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Lysine-specific histone demethylase 1 (LSD1) is a histone demethylase that plays a critical role in epigenetic regulation by removing the methyl group from mono- and di-methylated lysine 4 on histone H3 (H3K4me1/2), acting as a repressor of gene expression. Recently, catalytically independent functions of LSD1, serving as a scaffold for assembling chromatin-regulator and transcription factor complexes, have been identified. Herein, we show for the first time that LSD1 interacts with chromodomain-helicase-DNA-binding protein 7 (CHD7) in mouse embryonic stem cells (ESCs). To further investigate the CHD7-LSD1 crosstalk, we engineered Chd7 and Chd7/Lsd1 knockout (KO) mouse ESCs. We show that CHD7 is dispensable for ESC self-renewal and survival, while Chd7 KO ESCs can differentiate towards embryoid bodies (EBs) with defective expression of ectodermal markers. Intriguingly, Chd7/Lsd1 double KO mouse ESCs exhibit proliferation defects similar to Lsd1 KO ESCs and have lost the capacity to differentiate properly. Furthermore, the increased co-occupancy of H3K4me1 and CHD7 on chromatin following Lsd1 deletion suggests that LSD1 is required for facilitating the proper binding of CHD7 to chromatin and regulating differentiation. Collectively, our results suggest that LSD1 and CHD7 work in concert to modulate gene expression and influence proper cell fate determination., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The TET family is well known for active DNA demethylation and plays important roles in regulating transcription, the epigenome and development. Nevertheless, previous studies using knockdown (KD) or knockout (KO) models to investigate the function of TET have faced challenges in distinguishing its enzymatic and nonenzymatic roles, as well as compensatory effects among TET family members, which has made the understanding of the enzymatic role of TET not accurate enough. To solve this problem, we successfully generated mice catalytically inactive for specific Tet members (Tet m/m ). We observed that, compared with the reported KO mice, mutant mice exhibited distinct developmental defects, including growth retardation, sex imbalance, infertility, and perinatal lethality. Notably, Tet m/m mouse embryonic stem cells (mESCs) were successfully established but entered an impaired developmental program, demonstrating extended pluripotency and defects in ectodermal differentiation caused by abnormal DNA methylation. Intriguingly, Tet3, traditionally considered less critical for mESCs due to its lower expression level, had a significant impact on the global hydroxymethylation, gene expression, and differentiation potential of mESCs. Notably, there were common regulatory regions between Tet1 and Tet3 in pluripotency regulation. In summary, our study provides a more accurate reference for the functional mechanism of Tet hydroxymethylase activity in mouse development and ESC pluripotency regulation., (© 2024. Science China Press.)

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., (© 2024. The Author(s).)

The mammalian DNA methylome is formed by two antagonizing processes, methylation by DNA methyltransferases (DNMT) and demethylation by ten-eleven translocation (TET) dioxygenases. Although the dynamics of either methylation or demethylation have been intensively studied in the past decade, the direct effects of their interaction on gene expression remain elusive. Here, we quantify the concurrence of DNA methylation and demethylation by the percentage of unmethylated CpGs within a partially methylated read from bisulfite sequencing. After verifying 'methylation concurrence' by its strong association with the co-localization of DNMT and TET enzymes, we observe that methylation concurrence is strongly correlated with gene expression. Notably, elevated methylation concurrence in tumors is associated with the repression of 40~60% of tumor suppressor genes, which cannot be explained by promoter hypermethylation alone. Furthermore, methylation concurrence can be used to stratify large undermethylated regions with negligible differences in average methylation into two subgroups with distinct chromatin accessibility and gene regulation patterns. Together, methylation concurrence represents a unique methylation metric important for transcription regulation and is distinct from conventional metrics, such as average methylation and methylation variation., (© 2021. The Author(s).)

During late embryonic development of the cerebral cortex, the major class of cortical output neurons termed subcerebral projection neurons (SCPN; including the predominant population of corticospinal neurons, CSN) and the class of interhemispheric callosal projection neurons (CPN) initially express overlapping molecular controls that later undergo subtype-specific refinements. Such molecular refinements are largely absent in heterogeneous, maturation-stalled, neocortical-like neurons (termed "cortical" here) spontaneously generated by established embryonic stem cell (ES) and induced pluripotent stem cell (iPSC) differentiation. Building on recently identified central molecular controls over SCPN development, we used a combination of synthetic modified mRNA (modRNA) for Fezf2, the central transcription factor controlling SCPN specification, and small molecule screening to investigate whether distinct chromatin modifiers might complement Fezf2 functions to promote SCPN-specific differentiation by mouse ES (mES)-derived cortical-like neurons. We find that the inhibition of a specific histone deacetylase, Sirtuin 1 (SIRT1), enhances refinement of SCPN subtype molecular identity by both mES-derived cortical-like neurons and primary dissociated E12.5 mouse cortical neurons. In vivo, we identify that SIRT1 is specifically expressed by CPN, but not SCPN, during late embryonic and postnatal differentiation. Together, these data indicate that SIRT1 has neuronal subtype-specific expression in the mouse cortex in vivo, and that its inhibition enhances subtype-specific differentiation of highly clinically relevant SCPN / CSN cortical neurons in vitro., Competing Interests: The authors have declared that no competing interests exist.

Pericentromeric heterochromatin (PCH), the constitutive heterochromatin of pericentromeric regions, plays crucial roles in various cellular events, such as cell division and DNA replication. PCH forms chromocenters in the interphase nucleus, and chromocenters cluster at the prophase of meiosis. Chromocenter clustering has been reported to be critical for the appropriate progression of meiosis. However, the molecular mechanisms underlying chromocenter clustering remain elusive. In this study, we found that global DNA hypomethylation, 5hmC enrichment in PCH, and chromocenter clustering of Dnmt1-KO ESCs were similar to those of the female meiotic germ cells. Tet1 is essential for the deposition of 5hmC and facultative histone marks of H3K27me3 and H2AK119ub at PCH, as well as chromocenter clustering. RING1B, one of the core components of PRC1, is recruited to PCH by TET1, and PRC1 plays a critical role in chromocenter clustering. In addition, the rearrangement of the chromocenter under DNA hypomethylated condition was mediated by liquid-liquid phase separation. Thus, we demonstrated a novel role of Tet1 in chromocenter rearrangement in DNA hypomethylated cells., Competing Interests: The authors have declared that no competing interests exist.

