Decoding the universal human chromatin landscape through teratoma-based profiling.

Teratoma formation is key for evaluating differentiation of human pluripotent stem cells into embryonic germ layers and serves as a model for understanding stem cell differentiation and developmental processes. Its potential for insights into epigenome and transcriptome profiling is significant. This study integrates the analysis of the epigenome and transcriptome of hESC-generated teratomas, comparing transcriptomes between hESCs and teratomas. It employs cell type-specific expression patterns from single-cell data to deconvolve RNA-Seq data and identify cell types within teratomas. Our results provide a catalog of activating and repressive histone modifications, while also elucidating distinctive features of chromatin states. Construction of an epigenetic signature matrix enabled the quantification of diverse cell populations in teratomas and enhanced the ability to unravel the epigenetic landscape in heterogeneous tissue contexts. This study also includes a single cell multiome atlas of expression (scRNA-Seq) and chromatin accessibility (scATAC-Seq) of human teratomas, further revealing the complexity of these tissues. A histology-based digital staining tool further complemented the annotation of cell types in teratomas, enhancing our understanding of their cellular composition. This research is a valuable resource for examining teratoma epigenomic and transcriptomic landscapes and serves as a model for epigenetic data comparison.

Identifying Cancer Type-Specific Transcriptional Programs through Network Analysis.

Identifying cancer type-specific genes that define cell states is important to develop effective therapies for patients and methods for detection, early diagnosis, and prevention. While molecular mechanisms that drive malignancy have been identified for various cancers, the identification of cell-type defining transcription factors (TFs) that distinguish normal cells from cancer cells has not been fully elucidated. Here, we utilized a network biology framework, which assesses the fidelity of cell fate conversions, to identify cancer type-specific gene regulatory networks (GRN) for 17 types of cancer. Through an integrative analysis of a compendium of expression data, we elucidated core TFs and GRNs for multiple cancer types. Moreover, by comparing normal tissues and cells to cancer type-specific GRNs, we found that the expression of key network-influencing TFs can be utilized as a survival prognostic indicator for a diverse cohort of cancer patients. These findings offer a valuable resource for exploring cancer type-specific networks across a broad range of cancer types.

Identification of actionable targets for breast cancer intervention using a diversity outbred mouse model.

HER2-targeted therapy has improved breast cancer survival, but treatment resistance and disease prevention remain major challenges. Genes that enable HER2/Neu oncogenesis are the next intervention targets. A bioinformatics discovery platform of HER2/Neu-expressing Diversity Outbred (DO) F1 Mice was established to identify cancer-enabling genes. Quantitative Trait Loci (QTL) associated with onset ages and growth rates of spontaneous mammary tumors were sought. Twenty-six genes in 3 QTL contain sequence variations unique to the genetic backgrounds that are linked to aggressive tumors and 21 genes are associated with human breast cancer survival. Concurrent identification of TSC22D3, a transcription factor, and its target gene LILRB4, a myeloid cell checkpoint receptor, suggests an immune axis for regulation, or intervention, of disease. We also investigated TIEG1 gene that impedes tumor immunity but suppresses tumor growth. Although not an actionable target, TIEG1 study revealed genetic regulation of tumor progression, forming the basis of the genetics-based discovery platform.

FOXQ1 recruits the MLL complex to activate transcription of EMT and promote breast cancer metastasis.

Aberrant expression of the Forkhead box transcription factor, FOXQ1, is a prevalent mechanism of epithelial-mesenchymal transition (EMT) and metastasis in multiple carcinoma types. However, it remains unknown how FOXQ1 regulates gene expression. Here, we report that FOXQ1 initiates EMT by recruiting the MLL/KMT2 histone methyltransferase complex as a transcriptional coactivator. We first establish that FOXQ1 promoter recognition precedes MLL complex assembly and histone-3 lysine-4 trimethylation within the promoter regions of critical genes in the EMT program. Mechanistically, we identify that the Forkhead box in FOXQ1 functions as a transactivation domain directly binding the MLL core complex subunit RbBP5 without interrupting FOXQ1 DNA binding activity. Moreover, genetic disruption of the FOXQ1-RbBP5 interaction or pharmacologic targeting of KMT2/MLL recruitment inhibits FOXQ1-dependent gene expression, EMT, and in vivo tumor progression. Our study suggests that targeting the FOXQ1-MLL epigenetic axis could be a promising strategy to combat triple-negative breast cancer metastatic progression.

