TEAD2 initiates ground-state pluripotency by mediating chromatin looping.

The transition of mouse embryonic stem cells (ESCs) between serum/LIF and 2i(MEK and GSK3 kinase inhibitor)/LIF culture conditions serves as a valuable model for exploring the mechanisms underlying ground and confused pluripotent states. Regulatory networks comprising core and ancillary pluripotency factors drive the gene expression programs defining stable naïve pluripotency. In our study, we systematically screened factors essential for ESC pluripotency, identifying TEAD2 as an ancillary factor maintaining ground-state pluripotency in 2i/LIF ESCs and facilitating the transition from serum/LIF to 2i/LIF ESCs. TEAD2 exhibits increased binding to chromatin in 2i/LIF ESCs, targeting active chromatin regions to regulate the expression of 2i-specific genes. In addition, TEAD2 facilitates the expression of 2i-specific genes by mediating enhancer-promoter interactions during the serum/LIF to 2i/LIF transition. Notably, deletion of Tead2 results in reduction of a specific set of enhancer-promoter interactions without significantly affecting binding of chromatin architecture proteins, CCCTC-binding factor (CTCF), and Yin Yang 1 (YY1). In summary, our findings highlight a novel prominent role of TEAD2 in orchestrating higher-order chromatin structures of 2i-specific genes to sustain ground-state pluripotency.

YTHDF1 facilitates PRC1-mediated H2AK119ub in human ES cells.

Polycomb repressive complexes (PRCs) play critical roles in cell fate decisions during normal development as well as disease progression through mediating histone modifications such as H3K27me3 and H2AK119ub. How exactly PRCs recruited to chromatin remains to be fully illuminated. Here, we report that YTHDF1, the N6-methyladenine (m6 A) RNA reader that was previously known to be mainly cytoplasmic, associates with RNF2, a PRC1 protein that mediates H2AK119ub in human embryonic stem cells (hESCs). A portion of YTHDF1 localizes in the nuclei and associates with RNF2/H2AK119ub on a subset of gene loci related to neural development functions. Knock-down YTHDF1 attenuates H2AK119ub modification on these genes and promotes neural differentiation in hESCs. Our findings provide a noncanonical mechanism that YTHDF1 participates in PRC1 functions in hESCs.

Functional dissection of PRC1 subunits RYBP and YAF2 during neural differentiation of embryonic stem cells.

Polycomb repressive complex 1 (PRC1) comprises two different complexes: CBX-containing canonical PRC1 (cPRC1) and RYBP/YAF2-containing variant PRC1 (vPRC1). RYBP-vPRC1 or YAF2-vPRC1 catalyzes H2AK119ub through a positive-feedback model; however, whether RYBP and YAF2 have different regulatory functions is still unclear. Here, we show that the expression of RYBP and YAF2 decreases and increases, respectively, during neural differentiation of embryonic stem cells (ESCs). Rybp knockout impairs neural differentiation by activating Wnt signaling and derepressing nonneuroectoderm-associated genes. However, Yaf2 knockout promotes neural differentiation and leads to redistribution of RYBP binding, increases enrichment of RYBP and H2AK119ub on the RYBP-YAF2 cotargeted genes, and prevents ectopic derepression of nonneuroectoderm-associated genes in neural-differentiated cells. Taken together, this study reveals that RYBP and YAF2 function differentially in regulating mESC neural differentiation.

3D genome perspective on cell fate determination, organ regeneration, and diseases.

The nucleosome is the fundamental subunit of chromatin. Nucleosome structures are formed by the combination of histone octamers and genomic DNA. Through a systematic and precise process of folding and compression, these structures form a 30-nm chromatin fibre that is further organized within the nucleus in a hierarchical manner, known as the 3D genome. Understanding the intricacies of chromatin structure and the regulatory mode governing chromatin interactions is essential for unravelling the complexities of cellular architecture and function, particularly in relation to cell fate determination, regeneration, and the development of diseases. Here, we provide a general overview of the hierarchical structure of chromatin as well as of the evolution of chromatin conformation capture techniques. We also discuss the dynamic regulatory changes in higher-order chromatin structure that occur during stem cell lineage differentiation and somatic cell reprogramming, potential regulatory insights at the chromatin level in organ regeneration, and aberrant chromatin regulation in diseases.

RBBP4 is an epigenetic barrier for the induced transition of pluripotent stem cells into totipotent 2C-like cells.