Mammalian genomes contain long domains with distinct average compositions of A/T versus G/C base pairs. In a screen for proteins that might interpret base composition by binding to AT-rich motifs, we identified the stem cell factor SALL4, which contains multiple zinc fingers. Mutation of the domain responsible for AT binding drastically reduced SALL4 genome occupancy and prematurely upregulated genes in proportion to their AT content. Inactivation of this single AT-binding zinc-finger cluster mimicked defects seen in Sall4 null cells, including precocious differentiation of embryonic stem cells (ESCs) and embryonic lethality in mice. In contrast, deletion of two other zinc-finger clusters was phenotypically neutral. Our data indicate that loss of pluripotency is triggered by downregulation of SALL4, leading to de-repression of a set of AT-rich genes that promotes neuronal differentiation. We conclude that base composition is not merely a passive byproduct of genome evolution and constitutes a signal that aids control of cell fate., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Site-specific replication fork barriers (RFBs) have proven valuable tools for studying mechanisms of repair at sites of replication fork stalling in prokaryotes and yeasts. We adapted the Escherichia coli Tus-Ter RFB for use in mammalian cells and used it to trigger site-specific replication fork stalling and homologous recombination (HR) at a defined chromosomal locus in mammalian cells. By comparing HR responses induced at the Tus-Ter RFB with those induced by a site-specific double-strand break (DSB), we have begun to uncover how the mechanisms of mammalian stalled fork repair differ from those underlying the repair of a replication-independent DSB. Here, we outline how to transiently express the Tus protein in mES cells, how to use flow cytometry to score conservative and aberrant repair outcomes, and how to quantify distinct repair outcomes in response to replication fork stalling at the inducible Tus-Ter chromosomal RFB.

Distinct cell types emerge from embryonic stem cells through a precise and coordinated execution of gene expression programs during lineage commitment. This is established by the action of lineage specific transcription factors along with chromatin complexes. Numerous studies have focused on epigenetic factors that affect embryonic stem cells (ESC) self-renewal and pluripotency. However, the contribution of chromatin to lineage decisions at the exit from pluripotency has not been as extensively studied. Using a pooled epigenetic shRNA screen strategy, we identified chromatin-related factors critical for differentiation toward mesodermal and endodermal lineages. Here we reveal a critical role for the chromatin protein, ARID4B. Arid4b-deficient mESCs are similar to WT mESCs in the expression of pluripotency factors and their self-renewal. However, ARID4B loss results in defects in up-regulation of the meso/endodermal gene expression program. It was previously shown that Arid4b resides in a complex with SIN3A and HDACS 1 and 2. We identified a physical and functional interaction of ARID4B with HDAC1 rather than HDAC2, suggesting functionally distinct Sin3a subcomplexes might regulate cell fate decisions Finally, we observed that ARID4B deficiency leads to increased H3K27me3 and a reduced H3K27Ac level in key developmental gene loci, whereas a subset of genomic regions gain H3K27Ac marks. Our results demonstrate that epigenetic control through ARID4B plays a key role in the execution of lineage-specific gene expression programs at pluripotency exit., (Copyright © 2020 © 2020 Terzi Cizmecioglu et al. Published by Elsevier Inc. All rights reserved.)

The multifunctional histone chaperone, SET, is essential for embryonic development in the mouse. Previously, we identified SET as a factor that is rapidly downregulated during embryonic stem cell (ESC) differentiation, suggesting a possible role in the maintenance of pluripotency. Here, we explore SET's function in early differentiation. Using immunoprecipitation coupled with protein quantitation by LC-MS/MS, we uncover factors and complexes, including P53 and β-catenin, by which SET regulates lineage specification. Knockdown for P53 in SET-knockout (KO) ESCs partially rescues lineage marker misregulation during differentiation. Paradoxically, SET-KO ESCs show increased expression of several Wnt target genes despite reduced levels of active β-catenin. Further analysis of RNA sequencing datasets hints at a co-regulatory relationship between SET and TCF proteins, terminal effectors of Wnt signaling. Overall, we discover a role for both P53 and β-catenin in SET-regulated early differentiation and raise a hypothesis for SET function at the β-catenin-TCF regulatory axis., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a crucial role in mouse embryonic stem cells (ESCs). In RNA also, 5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its physiological roles are still largely unknown. Here we show the contribution and function of this mark in mouse ESCs and differentiating embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of messenger RNAs marked by 5hmC at sites characterized by a defined unique consensus sequence and particular features. During differentiation a large number of transcripts, including many encoding key pluripotency-related factors (such as Eed and Jarid2), show decreased cytosine hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites showing a topology similar to that of 5hmC sites. Tet-mediated RNA hydroxymethylation is found to reduce the stability of crucial pluripotency-promoting transcripts. We propose that RNA cytosine 5-hydroxymethylation by Tets is a mark of transcriptome flexibility, inextricably linked to the balance between pluripotency and lineage commitment.

RNA decay is crucial for mRNA turnover and surveillance and misregulated in many diseases. This complex system is challenging to study, particularly in mammals, where it remains unclear whether decay pathways perform specialized versus redundant roles. Cytoplasmic pathways and links to translation are particularly enigmatic. By directly profiling decay factor targets and normal versus aberrant translation in mouse embryonic stem cells (mESCs), we uncovered extensive decay pathway specialization and crosstalk with translation. XRN1 (5'-3') mediates cytoplasmic bulk mRNA turnover whereas SKIV2L (3'-5') is universally recruited by ribosomes, tackling aberrant translation and sometimes modulating mRNA abundance. Further exploring translation surveillance revealed AVEN and FOCAD as SKIV2L interactors. AVEN prevents ribosome stalls at structured regions, which otherwise require SKIV2L for clearance. This pathway is crucial for histone translation, upstream open reading frame (uORF) regulation, and counteracting ribosome arrest on small ORFs. In summary, we uncovered key targets, components, and functions of mammalian RNA decay pathways and extensive coupling to translation., Competing Interests: Declaration of Interests The Friedrich Miescher Institute for Biomedical Research (FMI) receives significant financial contributions from the Novartis Research Foundation., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Tead4 is critical for blastocyst development and trophoblast differentiation. We assayed long-range chromosomal interactions on the Tead4 promoter in mouse embryonic stem (ES) cells and trophoblast stem (TS) cells. Using luciferase reporter assays with ES and TS cells for 34 candidate enhancer regions, we identified five genomic fragments that increased Tead4 promoter activity in a TS-specific manner. The five loci consisted of three intra- and two inter-chromosomal loci relative to Tead4 on chromosome 6. We established five mouse lines with one of the five enhancer elements deleted and evaluated the effect of each deletion on Tead4 expression in blastocysts. By quantitative RT-PCR, we measured a 42% decrease in Tead4 expression in the blastocysts with a homozygous deletion with a 1.5 kb genomic interval on chromosome 19 (n = 14) than in wild-type blastocysts. By conducting RNA-seq analysis, we confirmed the trans effect of this enhancer deletion on Tead4 without significant cis effects on its neighbor genes at least within a 1.7 Mb distance. Our results demonstrated that the genomic interval on chromosome 19 is required for the appropriate level of Tead4 expression in blastocysts and suggested that an inter-chromosomal enhancer-promoter interaction may be the underlying mechanism., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The epiblast is a pluripotent cell population first formed in preimplantation embryos, and its quality is important for proper development. Here, we examined the mechanisms of epiblast formation and found that the Hippo pathway transcription factor TEAD and its coactivator YAP regulate expression of pluripotency factors. After specification of the inner cell mass, YAP accumulates in the nuclei and activates TEAD. TEAD activity is required for strong expression of pluripotency factors and is variable in the forming epiblast. Cells showing low TEAD activity are eliminated from the epiblast through cell competition. Pluripotency factor expression and MYC control cell competition downstream of TEAD activity. Cell competition eliminates unspecified cells and is required for proper organization of the epiblast. These results suggest that induction of pluripotency factors by TEAD activity and elimination of unspecified cells via cell competition ensure the production of an epiblast with naive pluripotency., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Multiple pathways counteract DNA replication stress to prevent genomic instability and tumorigenesis. The recently identified human SDE2 is a genome surveillance protein regulated by PCNA, a DNA clamp and processivity factor at replication forks. Here, we show that SDE2 cleavage after its ubiquitin-like domain generates Lys-SDE2Ct, the C-terminal SDE2 fragment bearing an N-terminal Lys residue. Lys-SDE2Ct constitutes a short-lived physiological substrate of the Arg/N-end rule proteolytic pathway, in which UBR1 and UBR2 ubiquitin ligases mediate the degradation. The Arg/N-end rule and VCP/p97UFD1-NPL4 segregase cooperate to promote phosphorylation-dependent, chromatin-associated Lys-SDE2Ct degradation upon UVC damage. Conversely, cells expressing the degradation-refractory K78V mutant, Val-SDE2Ct, fail to induce RPA phosphorylation and single-stranded DNA formation, leading to defects in PCNA-dependent DNA damage bypass and stalled fork recovery. Together, our study elucidates a previously unappreciated axis connecting the Arg/N-end rule and the p97-mediated proteolysis with the replication stress response, working together to preserve replication fork integrity., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