H3K4 demethylase KDM5B regulates cancer cell identity and epigenetic plasticity.

The H3K4 demethylase KDM5B is overexpressed in multiple cancer types, and elevated expression levels of KDM5B is associated with decreased survival. However, the underlying mechanistic contribution of dysregulated expression of KDM5B and H3K4 demethylation in cancer is poorly understood. Our results show that loss of KDM5B in multiple types of cancer cells leads to increased proliferation and elevated expression of cancer stem cell markers. In addition, we observed enhanced tumor formation following KDM5B depletion in a subset of representative cancer cell lines. Our findings also support a role for KDM5B in regulating epigenetic plasticity, where loss of KDM5B in cancer cells with elevated KDM5B expression leads to alterations in activity of chromatin states, which facilitate activation or repression of alternative transcriptional programs. In addition, we define KDM5B-centric epigenetic and transcriptional patterns that support cancer cell plasticity, where KDM5B depleted cancer cells exhibit altered epigenetic and transcriptional profiles resembling a more primitive cellular state. This study also provides a resource for evaluating associations between alterations in epigenetic patterning upon depletion of KDM5B and gene expression in a diverse set of cancer cells.

Integrative pan cancer analysis reveals epigenomic variation in cancer type and cell specific chromatin domains.

Epigenetic mechanisms contribute to the initiation and development of cancer, and epigenetic variation promotes dynamic gene expression patterns that facilitate tumor evolution and adaptation. While the NCI-60 panel represents a diverse set of human cancer cell lines that has been used to screen chemical compounds, a comprehensive epigenomic atlas of these cells has been lacking. Here, we report an integrative analysis of 60 human cancer epigenomes, representing a catalog of activating and repressive histone modifications. We identify genome-wide maps of canonical sharp and broad H3K4me3 domains at promoter regions of tumor suppressors, H3K27ac-marked conventional enhancers and super enhancers, and widespread inter-cancer and intra-cancer specific variability in H3K9me3 and H4K20me3-marked heterochromatin domains. Furthermore, we identify features of chromatin states, including chromatin state switching along chromosomes, correlation of histone modification density with genetic mutations, DNA methylation, enrichment of DNA binding motifs in regulatory regions, and gene activity and inactivity. These findings underscore the importance of integrating epigenomic maps with gene expression and genetic variation data to understand the molecular basis of human cancer. Our findings provide a resource for mining epigenomic maps of human cancer cells and for identifying epigenetic therapeutic targets.

H4K20me3 methyltransferase SUV420H2 shapes the chromatin landscape of pluripotent embryonic stem cells.

Heterochromatin, a densely packed chromatin state that is transcriptionally silent, is a critical regulator of gene expression. However, it is unclear how the repressive histone modification H4K20me3 or the histone methyltransferase SUV420H2 regulates embryonic stem (ES) cell fate by patterning the epigenetic landscape. Here, we report that depletion of SUV420H2 leads to a near-complete loss of H4K20me3 genome wide, dysregulated gene expression and delayed ES cell differentiation. SUV420H2-bound regions are enriched with repetitive DNA elements, which are de-repressed in SUV420H2 knockout ES cells. Moreover, SUV420H2 regulation of H4K20me3-marked heterochromatin controls chromatin architecture, including fine-scale chromatin interactions in pluripotent ES cells. Our results indicate that SUV420H2 plays a crucial role in stabilizing the three-dimensional chromatin landscape of ES cells, as loss of SUV420H2 resulted in A/B compartment switching, perturbed chromatin insulation, and altered chromatin interactions of pericentric heterochromatin and surrounding regions, indicative of localized decondensation. In addition, depletion of SUV420H2 resulted in compromised interactions between H4K20me3 and gene-regulatory regions. Together, these findings describe a new role for SUV420H2 in regulating the chromatin landscape of ES cells.

