally involve acetylation, methylation and ubiquitylation, among others (54). Alongside DNA methylation, DNMTs directly interact with enzymes involved in modifying histone tails, including histone deacetylases (HDACs) that remove acetylation and histone methyltransferases (HMTs) that methylate histones (57-59). It is widely recognized that both DNA methylation and HDAC-mediated histone deacetylation repress gene transcription. Histones deacetylated by HDACs exhibit stronger electrostatic interactions with DNA, leading to compacted chromatin and reduced transcriptional activity (60). Conversely, when histones are acetylated, chromatin structure is relaxed, promoting transcription (61). Recent studies show the interplay between DNMT1 and euchromatic histone methyltransferase EHMT2, also known as G9a, that together with UHRF2 ubiquitin ligase downregulate expression of osteogenic genes contributing to bone loss in a hind-limb unloading rat model (62). DNMT3A, in turn, has been shown to upregulate HDAC9 that subsequently deacetylates TBK1 kinase to activate the antiviral innate immunity (63). Furthermore, DNMTs, including de novo DNMT3A and DNMT3B, are capable of interacting with histone modifications. In details, pre-existing histone methylation can potentially influence the specific targeting of de novo DNMTs and the distribution of DNA methylation in the genome (59). A direct interaction has been observed between DNMT3B and SIRT1 in the context of restricting 10 proinflammatory gene activation (64). SIRT1 can also recruit DNMT3B to SIRT1-associated chromatin for the subsequent DNA methylation (65). 1.3 SIRT1 Sirtuins are a family of proteins with distinctive traits that have been conserved across all domains of life, including bacteria, archaea, and eukaryotes. There are seven mammalian sirtuins (SIRT1-SIRT7). SIRT1 is mainly localized in the nucleus and cytosol, where it interacts with various partners and targets for deacetylation. SIRT2 is typically located in the cytoplasm but can also be present in the nucleus for chromatin binding during mitosis. SIRT3, SIRT4, and SIRT5 are primarily located in the mitochondria and regulate many mitochondrial proteins. SIRT6 and SIRT7 are both nuclear proteins, with SIRT6 found in chromatin and SIRT7 localized to the nucleolus (66). Sirtuins are nicotinamide adenosine dinucleotide (NAD)-dependent deacetylases that can remove acetyl groups from both histone and non-histone proteins. SIRT1 is the most extensively studied among sirtuins (67). It was initially identified as the homolog of Sir2 in the budding yeast Saccharomyces cerevisiae with critical roles in transcriptional silencing, genome stability, and longevity (68). 1.3.1 SIRT1 in epigenetic regulation SIRT1 is involved in numerous cellular functions, owing to its role as an epigenetic regulator closely linked to epigenetic mechanisms. Its impact on epigenetics is multifaceted: SIRT1 operates through both SIRT1-dependent deacetylation processes and pathways independent of its 11 deacetylase activity. While its deacetylase function directly modulates histone acetylation levels, influencing chromatin organization and gene expression, SIRT1 is also engaged in various signaling pathways where its actions extend beyond histone deacetylation. 1.3.1.1 Deacetylation-dependent action of SIRT1 1.3.1.1.1 SIRT1 and histone targets SIRT1 acts as a histone modifier, which directly deacetylates lysine on different residues of histone proteins, for example at positions 9 and 26 of histone H1 (H1K9; H1K26), 14 of histone 3 (H3K14), and 16 of histone 4 (H4K16) (69). As mentioned in paragraphs above, histone deacetylation contributes to formation of a closed chromatin structure, heterochromatin, due to the increase in interactions between DNA and histones (70). This reduces the accessibility of DNA-interacting proteins and transcription-related proteins to DNA loci, eventually repressing the transcriptional machinery. 1.3.1.1.2 SIRT1 and non-histones (i.e., histone modifier) SIRT1 is known to indirectly modulate the chromatin structure through the interaction and deacetylation of other non-histone targets (71). Specifically, studies have demonstrated that acetylated DNMT1 is physically associated with SIRT1 and subsequently deacetylated by SIRT1 both in vitro and in vivo. Inhibiting SIRT1, in turn, enhances the activity of DNMT1 and DNMT1-mediated methylation and silencing of tumor suppressor genes in breast cancer cell lines (72). Furthermore, SIRT1 binds to and deacetylates histone acetyltransferases (HATs) such as p300, inhibiting its function and thus resulting in the removal of acetyl marks on histones and the formation of heterochromatin (73, 74). SIRT1 also interacts with the histone methyltransferase 12 SUV39H1, which catalyzes the addition of methyl groups to specific sites on histones (75). The activity of SUV39H1 can be enhanced through deacetylation of its catalytic domain by SIRT1. This leads to an increase in H3K9me3 marks and a reduction in transcriptional activity at SIRT1 target sites (76). 1.3.1.1.3 SIRT1 and non-histones (i.e., transcription factors and coregulators) Beyond the influence on chromatin, SIRT1 can positively or negatively regulate the expression of target genes through deacetylation of various transcription factors and coregulators (77). This SIRT1-dependent gene regulation is fundamental to its various biological functions, including DNA repair, cellular senescence, inflammation, energy metabolism, autophagy, mitochondrial function, and tumorigenesis (70) (78). Studies on the human SIRT1 homolog in yeast, Sir2 protein, have demonstrated its ability to modulate cellular senescence and promote lifespan extension in invertebrates (79). For example, the expression of Sir2 has been shown to positively impact the lifespan of Drosophila (80). Sir2 achieves this by stabilizing ribosomal DNA (rDNA) through the repression of homologous recombination between rDNA repeats and the subsequent formation of toxic extrachromosomal rDNA circles (ERCs) (81, 82). A study by Kilic et al., demonstrates that expression of SIRT1 increases in correlation with age and was found to be significantly higher in the elderly individuals aged over 73.5 years, based on the analysis of blood samples (83). This evidence underscores the importance of SIRT1 in longevity-related functions, highlighting its potential to maintain the genomic integrity. Through direct interactions and deacetylation of the human p53 protein, specifically at the K382 residue, SIRT1 attenuates p53 activity and p53-mediated apoptotic 13 response. The balance between p53-mediated cellular responses maintained by SIRT1 prevents excessive activation and promotes efficient DNA repair (84, 85). Additionally, SIRT1 directly deacetylates Mcm10, a DNA replication factor, to stabilize its activity in DNA replication in human HCT116 cell lines (86). In response to oxidative stress, SIRT1 provides protective effects by deacetylating FOXO family of transcription factors. This action enhances their participation in antioxidant processes, apoptosis, and cell proliferation (87). SIRT1 also regulates the inflammatory response through inhibiting NF-κB signaling directly by deacetylating the p65 subunit of NF-κB complex (88). Moreover, SIRT1 influences energy homeostasis by promoting deacetylation of multiple lysine residues on a transcription coactivator, PGC-1α, during fasting. This process upregulates gluconeogenic genes and suppresses genes involved in glycolysis (89). 1.3.1.2 Deacetylation-independent action of SIRT1 In addition to its well-established role in health maintenance through deacetylation activity, SIRT1 is implicated in various pathways that contribute to overall well-being. Increased expression of SIRT1 has been linked to reduced inflammatory response via the AMPK/NF-κB signaling pathway (90). SIRT1 can directly influence NF-κB signaling via direct activation of AMPK, which serves as a key suppressor of NF-κB (91). Moreover, increased SIRT1 expression has been shown to inhibit pro-inflammatory cytokines as well as mitigate effects of ROS in astrocytes, thereby promoting neuroprotection (92). In response to DNA damage, the recruitment of SIRT1 to specific damage sites has been suggested to play a crucial role in facilitating lesion repair and promoting genomic stability, with evidence of concomitant recruitment of Rad51, a key protein in 14 homologous recombination repair (93). This pathway possibly depends on phosphorylation of histone H2AX, as deficiency in H2AX resulted in reduced binding of SIRT1 to DNA damage sites (93). In the context of gastric cancer, SIRT1 has shown promise in reversing chemoresistance through activation of the AMPK/FOXO3a pathway (94). An interesting finding in mice demonstrates that SIRT1 is necessary for mammary gland development especially ductal morphogenesis via IGF1-signaling, while the mice lacking SIRT1 show lactation failure instead (95). 1.3.2 SIRT1 activation by natural phytochemicals Scientific evidence increasingly suggests that environmental factors, including diet, physical activity, smoking, and alcohol consumption, can influence epigenetic modifications and subsequently impact human health. Diet, as one of the most significant and modifiable risk factors for disease, is of particular interest. Nutrients and bioactive compounds appear to regulate epigenetic mechanisms during the entire lifespan starting with embryogenesis (96). During early fetal development, the adequate intakes of folic acid and vitamin B12 are essential for synthesizing S-adenosylmethionine (SAM), a key methyl donor crucial for maintaining DNA methylation patterns and preventing neural tube defects (97). As development progresses, bioactive compounds have been shown to directly modulate human health by preventing and/or reversing aberrant epigenetic patterns (30). One of the largest and most studied groups of bioactive plant-based compounds are polyphenols. A variety of compounds from different classes of polyphenols have been demonstrated to have the ability to reprogram the epigenetic landscapes in different tissues, thus preventing the occurrence of certain pathologies (98). For example, polyphenols reverse promoter hypermethylation of the transcription factor NRF2, which reactivates NRF2 expression 15 and helps to prevent cellular homeostatic imbalance and chronic disorders such as neurodegeneration, cardiovascular disease, and diabetes (99). SIRT1 expression/activity has been shown to be regulated by naturally occurring dietary polyphenols, exerting beneficial effects in various cellular functions (100). One of the most studied polyphenols, resveratrol, is the first to be shown to activate SIRT1 and enhance SIRT1-dependent cellular processes in yeast. Specifically, resveratrol can directly bind to SIRT1 and induce a conformational change, resulting in a significant 8-fold increase in SIRT1 activity (101). Although the direct interaction between resveratrol and SIRT1 was later challenged (102, 103), changes in SIRT1 in response to resveratrol have been widely reported. For example, treatment with resveratrol led to SIRT1-dependent effects on deacetylation and activation of PGC-1α, enhancing mitochondrial biogenesis in several tissues, as well as increasing insulin sensitivity and exercise performance in mice (104). Another polyphenol, quercetin, has been shown to up-regulate SIRT1 protein expression, thereby ameliorating diabetic-induced cognitive dysfunction in mice (105). Quercetin is also suggested as a treatment for the autoimmune disease rheumatoid arthritis by activating SIRT1 and modulating SIRT1-dependent pathways that can closely regulate mitochondrial biogenesis (106). Furthermore, through the activation and involvement of SIRT1, curcumin has been reported to mediate additional beneficial effects, including anticancer activity, neuroprotection, diabetes treatment, and cardiac protection in various models (107). Recent evidence demonstrates that SIRT1 is a crucial mechanistic player in genistein (GEN)-mediated suppression of lipid droplet accumulation in hepatocytes (108). Importantly, GEN belongs to the same group of phenolic compounds as resveratrol and its analog pterostilbene (PTS), namely to phytoestrogens, which will be described in paragraphs to follow. 