SOX2 is a dose-dependent master stem cell protein that controls the self-renewal and pluripotency or multipotency of embryonic stem (ES) cells and many adult stem cells. We have previously found that SOX2 protein is monomethylated at lysine residues 42 and 117 by SET7 methyltransferase to promote SOX2 proteolysis, whereas LSD1 and PHF20L1 act on both methylated Lys-42 and Lys-117 to prevent SOX2 proteolysis. However, the mechanism by which the methylated SOX2 protein is degraded remains unclear. Here, we report that L3MBTL3, a protein with the m alignant- b rain- t umor (MBT) methylation-binding domain, is required for SOX2 proteolysis. Our studies showed that L3MBTL3 preferentially binds to the methylated Lys-42 in SOX2, although mutation of Lys-117 also partially reduces the interaction between SOX2 and L3MBTL3. The direct binding of L3MBTL3 to the methylated SOX2 protein leads to the recruitment of the CRL4 DCAF5 ubiquitin E3 ligase to target SOX2 protein for ubiquitin-dependent proteolysis. Whereas loss of either LSD1 or PHF20L1 destabilizes SOX2 protein and impairs the self-renewal and pluripotency of mouse ES cells, knockdown of L3MBTL3 or DCAF5 is sufficient to restore the protein levels of SOX2 and rescue the defects of mouse ES cells caused by LSD1 or PHF20L1 deficiency. We also found that retinoic acid-induced differentiation of mouse ES cells is accompanied by the enhanced degradation of the methylated SOX2 protein at both Lys-42 and Lys-117. Our studies provide novel insights into the mechanism by which the methylation-dependent degradation of SOX2 protein is controlled by the L3MBTL3-CRL4 DCAF5 ubiquitin ligase complex., (© 2019 Zhang et al.)

The nucleosome remodeling and deacetylase (NuRD) complex plays an important role in gene expression regulation, stem cell self-renewal, and lineage commitment. However, little is known about the dynamics of NuRD during cellular differentiation. Here, we study these dynamics using genome-wide profiling and quantitative interaction proteomics in mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs). We find that the genomic targets of NuRD are highly dynamic during differentiation, with most binding occurring at cell-type specific promoters and enhancers. We identify ZFP296 as an ESC-specific NuRD interactor that also interacts with the SIN3A complex. ChIP-sequencing in Zfp296 knockout (KO) ESCs reveals decreased NuRD binding both genome-wide and at ZFP296 binding sites, although this has little effect on the transcriptome. Nevertheless, Zfp296 KO ESCs exhibit delayed induction of lineage-specific markers upon differentiation to embryoid bodies. In summary, we identify an ESC-specific NuRD-interacting protein which regulates genome-wide NuRD binding and cellular differentiation.

Active DNA demethylation, mediated by O 2 -dependent ten-eleven translocation (TET) enzymes, has essential roles in regulating gene expression. TET kinetics assay is vital for revealing mechanisms of demethylation process. Here, by a metal-organic framework (MOF)-based optical O 2 sensor, we present the first demonstration on real-time TET2 kinetics assay in vitro. A series of luminescent Cu(I) dialkyl-1,2,4-triazolate MOFs were synthesized, which were noble-metal-free and able to intuitively response to dissolved O 2 in a wide range from cellular hypoxia (≤15 μM) to ambient condition (∼257 μM). By further immobilization of the MOFs onto transparent silicon rubber (MOF@SR) to construct O 2 film sensors, and real-time monitoring of O 2 consumption on MOF@SR over the reaction time, the complete TET2-mediated 5-methylcytosine (5mC) oxidation process were achieved. The method overcomes the limitations of the current off-line methods by considerably shortening the analytical time from 0.5-18 h to 10 min, and remarkably reducing the relative standard deviation from 10%-68% to 0.68%-4.2%. As a result, the Michaelis-Menten constant ( K m ) values of TET2 for 5mC and O 2 in ascorbic acid-free (AA - ) condition were precisely evaluated to be 24 ± 1 and 43.8 ± 0.3 μM, respectively. By comparative study on AA-containing (AA + ) conditions, and further establishing kinetics models, the stem-cell behavior of TETs was successfully predicted, and the effects of key factors (AA, O 2 , Fe 2+ ) on TETs were revealed, which were fully verified in mouse embryonic stem (mES) cells. The method is promising in wide application in kinetics analysis and cell behavior prediction of other important O 2 -related enzymes.

Haematopoietic cells arise from endothelial cells within the dorsal aorta of the embryo via a process called the endothelial-haematopoietic transition (EHT). This process crucially depends on the transcription factor RUNX1 which rapidly activates the expression of genes essential for haematopoietic development. Using an inducible version of RUNX1 in a mouse embryonic stem cell differentiation model we showed that prior to the EHT, haematopoietic genes are primed by the binding of the transcription factor FLI1. Once expressed, RUNX1 relocates FLI1 towards its binding sites. However, the nature of the transcription factor assemblies recruited by RUNX1 to reshape the chromatin landscape and initiate mRNA synthesis are unclear. Here, we performed genome-wide analyses of RUNX1-dependent binding of factors associated with transcription elongation to address this question. We demonstrate that RUNX1 induction moves FLI1 from distal ETS/GATA sites to RUNX1/ETS sites and recruits the basal transcription factors CDK9, BRD4, the Mediator complex and the looping factor LDB1. Our study explains how the expression of a single transcription factor can drive rapid and replication independent transitions in cellular shape which are widely observed in development and disease.