Derivation of Maternal Epiblast Stem Cells from Haploid Embryos.

Pluripotent cells in the inner cell mass (ICM) or epiblast of mammalian embryos exhibit the capacity to differentiate into all cells represented in the three germ layers. Embryonic stem (ES) cells can be derived from the ICM of preimplantation stage blastocysts, while epiblast stem cells (EpiSCs) can be derived from the epiblast of postimplantation embryos or preimplantation stage embryos. The ability to derive distinct types of pluripotent cells from blastocyst-stage embryos suggests that optimization of culture conditions can promote self-renewal of various stem cell populations. Moreover, because mouse EpiSCs resemble human pluripotent stem (hPS) cells, EpiSCs are a useful model to study common and divergent mechanisms of self-renewal between orthologous species. In addition, studies have demonstrated that haploid embryos and ES cells can be derived from chemically activated oocytes. Here, we describe a protocol for deriving maternal (parthenogenetic/gynogenetic) EpiSCs (maEpiSCs) from haploid blastocyst-stage embryos. This protocol is suitable to establish an experimental model for the study of mechanisms of EpiSC self-renewal and differentiation.

Derivation of LIF-Independent Embryonic Stem Cells Using Inducible OCT4 Expression.

Pluripotent mouse embryonic stem (ES) cells, which are derived from the inner cell mass (ICM) of preimplantation stage embryos, are capable of self-renewing indefinitely in the presence of the external signal leukemia inhibitory factor (LIF), activation of Wnt signaling through inhibition of GSK3, and inhibition of MAP kinase/ERK kinase signaling. The OCT4 transcription factor is expressed highly in pluripotent cells and is a central transcriptional regulator of the pluripotent state. Here, we describe a protocol to culture ES cells in LIF-independent and serum-free media using an inducible OCT4 (iOCT4) ES cell model system. This protocol is sufficient to sustain ES cell self-renewal in vitro in defined conditions in the absence of external signals. LIF-independent iOCT4 ES cells are fully capable of differentiating following deactivation of the inducible OCT4 transgene.

Simultaneous Derivation of Embryonic and Trophoblast Stem Cells from Mouse Blastocysts.

The formation of the blastocyst during mammalian development involves the segregation of two populations of cells with unequal potential: pluripotent cells of the inner cell mass (ICM) and multipotent cells of the trophectoderm (TE). ICM cells maintain the capacity to give rise to all cells represented in the organism, while TE cells, which represent the first lineage to emerge during development, are capable of differentiating into trophoblast lineages of the placenta. The ICM and TE are both essential for development. The ICM is genetically programmed to generate all cells of the embryo proper, while the TE forms extraembryonic trophoblast lineages and is required for implantation of the embryo and maternal-fetal exchange of nutrients and waste. Embryonic stem (ES) cells, which can be derived from the ICM of blastocysts in the presence of external signals such as LIF, can self-renewal indefinitely, and because they can differentiate into all cells of the organism, ES cells are a widely used in vitro model to study genetics and development. Trophoblast stem (TS) cells can be derived from the TE of blastocyst stage embryos in the presence of FGF4, and like ES cells, TS cells are also able to self-renew indefinitely. Because TS cells can differentiate into epithelial lineages of the trophoblast, TS cells are an ideal in vitro model to study the biology of the trophoblast. In this chapter, we describe protocols for simultaneous derivation of ES cells and TS cells from mouse blastocysts and culture conditions that promote self-renewal of hybrid ESC/TSC colonies. These protocols are sufficient for efficient derivation of hybrid ESC/TSC colonies.