16 1.4 Polyphenols Polyphenols are indeed one of the largest groups of bioactive compounds produced naturally by plants. They are organic compounds distinguished by the presence of single or multiple hydroxyl groups attached to aromatic rings (109). The primary classes of polyphenols encompass flavonoids, phenolic acids, lignans, and stilbenes, each classified according to their shared structural backbone. Beyond these, there are additional subclasses of polyphenols, which are curcuminoids, phenolic terpenes, tannins, hydroxybenzaldehydes, and tyrosols (110). These compounds are widely and naturally present in a variety of plants accessible in the human diet, exhibiting potential anti-inflammatory and antioxidant properties. The well-known sources are: fruits, vegetables, tea, cereals, and wine. The long-term intake of them is associated with health benefits. However, the bioavailability and bioactivity of polyphenols can significantly differ based on a variety of factors, including chemical structure, molecular size, degree of polymerization, conjugation with other compounds, and the influence of gut microflora that facilitates metabolic processes (111, 112). 1.4.1 Phytoestrogens 1.4.1.1 Characteristics Phytoestrogens are another group of polyphenolic and non-steroid compounds that naturally occur in more than 300 plants (113). In a natural environment, phytoestrogens are considered as phytoalexins, offering protection against pathogens, bacterial and fungal growth along with defense against harmful effects of UV (114, 115). Additionally, these compounds exhibit structural similarities to human endogenous hormone estrogen, thus enabling them to exert estrogenic activities via binding to estrogen receptors (116). From the structures below, it is clear to see both 17 estrogens and phytoestrogens feature phenolic rings with attached side chains such as methyl and hydroxyl groups. Figure 1.2 Chemical structure of 17β-estradiol. 1.4.1.2 Classification and chemical structures Phytoestrogens comprise four groups of phenolic compounds: (1) stilbenes (resveratrol and pterostilbene), (2) isoflavones (genistein, daidzein, glycitein, biochanin A, and formononetin), (3) lignans (pinoresinol, lariciresinol, secoisolariciresinol, matairesinol, and enterolignans) and (4) coumestans (coumestrol, wedelolactone, plicadin) (Figure 1.3) (117). 18 Figure 1.3 Four subclasses of phytoestrogens. 1.4.1.3 Estrogenic signaling pathway The hormone estrogen comprises a group of hormones, including estrone, estradiol, estriol, and estetrol. Synthesized primarily in the ovaries, they are also produced in the adrenal glands and adipose tissue. Throughout women’s life, estriol and estetrol are predominantly found during pregnancy, whereas estrone levels typically rise during menopause. Importantly, 17β-estradiol is the most referred to as estrogen due to its abundance, potency, and physiological significance. These estrogens exert their effects by binding to estrogen receptors and activating specific signaling pathways. Phytoestrogen ClassificationsStilbenes Isoflavones Lignans CoumestansResveratrolPterostilbeneGenisteinDaidzeinGlyciteinBiochanin AFormononetinPinoresinolLariciresinolSecoisolariciresinolMatairesinolEnterolignansCoumestrolWedelolactonePlicadinStructure Example Structure Example Structure Example Structure Example(Genistein)(Coumestrol)(Pinoresinol)(Pterostilbene)19 In mammary glands, estrogen signaling plays a crucial role in maintaining normal cellular processes. It regulates gene expression; thereby cell proliferation, differentiation, and survival in target tissues. The pathway involves two classical receptors: estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). Upon binding of estradiol (E2) in the cytoplasm, these receptors undergo a conformational change, followed by translocation to the nucleus where they interact with specific DNA sequences called estrogen response elements (EREs), thereby regulating the transcription of target genes. In breast tissue, ERα signaling is particularly prominent and pivotal in controlling mammary gland development. Dysregulation of ERα signaling is strongly associated with the development of estrogen-dependent cancers. It has been shown that higher expression of ERα in normal breast epithelium correlates with an increased risk of breast cancer (118). Another novel membrane-bound receptor known as the G protein-coupled estrogen receptor (GPER), predominantly located on intracellular membranes, also mediates estrogen signaling. Upon binding of estradiol (E2), selective agonists, selective ER modulators, or selective ER downregulators/degraders, GPER is activated followed by a rapid activation of several heterotrimeric G proteins which initiates intracellular signaling pathways, including kinase activation, cAMP production, calcium mobilization, and transcriptional activation (119). GPER plays a pivotal role in mediating estrogen-mediated effects in diverse physiological systems, including the cardiovascular, reproductive, nervous, metabolic systems, and in cancer (120). The involvement of SIRT1 in ER and GPER signaling pathways was investigated in breast cancer cells. ERα physically cooperates with SIRT1 and binds to SIRT1 promoter increasing its transcription. Upregulated SIRT1 inactivates tumor suppressor genes, including p53, and enhances 20 ERα oncogenic potential in breast cancer cells (121). Inhibition of SIRT1 abrogates ERα pro-proliferative effect and induces apoptosis of breast cancer cells (121). In addition, SIRT1 can also upregulate ERα expression, demonstrating a bidirectional relationship between ERα and SIRT1 (122). Upon SIRT1 inhibition, ERα expression is disrupted and consequently estrogen-responsive genes are suppressed (122). Importantly, 17β-estradiol, acting through GPER and subsequent activation of the EGFR/ERK/c-fos/AP-1 transduction pathway, induces SIRT1 expression in ER-negative cancer cells (123). This effect is abolished by silencing either GPER or SIRT1(123). Despite the evidence in breast cancer cells, a functional cooperation between SIRT1 and ERα or GPER in normal mammary epithelial cells has not been well elucidated. 1.5 Pterostilbene 1.5.1 Food sources PTS is a naturally occurring stilbene compound found in a variety of foods, most abundantly in blueberries as well as in grapes, cranberries, peanuts, pistachios, and cocoa (124). Depending on a dietary source, the content of PTS is highly variable (Table 1). For the rabbit-eye blueberries, the concentration of PTS is between 99 -151 ng/g of dry samples (125). In different species of grape berries, the estimated concentration ranges between 14 – 530 ng/g (Table 1-1) (125, 126).\ 21 Table 1.1 The Concentration of Pterostilbene (PTS) in Various Types of Foods Food Types (fresh/dry samples) Concentration Range Fungus-infected grapes (Fresh) 0.2 - 4.7 µg/g Rabbit-eye blueberries (Dry) 99 -151 ng/g Deer-berries (Dry) 520 ng/g Grape berries (Fresh) 14 – 530 ng/g 1.5.2 Metabolism and bioavailability PTS is a dimethyl ether analog of resveratrol. Resveratrol has been well studied in terms of a variety of beneficial health effects since the first discovery of its cardioprotective effects upon red wine consumption (“the French Paradox”) (127, 128). Although PTS is not present in wine due to its degradation during wine production, its 60% higher in vivo bioavailability compared with resveratrol, as shown in animal models, has attracted a lot of attention in recent years (129, 130). PTS has two methoxyl group and one hydroxyl group compared to resveratrol that contains three hydroxyl groups (124). These methoxyl substitutes on PTS enhance the lipophilicity of a compound, thus improving its absorption through cell membrane, while hydroxyl groups make resveratrol more hydrophilic and impede its penetration through cell membranes (130, 131). The bioavailability of PTS can be further enhanced by resuspending it in a 2-hydroxypropyl-β-cyclodextrin (15 mg/kg) solution (132). Both PTS and resveratrol share a similar metabolic pathway. Within the small intestine, PTS undergoes several processes including demethylation, hydroxylation and conjugation (133). After absorption, it undergoes metabolism such as oxidation, glucuronidation and sulfation in the liver, followed by extensive deconjugation in colon (134). The resulting metabolites will be further 22 excreted through urinary excretion or biliary excretion (128). Recent progress in gut microbiome research delivers new insights into microbial metabolites of bioactive compounds. PTS has been shown to be metabolized by gut bacteria to pinostilbene (PIN) (135). Both PTS and PIN were demonstrated to exert protective effects on disrupted colonic tight junctions induced by 16-week of western diet feeding in mice (136). Both compounds had also inhibitory effects on human colon cancer growth (137). 1.5.3 Toxicity PTS appears to be very safe lacking significant toxic effects. In a clinical trial, PTS taken at a dosage of 125 mg twice daily was found to decrease blood pressure although exhibiting a minor weight loss effect (138). Feeding PTS at a high dosage of 3g/kg body weight per day over a 28-day period was demonstrated to be safe in mice, with no observed signs of toxicity considering body weight and biochemical parameters (139). Moreover, a well-tolerated PTS dosage of up to 250 mg per day was shown in a study involving 80 healthy volunteers over a period of 6–8 weeks (140) Even the intravenous administration of PTS has been observed to be safe without exhibiting any organ-specific toxicity in clinical studies (140). 1.5.4 Health related effects Numerous beneficial biological activities have been studied regarding PTS, including anti-aging, anti-oxidation, anti-inflammation, anti-cancer, anti-diabetic, cholesterol-lowering, neuroprotection, and nephroprotection (141-146). The majority of these effects are primarily attributed to its potent antioxidant activity, driven by the hydroxyl group that creates dense electrons, neutralizing free radicals and thereby reducing oxidative stress (147, 148). Importantly, 23 antioxidants can potentially impact DNA methylation landscapes by reducing oxidative stress (149). In response to oxidative stress, DNMT1 or DNMT3B along with SIRT1 and EZH2 are recruited to sites of oxidative DNA damage to participate in DNA repair. However, a repetitive mobilization of these epigenetic enzymes upon chronic oxidative stress may consequently bring about stable aberrations in epigenetic marks and lead to disease (149). 1.5.5 Epigenetic regulation of PTS 1.5.5.1 PTS and DNA methylation As previously discussed, DNA methylation is reversible and can be actively influenced by external and internal signals or exposures. In breast cancer cells, treatment with PTS led to the activation of methylation-silenced tumor suppressor genes, e.g., SEMA3A, which was accompanied by a decrease in DNMT3A binding and restoration of NF1C transcription factor occupancy at SEMA3A promoter (150). Simultaneously, treatment with PTS resulted in DNA methylation within regulatory regions of key oncogenes, e.g., PRKCA, and their suppression (151, 152). Importantly, using CRISPR-Cas9 technology, DNMT3B was proven to be required for PTS-mediated epigenetic suppression of oncogenes in breast cancer (151). Furthermore, PTS treatment induces overall hypomethylation by reducing DNMT enzymatic activity and can restore expression of ERα, which is typically absent in MDA-MB-157 breast cancer cells (153). This reactivation of estrogen receptors provides opportunities for hormone-related therapies in ER-negative breast cancer cell lines. In Caco-2 intestinal cells, PTS has demonstrated effects in reversing arsenic-induced damage and DNA demethylation (154). 