Cellular totipotency is critical for whole-organism generation, yet how totipotency is established remains poorly illustrated. Abundant transposable elements (TEs) are activated in totipotent cells, which is critical for embryonic totipotency. Here, we show that the histone chaperone RBBP4, but not its homolog RBBP7, is indispensable for maintaining the identity of mouse embryonic stem cells (mESCs). Auxin-induced degradation of RBBP4, but not RBBP7, reprograms mESCs to the totipotent 2C-like cells. Also, loss of RBBP4 enhances transition from mESCs to trophoblast cells. Mechanistically, RBBP4 binds to the endogenous retroviruses (ERVs) and functions as an upstream regulator by recruiting G9a to deposit H3K9me2 on ERVL elements, and recruiting KAP1 to deposit H3K9me3 on ERV1/ERVK elements, respectively. Moreover, RBBP4 facilitates the maintenance of nucleosome occupancy at the ERVK and ERVL sites within heterochromatin regions through the chromatin remodeler CHD4. RBBP4 depletion leads to the loss of the heterochromatin marks and activation of TEs and 2C genes. Together, our findings illustrate that RBBP4 is required for heterochromatin assembly and is a critical barrier for inducing cell fate transition from pluripotency to totipotency.

CTCF acetylation at lysine 20 is required for the early cardiac mesoderm differentiation of embryonic stem cells.

The CCCTC-binding factor (CTCF) protein and its modified forms regulate gene expression and genome organization. However, information on CTCF acetylation and its biological function is still lacking. Here, we show that CTCF can be acetylated at lysine 20 (CTCF-K20) by CREB-binding protein (CBP) and deacetylated by histone deacetylase 6 (HDAC6). CTCF-K20 is required for the CTCF interaction with CBP. A CTCF point mutation at lysine 20 had no effect on self-renewal but blocked the mesoderm differentiation of mouse embryonic stem cells (mESCs). The CTCF-K20 mutation reduced CTCF binding to the promoters and enhancers of genes associated with early cardiac mesoderm differentiation, resulting in diminished chromatin accessibility and decreased enhancer-promoter interactions, impairing gene expression. In summary, this study reveals the important roles of CTCF-K20 in regulating CTCF genomic functions and mESC differentiation into mesoderm.

YY1 safeguard multidimensional epigenetic landscape associated with extended pluripotency.

Although extended pluripotent stem cells (EPSCs) have the potential to form both embryonic and extraembryonic lineages, how their transcriptional regulatory mechanism differs from that of embryonic stem cells (ESCs) remains unclear. Here, we discovered that YY1 binds to specific open chromatin regions in EPSCs. Yy1 depletion in EPSCs leads to a gene expression pattern more similar to that of ESCs than control EPSCs. Moreover, Yy1 depletion triggers a series of epigenetic crosstalk activities, including changes in DNA methylation, histone modifications and high-order chromatin structures. Yy1 depletion in EPSCs disrupts the enhancer-promoter (EP) interactions of EPSC-specific genes, including Dnmt3l. Yy1 loss results in DNA hypomethylation and dramatically reduces the enrichment of H3K4me3 and H3K27ac on the promoters of EPSC-specific genes by upregulating the expression of Kdm5c and Hdac6 through facilitating the formation of CCCTC-binding factor (CTCF)-mediated EP interactions surrounding their loci. Furthermore, single-cell RNA sequencing (scRNA-seq) experiments revealed that YY1 is required for the derivation of extraembryonic endoderm (XEN)-like cells from EPSCs in vitro. Together, this study reveals that YY1 functions as a key regulator of multidimensional epigenetic crosstalk associated with extended pluripotency.

CTCF functions as an insulator for somatic genes and a chromatin remodeler for pluripotency genes during reprogramming.

CTCF mediates chromatin insulation and long-distance enhancer-promoter (EP) interactions; however, little is known about how these regulatory functions are partitioned among target genes in key biological processes. Here, we show that Ctcf expression is progressively increased during induced pluripotency. In this process, CTCF first functions as a chromatin insulator responsible for direct silencing of the somatic gene expression program and, interestingly, elevated Ctcf expression next ensures chromatin accessibility and contributes to increased EP interactions for a fraction of pluripotency-associated genes. Therefore, CTCF functions in a context-specific manner to modulate the 3D genome to enable cellular reprogramming. We further discover that these context-specific CTCF functions also enlist SMARCA5, an imitation switch (ISWI) chromatin remodeler, together rewiring the epigenome to facilitate cell-fate switch. These findings reveal the dual functions of CTCF in conjunction with a key chromatin remodeler to drive reprogramming toward pluripotency.