CpG islands are gene regulatory elements associated with the majority of mammalian promoters, yet how they regulate gene expression remains poorly understood. Here, we identify FBXL19 as a CpG island-binding protein in mouse embryonic stem (ES) cells and show that it associates with the CDK-Mediator complex. We discover that FBXL19 recruits CDK-Mediator to CpG island-associated promoters of non-transcribed developmental genes to prime these genes for activation during cell lineage commitment. We further show that recognition of CpG islands by FBXL19 is essential for mouse development. Together this reveals a new CpG island-centric mechanism for CDK-Mediator recruitment to developmental gene promoters in ES cells and a requirement for CDK-Mediator in priming these developmental genes for activation during cell lineage commitment., Competing Interests: ED, TK, AF, MN, YK, RK, BK, HK, RK No competing interests declared, (© 2018, Dimitrova et al.)

H3K4me3 is an important epigenetic modification that plays a critical role in maintaining self-renewal of mouse embryonic stem cells (mESCs). H3K4me3 is catalyzed mainly by the mixed lineage leukemia (MLL) methyl-transferase complex. ASH2L, a core subunit of the MLL complex, participates in regulating the open state of chromatin in mESCs. There are two isoforms of the ASH2L protein: ASH2L-1 (80 kDa), which only exists in mouse embryonic fibroblasts and ASH2L-2 (65 kDa), which is the predominant isoform in mESCs. The roles of Ash2l-1 and Ash2l-2 in mESCs have not yet been elucidated. In this study, we established Ash2l-1 -/- and Ash2l-2 -/- knockout mESCs using CRISPR/Cas9. Alkaline phosphatase (AP) staining, immunofluorescence staining, and qRT-PCR showed that there were no obvious differences on the expression level of AP and pluripotent transcription factors (Nanog, Oct4, sox2 and Klf4) among Ash2l-1 -/- mESCs, Ash2l-2 -/- mESCs and wild type (WT) mESCs. However, analysis of embryoid body (EB) differentiation showed that the expression level of Snai2 (ectoderm gene) and Gata4 (endoderm gene) in Ash2l-1 -/- EBs was significantly lower than that in WT EBs (P<0.01). Western blotting assay revealed that the expression of ASH2L-2 was significantly increased (P<0.01) in Ash2l-1 -/- mESCs and vice versa. However, there were no obvious differences on the genomic H3K4me3 level among Ash2l-1 -/- mESCs, Ash2l-2 -/- mESCs and WT mESCs. These results indicate that there exist compensation effects between Ash2l-1 and Ash2l-2. Bioinformatic analysis predicted that there were three and 16 potential binding sites for pluripotency transcription factors located in the promoter of Ash2l-1 and Ash2l-2, respectively. Theses transcription factors may mediate the compensation effect between Ash2l-1 and Ash2l-2. Collectively, these results indicate that the compensation effects between Ash2l-1 and Ash2l-2 may be involved in the maintenance of mESCs pluripotency and the regulation of genomic H3K4me3.

Background: The KZFP/KAP1 (KRAB zinc finger proteins/KRAB-associated protein 1) system plays a central role in repressing transposable elements (TEs) and maintaining parent-of-origin DNA methylation at imprinting control regions (ICRs) during the wave of genome-wide reprogramming that precedes implantation. In naïve murine embryonic stem cells (mESCs), the genome is maintained highly hypomethylated by a combination of TET-mediated active demethylation and lack of de novo methylation, yet KAP1 is tethered by sequence-specific KZFPs to ICRs and TEs where it recruits histone and DNA methyltransferases to impose heterochromatin formation and DNA methylation., Results: Here, upon removing either KAP1 or the cognate KZFP, we observed rapid TET2-dependent accumulation of 5hmC at both ICRs and TEs. In the absence of the KZFP/KAP1 complex, ICRs lost heterochromatic histone marks and underwent both active and passive DNA demethylation. For KAP1-bound TEs, 5mC hydroxylation correlated with transcriptional reactivation. Using RNA-seq, we further compared the expression profiles of TEs upon Kap1 removal in wild-type, Dnmt and Tet triple knockout mESCs. While we found that KAP1 represents the main effector of TEs repression in all three settings, we could additionally identify specific groups of TEs further controlled by DNA methylation. Furthermore, we observed that in the absence of TET proteins, activation upon Kap1 depletion was blunted for some TE integrants and increased for others., Conclusions: Our results indicate that the KZFP/KAP1 complex maintains heterochromatin and DNA methylation at ICRs and TEs in naïve embryonic stem cells partly by protecting these loci from TET-mediated demethylation. Our study further unveils an unsuspected level of complexity in the transcriptional control of the endovirome by demonstrating often integrant-specific differential influences of histone-based heterochromatin modifications, DNA methylation and 5mC oxidation in regulating TEs expression.

Unipotent spermatogonial stem cells (SSCs) can be efficiently reprogrammed into pluripotent stem cells only by manipulating the culture condition, without introducing exogenous reprogramming factors. This phenotype raises the hypothesis that the endogenous transcription factors (TFs) in SSCs may facilitate reprogramming to acquire pluripotency. In this study, we screened a pool of SSCs TFs (Bcl6b, Lhx1, Foxo1, Plzf, Id4, Taf4b, and Etv5), and found that oncogene Etv5 could dramatically increase the efficiency of induced pluripotent stem cells (iPSCs) generation when combined with Yamanaka factors. We also demonstrated that Etv5 could promote mesenchymal-epithelial transition (MET) at the early stage of reprogramming by regulating Tet2-miR200s-Zeb1 axis. In addition, Etv5 knockdown in mouse embryonic stem cells (mESCs) could decrease the genomic 5hmC level by downregulating Tet2. Furthermore, the embryoid body assay revealed that Etv5 could positively regulate primitive endoderm specification through regulating Gata6 and negatively regulate epiblast specification by inhibiting Fgf5 expression. In summary, our findings provide insights into understanding the regulation mechanisms of Etv5 under the context of somatic reprogramming, mESCs maintenance, and differentiation.

The human Baf (Brg1/Brm associated factor) complex, also known as the mammalian SWI/SNF chromatin-remodeling complex, is involved in a variety of cellular processes. The pluripotency and self-renewal abilities are major characteristics of embryonic stem (ES) cells and are regulated by the ES cell-specific BAF (esBAF) complex. Baf53a is one of the subunits of the esBAF complex. Here, we found that Baf53a was expressed in undifferentiated ES cells and that it interacted with Oct3/4. Analyses of tetracycline-inducible Baf53a conditional knockout ES cells revealed that the undifferentiated markers, including Nanog and Oct3/4, were expressed in Baf53a-deficient ES cells; however, growth of the cells was repressed, and expression of p53, p21, and cleaved Caspase 3 was increased. Cell death of Baf53a-deficient ES cells was rescued by overexpression of Baf53a, but not by the Baf53a M3 mutant (E388A/R389A/R390A). Interestingly, Baf53b, a homologue of Baf53a, rescued cell death of Baf53a-deficient ES cells. Baf53a-deficient ES cells overexpressing exogenous Baf53a or Baf53b remained in the undifferentiated state, proliferated, and repressed expression of p21. In summary, our findings suggest that Baf53a is involved in the survival of ES cells by regulating p53 and Caspase3, and that Baf53b is able to compensate for this functional aspect of Baf53a.