24 1.5.5.2 PTS and SIRT1 Health benefits of PTS have been associated, at least partially, with enhancing expression of SIRT1. Considering neuroprotection, PTS improves neuroplasticity and reduces neuronal inflammation by increasing SIRT1/Nrf2 expression, which inhibits mitochondria-dependent apoptosis (155). It also alleviates microglial inflammation and oxidative stress-induced neuronal injury by enhancing SIRT1 signaling (156). In addition, enhanced SIRT1 expression in response to PTS is crucial in inhibiting oxidative stress and abnormal mitochondrial morphologies induced by acute doxorubicin exposure in cardiomyocytes (157). Activation of SIRT1 by PTS prevents obesity by promoting mitochondrial biogenesis and thermogenesis, thereby helping to reduce body weight in rats with western-diet induced obesity (158). On the contrary, in cancer models, a pro-survival action of SIRT1 could actually enhance cancerous properties. Hence, strategies aimed at reducing SIRT1 are beneficial (159). Indeed, in breast cancer, treatment with PTS in combination with RSV induces tumor cell apoptosis and reverses DNA damage responses through significant inhibition of SIRT1 (160). 1.6 Genistein 1.6.1 Food sources GEN is an isoflavonoid compound found abundantly in legumes and constitutes approximately 60% of soy isoflavones. It is present in soybeans and soy-based products such as soy milk, tofu, and fermented foods, but is barely found in soy sauce (161). The concentration of GEN varies widely across different food products (Table 1-2) (162-164). 25 Table 1.2 The Concentration of Genistein (GEN) in Various Types of Foods Food Types Concentration Range (mg/100 g food) Textured soy flour 89.42 ± 26.96 mg Instant soy milk powder 62.18 ± 3.69 mg Miso 23.24 ± 8.37 mg Cooked firm tofu 10.83 ± 3.98 mg American soy cheese 8.7 mg Soy sauce 0.28 mg 1.6.2 Metabolism and bioavailability Being a lipophilic compound with low molecular weight, GEN is absorbed rapidly and completely in the gastrointestinal tract. However, it paradoxically has low oral bioavailability that is estimated to be around 10% as reported in the human prostate tissue (115, 165). During metabolism, genistin is primarily the ingested glycoside form of GEN, which will be hydrolyzed back into the GEN form by intestinal bacteria before absorption. The presence of efflux transporters in the intestine can actively pump GEN back into the intestinal lumen, which might be one reason contributing to its low bioavailability (166). After that, GEN undergoes extensive metabolism through glucuronidation and sulfotransferase reactions, resulting in the excretion of its derivatives through intestinal, biliary, and renal pathways (167). Human pharmacokinetic investigations reveal that the plasma concentrations of GEN typically fall within the hundred nano-molar range, considerably lower than the concentrations commonly utilized in in-vitro studies (168). Despite this, various strategies have been explored to enhance its bioavailability. Concurrent consumption of soluble soybean polysaccharides with GEN, or the utilization of chemically modified GEN incorporating a sugar moiety, have shown favorable outcomes in augmenting both the bioavailability and 26 bioactivity of the compound (169, 170). It is important to note that the gut microbial metabolites of GEN have been suggested to exert estrogen-like effects in the body (171, 172). However, this estrogenic potency is unique to every individual due to the dynamic composition of gut microbiota in humans (172). Further studies are required to fully understand how these metabolites contribute to the beneficial health outcomes associated with GEN. 1.6.3 Toxicity The ingestion of 25 to 50 g/d of soy protein has been established as both safe and effective in reducing LDL cholesterol by approximately 4% to 8% (173). Nevertheless, adverse effects, potentially attributable to GEN intake, have been noted at different dosage levels from 2-16 mg/kg body weight (174). More evidence is needed to prove and establish the upper toxic level of GEN. 1.6.4 Health related effects GEN has been linked to a decreased risk particularly in cardiovascular diseases, but also has been implicated to confer positive effects in conditions such as osteoporosis, obesity, diabetes, cognitive decline, and specific types of cancer such as breast and prostate cancer (175). GEN anti-oxidant activity is one of the contributors to its health promoting effects, similarly to PTS. For instance, GEN treatment protects neuronal cells by reducing oxidative stress in Alzheimer's disease (176). Moreover, GEN protects human mammary epithelial cells from carcinogens by activating the antioxidant and detoxification system of glutathione and glutathione S-transferase (177). As described for PTS, GEN as an antioxidant compound may modify DNA methylation by reducing oxidative stress and changing the localization of DNA damage repair complexes that are formed by epigenetic enzymes, among other proteins (149). It is worth noting that the anti-cancer 27 properties of GEN, when studied in the context of altering epigenetic/DNA methylation profiles, yield mixed results which might be linked to GEN dosage (178, 179). 1.6.5 Epigenetic regulation of GEN 1.6.5.1 GEN and DNA methylation The epigenetic action of GEN in modifying DNA methylation marks was shown in difference types of cells. In prostate cancer, treatment with GEN induces genome-wide differential DNA methylation changes and reverses hypermethylation and silencing of the tumor suppressor gene BTG3, thereby exhibiting a therapeutic role against cancer (178, 180). Similarly, exposure to 50 µM GEN for 48 hours reduces the DNA methylation levels of several tumor suppressor genes, leading to a restoration of their transcription levels in HeLa cervical cancer cells (181). In response to GEN, breast cancer cell growth was inhibited and accompanied by a reduction in CpG methylation at the promoter regions of tumor suppressor genes such as BRCA1 (182-184). GEN has been demonstrated to modify expression of epigenetic enzymes, including DNMT3B and TETs (185), which was linked to differential methylation of tumor-regulated genes in triple negative breast cancer (185) and to demethylation and activation of tumor suppressors in neuroblastoma (185). These findings suggest that beneficial effects of GEN are exerted, at least partially, via the epigenetic changes. 1.6.5.2 GEN and SIRT1 A protective role of GEN against health disorders has been linked to regulation of SIRT1 expression. As already mentioned in preceding sections, SIRT1 is mechanistically involved in GEN-mediated suppression of the accumulation of lipid droplets in hepatocytes (108). GEN 28 activated SIRT1 upon an increase in NAD+ levels via estrogen receptor-dependent pathway which was followed by AMPK activation and thus regulation of lipid metabolism (108). GEN also induces SIRT1 expression followed by NRF2 activation to reduce oxidative stress in liver injury (186). SIRT1 upregulation also accompanies GEN-mediated amelioration of renal injury (187). The cooperation between GEN and SIRT1 also contributes to ameliorating hepatic fibrosis by downregulation of α-SMA and TGF-β (188). In breast cancer, GEN treatment leads to a 1.6-fold increase in SIRT1 expression, which mechanistically is linked to suppressing secreted osteopontin, a potential biomarker that promotes the progression and malignancy of breast tumor cells (189). 1.7 Research question 1.7.1 Knowledge gaps In summary, there is evidence for the functional association between PTS and SIRT1 as well as between PTS and DNMT3B in breast cancer cells, but not in normal mammary epithelial cells. PTS treatment has been shown to downregulate SIRT1 expression and activity, leading to the inhibition of breast cancer cell proliferation (160). DNMT3B is mechanistically required for PTS-mediated DNA methylation and de-activation of oncogenes in breast cancer (151). Simultaneously, studies on SIRT1 and DNMT3B have indicated that the two proteins co-immunoprecipitate and cooperate to initiate DNA methylation (65). However, the role of SIRT1 and DNMT3B interaction in PTS-mediated epigenetic effects remains unknown in normal mammary epithelial cells. Although limited, published reports imply that GEN-promoting health benefits are mechanistically linked, at least partially, to SIRT1 (108, 186, 188, 189). Furthermore, GEN has been shown to 29 modify DNA methylation patterns and expression of epigenetic enzymes (i.e., DNMT3B) in different models, including breast cancer (185). Considering the structural differences between PTS and GEN and their classification to different subgroups of phytoestrogens, an interesting question arises whether the epigenetic regulation under the influence of both compounds is distinct. 1.7.2 Hypothesis We hypothesize that SIRT1 and its interaction with DNMT3B are involved in phytoestrogen-mediated epigenetic regulation of gene expression in MCF10A human mammary epithelial cells. 1.7.3 Objectives 1. Determine if PTS- and GEN-mediated increase in DNA methylation at target genes is accompanied by SIRT1 occupancy. 2. Determine if DNMT3B co-binds to SIRT1-occupied hypermethylated targets of PTS and GEN. 3. Assess if SIRT1 is required for PTS- and GEN-mediated epigenetic regulation of gene transcription. 30 Figure 1.4 Diagram illustrating research objectives. 31 Chapter 2: Materials and Methods 2.1 Cell Culture and Treatment with Pterostilbene and Genistein The MCF10A human mammary epithelial cell line was obtained from ATCC (CRL-10317, USA). MCF10A cells were cultured in DMEM/F12 (1:1) medium (Gibco), supplemented with the following: 1) 20 ng/ml epidermal growth factor (Sigma-Aldrich, St. Louis, MO, USA); 2) 100 ng/ml cholera toxin (Calbiochem, EMD Millipore, Billerica, MA, USA); 3) 0.01 mg/ml insulin (Sigma-Aldrich, St. Louis, MO, USA); 4) 500 ng/ml hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA); 5) 5% horse serum (Gibco); 6) 1U/ml penicillin; and 7) 1µg/ml streptomycin (Gibco). Pterostilbene (PTS, Cayman Chemical Company, Michigan, USA) and genistein (GEN, Cayman Chemical Company, Michigan, USA) were resuspended in ethanol and DMSO, respectively, to create a 50 mM stock solution and was stored at -20°C for no more than one month. We freshly prepared dilutions of each compound before the compound treatment. Cell cultures were maintained in a 37°C incubator with humidified atmosphere of 5% carbon dioxide. Cells were seeded at a density of 4 x 10^5 cells in each 10-cm tissue culture dish and allowed to attach for 24 hours before refreshing the medium with media containing PTS or GEN. A range of PTS concentrations (0 to 20 µM) and GEN concentrations (0 to 40 µM) were applied to the cells. After 4 days of incubation, we split the cells in a ratio of 1:20 and allowed them to attach overnight followed by exposure to PTS or GEN for an additional 4 days (totaling 9 days). On the ninth day, cell pellets were collected by washing with 2 ml of PBS (Sigma-Aldrich, St. Louis, MO, USA) and treating with 1 ml of trypsin (Gibco, Canada) for cell dissociation, followed by centrifugation. 32 2.2 Trypan Blue Exclusion Test Cell viability was assessed using the trypan blue exclusion test (Sigma-Aldrich). Cells were collected after exposure to PTS or GEN on both day 4 and day 9. After a 3-5-minute incubation with trypan blue, viable and dead cells were enumerated under a microscope. Of note, only dead cells take up the dye and stain blue. Similarly, cell viability post-treatment with siRNAs was determined using the trypan blue exclusion test. 2.3 RNA Isolation and Real-Time qPCR Total RNA was isolated using TRIzol (Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. Isolated RNA was resuspended, and its concentration was measured using NanoDrop OneC (Thermo Scientific, Waltham, MA, USA), then stored at -80°C. 1000 mg of isolated RNA from each sample/condition was used for cDNA synthesis using AMV reverse transcriptase (Roche Diagnostics GmbH, Mannheim, Germany), following the manufacturer’s protocol. The reaction mixture was incubated in a thermocycler Biometra TRIO (MBI Lab Equipment) followed the protocol of 10 minutes at 25°C, 1 hour at 37°C, and 10 minutes at 72°C. The cDNA was kept at -20°C. The candidate genes were amplified in CFX96 Touch Real-Time PCR detection System (Bio-Rad) using 2 µl of cDNA, 400 nM forward and reverse primers (please see Table 2-1 for primer sequences), 10 µl of SsoFast EvaGreen Supermix (Bio-Rad) in a final volume of 20 µl. Amplification was conducted according to the following conditions: 1) denaturation at 95 °C for 10 min; 2) amplification for 60 cycles at 95 °C for 10s; 3) annealing temperature for 10s; 4) 72 °C for 10s; and 5) final extension at 72 °C for 10 min. To quantify mRNA levels of the tested genes, 33 we used the ∆CT method using a reference gene, GAPDH, which is a variation of the Livak method. The results are presented as gene of interest/GAPDH, based on the formula 2−[𝐶𝑇(𝑡𝑎𝑟𝑔𝑒𝑡)−𝐶𝑇(𝑟𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒)] (190). 