RNA helicase DDX5 acts as a critical regulator for survival of neonatal mouse gonocytes.

OBJECTIVES: Mammalian spermatogenesis is a biological process of male gamete formation. Gonocytes are the only precursors of spermatogonial stem cells (SSCs) which develop into mature spermatozoa. DDX5 is one of DEAD-box RNA helicases and expresses in male germ cells, suggesting that Ddx5 plays important functions during spermatogenesis. Here, we explore the functions of Ddx5 in regulating the specification of gonocytes. MATERIALS AND METHODS: Germ cell-specific Ddx5 knockout (Ddx5-/- ) mice were generated. The morphology of testes and epididymides and fertility in both wild-type and Ddx5-/- mice were analysed. Single-cell RNA sequencing (scRNA-seq) was used to profile the transcriptome in testes from wild-type and Ddx5-/- mice at postnatal day (P) 2. Dysregulated genes were validated by single-cell qRT-PCR and immunofluorescent staining. RESULTS: In male mice, Ddx5 was expressed in germ cells at different stages of development. Germ cell-specific Ddx5 knockout adult male mice were sterile due to completely devoid of germ cells. Male germ cells gradually disappeared in Ddx5-/- mice from E18.5 to P6. Single-cell transcriptome analysis showed that genes involved in cell cycle and glial cell line-derived neurotrophic factor (GDNF) pathway were significantly decreased in Ddx5-deficient gonocytes. Notably, Ddx5 ablation impeded the proliferation of gonocytes. CONCLUSIONS: Our study reveals the critical roles of Ddx5 in fate determination of gonocytes, offering a novel insight into the pathogenesis of male sterility.

DNA Damage Induces Dynamic Associations of BRD4/P-TEFb With Chromatin and Modulates Gene Transcription in a BRD4-Dependent and -Independent Manner.

The bromodomain-containing protein BRD4 has been thought to transmit epigenetic information across cell divisions by binding to both mitotic chromosomes and interphase chromatin. UV-released BRD4 mediates the recruitment of active P-TEFb to the promoter, which enhances transcriptional elongation. However, the dynamic associations between BRD4 and P-TEFb and BRD4-mediated gene regulation after UV stress are largely unknown. In this study, we found that BRD4 dissociates from chromatin within 30 min after UV treatment and thereafter recruits chromatin. However, P-TEFb binds tightly to chromatin right after UV treatment, suggesting that no interactions occur between BRD4 and P-TEFb within 30 min after UV stress. BRD4 knockdown changes the distribution of P-TEFb among nuclear soluble and chromatin and downregulates the elongation activity of RNA polymerase II. Inhibition of JNK kinase but not other MAP kinases impedes the interactions between BRD4 and P-TEFb. RNA-seq and ChIP assays indicate that BRD4 both positively and negatively regulates gene transcription in cells treated with UV stress. These results reveal previously unrecognized dynamics of BRD4 and P-TEFb after UV stress and regulation of gene transcription by BRD4 acting as either activator or repressor in a context-dependent manner.

Systematic screening of CTCF binding partners identifies that BHLHE40 regulates CTCF genome-wide distribution and long-range chromatin interactions.

CTCF plays a pivotal role in mediating chromatin interactions, but it does not do so alone. A number of factors have been reported to co-localize with CTCF and regulate CTCF loops, but no comprehensive analysis of binding partners has been performed. This prompted us to identify CTCF loop participants and regulators by co-localization analysis with CTCF. We screened all factors that had ChIP-seq data in humans by co-localization analysis with human super conserved CTCF (hscCTCF) binding sites, and identified many new factors that overlapped with hscCTCF binding sites. Combined with CTCF loop information, we observed that clustered factors could promote CTCF loops. After in-depth mining of each factor, we found that many factors might have the potential to promote CTCF loops. Our data further demonstrated that BHLHE40 affected CTCF loops by regulating CTCF binding. Together, this study revealed that many factors have the potential to participate in or regulate CTCF loops, and discovered a new role for BHLHE40 in modulating CTCF loop formation.

R-loops coordinate with SOX2 in regulating reprogramming to pluripotency.