LMO2 is a bridging factor within a DNA binding complex and is required for definitive haematopoiesis to occur. The developmental stage of the block in haematopoietic specification is not known. We show that Lmo2-/- mouse embryonic stem cells differentiated to Flk-1+ haemangioblasts, but less efficiently to haemogenic endothelium, which only produced primitive haematopoietic progenitors. Genome-wide approaches indicated that LMO2 is required at the haemangioblast stage to position the TAL1/LMO2/LDB1 complex to regulatory elements that are important for the establishment of the haematopoietic developmental program. In the absence of LMO2, the target site recognition of TAL1 is impaired. The lack of LMO2 resulted in altered gene expression levels already at the haemangioblast stage, with transcription factor genes accounting for ∼15% of affected genes. Comparison of Lmo2-/- with Tal1-/- Flk-1+ cells further showed that TAL1 was required to initiate or sustain Lmo2 expression., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The Cys2/His2-type zinc finger protein Zfp296 has been implicated in stem cell pluripotency and tumor pathogenesis. However, its mechanisms remain elusive. Here, we demonstrated that a Zfp296 deficiency in mice impairs germ-cell development and embryonic growth. Zfp296 was intracellularly localized to heterochromatin in embryos. A GST-Zfp296 pull-down experiment using ES cell nuclear extract followed by LC-MS/MS showed that Zfp296 interacts with component proteins of heterochromatin (such as HP1, Dnmt1, Dnmt3b, and ATRX) and the NuRD complex. We focused on H3K9 methylation as a hallmark of heterochromatin, and found that Zfp296 overexpression in cultured cells reduces the Suv39h1-mediated H3K9 methylation. Consistent with this finding, in Zfp296 -/- mouse embryos, we observed a global increase in H3K9 methylation in a developmental stage-dependent manner, and showed, by ChIP-qPCR, that the H3K9me3 levels at major satellite repeats were elevated in Zfp296 -/- embryos. Our results demonstrate that Zfp296 is a component of heterochromatin that affects embryonic development by negatively regulating H3K9 methylation.

THAP1 (THAP [Thanatos-associated protein] domain-containing, apoptosis-associated protein 1) is a ubiquitously expressed member of a family of transcription factors with highly conserved DNA-binding and protein-interacting regions. Mutations in THAP1 cause dystonia, DYT6, a neurologic movement disorder. THAP1 downstream targets and the mechanism via which it causes dystonia are largely unknown. Here, we show that wild-type THAP1 regulates embryonic stem cell (ESC) potential, survival, and proliferation. Our findings identify THAP1 as an essential factor underlying mouse ESC survival and to some extent, differentiation, particularly neuroectodermal. Loss of THAP1 or replacement with a disease-causing mutation results in an enhanced rate of cell death, prolongs Nanog, Prdm14, and/or Rex1 expression upon differentiation, and results in failure to upregulate ectodermal genes. ChIP-Seq reveals that these activities are likely due in part to indirect regulation of gene expression., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cytosine modifications diversify and structure the genome thereby controlling proper development and differentiation. Here, we focus on the interplay of the 5-methylcytosine reader Mbd1 and modifier Tet1 by analyzing their dynamic subcellular localization and the formation of the Tet oxidation product 5-hydroxymethylcytosine in mammalian cells. Our results demonstrate that Mbd1 enhances Tet1-mediated 5-methylcytosine oxidation. We show that this is due to enhancing the localization of Tet1, but not of Tet2 and Tet3 at heterochromatic DNA. We find that the recruitment of Tet1 and concomitantly its catalytic activity eventually leads to the displacement of Mbd1 from methylated DNA. Finally, we demonstrate that increased Tet1 heterochromatin localization and 5-methylcytosine oxidation are dependent on the CXXC3 domain of Mbd1, which recognizes unmethylated CpG dinucleotides. The Mbd1 CXXC3 domain deletion isoform, which retains only binding to methylated CpGs, on the other hand, blocks Tet1-mediated 5-methylcytosine to 5-hydroxymethylcytosine conversion, indicating opposite biological effects of Mbd1 isoforms. Our study provides new insights on how cytosine modifications, their modifiers and readers cross-regulate themselves., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Histone H2B lysine 120 mono-ubiquitination (H2Bub1) catalyzed by Rnf20 has been implicated in normal differentiation of embryonic stem (ES) and adult stem cells. However, it remains unknown how Rnf20 is recruited to its specific target chromosomal loci for the establishment of H2Bub1. Here, we reveal that Fbxl19, a CxxC domain-containing protein, promotes H2Bub1 at the promoters of CpG island-containing genes by interacting with Rnf20. We show that up-regulation of Fbxl19 increases the level of global H2Bub1 in mouse ES cells, while down-regulation of Fbxl19 reduces the level of H2Bub1. Our genome-wide target mapping unveils the preferential occupancy of Fbxl19 on CpG island-containing promoters, and we further discover that chromosomal binding of Fbxl19 is required for H2Bub1 of its targets. Moreover, we reveal that Fbxl19 is critical for proper differentiation of ES cells in collaboration with Rnf20. Altogether, our results demonstrate that Fbxl19 recruitment to CpG islands is required for Rnf20-mediated H2B mono-ubiquitination., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bag1 transcribes a multifunctional protein that participates in many important biological processes such as cell apoptosis, proliferation, differentiation and embryo development. Despite numerous published studies, the role of Bag1 in the context of embryonic stem (ES) cells, has not been explored. To investigate the function of Bag1 in ES cells, we generated mutant Bag1 -/- ES cells using the CRISPR/Cas9 system. We established that the Bag1 double knockout ES cell line maintained their pluripotency, possessed a normal karyotype and the ability to differentiate into all three germ layers., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Transition from symmetric to asymmetric cell division requires precise coordination of differential gene expression. We show that embryonic stem cells (ESCs) mainly express DIDO3 and that their differentiation after leukemia inhibitory factor withdrawal requires DIDO1 expression. C-terminal truncation of DIDO3 (Dido3ΔCT) impedes ESC differentiation while retaining self-renewal; small hairpin RNA-Dido1 ESCs have the same phenotype. Dido3ΔCT ESC differentiation is rescued by ectopic expression of DIDO3, which binds the Dido locus via H3K4me3 and RNA POL II and induces DIDO1 expression. DIDO1, which is exported to cytoplasm, associates with, and is N-terminally phosphorylated by PKCiota. It binds the E3 ubiquitin ligase WWP2, which contributes to cell fate by OCT4 degradation, to allow expression of primitive endoderm (PE) markers. PE formation also depends on phosphorylated DIDO3 localization to centrosomes, which ensures their correct positioning for PE cell polarization. We propose that DIDO isoforms act as a switchboard that regulates genetic programs for ESC transition from pluripotency maintenance to promotion of differentiation., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Geminin is implicated in regulation of the cell cycle and differentiation. Although loss of Geminin triggers unscheduled DNA rereplication as a result of interruption of its interaction with Cdt1 in some somatic cancer cells, whether such cell cycle regulation also operates in embryonic stem cells (ESCs) has remained unclear. To characterize the Geminin-Cdt1 axis in ESCs and compare it with that in somatic cells, we established conditional knockout (KO) of Geminin in mouse ESCs and mouse embryonic fibroblasts (MEFs). Geminin KO ESCs manifest a large flattened morphology, develop polyploidy accompanied by DNA damage and G 2 -M checkpoint activation, and subsequently undergo apoptosis. Rereplication in Geminin KO ESCs was attenuated by inhibition of G 2 -M checkpoint signaling or by expression of wild-type Geminin, but not by expression of a Geminin mutant that does not bind to Cdt1, indicating the importance of sequestration of Cdt1 by Geminin in G 2 phase. In contrast, Geminin KO MEFs did not manifest disturbance of the cell cycle unless they were treated to force abnormal accumulation of Cdt1. Together, our results indicate that Geminin is a key inhibitor of Cdt1 in mouse ESCs, but that it plays a backup role in MEFs to compensate for accidental up-regulation of Cdt1., (© 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