2.4 Whole-cell Protein Extraction Total protein extract was obtained using RIPA buffer supplemented with protease inhibitor (Roche Diagnostics GmbH, Mannheim, Germany). NanoDrop OneC helps to measure sample protein concentrations using the Dye Reagent Concentrate (BioRad). Bovine Serum Albumin (BSA) dilutions ranging from concentration 1 mg/ml to 33.34 mg/ml were used to set up a standard curve. Samples with protein extracts were stored at -80°C. 2.5 Western Blot Analysis The amount of 25 µg of each extracted protein sample was loaded onto fast cast SDS-PAGE gels based on the manufacturer’s protocol (Bio-Rad). Next, the gel was carefully positioned onto a TransBlot Turbo Polyvinylidene Fluoride (PVDF) membrane for protein transfer. This was done using Trans-Blot Turbo Transfer System (Bio-Rad). The Bio-Rad ChemiDoc imaging system was utilized to confirm successful protein transfer. Prior to overnight incubation at 4 °C with the primary monoclonal antibody, anti-SIRT1 (diluted 1:10000, MilliporeSigma, #07-131), the membrane was blocked with EveryBlot Blocking Buffer for 5 minutes at room temperature. On the subsequent day, the membrane underwent 5-6 washes with TBS-Tween (TBST) buffer, following which it was subjected to another hour of incubation with the secondary antibody horseradish peroxidase-conjugated anti-rabbit IgG (diluted 1:1500; Cell Signaling, #7077) at room temperature. Through imaging the membrane using the Bio-Rad ChemiDoc imaging system, the 34 total protein content was quantified to serve as a loading control for protein normalization as previously explained (191, 192). Chemiluminescent signals were captured on the ChemiDoc MP (Bio-Rad) after soaking the membrane with a 1:1 mixture of Clarity Western ECL Substrate (Bio-Rad) for 10 minutes. Band intensities and protein expressions were analyzed using Bio-Rad's Image Lab software. 2.6 DNA Extraction and Pyrosequencing Genomic DNA from MCF10A cells was isolated after 20 hours of incubation of a cell lysate with proteinase K at 55°C, followed by extraction using a standard phenol:chloroform mixture (250 µl:250 µl), according to the manufacturer’s protocol. Precipitated pure DNA was resuspended in TE buffer and stored at -20°C. DNA bisulfite conversion was performed as previously described (151). The process of DNA bisulfite conversion commenced with the incubation of DNA with a restriction enzyme EcoRI, followed by purification using the GeneJet PCR Purification Kit (Thermo fisher scientific, Waltham, MA, USA). Upon treatment with sodium bisulfite, unmethylated cytosines are converted into uracils whereas methylated cytosines are preserved unaltered. The converted DNA was further amplified using HotStar Taq DNA polymerase (Qiagen) and biotinylated primers for tested gene regulatory regions. The biotinylated primers were designed using PyroMark Assay Design software and purchased from Integrated DNA Technologies (please see Table 2-1 for primer sequences). The biotinylated DNA strands were pyrosequenced using the PyroMark Q48 AutoPrep instrument (Biotage, Qiagen) according to the manufacturer’s manual. Primers and associated reagents were manually loaded into each well, with quantities calculated based on the sequence to 35 analyze. 10 µL of biotinylated PCR products were dispensed into disk wells along with 3 µL of magnetic beads. The quantification of methylation percentage at single CpG site resolution was conducted based on the samples added to the disk wells and analyzed using PyroMark Q48 software. 2.7 Chromatin Immunoprecipitation and Quantitative ChIP (qChIP) Chromatin immunoprecipitation (ChIP) assay was performed to determine DNA-protein interactions, as previously described (193). Both control and PTS/GEN exposed-human mammary epithelial MCF10A cells were fixed with 1% formaldehyde for 15 minutes (approximately 5 million cells), incubated with PBS containing protease inhibitors, lysed and subjected to 20 cycles of sonication at amplitude of 40. Samples were precleared with protein G agarose followed by centrifugation. The supernatants were divided into three sub-samples. The first sub-sample served as the input, while the second (negative control) and third sub-samples (experimental samples) were separately incubated overnight at 4°C with rabbit IgG non-specific antibody and with anti-SIRT1 or anti-DNMT3B antibodies, respectively. Next, all sub-samples were washed, eluted, and utilized as a template for qPCR (qChIP), where the starting materials in all conditions consisted of 25 ng of input, antibody-bound and IgG-bound DNA. The occupancy level of the proteins were shown as (Bound-IgG)/Input in each figure. We used the following antibodies: anti-SIRT1 rabbit polyclonal (Millipore, #07-131), anti-DNMT3B rabbit mAb (Cell Signaling, #72335) and normal rabbit IgG (Cell Signaling, #2729). Primers used in qChIP are listed in Table 2-1. 36 2.8 Double Chromatin Immunoprecipitation Assay (Double-ChIP) Double ChIP assay was conducted following the procedure outlined previously (194). In summary, 10 million MCF10A cells, exposed to 22 μM GEN for 9 days, were fixed using 1% formaldehyde (for 15 minutes) and divided into two equal samples. The first sample underwent immunoprecipitation with the anti-SIRT1 antibody followed by the anti-DNMT3B antibody, while the second sample was initially immunoprecipitated with the anti-DNMT3B antibody and subsequently with the anti-SIRT1 antibody. The DNA bound to Protein G agarose beads was dissociated from the first antibody, and subsequently eluted. It was then incubated overnight at 4°C with the second antibody. The subsequent steps followed the same workflow as outlined in the single ChIP protocol mentioned in Chapter 2.7. 2.9 Small-interfering RNA (siRNA) Transfection Cells were plated at a density of 6 x 105 per a 10-cm tissue culture dish, 24h prior to siRNA treatment. siRNA sequences, including control siRNA (si-CTRL, custom, antisense strand: 5’-UCGCCUAGGCUGCCAAGGCUU-3’) and human SIRT1 siRNA (si-SIRT1, SMARTpool M-003540-01-0005), were purchased from Dharmacon. The cells were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) prepared in serum-free Opti-MEM. In brief, 15 µl of Lipofectamine was incubated in 500 µl of Opti-MEM for 45 minutes at room temperature. A concentration of 56 nM siRNAs was added to the Lipofectamine-Opti-MEM solution followed by a 15-minute incubation at room temperature. After rinsing attached cells twice with 2 ml of PBS, 4 ml of Opti-MEM with the above 1 ml mixture solution was transferred into the plates with cells. The transfection solution was removed after 5 hours of incubation at 37°C and replaced with a fresh medium. GEN was added in the morning at 22 μM concentration. 37 After 2 days, cells were split 1:2 and 24h later the second transfection and treatment with GEN were performed following the same protocol. Cells were harvested and subjected to RNA isolation for further analyses. 2.10 Data Analysis The genes listed in Figure 1 were selected from genome-wide data obtained from two existing datasets in our laboratory. The first dataset originated from the Infinium HumanMethylation 450K BeadChip DNA methylation analysis of MCF10A control cells (treated with vehicle-ethanol) and MCF10A cells cultured with 15 µM RSV for 9 days. The second dataset involves quantified gene expression analyzed through RNA-sequencing from the blood of healthy Fischer 344 rats exposed to a PTS-supplemented diet for 3 weeks (134 mg/kg of body weight per day, between 5-8 weeks of age), compared to healthy rats fed with chow diet. The two datasets, differentially methylated and differentially expressed genes, were initially analyzed and categorized based on functional analyses using the DAVID knowledgebase bioinformatics tool. The detailed function of each gene was further investigated using Gene Cards and relevant published literature in PubMed. The ultimately selected genes were categorized into different functions that have the potential to promote health maintenance/longevity. 2.11 Statistical Analysis The statistical analysis was performed using the unpaired t-test with a two-tailed distribution for comparisons between two groups. One-way analysis of variance (ANOVA) with Tukey post-hoc test was used to analyze and compare multiple groups in western blot, qRT-PCR, pyrosequencing, 38 and double-ChIP. All values are presented as the mean ± S.D. of three independent experiments. All experimental groups were compared with the control group in each figure. Results were considered statistically significant when p < 0.05. Statistical tests were performed in Excel. Table 2.1 Pyrosequencing, qRT-PCR and Quantitative ChIP (qChIP) Primer Sequences for the Selected Gene Targets. Gene Primer sequences Annealing temperature [°C] qRT-PCR SIRT1 FW 5'-GTAGGCGGCTTGATGGTAAT-3' RV 5'-CTGTTACTGCCACAAGAACTAGA-3' 59 RNF114 FW 5'-ACATCATGGAAGGTGTGAAGG-3' RV 5'-GGACAGTAAGGACAAGGAAAGG-3' 59 RNF169 FW 5'-CATCGCTCGGCATTTGTTTC-3' RV 5'-CACTACAGCTCTGACTCCTTTC-3' 59 rDNA FW 5'-CTGAGAAACGGCTACCACATC-3' RV 5'-GCCTCGAAAGAGTCCTGTATTG-3' 59 Pyrosequencing RNF169 FW 5'-TGGATTTAGGAGGTTTGGGTTGTA-3' RVBio 5'-ATAACTTTATAACTAAAACCAAACCCAC-3' Seq1 5'-AGAAAATGTGGATTAGTGTA-3' (CpGs 1-2) 5939 Seq2 5'-AGGTTGTTTTTTTTGGTAG-3' (CpG 3) rDNA FWBio 5'- GTGTGTTTTGGGGTTGATTAGAG-3' RV 5'- ATCCCAAAACCCAACCTCTCCA-3' Seq1 5'-ACCCAACCTCTCCAA-3’ 56 qChIP RNF114 FW 5'-AAGCAAGACCTTGTCCCAAA-3' RV 5'-GGCACCACTTGCACTATCA-3' 59 RNF169 FW 5'-CACATGGAAGCACCAAACAC-3' RV 5'-AATCCTACCTCCTCATCCTCTC-3' 59 rDNA FW 5’-CTGCGATGGTGGCGTTT-3’ RV 5’-CGACTCGGAGCGAAAGATATAC-3’ 59 40 Chapter 3: Results 3.1 Putative Phytoestrogen-target Genes were selected from Existing Datasets In our previous study, we observed substantial changes in the DNA methylation patterns in MCF10A cells upon exposure to 15 µM RSV for 9 days (195) as well as significant changes in gene-specific expression profile in the blood of healthy rats fed a PTS-supplemented diet for 3 weeks (data not published) (Table 3-1). Based on bioinformatic analyses of statistically significant differential changes in these two datasets, we have identified genes that are categorized into biological processes, pathways and functions related to health maintenance and/or longevity (Figure 3.1 and Table 3-1). We refer to these genes as “phytoestrogen-target genes”. Some of the most enriched processes and pathways include decreased oxidative stress, increased anti-inflammation, decreased pathogenesis and inflammation, and increased genomic stability (Figure 3.1). One of the identified functions was increased mitochondrial biogenesis that involves the increased generation of new mitochondria while removing damaged ones (196). Maintaining the homeostasis of a healthy cell requires controlled regulation of mitochondrial mass and functions. Among hypomethylated phytoestrogen-target genes, we identified 1 encoding for a well-studied mitochondrial fusion protein, which is crucial for proper mitochondrial metabolism and supports biogenesis (197). The deficiency of OPA1 in mouse models has been shown to alter mitochondrial morphology and impair functions (198, 199). Mitochondria also help with cell homeostasis via the metabolism of ROS. The accumulation of ROS due to oxidative stress can induce cellular damage, thus contributing to age-related decline in cellular functions (200). Indeed, research indicates that centenarians, a population of individuals who reach 100 years or more, exhibited significantly 41 reduced oxidative damage and lower levels of oxidative stress markers than elderly controls (201). Among hypermethylated phytoestrogen-target genes, we identified NOX5, encoding for NADPH oxidase 5, whose downregulation has been associated with decreased intracellular ROS generation in human hepatic cells (202). In terms of cell survival, we found TERT as one of