Chapter 1: The pluripotent state is maintained by a network of “core” transcription factors (TF). REX1 (Reduced Expression-1) is a pluripotency related TF derived from retrotransposon-mediated duplication of the zinc finger TF Yin Yang 1 (YY1). Furthermore, expression of REX1 and YY1 induces changes in genes regulated by endogenous retroviral elements (ERV), suggesting an evolutionary origin of REX1 for ERV regulation. Studies suggest that murine REX1 may act in epigenetic regulation of gene expression and ERVs, but the precise mechanism remains unelucidated, so we generated FLAG-tagged REX1 pluripotent stem cell (PSC) lines, as well as a series of truncation mutants to explore the REX1 function. Our studies indicate the presence of previously undescribed isoforms of the full-length REX1 protein, suggesting that regulation by REX1 may be more complex than initially appreciated. We hypothesize that REX1 regulates the expression of a sub-set of ERVs and REX1 isoforms regulate REX1 target genes in pluripotent stem cells. Previously, we performed REX1 ChIP-seq and found enrichment for REX1 binding at specific ERVs. Here, we show that differential expression of REX1 isoforms do not change the expression of ERVs. Furthermore, our REX1 KO lines show changes in expression of ERV family members and together with the ChIP data, suggest that REX1 may act as a negative regulator of some retroviral elements. However, further experiments reveal a potential compensation of REX1 KO, possibly by the homologous factors YY1 and YY2. Due to the limited nature and time constrain of our study, we did not find conclusive evidence to further elucidate the potential compensation mechanism and the characteristics of the REX1 isoforms. Our work provided a new avenue for exploring the functional importance of REX1 isoforms and the potential, YY1 and YY2 independent, regulatory role REX1.
Chapter 2: Mitotic bookmarking describes a potential mechanism involved in the stable propagation of cellular identity through the cell cycles. Candidate based studies have identified mitotic bookmarking factors (MBFs) that are retained on the mitotic chromatin and preserve the transcriptional memory of the cell. Nevertheless, there is a poor understanding of which proteins can serve as MBFs, as well as the chromatin dynamics of bookmarked sites during mitosis and the start of G1 phase. Previously, we designed a chromatin immunoprecipitation followed by mass spectrometry (ChIP-MS) assay to develop a global unbiased approach for identifying and characterizing novel MBFs. Using ChIP-MS, we identifed putative MBFs associated with the mitotic chromatin in pluripotent stem cells (PSCs) and used ATAC-seq to identify subsets of pluripotency-associated accessible gene regions that appear to be bookmarked by a variety of transcription factors, including PARP1, PSIP1, and HDGF. Here, we characterize the interaction of a putative MBF, not found in our ChIP-MS screen, NFYa, with PARP1 and, inconclusively, another putative MBF, DNMT1. Furthermore, we found that PWWP containing putative MBF, HDGF, has a potential role in pluripotency maintenance but it is not mitosis-specific. Due to the limited nature and time constrain of our study, we did not find conclusive evidence to establish the role of PSIP1 in PSC mitotic bookmarking. Our work provided a new avenue for exploring the functional importance of mitotic bookmarks in pluripotent maintenance. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/24930 |
| Date | January 2019 |
| Creators | Alam, Mohammad |
| Contributors | Draper, Jonathan, Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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