The spatiotemporal regulation of gene expression is central for cell-lineage specification during embryonic development and is achieved through the combinatorial action of transcription factors/co-factors and epigenetic states at cis-regulatory elements. Here, we show that in addition to implementing H3K4me3 at promoters of bivalent genes, Mll2 (KMT2B)/COMPASS can also implement H3K4me3 at a subset of non-TSS regulatory elements, a subset of which shares epigenetic signatures of active enhancers. Our mechanistic studies reveal that association of Mll2's CXXC domain with CpG-rich regions plays an instrumental role for chromatin targeting and subsequent implementation of H3K4me3. Although Mll2/COMPASS is required for H3K4me3 implementation on thousands of loci, generation of catalytically mutant MLL2/COMPASS demonstrated that H3K4me3 implemented by this enzyme was essential for expression of a subset of genes, including those functioning in the control of transcriptional programs during embryonic development. Our findings suggest that not all H3K4 trimethylations implemented by MLL2/COMPASS are functionally equivalent., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The methylcytosine oxidase TET proteins play important roles in DNA demethylation and development. However, it remains elusive how exactly they target substrates and execute oxidation. Interestingly, we found that, in mice, the full-length TET1 isoform (TET1e) is restricted to early embryos, embryonic stem cells (ESCs), and primordial germ cells (PGCs). By contrast, a short isoform (TET1s) is preferentially expressed in somatic cells, which lacks the N terminus including the CXXC domain, a DNA-binding module that often recognizes CpG islands (CGIs) where TET1 predominantly occupies. Unexpectedly, TET1s can still bind CGIs despite the fact that its global chromatin binding is significantly reduced. Interestingly, global chromatin binding, but not targeted binding at CGIs, is correlated with TET1-mediated demethylation. Finally, mice with exclusive expression of Tet1s failed to erase imprints in PGCs and displayed developmental defects in progeny. These data show that isoform switch of TET1 regulates epigenetic memory erasure and mouse development., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cytosine methylation is an epigenetic and regulatory mark that functions in part through recruitment of chromatin remodeling complexes containing methyl-CpG binding domain (MBD) proteins. Two MBD proteins, Mbd2 and Mbd3, were previously shown to bind methylated or hydroxymethylated DNA, respectively; however, both of these findings have been disputed. Here, we investigated this controversy using experimental approaches and re-analysis of published data and find no evidence for methylation-independent functions of Mbd2 or Mbd3. We show that chromatin localization of Mbd2 and Mbd3 is highly overlapping and, unexpectedly, we find Mbd2 and Mbd3 are interdependent for chromatin association. Further investigation reveals that both proteins are required for normal levels of cytosine methylation and hydroxymethylation in murine embryonic stem cells. Furthermore, Mbd2 and Mbd3 regulate overlapping sets of genes that are also regulated by DNA methylation/hydroxymethylation factors. These findings reveal an interdependent regulatory mechanism mediated by the DNA methylation machinery and its readers., Competing Interests: The authors declare that no competing interests exist.

Many RNA binding proteins (RBPs) bind specific RNA sequence motifs, but only a small fraction (∼15%-40%) of RBP motif occurrences are occupied in vivo. To determine which contextual features discriminate between bound and unbound motifs, we performed an in vitro binding assay using 12,000 mouse RNA sequences with the RBPs MBNL1 and RBFOX2. Surprisingly, the strength of binding to motif occurrences in vitro was significantly correlated with in vivo binding, developmental regulation, and evolutionary age of alternative splicing. Multiple lines of evidence indicate that the primary context effect that affects binding in vitro and in vivo is RNA secondary structure. Large-scale combinatorial mutagenesis of unfavorable sequence contexts revealed a consistent pattern whereby mutations that increased motif accessibility improved protein binding and regulatory activity. Our results indicate widespread inhibition of motif binding by local RNA secondary structure and suggest that mutations that alter sequence context commonly affect RBP binding and regulation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Nono is a component of the para-speckle, which stores and processes RNA. Mouse embryonic stem cells (mESCs) lack para-speckles, leaving the function of Nono in mESCs unclear. Here, we find that Nono functions as a chromatin regulator cooperating with Erk to regulate mESC pluripotency. We report that Nono loss results in robust self-renewing mESCs with epigenomic and transcriptomic features resembling the 2i (GSK and Erk inhibitors)-induced "ground state." Erk interacts with and is required for Nono localization to a subset of bivalent genes that have high levels of poised RNA polymerase. Nono loss compromises Erk activation and RNA polymerase poising at its target bivalent genes in undifferentiated mESCs, thus disrupting target gene activation and differentiation. These findings argue that Nono collaborates with Erk signaling to regulate the integrity of bivalent domains and mESC pluripotency., Competing Interests: Statement F.L. is a shareholder of Constellation Pharmaceuticals. Y.S. is a co-founder of Constellation Pharmaceuticals, Inc., as well as a member of its scientific advisory board. F.L. and Y.S. are also consultants for Active Motif, Inc., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Pluripotency is increasingly recognized as a spectrum of cell states defined by their growth conditions. Although naive and primed pluripotency states have been characterized molecularly, our understanding of events regulating state acquisition is wanting. Here, we performed comparative RNA sequencing of mouse embryonic stem cells (ESCs) and defined a pluripotent cell fate (PCF) gene signature associated with acquisition of naive and primed pluripotency. We identify Zfp281 as a key transcriptional regulator for primed pluripotency that also functions as a barrier toward achieving naive pluripotency in both mouse and human ESCs. Mechanistically, Zfp281 interacts with Tet1, but not Tet2, and its direct transcriptional target, miR-302/367, to negatively regulate Tet2 expression to establish and maintain primed pluripotency. Conversely, ectopic Tet2 alone, but not Tet1, efficiently reprograms primed cells toward naive pluripotency. Our study reveals a molecular circuitry in which opposing functions of Tet1 and Tet2 control acquisition of alternative pluripotent states., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Heat shock factor 1 (HSF1) monitors the structural integrity of the proteome. Phosphorylation at S326 is a hallmark for HSF1 activation, but the identity of the kinase(s) phosphorylating this site has remained elusive. We show here that the dietary agent phenethyl isothiocyanate (PEITC) inhibits heat shock protein 90 (Hsp90), the main negative regulator of HSF1; activates p38 mitogen-activated protein kinase (MAPK); and increases S326 phosphorylation, trimerization, and nuclear translocation of HSF1, and the transcription of a luciferase reporter, as well as the endogenous prototypic HSF1 target Hsp70. In vitro, all members of the p38 MAPK family rapidly and stoichiometrically catalyze the S326 phosphorylation. The use of stable knockdown cell lines and inhibitors indicated that among the p38 MAPKs, p38γ is the principal isoform responsible for the phosphorylation of HSF1 at S326 in cells. A protease-mass spectrometry approach confirmed S326 phosphorylation and unexpectedly revealed that p38 MAPK also catalyzes the phosphorylation of HSF1 at S303/307, previously known repressive posttranslational modifications. Thus, we have identified p38 MAPKs as highly efficient catalysts for the phosphorylation of HSF1. Furthermore, our findings suggest that the magnitude and persistence of activation of p38 MAPK are important determinants of the extent and duration of the heat shock response., (Copyright © 2016 Dayalan Naidu et al.)

