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search.filters.applied.f.advisor: Fan, Yuhong × End date: 2017 × Start date: 2013 × Item Type: Publication ×

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Dissecting the role of linker histone H1 variants in embryonic stem cells

2017-08-28 , Ho, Po-Yi

To further dissect the role of linker histone H1 in embryonic stem cells (ESCs), here, we have utilized a functional reconstitution approach to identify the regions of H1 proteins that are important to mediate neurite outgrowth during neural differentiation of ESCs. We first generated H1 reconstituted ESC lines by overexpressing exogenous H1d proteins in H1 depleted ESCs. We find that overexpression of H1d in H1 depleted ESCs significantly restored the neurite outgrowth capacity of embryoid bodies (EBs) formed from ESCs. Next, to dissect the role of individual domains of H1d in ESC differentiation, we constructed a series of vectors to express H1 deletion mutants in H1 depleted ESCs. Our results show that reconstitution with H1d-GD and H1d-NTD-GD increases neurite outgrowth of EBs, suggesting that the globular domain of H1d is critical in mediating the neurite outgrowth during neural differentiation of ESCs. Lastly, we investigated the potential role of H1 modifications in ESC differentiation. We constructed an expression vector encoding the H1d mutant (H1dK46R) containing a lysine-to-arginine mutation at site K46, and expressed H1dK46R in H1c/H1d/H1e triple knockout (H1 TKO) ESCs by stable transfection and analyzed chromatin binding and biochemical properties through HPLC analysis. Our results suggest that K46R mutation of H1d disrupts the function of H1d in mediating the neurite outgrowth of EBs, suggesting a critical role of post-translational modification(s) on H1d K46 in ESC differentiation. To further dissect the potential mechanisms underlying the defects of H1dK46R mutant, we set out to characterize and compare the mobility of H1dK46R and H1d in ESCs in vivo using fluorescent recovery after photobleaching (FRAP) assay. Results from FRAP assay suggest that K46R mutation decreases dynamic mobility of H1d in ESCs, which may partially contribute to the defects of H1dK46R in mediating proper ESC differentiation. In summary, through a series of studies aimed at dissection of different regions and sites of H1d, we pinpoint GD as a key domain of H1 in mediating neurite outgrowth during neural differentiation of ESCs. Furthermore, our results suggest that the modification(s) on K46 of H1d are critical for proper ESC differentiation and are likely to increase the dynamic plasticity of H1d.

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Genome-wide profiling of H1 linker histone variants in mouse embryonic stem cells

2014-01-14 , Cao, Kaixiang

H1 linker histone facilitates the formation of higher order chromatin structure and is essential for mammalian development. Mice have 11 H1 variants which are differentially regulated and conserved in human. Previous research indicates that H1 regulates the expression of specific genes in mouse embryonic stem cells (ESCs). However, whether individual variants have distinct functions and how H1 participates in gene regulation remain elusive. An investigation of the precise localization of individual H1 variants in vivo would facilitate the elucidation of mechanisms underlying chromatin compaction regulated gene expression, while it has been extremely difficult due to the lacking of specific antibodies toward H1 variants. In this dissertation, I have generated a knock-in system in ESCs and shown that the N-terminally tagged H1 proteins are functionally interchangeable to their endogenous counterparts in vivo. H1d and H1c are depleted from GC- and gene-rich regions and active promoters, inversely correlated with H3K4me3, but positively correlated with H3K9me3 and associated with characteristic sequence features. Surprisingly, both H1d and H1c are significantly enriched at major satellites, which display increased nucleosome spacing compared with bulk chromatin. While also depleted at active promoters and enriched at major satellites, overexpressed H10 displays differential binding patterns in specific repetitive sequences compared with H1d and H1c. Depletion of H1c, H1d ,and H1e causes pericentric chromocenter clustering and de-repression of major satellites. Collectively, these results integrate the localization of an understudied type of chromatin proteins, namely the H1 variants, into the epigenome map of mouse ESCs, and demonstrate significant changes at pericentric heterochromatin upon depletion of this epigenetic mark.

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Role of histone H1 in neural differentiation of embryonic stem cells

2015-07-27 , Pan, Chenyi

Linker histone H1 is a key structural protein facilitating the formation of higher order chromatin structures and regulates specific gene expression. In mammals, there exist 11 closely related H1 variants. Previous studies show that H1 depletion by 50% impairs specific gene regulation and differentiation of embryonic stem cells (ESCs). However, the mechanisms by which H1 and its variants regulate ESC differentiation remain elusive. Here, we demonstrate a dosage effect of H1 variants in mouse ESCs through severe H1 depletion and mutation analysis. We establish ultra-low H1 ESCs by sequential depletion of six somatic H1 variants. These cells exhibit normal ESC morphology and self-renewal. During neural differentiation, the total H1 level gradually increases, and H1 depletion reveals a dosage effect in neurite formation, induction of neural lineage-specific genes, and silencing of pluripotency-associated genes such as Oct4 and Nanog. In addition, severe H1 depletion causes reduced cell proliferation and cellular senescence in neural lineages. Significantly, Oct4 knockdown effectively restores neural differentiation and partially rescues the reduction in cell proliferation and cellular senescence. These results suggest that H1 is crucial for neural differentiation of ESCs and its regulation in the process acts in a dosage dependent, rather than a variant specific, manner. Another part of this thesis centers on analysis of H1 mutations frequently occurred in follicular lymphoma or transformed follicular lymphoma. These mutations in H1 are clustered in the globular and C-terminal domains directly involved in chromatin binding. By comparing the properties of wild-type human H1c (hH1c) and mutant hHcS102F expressed in H1c/H1d/H1e triple knockout mouse ESCs, we find that S102F mutation dramatically impairs the association of hH1c with chromatin. These results suggest that the identified H1 mutations in follicular lymphoma most likely result in a loss-of-function phenotype by reducing the binding affinity of H1 for chromatin, thus compromising chromatin compaction and the regulation of specific genes.

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Role of linker histone H1 in epigenetic regulation of pluripotency genes and Hox genes

2014-04-04 , Zhang, Yunzhe

Linker histone H1 plays a key role in facilitating folding of higher order chromatin structure. Previous studies have shown that deletion of three somatic H1 subtypes together leads to embryonic lethality and that H1c/H1d/H1e triple knockout (TKO) embryonic stem cells (ESCs) display bulk chromatin decompaction. Following this initial work, we investigated the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, as well as the regulation of Hox genes expression. We find that H1 TKO ESCs are more resistant to spontaneous differentiation, impaired in embryoid body differentiation, and largely blocked in neural differentiation. We present evidence that H1 contributes to efficient repression of the expression of pluripotency factors, Oct4 and Nanog, and participates in establishment and maintenance of DNA methylation and histone modification necessary for silencing pluripotency genes during stem cell differentiation and embryogenesis. In addition, we find reduced expression of a distinct set of Hox genes in embryos and ESCs, respectively. Furthermore, by characterizing H1c−/−; H1d−/−; and H1e−/− single-H1 null ESCs established in this study, we showed that individual H1 subtypes regulated specific Hox genes in ESCs. Finally, we demonstrate that the levels of H3K4me3 were significantly diminished at the affected Hox genes in H1 TKO- and single-H1 KO- ESCs, whereas H3K27me3 occupancy is modestly increased at specific Hox genes. Our results suggest that marked reduction of H1 levels and decondensation of bulk chromatin affect the expression of pluripotency genes and Hox genes in embryos and ESCs, which may be in part mediated through establishment and maintenance of epigenetic marks.

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