the hypomethylated phytoestrogen-target genes. TERT1 plays a crucial role in activating and extending telomeres, essential for supporting cell divisions during development. Animal studies have demonstrated that deficiency in TERT leads to shorter telomeres and a corresponding decrease in lifespan (203, 204). Another functional category was the increased genomic stability (Figure 3.1) with RAD50 as a hypomethylated target gene. RAD50 encodes DNA repair protein, which is a part of MRN complex involved in double-strand break repair (205). Patients who contain inactive RAD50 haven been shown to exhibit low RAD50 expression accompanied by chromosomal instability (206). The loss of RAD50 function leads to a failure in detection in the DNA damage response and maintaining genomic stability (207-209). Cell transformation is characterized by progressive disruptions in cellular homeostasis, eventually leading to the development of a pathological state of disease and reduced life expectancy (210). PTPRD, one of the hypomethylated phytoestrogen-target genes falling into the category of decreased cellular transformation, is a tumor suppressor, which impedes cancer cell migration in liver carcinoma and gliomas (211, 212). Indeed, the loss of PTPRD can promote glioma progression by increasing the activity of oncogenic transcription factors, such as STAT3 (213). Interestingly, another hypomethylated target gene, PIAS1, from the decreased pathogenesis 42 category (Figure 3.1), acts as a protein inhibitor of activated STATs, which enhances the anti-inflammatory response and protects against severe acute pancreatitis associated with acute lung injury (214). In the same functional category as PIAS1, we found PDCD6IP, also known as AIP1. Upregulation of PDCD6IP has been shown to reduce Aβ levels and protect against Aβ toxicity (215). The accumulation of Aβ peptides, a hallmark of Alzheimer’s disease, interferes with synaptic functions and communications between neurons (216). Additionally, the presence of these peptides can lead to the generation of reactive oxygen species (ROS), further contributing to neural injury in Alzheimer’s disease (217). Phytoestrogen-target genes were also enriched with functions linked to increased anti-inflammatory processes (Figure 3.1). Inflammation is widely recognized as one of the major factors in the process of aging and the development of disease (218). For instance, disruption of the inflammatory response significantly impacts tissue homeostasis, leading to tissue dysfunction followed by the onset of disease (219). Moreover, chronic inflammation accelerates the immune cell senescence, resulting in compromised immune functions and a reduced capability to clear old cells and other inflammatory mediators, which further exacerbates inflammation and may trigger disease (220). Hypermethylated, and thereby potentially downregulated, PDE10A is a very interesting phytoestrogen target in the category of “increased anti-inflammation”. This phosphodiesterase regulates levels of cyclic nucleotides, e.g., cAMP, and thus plays a role in signal transduction. PDE10A was shown to promote inflammation in the lung tissue and its knockdown suppressed inflammatory cell infiltration and levels of inflammatory molecules (221). Recent studies demonstrate that inhibition of PDE10A decreases Wnt signaling activity, along with Ras/MAPK and AKT pathways, which are believed to interact with inflammatory signaling 43 cascades (222) As a result, cell proliferation, migration and invasion are inhibited in ovarian cancer models (222). Among genes with the highest magnitude of changes, RNF114, RNF169 and rDNA were of special interest due to their direct connection to cellular homeostasis and thus were selected as target genes for further experiments. RNF114 belongs to a family of RING domain E3 ubiquitin ligases, exhibiting a regulatory role in immune response. Its downregulation has been associated with both induction of T-cell activation and reduction in the ubiquitination of inflammatory mediators (223-225). RNF169 is an E3 ubiquitin-protein ligase known to negatively regulate double-stranded DNA break repair processes. It competitively interferes with the recruitment of DNA repair factors mediated by RNF168, thus leading to a delay in the repair processes of double-strand breaks (226). Gene rDNA encoding for ribosomal RNA (18S, 5.8S and 28S rRNA) is responsible for ribosomal biogenesis, which is inversely correlated with longevity in post-developmental phase (227-229). Ribosome biogenesis refers to the process of production of ribosomes which are multi-units responsible for protein synthesis. Throughout fetal growth and development, ribosome biogenesis is essential for cell growth and tissue formation (228). However, studies show that a reduced ribosome biogenesis can increase lifespan in post-developmental stages (230, 231). Indeed, elevated levels of ribosomal proteins were detected during aging in human primary fibroblasts (232). Thus, downregulation of RNF114, RNF169 and rDNA can potentially promote health maintenance/longevity via enhanced anti-inflammatory signaling, increased DNA repair, and decreased ribosomal biogenesis, respectively (Figure 3.1). 44 Figure 3.1 Putative phytoestrogen-target genes that are differentially methylated or expressed in healthy experimental models. The genes are implicated in processes that promote health maintenance and cellular homeostasis, i.e., supporting cell survival, increasing anti-inflammation, DNA repair, nutrient restriction, genomic stability, and mitochondrial biogenesis, while decreasing ribosomal biogenesis, pathogenesis (referring to diseases occurrences), transformation, and oxidative stress. Figure prepared in Canva. 45 Table 3.1 Bioinformatics Data for Selected Top Genes from our Existing Databases DNA Methylation – Illumina Gene Names Methylation Difference (RSV-Ctrl in MCF10A) RNF169 0.127169 CA8 0.072988 ENO1 0.072965 ENMT1 0.070572 DPP4 0.069865 PDE10A 0.064743 PTPRD -0.05053 AGTPBP1 -0.0531 TERT -0.05433 PCDHB4 -0.05491 OPA1 -0.05588 RAD50 -0.06437 Gene Expression – RNA Seq Gene Names Log2 Fold Change (Healthy Rat Blood) Rn5-8s (rDNA) -3.289901015 Slc7a5 -1.062785377 Rnf114 -0.62497611 Etaa1 0.396941003 46 3.2 Growth Rate of MCF10A Human Mammary Epithelial Cells in Response to Pterostilbene or Genistein. We established non-toxic doses of PTS and GEN that exhibited effect on MCF10A mammary epithelial cell growth. We initially subjected MCF10A cells to PTS or GEN at increasing concentrations, ranging from 0 to 20 µM or 0 to 40 µM, respectively (Figure 3.2). With increasing concentrations, we observed a dose-dependent effect of PTS and GEN on MCF10A cell growth inhibition after 4-day and 9-day exposure (Figure 3.2A and 3.2C). Our aim was to subject cells to an extended exposure to PTS or GEN in order to increase the translation of the results to habitual dietary intakes in humans. Considering the impact of a contact inhibition on cell proliferation (233), we implemented a strategy to split and re-seed cells following a 4-day treatment based on the observed confluency of control-vehicle treated cells. The cells were re-seeded at a ratio of 1:20, followed by an additional 4 days of treatment to achieve our target 9-day exposure time. Cell counts were recorded at both time points, day 4 and day 9, providing a basis for a comparison how the prolonged exposure to PTS or GEN further influences cell growth. As expected, higher concentrations of the compounds were needed at day 4 to achieve the same inhibitory effect on cell growth as upon 9-day exposure (Figure 3.2). To apply the lowest doses possible that resemble ranges of peak plasma concentrations in blood upon consumption of foods reach in PTS or GEN (please see Discussion for details on peak plasma concentrations), we proceeded with lower doses and longer 9-day exposure time for further experiments. We determined that PTS at 7 µM and GEN at 8 µM decrease cell number by 25% compared to vehicle-treated control cells at day 9 of exposure. Hence, these doses represent a cytostatic index IC25, an inhibitory concentration that causes 25% decrease in viable cells vs. vehicle-treated control cells, and results in less than 10% of dead cells. On the other hand, PTS at 11 µM and GEN at 22 µM were established as IC50 47 values, which produce around 50% inhibition of cell growth with low cytotoxic effect (less than 10% of dead cells). Both IC25 and IC50 concentrations were reassessed for their effects on MCF10A cell growth by another series of treatments. Cell growth was significantly inhibited by approximately 22% at 7 µM PTS and by 52% at 11 µM PTS after 9 days compared with control cells (Figure 3.2B). GEN at 8 µM and 22 µM led to approximately 20% and 50% inhibition, respectively, after 9 days compared with control cells (Figure 3.2D). Therefore, these concentrations were used in further experiments to determine their epigenetic effects in MCF10A cells. 48 Figure 3.2 MCF10A cell growth in response to PTS or GEN. (A) MCF10A cell growth in response to different PTS concentrations within 0-20 μM range upon 4- and 9-day exposure. (B) MCF10A cell growth in response to 9-day exposure to 7μM and 11μM PTS, as compared with vehicle-treated cells (ethanol, Ctrl). (C) MCF10A cell growth in response to different GEN concentrations within 0-40 μM range upon 4- and 9-day exposure. (D) MCF10A cell growth in response to 9-day exposure to 8 μM and 22 μM GEN, as compared with vehicle-treated cells (DMSO, Ctrl). All results were determined by the trypan blue exclusion test, and cell number is expressed as a percentage of control. All results represent mean ± SD of three independent experiments. ***p<0.001, **p<0.01, *p<0.05. 020406080100120Ctrl GEN 8 μM GEN 22 μM% ControlMCF10A_Day 9_ Cell Growth01020304050607080901000 5 10 15 20% ControlPTS Concentration (μM)MCF10A Cell Growth4 Days 9 Days01020304050607080901000 10 20 30 40% Control GEN Concentration (μM)MCF10A Cell Growth4 days 9 days020406080100120Ctrl PTS 7 μM PTS 11 μM% ControlMCF10A_Day 9_Cell Growth8 22IC25IC50IC25IC507 11.5**********A BC D49 3.3 SIRT1 Level is Upregulated at IC50 concentration of Pterostilbene or Genistein in MCF10A Human Mammary Epithelial Cells, Despite Unchanged mRNA Levels. We subsequently investigated the impact of compounds PTS and GEN on SIRT1 expression at both mRNA and protein levels in MCF10A cells. Following a 9-day exposure to PTS, no significant changes in SIRT1 expression were observed at both doses of 7 μM and 11 μM, compared to the control MCF10A cells (Figure 3.3A). A similar trend was noted in response to GEN (Figure 3.3B). Next, we quantified SIRT1 protein level by normalizing protein samples with respect to total sample protein loaded using free stain gels. No change of SIRT1 protein level was observed at the lower doses of both PTS and GEN after 9 days in MCF10A cells. However, the higher doses produced significant effects. Treatment with PTS at 11 μM resulted in a 59% increase in SIRT1 protein expression, while 22 μM GEN led to approximately a 68% SIRT1 increase, compared to vehicle-treated control cells (Figure 3.3C and 3.3D). Therefore, we concluded that both PTS and GEN at IC50 doses led to the increase in SIRT1 protein level, without changes in the mRNA level. 50 Figure 3.3 Impact of PTS or GEN on SIRT1 expression in MCF10A cells. (A, C) SIRT1 gene expression at mRNA (A) and protein levels (C) in MCF10A cells upon 9-day exposure to PTS at 7 and 11 μM. (B, D) SIRT1 mRNA expression (B) and protein level (D) in MCF10A cells upon 9-day exposure to GEN at 8 and 22 μM. Results were determined by qRT-PCR and western blot. In western blot analyses, reactive bands were visualized using the chemiluminescent protocol in the ChemiDoc MP Imaging System (Bio-Rad) and analyzed using the Image Lab software. Protein loading was normalized with respect to total sample protein loaded using free stain gels. All results represent mean ± SD of three independent experiments. ***p<0.001, **p<0.01, *p<0.05. 