R-loops modulate genome stability and regulate gene expression, but the functions and the regulatory mechanisms of R-loops in stem cell biology are still unclear. Here, we profiled R-loops during somatic cell reprogramming and found that dynamic changes in R-loops are essential for reprogramming and occurred before changes in gene expression. Disrupting the homeostasis of R-loops by depleting RNaseH1 or catalytic inactivation of RNaseH1 at D209 (RNaseH1D209N) blocks reprogramming. Sox2, but not any other factor in the Yamanaka cocktail, overcomes the inhibitory effects of RNaseH1 activity loss on reprogramming. Sox2 interacts with the reprogramming barrier factor Ddx5 and inhibits the resolvase activity of Ddx5 on R-loops and thus facilitates reprogramming. Furthermore, reprogramming efficiency can be modulated by dCas9-mediated RNaseH1/RNaseH1D209N targeting the specific R-loop regions. Together, these results show that R-loops play important roles in reprogramming and shed light on the regulatory module of Sox2/Ddx5 on R-loops during reprogramming.

Generation of Rybp homozygous knockout murine ES cell line GIBHe001-A-1 by using CRISPR/Cas9 technology.

RYBP (Ring1 and YY1 Binding Protein) is critical for pluripotency and differentiation of embryonic stem cells (ESCs). RYBP depletion disturbs both neural and myocardial differentiation of ESCs. Moreover, low level of RYBP is correlated with diseases such as glioblastoma. To study the biological function of RYBP in neural differentiation of ESCs, here we generated Rybp homozygous knockout murine ESC line based on Sox1-GFP reporter using CRISPR/Cas9 genome editing technology. The last two exons of Rybp gene in which contain 115 amino acids have been replaced with PGK-Pruo by homologous recombination.

An alternative CTCF isoform antagonizes canonical CTCF occupancy and changes chromatin architecture to promote apoptosis.

CTCF plays key roles in gene regulation, chromatin insulation, imprinting, X chromosome inactivation and organizing the higher-order chromatin architecture of mammalian genomes. Previous studies have mainly focused on the roles of the canonical CTCF isoform. Here, we explore the functions of an alternatively spliced human CTCF isoform in which exons 3 and 4 are skipped, producing a shorter isoform (CTCF-s). Functionally, we find that CTCF-s competes with the genome binding of canonical CTCF and binds a similar DNA sequence. CTCF-s binding disrupts CTCF/cohesin binding, alters CTCF-mediated chromatin looping and promotes the activation of IFI6 that leads to apoptosis. This effect is caused by an abnormal long-range interaction at the IFI6 enhancer and promoter. Taken together, this study reveals a non-canonical function for CTCF-s that antagonizes the genomic binding of canonical CTCF and cohesin, and that modulates chromatin looping and causes apoptosis by stimulating IFI6 expression.

The chromatin remodeling protein INO80 contributes to the removal of H2A.Z at the p53-binding site of the p21 gene in response to doxorubicin.

Transcriptional activation of p21 (cyclin-dependent kinase inhibitor 1A) due to DNA damage often alters the distribution of histone variant H2A.Z at the p21 gene. However, whether the human INO80 complex regulates changes in H2A.Z at the p21 promoter is unclear. We show here that activation of p21 expression by doxorubicin (Doxo) in U2OS cells is required for removal of H2A.Z by INO80 at the p53-binding site proximal region (-2.2 kb) of the p21 promoter. A purified INO80 complex, but not the INO80E653Q mutant-complex, which lost DNA-sliding activity, is mainly responsible for removing H2A.Z from reconstituted nucleosomes in vitro. This activity was enhanced with MOF-mediated histone acetylation, suggesting that INO80 more readily removes H2A.Z from loosened nucleosomes. Also, co-occupancy of INO80 and H2A.Z -2.2 kb upstream of the p21 transcriptional start site (TSS) was observed. H2A.Z at this region was removed in a short time after Doxo treatment and activated p21 expression. However, p21 induction was inhibited by INO80 knockdown by delaying H2A.Z removal, indicating the need for INO80. Moreover, shMOF-mediated histone acetylation reduced recruitment of INO80 -2.2 kb upstream of p21 TSS and inhibited the removal of H2A.Z in Doxo-treated cells. These data provide new insights into the transcriptional regulation of p21 by the INO80 complex.

PCGF5 is required for neural differentiation of embryonic stem cells.

