Epigenetic plasticity of cultured female human embryonic stem cells and regulation of gene expression and chromatin by PR-SET7 mediated H4K20me1.

摘要

Epigenetics is the study of changes in gene expression that occur in cells without alterations to DNA sequence. Epigenetic modifications are critical components of eukaryotic gene regulation and chromatin organization. Different epigenetic mechanisms, including the post-translational modifications of DNA-associated histone proteins play a role in the activation or repression of genes.;One of my research goals was to define the epigenetic signature of cultured human embryonic stem cells (hESCs) and to determine how their epigenomes change during lineage commitment. Pluripotent hESCs are capable of self-renewal and have the capacity to differentiate into any lineage of the embryo. However, hESCs grown in culture are heterogeneous in nature, consisting of a mixture of pluripotent to differentiated cells, making investigation of pluripotent hESCs difficult. Therefore precise definition of pluripotent cells present in culture is critical in order to use these cells for future stem cell based therapies. Using a FACS based approach, I demonstrated that I was able to selectively isolate sub-populations from the bulk culture that expressed high levels of pluripotency factors (P2) and those that lacked these factors (P5). For both populations, I performed high resolution ChIP-sequencing for 2 different histone modifications, indicative of transcriptionally active regions (H3K4me3) and repressed regions (H3K27me3) in order to define, compare and contrast their epigenomes. In the widely used female H9 hESC line, I found that the X-chromosome of the P5 population was enriched for H3K27me3 but was not in the P2 population. These findings strongly suggest that P2 represents the more naïve pluripotent stem cells, whereas, P5 has committed to differentiate, consistent with X-chromosome inactivation (Xi). In a separate female hESC line, HES3, I discovered that low passage cells (LP) are devoid of H3K27me3 on the X-chromosome but high passage cells (HP) are enriched for H3K27me3 on the X-chromosome. These findings indicated that extended passage length of hESCs in culture can have a dramatic effect on their epigenetic signature. My detailed analysis of these data sets revealed many novel findings. In LP P2 cells, I defined for the first time, the presence of a non-canonical H3K4me3 profile which is characterized by lowly enriched H3K4me3 domains many kilobases long, and spanning protein families such as zinc finger, keratin, olfactory and extra cellular matrix protein families. The function of these long domains in hESCs are unknown and previously undefined. I have also detected many stochastic quantitative differences in H3K4me3 and/or H3K27me3 between P2 and P5 that are not conserved between cell types, suggesting that embryonic stem cells are epigenetically plastic. However, more importantly, I have identified a core subset of genes, promoters and regulatory regions that contain quantitative epigenetic differences between P2 and P5 and correlate with gene expression changes. These conserved regions may play a critical role in early differentiation or maintenance of pluripotency. Overall, I have defined a unique epigenetic signature of purified pluripotent stem cells and identified conserved epigenetic changes that likely play an important role in the maintenance of pluripotent state or play a role in the commitment to differentiation (Chapter 1).;My second research goal was to investigate the role of the PR-SET7 H4K20 mono-methyltransferase (H4K20me1) in the transcriptional regulation of specific genes. Although PR-SET7-mediated H4K20me1 was previously shown to be involved in several DNA-templated processes including chromatin compaction, DNA damage response, DNA replication and cell cycle progression, the role of H420me1 in transcriptional regulation remains controversial. Initial studies showed that H4K20me1 functioned as a repressor but newer studies suggested a role in activation. Using conventional molecular biology techniques, I found that PR-SET7 and H4K20me1 predominantly functions as a transcriptional repressor of specific sets of genes. Consistent with this, my bioinformatics analysis indicated that H4K20me1 associated genes are largely devoid of acetylated histones; marks of transcriptionally active genes. In addition, I discovered that H4K20me1-associated genes are typically cell-type specific, but also, ∼500 highly transcribed metabolic genes are conserved for H4K20me1 across cell types. Also, I showed that genes enriched with PR-SET7 and H4K20me1 were physically distinct on gene bodies and are highly expressed in the genome compared to genes modified for either H4K20me1 or PR-SET7. My results demonstrated that PR-SET7-mediated H4K20me1 functions to repress certain genes in a cell-type specific manner, regardless of basal levels of expression. However, my study also suggested that H4K20me1 together with localization of PR-SET7 might have a distinct function associated with highly expressed genes, compared with genes modified with only H4K20me1 or PR-SET7, which are expressed at lower levels (Chapter 2).