51 3.4 SIRT1 Occupancy is Consistently Enriched at All Target Genes in Response to Genistein, but not Pterostilbene, in MCF10A Human Mammary Epithelial Cells. PTS and GEN at IC50 concentrations led to an increase in SIRT1 protein level. However, changes in SIRT1 levels may not reflect a functional role of SIRT1 which is associated with SIRT1 binding to DNA. Thus, we used quantitative chromatin immunoprecipitation (qChIP) to measure SIRT1 occupancy at promoters of the selected target genes, namely RNF114, RNF169 and rDNA, in MCF10A cells upon 9-day exposure to IC25 and IC50 doses of PTS and GEN (Figure 3.4). Within the RNF114 promoter region, our analysis revealed that SIRT1 occupancy exhibited no significant increase after exposure to 7 μM PTS (Figure 3.4A). However, there was a remarkable enrichment at 11 μM PTS, demonstrating an approximately 5.7-fold increase in SIRT1 binding compared to the control cells (Figure 3.4B). Interestingly, while SIRT1 binding was not detectable in control cells, PTS at both 7 μM and 11 μM concentrations resulted in a robust increase of SIRT1 occupancy at the RNF169 promoter region (Figure 3.4C and 3.4D). Conversely, no significant changes in SIRT1 binding were observed within the rDNA promoter in response to PTS (Figure 3.4E and 3.4F). Contrary to PTS, 9-day treatment with GEN resulted in SIRT1 increase at promoters of all tested genes, regardless of its concentration (Figure 3.4G-L). While there was no enrichment in control cells, we detected SIRT1 occupancy in response to lower 8 μM dose of GEN (Figure 3.4G, I & K). Upon exposure to 22 μM GEN, there was approximately a 2.7-, 5.6-, and 3.5-fold increase in SIRT1 occupancy at RNF114, RNF169, and rDNA promoters, respectively, as compared to control cells (Figure 3.4H, J & L). In summary, both PTS and GEN led to the increase in SIRT1 occupancy within the tested target genes, except for rDNA in response to PTS. 52 Figure 3.4 SIRT1 binding at RNF114, RNF169 and rDNA promoters upon 9-day exposure to PTS or GEN in MCF10A cells. (A-F) SIRT1 binding after 9-day exposure to 7 μM and 11μM PTS at RNF114 (A, B), RNF169 (C, D), and rDNA (E, F) promoters. (G-L) SIRT1 binding after 9-day exposure to 8 μM and 22 μM GEN at RNF114 (G, H), RNF169 (I, J), and rDNA (K, L) promoters. All results were determined by quantitative ChIP and represent mean ± SD of three independent experiments. ***p<0.001, **p<0.01, *p<0.05. 00.00050.0010.00150.002PTS Ctrl PTS 7 µM(Bound-IgG)/INPUTRNF114_PTS_SIRT1 Binding00.0050.010.0150.020.0250.03PTS Ctrl PTS 11 µM(Bound-IgG)/INPUTRNF114_PTS_SIRT1 Binding00.0050.010.0150.02PTS Ctrl PTS 7 µM(Bound-IgG)/INPUTRNF169_PTS_SIRT1 Binding00.0050.010.0150.020.025PTS Ctrl PTS 11 µM(Bound-IgG)/INPUTRNF169_PTS_SIRT1 Binding00.00050.0010.00150.0020.00250.0030.00350.004PTS Ctrl PTS 7 µM(Bound-IgG)/INPUTrDNA_PTS_SIRT1 Binding00.0020.0040.0060.0080.010.012PTS Ctrl PTS 11 µM(Bound-IgG)/INPUTrDNA_PTS_SIRT1 Binding00.00010.00020.00030.00040.00050.0006GEN Ctrl GEN 8 µM(Bound-IgG)/INPUTRNF169_GEN_SIRT1 Binding00.020.040.060.080.10.12GEN Ctrl GEN 22 µM(Bound-IgG)/INPUTRNF169_GEN_SIRT1 Binding00.00020.00040.00060.0008GEN Ctrl GEN 8 µM(Bound-IgG)/INPUTrDNA_GEN_SIRT1 Binding00.0050.010.0150.020.0250.03GEN Ctrl GEN 22 µM(Bound-IgG)/INPUTrDNA_GEN_SIRT1 Binding00.00050.0010.00150.002GEN Ctrl GEN 8 µM(Bound-IgG)/INPUTRNF114_GEN_SIRT1 Binding00.010.020.030.040.050.060.07GEN Ctrl GEN 22 µM(Bound-IgG)/INPUTRNF114_GEN_SIRT1 BindingCDEFABI KJ LGHP=0.054ns ***nsnsP=0.069****************No BindingNo BindingNo Binding No Binding No Binding53 3.5 Expression of RNF169 and rDNA, but Not RNF114, is Downregulated in Response to Pterostilbene and Genistein. SIRT1 acts as a histone deacetylase which consequently may result in a condensed chromatin structure due to the interaction between positive charge of lysine in histone proteins and negatively charged DNA. To explore whether SIRT1 binding to gene promoters of RNF114, RNF169, and rDNA upon a 9-day exposure to PTS or GEN in MCF10A cells is accompanied by downregulation of gene expression, we assessed the mRNA levels of these target genes using qRT-PCR. While RNF114 expression exhibited no significant variations in response to either PTS or GEN (Figure 3.5A and 3.5D), a distinct pattern emerged for RNF169 and rDNA. RNF169 expression was significantly reduced by 40% and 21% in response to 11 µM PTS and 22 µM GEN, respectively (Figure 3.5B and 3.5E). Similarly, there was a marked downregulation by 25% and 32% for rDNA in response to 11 µM PTS and 22 µM GEN, respectively (Figure 3.5C and 3.5F). Notably, the lower 7 µM dose of PTS selectively led to RNF169 downregulation only, without influencing the expression level of other gene targets (Figure 3.5B). The lower 8 µM dose of GEN did not produce any statistically significant downregulation of the tested genes. Taking into consideration changes in expression levels, only RNF169 and rDNA were further investigated to elucidate the role of SIRT1 in PTS and GEN-mediated epigenetic regulation, as no changes in RNF114 expression were detected. This finding suggests that there is a gene-dependent effect mediated by PTS and GEN. 54 Figure 3.5 Expression of RNF114, RNF169 and rDNA at mRNA levels in response to 9-day exposure to PTS or GEN in MCF10A cells. (A-F) Expression at mRNA levels for RNF114, RNF169 and rDNA upon 9-day exposure to 7 and 11 μM PTS (A-C), and at 8 and 22 μM GEN (D-F) in MCF10A cells as determined by qRT-PCR. All results represent mean ± SD of three independent experiments. ***p<0.001, **p<0.01, *p<0.05. -0.0020.0020.0060.010.0140.0180.0220.026PTS Ctrl PTS 7µM PTS 11µMTarget/GAPDHRNF114_Expression_PTS00.0050.010.0150.020.0250.030.0350.040.045PTS Ctrl PTS 7µM PTS 11µMTarget/GAPDHRNF169_Expression_PTS0100200300400500600700PTS Ctrl PTS 7µM PTS 11µMTarget/GAPDHrDNA_Expression_PTS0.0000.0100.0200.0300.040GEN Ctrl GEN 8µM GEN 22µMTarget/GAPDHRNF114_Expression_GEN0.0000.0100.0200.0300.0400.050GEN Ctrl GEN 8µM GEN 22µMTarget/GAPDHRNF169_Expression_GEN0100200300400500600700GEN Ctrl GEN 8µM GEN 22µMTarget/GAPDHrDNA_Expression_GENABDEFCns nsns nsnsnsns*** ** *55 3.6 Differential DNA Methylation within Promoters of the Target Genes in Response to Pterostilbene or Genistein in MCF10A Human Mammary Epithelial Cells. Since SIRT1 was shown to interact with DNMT3B at gene loci (65), a vital enzyme responsible for de novo DNA methylation, we next determined whether SIRT1 binding at RNF169 and rDNA promoters and decreased expression of RNF169 and rDNA in response to PTS or GEN are associated with the increased promoter DNA methylation. We used the pyrosequencing technology which is a quantitative method that efficiently assesses the percentage of DNA methylation at a single CpG site resolution. Maps of promoter regions of RNF169 and rDNA are shown in Figure 3.6A and 3.6B, where the light blue area highlights the targeted CpG sites tested via pyrosequencing. Three CpG sites and seven CpG sites were assessed within the RNF169 and rDNA promoter regions, respectively, allowing us to obtain a pattern of methylation within broader DNA regions (Figure 3.6A and 3.6B). Exposure of MCF10A cells to 22 µM GEN resulted in a significant 22% increase in methylation at CpG 2 of RNF169 on day 9, whereas no such effect was observed for PTS (Figure 3.6C and 3.6E). With respect to rDNA, both PTS and GEN resulted in hypermethylation across different CpG sites (Figure 3.6D and 3.6F). After exposing MCF10A cells to 11µM PTS for 9 days, we noted a ~24% increase in DNA methylation at CpG 2 and CpG 5 within rDNA promoter (Figure 3.6D). Upon 22µM GEN, in turn, significant increases in DNA methylation of rDNA promoter were observed at CpG 4 (by 30%) and CpG 7 (by 50%) (Figure 3.6F). In summary, GEN consistently led to DNA hypermethylation of both RNF169 and rDNA, whereas PTS treatment resulted in rDNA hypermethylation only. These hypermethylation events were detected only upon exposure to higher doses of the compounds (IC50 concentrations) and correlated with gene downregulation. 56 Figure 3.6 PTS- and GEN-mediated changes in DNA methylation of CpG loci within RNF169 and rDNA promoters in MCF10A cells. A map of promoter regions of RNF169 (A) and rDNA (B), where the light blue shaded area represents the CpG sites tested by pyrosequencing (numbered CpGs). Average methylation status of CpG sites within RNF169 (C,E) and rDNA (D,F) promoters in MCF10A cells after 9-day exposure to 7 μM and 11μM PTS (C-D) or 8 μM and 22 μM GEN (E-F). All results were determined by pyrosequencing and represent mean ± SD of three independent experiments. ***p<0.001, **p<0.01, *p<0.05. 051015202530CpG 1 CpG 2 CpG 3 CpG 4 CpG 5 CpG 6 CpG 7% MethylationrDNA_PTSPTS Ctrl PTS 7 µM PTS 11 µM051015202530CpG 1 CpG 2 CpG 3 CpG 4 CpG 5 CpG 6 CpG 7% MethylationrDNA_GENGEN Ctrl GEN 8 µM GEN 22 µM01020304050CpG 1 CpG 2 CpG 3% MethylationRNF169_GENGEN Ctrl GEN 8 µM GEN 22 µM01020304050CpG 1 CpG 2 CpG 3% MethylationRNF169_PTSPTS Ctrl PTS 7 µM PTS 11 µMC DE FABnsns*******ns2 3ISGF-3NF-κβPIT-1a +1-691 -628 -476 -325 -305 -231 -223 -212 -120 -82 -72 -54 -46 -30 -15 -31SRFYY1Sp1SP1NF-1HbGRSP1AP-1MIG-1FTZOCT-11C/EBPαSP1WT-1ERG-1NF-muE1MYO PRAP-2HBP-1PENKTF-1+1-149 -137 -128 -103-101-99 -96 -87-84-81 -75 -69-67 -59 -53 -35 -30 -25 -12 -9 +9 +22 SP1AP-2αSP1AP-2α18S 5.8S 28S1 2 3 4 5 6 7RNF169rDNA57 3.7 DNMT3B Occupancy is Enriched at RNF169 and rDNA Promoters in Response to Genistein in MCF10A Human Mammary Epithelial Cells. Since we observed DNA hypermethylation at several SIRT1-occupied gene regions following 9-day exposure of MCF10A cells to PTS or GEN, we proceeded with the evaluation of DNMT3B binding at the regulatory regions of the tested genes using qChIP. DNMT3B was selected among DNA methyltransferases as it was previously shown that SIRT1 facilitates DNMT3B recruitment to DNA and subsequent DNA methylation (64). In addition, we previously reported on DNMT3B as a required enzyme for PTS-mediated hypermethylation of oncogenes in breast cancer cells (152). Interestingly, increased in DNMT3B occupancy was consistently observed for GEN, but not PTS (Figure 3.7). Strikingly, no binding of DNMT3B was detected for rDNA upon exposure to PTS (Figure 3.7C and 3.7D). Within the RNF169 promoter, 38% and 90% reduction, rather than an increase, in DNMT3B binding was observed in response to 7 μM and 11 μM PTS, respectively (Figure 3.7A and 3.7B). On the contrary, GEN at both 8 μM and 22 μM concentrations resulted in a robust enrichment in DNMT3B binding at rDNA, as compared with control cells where no binding was detected (Figure 3.7J and 3.7H). Within the RNF169 promoter region, a substantial 27-fold increase in DNMT3B occupancy was observed upon exposure to 22 μM GEN (Figure 3.7F), whereas a lower dose produced a statistically non-significant increase (Figure 3.7E). To conclude, DNA hypermethylation of the target genes was accompanied by DNMT3B binding only in response to GEN, but not PTS, which suggests a distinct mechanism of epigenetic regulation of gene transcription between GEN and PTS. 58 Figure 3.7 DNMT3B binding at RNF169 and rDNA promoters upon 9-day exposure to PTS or GEN in MCF10A cells. (A-D) DNMT3B binding after 9-day exposure to 7 μM and 11μM PTS at RNF169 (A,B), and rDNA (C,D) promoters, respectively. (E-F) DNMT3B binding after 9-day exposure to 8 μM and 22 μM GEN at RNF169 (E,F) and rDNA (J,H) promoters, respectively. All results were determined by quantitative ChIP and represent mean ± SD of three independent experiments. ***p<0.001, **p<0.01, *p<0.05. 