LIM domain-binding protein 1 (Ldb1) is a nuclear cofactor that interacts with LIM homeodomain proteins to form multiprotein complexes that are important for transcription regulation. Ldb1 has been shown to play essential roles in various processes during mouse embryogenesis. To determine the role of Ldb1 in mid- and hindbrain development, we have generated a conditional mutant with a specific deletion of the Ldb1 in the Engrailed-1-expressing region of the developing mid- and hindbrain. Our study showed that the deletion impaired the expression of signaling molecules, such as fibroblast growth factor 8 (FGF8) and Wnt1, in the isthmic organizer and the expression of Shh in the ventral midbrain. The midbrain and the cerebellum were severely reduced in size, and the midbrain dopaminergic (mDA) neurons were missing in the mutant. These defects are identical to the phenotype that has been observed previously in mice with a deletion of the LIM homeodomain gene Lmx1b. Our results thus provide genetic evidence supporting that Ldb1 and Lmx1b function cooperatively to regulate mid- and hindbrain development. In addition, we found that mouse embryonic stem cells lacking Ldb1 failed to generate several types of differentiated neurons, including the mDA neurons, serotonergic neurons, cholinergic neurons, and olfactory bulb neurons, indicating an essential cell-autonomous role for Ldb1 in the development of these neurons.

Background: Intrinsic factors and extrinsic signals which control unlimited self-renewal and developmental pluripotency in embryonic stem cells (ESCs) have been extensively investigated. However, a much smaller number of factors involved in extra-embryonic trophoblast differentiation from ESCs have been studied. In this study, we investigated the role of the single-stranded DNA binding protein, Ssbp3, for the induction of trophoblast-like differentiation from mouse ESCs., Methods: Gain- and loss-of-function experiments were carried out through overexpression or knockdown of Ssbp3 in mouse ESCs under self-renewal culture conditions. Expression levels of pluripotency and lineage markers were detected by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analyses. The global gene expression profile in Ssbp3-overexpressing cells was determined by affymetrix microarray. Gene ontology and pathway terms were analyzed and further validated by qRT-PCR and Western blotting. The methylation status of the Elf5 promoter in Ssbp3-overexpressing cells was detected by bisulfite sequencing. The trophoblast-like phenotype induced by Ssbp3 was also evaluated by teratoma formation and early embryo injection assays., Results: Forced expression of Ssbp3 in mouse ESCs upregulated expression levels of lineage-associated genes, with trophoblast cell markers being the highest. In contrast, depletion of Ssbp3 attenuated the expression of trophoblast lineage marker genes induced by downregulation of Oct4 or treatment with BMP4 and bFGF in ESCs. Interestingly, global gene expression profiling analysis indicated that Ssbp3 overexpression did not significantly alter the transcript levels of pluripotency-associated transcription factors. Instead, Ssbp3 promoted the expression of early trophectoderm transcription factors such as Cdx2 and activated MAPK/Erk1/2 and TGF-β pathways. Furthermore, overexpression of Ssbp3 reduced the methylation level of the Elf5 promoter and promoted the generation of teratomas with internal hemorrhage, indicative of the presence of trophoblast cells., Conclusions: This study identifies Ssbp3, a single-stranded DNA binding protein, as a regulator for mouse ESCs to differentiate into trophoblast-like cells. This finding is helpful to understand the regulatory networks for ESC differentiation into extra-embryonic lineages.

A few central transcription factors inside mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are believed to control the cells' pluripotency. Characterizations of pluripotent state were put forward on both transcription factor and epigenetic levels. Whereas core players have been identified, it is desirable to map out gene regulatory networks which govern the reprogramming of somatic cells as well as the early developmental decisions. Here we propose a multiple level model where the regulatory network of Oct4, Nanog and Tet1 includes positive feedback loops involving DNA-demethylation around the promoters of Oct4 and Tet1. We put forward a mechanistic understanding of the regulatory dynamics which account for i) Oct4 overexpression is sufficient to induce pluripotency in somatic cell types expressing the other Yamanaka reprogramming factors endogenously; ii) Tet1 can replace Oct4 in reprogramming cocktail; iii) Nanog is not necessary for reprogramming however its over-expression leads to enhanced self-renewal; iv) DNA methylation is the key to the regulation of pluripotency genes; v) Lif withdrawal leads to loss of pluripotency. Overall, our paper proposes a novel framework combining transcription regulation with DNA methylation modifications which, takes into account the multi-layer nature of regulatory mechanisms governing pluripotency acquisition through reprogramming.

Zfp57 is a master regulator of genomic imprinting in mouse embryos. To further test its functions, we have derived multiple Zfp57 mutant ES clones directly from mouse blastocysts. Indeed, we found DNA methylation imprint was lost at most examined imprinting control regions in these Zfp57 mutant ES clones, similar to what was observed in Zfp57 mutant embryos in the previous studies. This result indicates that these blastocyst-derived Zfp57 mutant ES clones can be employed for functional analyses of Zfp57 in genomic imprinting., (Copyright © 2016 University of Texas at Austin Dell Medical School. Published by Elsevier B.V. All rights reserved.)