Polycomb repressive complex 1 (PRC1) is an important regulator of gene expression and development. PRC1 contains the E3 ligases RING1A/B, which monoubiquitinate lysine 119 at histone H2A (H2AK119ub1), and has been sub-classified into six major complexes based on the presence of a PCGF subunit. Here, we report that PCGF5, one of six PCGF paralogs, is an important requirement in the differentiation of mouse embryonic stem cells (mESCs) towards a neural cell fate. Although PCGF5 is not required for mESC self-renewal, its loss blocks mESC neural differentiation by activating the SMAD2/TGF-ß signaling pathway. PCGF5 loss-of-function impairs the reduction of H2AK119ub1 and H3K27me3 around neural specific genes and keeps them repressed. Our results suggest that PCGF5 might function as both a repressor for SMAD2/TGF-ß signaling pathway and a facilitator for neural differentiation. Together, our findings reveal a critical context-specific function for PCGF5 in directing PRC1 to control cell fate.

Genome-wide DNA methylation analysis reveals that mouse chemical iPSCs have closer epigenetic features to mESCs than OSKM-integrated iPSCs.

Induced pluripotent stem cells can be derived from somatic cells through ectopic expression of transcription factors or chemical cocktails. Chemical iPSCs (C-iPSCs) and OSKM-iPSCs (4F-iPSCs) have been suggested to have similar characteristics to mouse embryonic stem cells (mESCs). However, their epigenetic equivalence remains incompletely understood throughout the genome. In this study, we have generated mouse C-iPSCs and 4F-iPSCs, and further compared the genome-wide DNA methylomes of C-iPSCs, 4F-iPSCs, and mESCs that were maintained in 2i and LIF. Three pluripotent stem cells tend to be low methylated overall, however, DNA methylations in some specific regions (such as retrotransposons) are cell type-specific. Importantly, C-iPSCs are more hypomethylated than 4F-iPSCs. Bisulfite sequencing indicated that DNA methylation status in several known imprinted clusters, such as: Dlk1-Dio3 and Peg12-Ube3a, in C-iPSCs are closer to those of mESCs than 4F-iPSCs. Overall, our data demonstrate the reprogramming methods-dependent epigenetic differences of C-iPSCs and 4F-iPSCs and reveal that C-iPSCs are more hypomethylated than OSKM-integrated iPSCs.

PRC2 specifies ectoderm lineages and maintains pluripotency in primed but not naïve ESCs.

Polycomb repressive complex 2 and the epigenetic mark that it deposits, H3K27me3, are evolutionarily conserved and play critical roles in development and cancer. However, their roles in cell fate decisions in early embryonic development remain poorly understood. Here we report that knockout of polycomb repressive complex 2 genes in human embryonic stem cells causes pluripotency loss and spontaneous differentiation toward a meso-endoderm fate, owing to de-repression of BMP signalling. Moreover, human embryonic stem cells with deletion of EZH1 or EZH2 fail to differentiate into ectoderm lineages. We further show that polycomb repressive complex 2-deficient mouse embryonic stem cells also release Bmp4 but retain their pluripotency. However, when converted into a primed state, they undergo spontaneous differentiation similar to that of hESCs. In contrast, polycomb repressive complex 2 is dispensable for pluripotency when human embryonic stem cells are converted into the naive state. Our studies reveal both lineage- and pluripotent state-specific roles of polycomb repressive complex 2 in cell fate decisions.Polycomb repressive complex 2 (PRC2) plays an essential role in development by modifying chromatin but what this means at a cellular level is unclear. Here, the authors show that ablation of PRC2 genes in human embryonic stem cells and in mice results in changes in pluripotency and the primed state of cells.

MyoD- and FoxO3-mediated hotspot interaction orchestrates super-enhancer activity during myogenic differentiation.

Super-enhancers (SEs) are cis-regulatory elements enriching lineage specific key transcription factors (TFs) to form hotspots. A paucity of identification and functional dissection promoted us to investigate SEs during myoblast differentiation. ChIP-seq analysis of histone marks leads to the uncovering of SEs which remodel progressively during the course of differentiation. Further analyses of TF ChIP-seq enable the definition of SE hotspots co-bound by the master TF, MyoD and other TFs, among which we perform in-depth dissection for MyoD/FoxO3 interaction in driving the hotspots formation and SE activation. Furthermore, using Myogenin as a model locus, we elucidate the hierarchical and complex interactions among hotspots during the differentiation, demonstrating SE function is propelled by the physical and functional cooperation among hotspots. Finally, we show MyoD and FoxO3 are key in orchestrating the Myogenin hotspots interaction and activation. Altogether our results identify muscle-specific SEs and provide mechanistic insights into the functionality of SE.