00.20.40.60.81PTS Ctrl PTS 7 µM(Bound-IgG)/InputrDNA_PTS_DNMT3B Binding00.20.40.60.81PTS Ctrl PTS 11 µM(Bound-IgG)/InputrDNA_PTS_DNMT3B Binding00.010.020.030.040.050.06PTS Ctrl PTS 7 µM(Bound-IgG)/INPUTRNF169_PTS_DNMT3B Binding00.010.020.030.040.050.06PTS Ctrl PTS 11 µM(Bound-IgG)/INPUTRNF169_PTS_DNMT3B Binding00.0020.0040.0060.0080.01GEN Ctrl GEN 8 µM(Bound-IgG)/INPUTRNF169_GEN_DNMT3B Binding00.010.020.030.040.050.06GEN Ctrl GEN 22 µM(Bound-IgG)/INPUTRNF169_GEN_DNMT3B Binding00.0010.0020.0030.004GEN Ctrl GEN 8 µM(Bound-IgG)/INPUTrDNA_GEN_DNMT3B Binding00.0020.0040.0060.008GEN Ctrl GEN 22 µM(Bound-IgG)/INPUTrDNA_GEN_DNMT3B BindingA BDFHJECNo Binding No Binding**********nsNo Binding No Binding59 3.8 SIRT1 and DNMT3B do Not Co-occupy RNF169 and rDNA Promoter Regions in Response to IC50 Genistein in MCF10A Human Mammary Epithelial Cells. Prior studies suggest that SIRT1 serves as a platform for recruiting DNA methylating enzymes like DNMT3B, ultimately facilitating DNA methylation (64). Our results have demonstrated an enrichment of both SIRT1 and DNMT3B binding at RNF169 and rDNA promoters when MCF10A cells are exposed to 22 μM GEN for 9 days. Intrigued by these findings, we sought to investigate whether DNMT3B and SIRT1 co-bind the same DNA loci in response to GEN to remodel gene transcription. To explore it further, we used a double ChIP method paired with quantitative PCR analysis of the input and bound samples with single ChIP for both proteins and double ChIP in both directions (protein 1-protein 2 and protein 2-protein 1) (194). In double ChIP, if levels of enrichment are higher than those of single ChIPs, full co-occupancy is concluded. As shown in Figure 3.8, in single ChIP analysis, SIRT1 (grey bars) and DNMT3B (black bars) bind to promoters of RNF169 and rDNA. However, in double ChIP analysis, SIRT1 and DNMT3B are not co-enriched at either of the gene promoters; dramatically lower levels of enrichment than those of single ChIPs are observed regardless of the order of performance of the two immunoprecipitations (white and pattern bars in Figure 3.8). Hence, we concluded that, although both SIRT1 and DNMT3B bind to RNF169 and rDNA promoters, the two proteins do not co-occupy the regions. This suggests a cooperation in regulation of gene transcription however without a physical interaction at the DNA loci. 60 Figure 3.8 Evaluation of a co-occupancy of SIRT1 and DNMT3B at RNF169 and rDNA promoter regions upon 9-day exposure to 22 μM GEN in MCF10A cells as measured by double ChIP. Two consecutive ChIP assays were conducted using antibodies specific to SIRT1 and DNMT3B, arranged in different orders. 'Double: SIRT1-DNMT3B' signifies SIRT1 binding first, followed by DNMT3B binding, while 'Double: DNMT3B-SIRT1' indicates the reverse order. The sequential binding orders at the same region can be identified. Results were quantified by quantitative PCR. All results represent mean ± SD of three independent experiments. ***p<0.001, **p<0.01, *p<0.05. A BA B61 3.9 SIRT1 is Required for Genistein-mediated Downregulation of rDNA, but Not RNF169, in MCF10A Human Mammary Epithelial Cells. To assess whether SIRT1 is required for GEN-mediated epigenetic regulation of gene transcription, we performed SIRT1-siRNA transfection in MCF10A cells to deplete SIRT1. We scheduled two rounds of siRNA transfection, each followed by 48 hr-treatment with GEN in order to extend the exposure time to GEN and align it with our standard long-term exposure protocol. Using qRT-PCR, we confirmed a significant 75% reduction of mRNA levels of SIRT1 upon SIRT1-targeting siRNA treatment, as compared with cells treated with control siRNA, si-Ctrl (Figure 3.9A). The level of SIRT1 expression remained low and unchanged even after GEN treatment. Interestingly, we found that SIRT1 depletion abrogated GEN-mediated downregulation of rDNA expression, providing a mechanistic proof for a requirement of SIRT1 in GEN-mediated rDNA transcriptional regulation (Figure 3.9C). On the other hand, despite SIRT1 knockdown, GEN still led to downregulation of RNF169, suggesting that there is an alternative mechanism of epigenetic regulation of RNF169 expression in response to GEN (Figure 3.9B). 62 Figure 3.9 Expression of SIRT1, RNF169 and rDNA upon exposure to 22 μM GEN in MCF10A cells with SIRT1 knockdown. (A-C) MCF10A cells transfected with control-siRNA and SIRT1-siRNA followed by exposure to 22 μM GEN, respectively. mRNA levels were determined by qRT-PCR at SIRT1 (A), RNF169 (B), and rDNA (C). All results represent mean ± SD of three independent experiments. ***p<0.001, **p<0.01, *p<0.05 (ns = not significant). 0.0000.0010.0020.0030.004si-CTRL si-CTRLGEN22si-SIRT1 si-SIRT1GEN22Target/GAPDHSIRT1_mRNA Expression0.0000.0050.0100.0150.0200.0250.0300.035si-CTRL si-CTRLGEN22si-SIRT1 si-SIRT1GEN22Target/GAPDHRNF169_mRNA Expression020406080100120140160si-CTRL si-CTRLGEN22si-SIRT1 si-SIRT1GEN22Target/GAPDHrDNA_mRNA ExpressionABC*** ns** **nsns****63 Chapter 4: Discussion & Conclusions In my MSc research project, we used healthy mammary epithelial cells, MCF10A, to discover how phytoestrogens epigenetically impact cell biology to maintain cellular homeostasis. In the present study, we found that both PTS and GEN resulted in decreased expression of RNF169 and rDNA, which was accompanied by increased SIRT1 occupancy at promoters of these two target genes (except for rDNA in response to PTS). Downregulation of RNF169 brings about higher efficiency in DNA damage repair whereas suppression of rDNA results in decreased ribosomal biogenesis, which are linked to longevity (228). This provides evidence supporting the health-related benefits of phytoestrogens in maintaining cellular health through potential epigenetic effects mediated, at least partially, by SIRT1. The action of GEN, but not PTS, on downregulation of the two target genes was accompanied by DNMT3B binding and hypermethylation of gene promoters. Furthermore, we mechanistically established that GEN-mediated epigenetic suppression of rDNA transcription requires SIRT1. Our findings demonstrate a distinct mechanism of action of GEN and PTS in epigenetic suppression of genes functionally linked to disturbed cellular homeostasis. Differential PTS doses in normal and tumor breast cells We established that both PTS and GEN exert a dose-dependent inhibitory effect on growth of MCF10A cells with IC25 and IC50 equal to 7 µM and 11 µM, respectively, for PTS, and 8 µM and 22 µM, respectively, for GEN, following a 9-day exposure to the compounds (Figure 3.2). In our previous study, 7 µM concentration of PTS decreased growth of MCF10CA1a breast cancer cells by 50%, indicating higher sensitivity of cancer cells to PTS as compared to normal cells (234). This difference in sensitivity can be attributed to the distinct responses of tumor and normal 64 human cell lines to the same treatment, with tumor cell lines generally exhibiting greater sensitivity (235, 236). Extended 9-day exposure time to PTS or GEN In most studies, cells are typically exposed to PTS or GEN for short duration, ranging from 6 h to 72 h (237-241) and with a higher doses of up to 100 µM for both compounds (242, 243). In contrast, our treatment schedule was extended to 9 days which allowed us to use low doses, better reflecting peak plasma concentrations in humans (115). The peak plasma concentration, or Cmax refers to maximum concentration of a compound in the bloodstream following consumption. For GEN, the Cmax is early reported as 1.26 ± 0.27 μM, which is determined from healthy adult women who consumed a single GEN dose at 50mg/kg body weight per day (115, 244). Unlike GEN, the Cmax of PTS has only been studied in rats, revealing levels around 2550 ng/mL (9.95 μM) after oral intake of 56 mg/kg body weight per day (130). Moreover, our extended exposure scheme more closely resembles a prolonged exposure to dietary polyphenols experienced by humans through habitual dietary intake. Habitual intakes may induce cumulative impacts of PTS or GEN on cellular responses and functions that may not occur with short-term treatments, such as alterations in signaling pathways and epigenetic modifications that require time to manifest. PTS and GEN induced increase in SIRT1 protein level with the unchanged transcript level Our hypothesis was that SIRT1 is involved in epigenetic effects mediated by PTS and GEN in mammary epithelial cells. The first step was to determine the effects of the phytoestrogens on SIRT1 expression. Surprisingly, we observed no changes in SIRT1 mRNA levels at either IC25 or IC50 concentrations of PTS and GEN (Figure 3.3A&B). However, a notable increase in SIRT1 65 protein levels was detected at the IC50 dose of both PTS and GEN (Figure 3.3C&D). Previous studies have shown that 17β-estradiol induces SIRT1 protein levels in breast cancer cells through ERα-dependent signaling (123, 245). In addition, 17β-estradiol can allosterically activate SIRT1 via binding to its N-terminal domain (123, 245). Considering the structural similarity between phytoestrogens and 17β-estradiol, it is plausible that phytoestrogens lead to allosteric activation f SIRT1 and/or induce SIRT1 protein level by mimicking the conformational structure of estradiol and activating estrogen signaling through GPER-1 in our cellular model, namely ER-negative MCF10A cells (246). Of note, MCF10A cells have active GPER signaling and estrogen-induced proliferation was shown to be mediated by GPER in this cell line (246, 247). GPER depletion abrogated pro-proliferative effects of estrogen in MCF10A cells providing a mechanistic link to GPER (246). The increase in SIRT1 protein level in response to phytoestrogens in our study may also be attributed to other regulatory mechanisms, including post-translational modifications like ubiquitination and post-transcriptional events such as regulation of mRNA stability (248). A decrease in SIRT1 protein ubiquitination, which involves the covalent attachment of ubiquitin to mark proteins for degradation, may reduce subsequent SIRT1 degradation and thus increase SIRT1 protein levels (249). On the other hand, studies have shown that the half-life of SIRT1 mRNA stability can range from 1 hour to 8 hours, depending on the presence of the RNA binding protein HuR that interacts with the 3’-untranslated region of SIRT1 mRNA and stabilizes it (250). Increased mRNA stability, in turn, enhances SIRT1 translation and thus protein levels. Indeed, a developing field of epitranscriptomics addresses regulation of RNA stability through post-transcriptional epigenetic modifications on RNA molecules (251). A recent study calls attention 66 to the crucial role of epitranscriptomic modifications in mediating dietary effects on individual health (252). Specifically, in mice fed high-fat diet, disrupted gut microbiome resulted in decreased m6A methylation levels of two key regulators of GLP1 expression, namely Gcg and Pc3 genes, leading to suppression of the gut hormone GLP1 in the small intestine (252). Of note, GLP1 exerts antiobesity effects through regulating insulin secretion and many gastrointestinal functions. Although the evidence for the impact of phytoestrogens on epitranscriptomics remains limited, GEN has been shown to increase expression of m6A RNA demethylase ALKBH5 in a mouse model of renal fibrosis (253). This effect led to reduced total m6A levels and suppression of Snail transcription factor, consequently inhibiting epithelial-mesenchymal transition. Unchanged methylation level of RNF169 despite reduced mRNA levels upon PTS DNA methylation at gene regulatory regions is associated with transcriptional silencing (47, 254). Indeed, downregulation of rDNA in response to GEN and PTS was accompanied by increased DNA methylation levels at rDNA promoter region (Figure 3.5C&F, Figure 3.6D&F). A similar correlation was observed for RNF169, however only in response to GEN (Figure 3.5E & Figure 3.6E). Although we detected decreased mRNA levels of RNF169 in response to both IC25 and IC50 PTS (Figure 3.5B), we found no significant changes in RNF169 promoter methylation (Figure 3.6C). This discrepancy may arise from the involvement of other regulatory DNA elements within the promoter regions such as silencers. Silencers function to inhibit gene expression by interacting with transcriptional repressors, thereby blocking the binding of transcriptional activators to DNA (255). Another epigenetic mechanism, including histone modifications, may also contribute to decreased transcription of RNF169. Our finding of a decreased DNMT3B occupancy (Figure 3.7A&B) at the RNF169 promoter suggests that reduced 67 mRNA levels may not solely depend on DNMT3B-mediated DNA methylation but potentially on SIRT1 deacetylase activity, as indicated by the observed enrichment in SIRT1 binding (Figure 3.4I&J). As mentioned in the introduction, SIRT1 is capable of directly deacetylating H3 and H4. Recent study shows that deacetylation of histone H4 inhibits the elongation of RNA polymerase II, thereby reducing the transcription activity of the target gene (256). Additionally, other repressive histone marks such as tri-methylation of lysine 27 at histone H3 (H3K27me3) may be deposited at