Spermatogonial stem cells (SSCs) can spontaneously dedifferentiate into embryonic stem cell (ESC)-like cells, which are designated as multipotent SSCs (mSSCs), without ectopic expression of reprogramming factors. Interestingly, SSCs express key pluripotency genes such as Oct4, Sox2, Klf4 and Myc. Therefore, molecular dissection of mSSC reprogramming may provide clues about novel endogenous reprogramming or pluripotency regulatory factors. Our comparative transcriptome analysis of mSSCs and induced pluripotent stem cells (iPSCs) suggests that they have similar pluripotency states but are reprogrammed via different transcriptional pathways. We identified 53 genes as putative pluripotency regulatory factors using an integrated systems biology approach. We demonstrated a selected candidate, Positive cofactor 4 (Pc4), can enhance the efficiency of somatic cell reprogramming by promoting and maintaining transcriptional activity of the key reprograming factors. These results suggest that Pc4 has an important role in inducing spontaneous somatic cell reprogramming via up-regulation of key pluripotency genes., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

ATP-dependent chromatin remodellers allow access to DNA for transcription factors and the general transcription machinery, but whether mammalian chromatin remodellers target specific nucleosomes to regulate transcription is unclear. Here we present genome-wide remodeller-nucleosome interaction profiles for the chromatin remodellers Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind one or both full nucleosomes that flank micrococcal nuclease (MNase)-defined nucleosome-free promoter regions (NFRs), where they separate divergent transcription. Surprisingly, large CpG-rich NFRs that extend downstream of annotated transcriptional start sites are nevertheless bound by non-nucleosomal or subnucleosomal histone variants (H3.3 and H2A.Z) and marked by H3K4me3 and H3K27ac modifications. RNA polymerase II therefore navigates hundreds of base pairs of altered chromatin in the sense direction before encountering an MNase-resistant nucleosome at the 3' end of the NFR. Transcriptome analysis after remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. Whereas at active genes individual remodellers have either positive or negative roles via altering nucleosome stability, at polycomb-enriched bivalent genes the same remodellers act in an opposite manner. These findings indicate that remodellers target specific nucleosomes at the edge of NFRs, where they regulate ES cell transcriptional programs.

Although mechanisms underlying early steps in cerebellar development are known, evidence is lacking on genetic and epigenetic changes during the establishment of the synaptic circuitry. Using metagene analysis, we report pivotal changes in multiple reactomes of epigenetic pathway genes in cerebellar granule cells (GCs) during circuit formation. During this stage, Tet genes are upregulated and vitamin C activation of Tet enzymes increases the levels of 5-hydroxymethylcytosine (5hmC) at exon start sites of upregulated genes, notably axon guidance genes and ion channel genes. Knockdown of Tet1 and Tet3 by RNAi in ex vivo cerebellar slice cultures inhibits dendritic arborization of developing GCs, a critical step in circuit formation. These findings demonstrate a role for Tet genes and chromatin remodeling genes in the formation of cerebellar circuitry., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Genotoxic stress activates checkpoint signaling pathways that activate the checkpoint kinases ATM and ATR, halt cell cycle progression, and promote DNA repair. A number of proteins act in concert with ATR to phosphorylate Chk1, including RAD17, the RAD9-RAD1-HUS1 complex, ATR/ATRIP and TopBp1. However, how these proteins involved act in concert with one another to propagate and maintain the checkpoint response is not well understood. Here, we reported that upregulation of RAD9 protein increased the quantity of ATRIP, suggesting that RAD9 activation will induce more efficient accumulation of ATRIP in vivo. Furthermore, the DNA damage-induced ATRIP foci formation was faster in the mRad9-/- ES cells. Also, ATRIP interacts specifically with RAD9, but not HUS1 and RAD1. Taken together, we suggested that RAD9 could affect both the ATRIP protein levels and DNA damage-induced ATRIP foci formation. Thus, we propose a role of RAD9 in the ATR-Chk1 pathway that is necessary for successful formation of the damage-sensing complex and DNA damage checkpoint signaling.

Ten eleven translocation (Tet) family-mediated DNA oxidation on 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) represents a novel epigenetic modification that regulates dynamic gene expression during embryonic stem cells (ESCs) differentiation. Through the role of Tet on 5hmC regulation in stem cell development is relatively defined, how the Tet family is regulated and impacts on ESCs lineage development remains elusive. In this study, we show non-coding RNA regulation on Tet family may contribute to epigenetic regulation during ESCs differentiation, which is suggested by microRNA-29b (miR-29b) binding sites on the Tet1 3' untranslated region (3' UTR). We demonstrate miR-29b increases sharply after embyoid body (EB) formation, which causes Tet1 repression and reduction of cellular 5hmC level during ESCs differentiation. Importantly, we show this miR-29b/Tet1 regulatory axis promotes the mesendoderm lineage formation both in vitro and in vivo by inducing the Nodal signaling pathway and repressing the key target of the active demethylation pathway, Tdg. Taken together, our findings underscore the contribution of small non-coding RNA mediated regulation on DNA demethylation dynamics and the differential expressions of key mesendoderm regulators during ESCs lineage specification. MiR-29b could potentially be applied to enrich production of mesoderm and endoderm derivatives and be further differentiated into desired organ-specific cells., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

For cells to exit from pluripotency and commit to a lineage, the circuitry of a core transcription factor (CTF) network must be extinguished in an orderly manner through epigenetic modifications. However, how this choreographed epigenetic remodeling at active embryonic stem cell (ESC) genes occurs during differentiation is poorly understood. In this study, we demonstrate that C-terminal binding protein 2 (Ctbp2) regulates nucleosome remodeling and deacetylation (NuRD)-mediated deacetylation of H3K27 and facilitates recruitment of polycomb repressive complex 2 (PRC2)-mediated H3K27me3 in active ESC genes for exit from pluripotency during differentiation. By genomewide analysis, we found that Ctbp2 resides in active ESC genes and co-occupies regions with ESC CTFs in undifferentiated ESCs. Furthermore, ablation of Ctbp2 effects inappropriate gene silencing in ESCs by sustaining high levels of H3K27ac and impeding H3K27me3 in active ESC genes, thereby sustaining ESC maintenance during differentiation. Thus, Ctbp2 preoccupies regions in active genes with the NuRD complex in undifferentiated ESCs that are directed toward H3K27me3 by PRC2 to induce stable silencing, which is pivotal for natural lineage commitment., (© 2015 AlphaMed Press.)

Actl6a (actin-like protein 6A, also known as Baf53a or Arp4) is a subunit shared by multiple complexes including esBAF, INO80, and Tip60-p400, whose main components (Brg1, Ino80, and p400, respectively) are crucial for the maintenance of embryonic stem cells (ESCs). However, whether and how Actl6a functions in ESCs has not been investigated. ESCs originate from the epiblast (EPI) that is derived from the inner cell mass (ICM) in blastocysts, which also give rise to primitive endoderm (PrE). The molecular mechanisms for EPI/PrE specification remain unclear. In this study, we provide the first evidence that Actl6a can protect mouse ESCs (mESCs) from differentiating into PrE. While RNAi knockdown of Actl6a, which appeared highly expressed in mESCs and downregulated during differentiation, induced mESCs to differentiate towards the PrE lineage, ectopic expression of Actl6a was able to repress PrE differentiation. Our work also revealed that Actl6a could interact with Nanog and Sox2 and promote Nanog binding to pluripotency genes such as Oct4 and Sox2. Interestingly, cells depleted of p400, but not of Brg1 or Ino80, displayed similar PrE differentiation patterns. Mutant Actl6a with impaired ability to bind Tip60 and p400 failed to block PrE differentiation induced by Actl6a dysfunction. Finally, we showed that Actl6a could target to the promoters of key PrE regulators (e.g., Sall4 and Fgf4), repressing their expression and inhibiting PrE differentiation. Our findings uncover a novel function of Actl6a in mESCs, where it acts as a gatekeeper to prevent mESCs from entering into the PrE lineage through a Yin/Yang regulating pattern., (© 2015 AlphaMed Press.)