Relaxed 3D genome conformation facilitates the pluripotent to totipotent-like state transition in embryonic stem cells

The 3D genome organization is crucial for gene regulation. Although recent studies have revealed a uniquely relaxed genome conformation in totipotent early blastomeres of both fertilized and cloned embryos, how weakened higher-order chromatin structure is functionally linked to totipotency acquisition remains elusive. Using low-input Hi-C, ATAC-seq and ChIP-seq, we systematically examined the dynamics of 3D genome and epigenome during pluripotent to totipotent-like state transition in mouse embryonic stem cells (ESCs). The spontaneously converted 2-cell-embryo-like cells (2CLCs) exhibited more relaxed chromatin architecture compared to ESCs, including global weakening of both enhancer-promoter interactions and TAD insulation. While the former correlated with inactivation of ESC enhancers and down-regulation of pluripotent genes, the latter might facilitate contacts between the putative new enhancers arising in 2CLCs and neighboring 2C genes. Importantly, disruption of chromatin organization by depleting CTCF or the cohesin complex promoted the ESC to 2CLC transition. Our results thus establish a critical role of 3D genome organization in totipotency acquisition.     

Histone h1 depletion impairs embryonic stem cell differentiation

Pluripotent embryonic stem cells (ESCs) are known to possess a relatively open chromatin structure; yet, despite efforts to characterize the chromatin signatures of ESCs, the role of chromatin compaction in stem cell fate and function remains elusive. Linker histone H1 is important for higher-order chromatin folding and is essential for mammalian embryogenesis. To investigate the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, we examine the differentiation of embryonic stem cells that are depleted of multiple H1 subtypes. H1c/H1d/H1e triple null ESCs are more resistant to spontaneous differentiation in adherent monolayer culture upon removal of leukemia inhibitory factor. Similarly, the majority of the triple-H1 null embryoid bodies (EBs) lack morphological structures representing the three germ layers and retain gene expression signatures characteristic of undifferentiated ESCs. Furthermore, upon neural differentiation of EBs, triple-H1 null cell cultures are deficient in neurite outgrowth and lack efficient activation of neural markers. Finally, we discover that triple-H1 null embryos and EBs fail to fully repress the expression of the pluripotency genes in comparison with wild-type controls and that H1 depletion impairs DNA methylation and changes of histone marks at promoter regions necessary for efficiently silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. In summary, we demonstrate that H1 plays a critical role in pluripotent stem cell differentiation, and our results suggest that H1 and chromatin compaction may mediate pluripotent stem cell differentiation through epigenetic repression of the pluripotency genes.     

Reduction of Hox gene expression by histone H1 depletion

The evolutionarily conserved homeotic (Hox) genes are organized in clusters and expressed collinearly to specify body patterning during embryonic development. Chromatin reorganization and decompaction are intimately connected with Hox gene activation. 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. To investigate the potential role of H1 and higher order chromatin folding in the regulation of Hox gene expression, we systematically analyzed the expression of all 39 Hox genes in triple H1 null mouse embryos and ESCs by quantitative RT-PCR. Surprisingly, we find that H1 depletion causes significant reduction in the expression of a broad range of Hox genes in embryos and ESCs. To examine if any of the three H1 subtypes (H1c, H1d and H1e) is responsible for decreased expression of Hox gene in triple-H1 null ESCs, we derived and characterized H1c(-/-), H1d(-/-), and H1e(-/-) single-H1 null ESCs. We show that deletion of individual H1 subtypes results in down-regulation of specific Hox genes in ESCs. Finally we demonstrate that, in triple-H1- and single-H1-null ESCs, the levels of H3K4 trimethylation (H3K4me3) and H3K27 trimethylation (H3K27me3) were affected at specific Hox genes with decreased expression. Our data demonstrate that marked reduction in total H1 levels causes significant reduction in both expression and the level of active histone mark H3K4me3 at many Hox genes and that individual H1 subtypes may also contribute to the regulation of specific Hox gene expression. We suggest possible mechanisms for such an unexpected role of histone H1 in Hox gene regulation.     

miR-371-3 expression predicts neural differentiation propensity in human pluripotent stem cells

The use of pluripotent stem cells in regenerative medicine and disease modeling is complicated by the variation in differentiation properties between lines. In this study, we characterized 13 human embryonic stem cell (hESC) and 26 human induced pluripotent stem cell (hiPSC) lines to identify markers that predict neural differentiation behavior. At a general level, markers previously known to distinguish mouse ESCs from epiblast stem cells (EPI-SCs) correlated with neural differentiation behavior. More specifically, quantitative analysis of miR-371-3 expression prospectively identified hESC and hiPSC lines with differential neurogenic differentiation propensity and in vivo dopamine neuron engraftment potential. Transient KLF4 transduction increased miR-371-3 expression and altered neurogenic behavior and pluripotency marker expression. Conversely, suppression of miR-371-3 expression in KLF4-transduced cells rescued neural differentiation propensity. miR-371-3 expression level therefore appears to have both a predictive and a functional role in determining human pluripotent stem cell neurogenic differentiation behavior.     

Differential contribution to gene expression prediction of histone modifications at enhancers or promoters

The ChIP-seq signal of histone modifications at promoters is a good predictor of gene expression in different cellular contexts, but whether this is also true at enhancers is not clear. To address this issue, we develop quantitative models to characterize the relationship of gene expression with histone modifications at enhancers or promoters. We use embryonic stem cells (ESCs), which contain a full spectrum of active and repressed (poised) enhancers, to train predictive models. As many poised enhancers in ESCs switch towards an active state during differentiation, predictive models can also be trained on poised enhancers throughout differentiation and in development. Remarkably, we determine that histone modifications at enhancers, as well as promoters, are predictive of gene expression in ESCs and throughout differentiation and development. Importantly, we demonstrate that their contribution to the predictive models varies depending on their location in enhancers or promoters. Moreover, we use a local regression (LOESS) to normalize sequencing data from different sources, which allows us to apply predictive models trained in a specific cellular context to a different one. We conclude that the relationship between gene expression and histone modifications at enhancers is universal and different from promoters. Our study provides new insight into how histone modifications relate to gene expression based on their location in enhancers or promoters.