the RNF169 locus, leading to a more condensed chromatin state (257). This process may depend on the action of the PRC2, which is involved in gene silencing through H3K27me3, and has been evaluated as a therapeutic target in breast cancer (258, 259). Importantly, Michalak et al. demonstrated that the disruption of PRC complexes led to depletion of both H3K27me3 and H3K27me2, thereby completely halting the development of mammary gland (260). Interaction between SIRT1 and DNMT3B The interaction between SIRT1 and DNMT3B has been elucidated in several studies. Initially, SIRT1 was found to co-immunoprecipitate with DNMT3B, contributing to the maintenance of a repressed state for pro-inflammatory genes during macrophage differentiation (64, 65). SIRT1 can also facilitate the recruitment of DNMT3B through histone deacetylation, leading to subsequent DNA methylation within the nucleus of embryonic stem cells (65). Furthermore, DNMT3B and SIRT1 were shown to be transiently recruited to the induced DNA break loci at the promoter of E-cadherin gene. However, the recruitment of DNMT3B appears to rely on the presence of SIRT1, as DNMT3B is not enriched at the break sites when SIRT1 is knocked down (261).While co-immunoprecipitation uses an antibody to purify its target antigen (protein of interest) along with its binding partners from a mixed sample, double chromatin immunoprecipitation (double ChIP), 68 which was applied in our study, allows for the investigation of the sequential binding of SIRT1 and DNMTB to the same DNA loci. Hence, double ChIP provides insights into the combinatorial patterns of protein-DNA interactions, which constitutes a strength of our study. Interestingly, although both SIRT1 and DNMT3B bind RNF169 and rDNA promoters in response to GEN, the double ChIP analysis shows that they do not co-occupy the same DNA loci (Figure 3.8). We could speculate that SIRT1 binds first and subsequent histone deacetylation is recognized by DNMT3B facilitating DNMT3B recruitment. Indeed, it was demonstrated that DNMT3B binding is enriched within hypoacetylated and condensed chromatin (65). Results depicted in Figure 3.4 & Figure 3.7 appear to support this scenario as IC25 dose of GEN is sufficient for SIRT1 binding however higher IC50 concentration is required to detect consistent DNMT3B enrichment. Another possibility is SIRT1-mediated increase in binding affinity of PRC4 polycomb complex that was shown to contain DNMT3B (262-264). SIRT1 associates with polycomb proteins in PRC4 (262), which may subsequently recruit DNMT3B (263). Considering SIRT1 capability to modify the activity of non-histone proteins through deacetylation, as demonstrated for numerous transcription factors and coregulators (71), it is probable that SIRT1 acts through regulating the activity of a transcriptional repressor interacting with DNMT3B. For instance, DNMT3B recruitment was dependent on binding of E2F6 transcriptional repressor in somatic tissues in a mouse model (91, 265). SIRT1-dependent epigenetic regulation of rDNA, but not of RNF169, in response to GEN Our findings provide evidence for SIRT1-dependent downregulation of rDNA and thus potential reduction in ribosomal biogenesis in response to GEN (Figure 3.9). When SIRT1 is depleted, GEN no longer leads to suppression of rDNA at mRNA levels. To our knowledge, our current study 69 delivers the first evidence that mechanistically connects SIRT1 to rDNA transcriptional activity upon GEN treatment. SIRT1-dependent action of GEN has been previously reported however without identifying gene targets or mechanisms (187, 266). GEN mitigated acetaminophen-induced liver toxicity by upregulating SIRT1 protein expression and this protective effect was abolished by SIRT1 knockdown (266). In mice with induced renal injury, the protective effects of GEN were eliminated after stable knockdown of SIRT1 (187). Furthermore, none of previous reports assessed a direct regulatory action of SIRT1 on rDNA expression. Existing studies on human normal and cancer cells of different origins report on the energy-dependent nucleolar silencing complex (eNoSC) which contains SIRT1, among other proteins, and downregulates rRNA transcription upon reduction of energy status (267). Nucleomethylin that mediates the assembly of the eNoSC complex binds SIRT1, which facilitates the formation of a condensed chromatin structure and results in repression of rDNA transcription by eNoSC (268). In another study on human cells, depletion of active regulator of SIRT1 (AROS), that binds and upregulates SIRT1, led to deficient ribosomal biogenesis and reduced ribosomal proteins (269). However, no connection to SIRT1 was investigated. Of note, in our model of normal mammary epithelial cells, SIRT1 depletion did not bring about a decrease in rDNA expression (Figure 3.9C), which one would expect based on AROS study. Yet, we still observed downregulation of RNF169 in response to GEN when SIRT1 was depleted (Figure 3.9B). We propose this might be due to the roles of other members from the sirtuin family, which could potentially take over SIRT1 roles. Similar to SIRT1, SIRT6 and SIRT7 are nuclear sirtuins that exert epigenetic influence on gene expression through the deacetylase activity (270). SIRT6 inhibits target gene expression by deacetylating H3K9 and H3K56, and regulates cellular 70 energy homeostasis, stress resistance, and genomic stability (271, 272). Studies have shown that SIRT6 activation acts as a DNA damage sensor and enhances DNA repair (273, 274). Notably, SIRT7 deacetylase activity, targeting H3K18 and ATM kinase, is also associated with DNA repair and genomic stability through transient recruitment to break sites (275-277). Therefore, given the function of gene RNF169 associated with DNA repair (Figure 3.1), there is a likelihood that either SIRT6 or SIRT7 may mediate downregulation of RNF169 even after SIRT1 knockdown. However, whether GEN acts through SIRT6 and/or SIRT7 remains to be elucidated. Distinct mechanisms of action of GEN and PTS In summary, our findings indicate that although both PTS and GEN result in downregulation of RNF169 and rDNA, they employ different mechanisms as revealed by the ChIP-qPCR results (Figure 3.7). We showed that only GEN treatment led to the enrichment of DNMT3B within SIRT1-occupied regions at the promoters of RNF169 and rDNA, whereas PTS either decreased DNMT3B binding or showed no binding, respectively (Figure 3.7). This finding corresponds to our initial hypothesis that phytoestrogens belonging to different subclasses and characterized by distinct structures, possibly due to methyl or hydroxyl side chains (Figure 1.3), act through different mechanisms in regulating the transcriptional activity of target genes. Further studies are warranted to investigate if compounds from the same group of phytoestrogens share the mechanisms of action or use alternative pathways (e.g., GEN and daidzein). Conclusions, limitations and future directions While the majority of studies focus on the therapeutic potential of phytoestrogens in disease models (278-280), our research examines their action, specifically epigenetic regulation of gene 71 expression, in normal cells. We found an increase in DNA methylation at the target genes, RNF169 and rDNA, which was accompanied by enhanced SIRT1 and DNMT3B occupancy following 9-day treatment with GEN, but not PTS, at IC50 concentration. This finding suggests a distinct mode of action of phytoestrogens with different chemical structure. Next, we discovered that while both SIRT1 and DNMT3B bind to promoter regions of RNF169 and rDNA, they do not co-occupy the same DNA locus. Finally, our results indicate that SIRT1 is required for GEN-mediated downregulation of rDNA, but not RNF169, implying gene-dependent effects of GEN. Discovering the role of the compounds in normal cells is crucial for learning about the impact of the environment, specifically diet, on maintaining cellular homeostasis. This new knowledge may facilitate the application of the compounds in health maintenance, which is especially important today when we face the aging of the world’s population and rising rates of age-related disease. Moreover, studying the impact of phytoestrogens on cell biology is especially important from a toxicology point of view (281). Namely, numerous environmental contaminants, including bisphenol, nonylphenols, parabens, and phthalates, among others, have estrogen-like effects and are potent endocrine disruptors, which impact human, animal and plant life (282). Hence, our findings propose mechanisms that could well be involved in the action of the synthetic environmental estrogens and better inform us about their potential health effects. MCF10A human breast epithelial cell line selected for our in-vitro models is widely utilized for studying normal breast cell biology and transformation (283). As previously mentioned, although MCF10A cells originate from benign proliferative breast cells, they lack expression of estrogen receptors alpha and beta (ER α/β) but have active GPER estrogen pathway (246). This allows us 72 to explore an alternative pathway that could have therapeutic implications for aggressive and devastating triple-negative breast cancer (TNBC) that lacks ER α/β (284). Given that GPER can modulate estrogenic actions in TNBC, targeting GPER with phytoestrogen treatment may present a viable therapeutic approach (119, 285). To delve deeper into the mechanistic roles of SIRT1 in PTS and GEN action in the ERα/β context, it might be important to consider using another cell line, for example MCF-12A non-tumorigenic mammary epithelial cells, which express both ERα and ERβ receptors (286). Moreover, using only MCF10A cells as an experimental model may pose challenges in generalizing to other cell lines, other study designs, and in vivo models. Therefore, further in vitro investigations are warranted to validate our findings, facilitating their translation to in vivo experiments and subsequent clinical trials. Another limitation of our current investigation is using solely in vitro approach. Although in vitro controlled environment allows for a precise investigation of cellular targets, it makes it impossible to capture effects of PTS or GEN metabolites. Our results deliver new evidence for distinct mechanisms of epigenetic action of PTS and GEN in regulation of gene transcription in human normal mammary epithelial cells. Although PTS downregulated RNF169 and rDNA expression and led to SIRT1 enrichment within promoters of these genes along with rDNA promoter hypermethylation, no DNMT3B occupancy was detected upon PTS treatment. This breakthrough findings indicate that PTS exerts its epigenetic effects through an alternative mechanism, which remains to be explored in future research. Thus, our novel discovery opens the door to further investigations in the nutritional epigenomics field. 73 It is also important to emphasize mixed evidence for GEN role in mammary gland health. Numerous studies have demonstrated the anti-cancer role of GEN in breast cancer (240, 287, 288). Importantly, exposure to GEN in early life was found to diminish the mammary terminal end buds, which comprise a diverse range of undifferentiated cells susceptible to carcinogens. This implies that GEN may play a role in lowering the incidence of breast cancer (289). However, a recent study also found GEN potential proliferative effects at lower doses in breast cancer (240). Thus, more studies are needed to confirm the benefits of GEN exposure in promoting health of normal mammary epithelial cells and reducing the incidence of breast disease. Our findings strengthen a beneficial mode of action of GEN in mammary epithelial cells via epigenetic mechanisms. Significance This research enhances our understanding of the pivotal role played by SIRT1 in epigenetic mechanisms that mediate the impact of the environment, such as dietary factors PTS and GEN, on cellular biology. 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Thesis/Dissertation
eng
Human Nutrition
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Stefanska, Barbara
Investigating the role of SIRT1 in epigenetic effects of dietary phytoestrogens in human mammary epithelial cells
Text