Direct Reprogramming of Mouse Embryonic Fibroblasts to Induced Trophoblast Stem Cells.

Trophoblast cells are the first committed lineage to emerge during mammalian preimplantation embryo development. Trophoblast stem (TS) cells can be derived from the trophectoderm (TE) of blastocyst-stage embryos and differentiate into extraembryonic trophoblast cells of the placenta. While mouse TS cells are an indispensable tool to study placental development, and reproductive diseases such as implantation failure and recurrent miscarriage, human TS cells have not been isolated. To model human trophoblast development and to investigate trophoblast-specific causes of reproductive diseases, it will be important to derive human induced trophoblast stem (iTS) cells. Recent studies have shown that fibroblasts can be reprogrammed to iTS cells by overexpressing four transcription factors (TFs) including TFAP2C, GATA3, EOMES, and ETS2. Here, we describe a protocol to directly convert mouse embryonic fibroblasts (MEFs) to iTS cells following overexpression of 10 TFs. iTS cells are capable of self-renewing using conventional TS cell culture media supplemented with the external signal FGF4 and heparin. iTS cells are also able to differentiate into trophoblast lineages.

Mdig promotes oncogenic gene expression through antagonizing repressive histone methylation markers.

The mineral dust-induced gene (mdig) is overexpressed in a number of human cancers, suggesting critical roles of this gene played on the pathogenesis of cancers. Unlike several other JmjC-domain containing proteins that exhibit histone demethylase activity, it remains enigmatic whether mdig is involved in the demethylation processes of the histone proteins. Methods: To provide direct evidence suggesting contribution of mdig to the demethylation of histone proteins, we recently examined the histone methylation profiles in human bronchial epithelial cells as well as two cancer cell lines with mdig knockout through CRISPR-Cas9 gene editing. Results: Global histone methylation analysis revealed a pronounced increase of the repressive histone trimethylation in three different cell types with mdig depletion, including trimethylation of lysines 9 and 27 on histone H3 (H3K9me3, H3K27me3) and trimethylation of lysine 20 of histone H4 (H4K20me3). Importantly, data from both ChIP-seq and RNA-seq suggested that genetic disruption of mdig enriches repressive histone trimethylation and inhibits expression of target genes in the oncogenic pathways of cell growth, stemness of the cells, tissue fibrosis, and cell motility. Conclusion: Taken together, our study provides the first insight into the molecular effects of mdig as an antagonist for repressive histone methylation markers and suggests that targeting mdig may represent a new area to explore in cancer therapy.

Contribution of H3K4 demethylase KDM5B to nucleosome organization in embryonic stem cells revealed by micrococcal nuclease sequencing.

BACKGROUND: Positioning of nucleosomes along DNA is an integral regulator of chromatin accessibility and gene expression in diverse cell types. However, the precise nature of how histone demethylases including the histone 3 lysine 4 (H3K4) demethylase, KDM5B, impacts nucleosome positioning around transcriptional start sites (TSS) of active genes is poorly understood. RESULTS: Here, we report that KDM5B is a critical regulator of nucleosome positioning in embryonic stem (ES) cells. Micrococcal nuclease sequencing (MNase-Seq) revealed increased enrichment of nucleosomes around TSS regions and DNase I hypersensitive sites in KDM5B-depleted ES cells. Moreover, depletion of KDM5B resulted in a widespread redistribution and disorganization of nucleosomes in a sequence-dependent manner. Dysregulated nucleosome phasing was also evident in KDM5B-depleted ES cells, including asynchronous nucleosome spacing surrounding TSS regions, where nucleosome variance was positively correlated with the degree of asynchronous phasing. The redistribution of nucleosomes around TSS regions in KDM5B-depleted ES cells is correlated with dysregulated gene expression, and altered H3K4me3 and RNA polymerase II occupancy. In addition, we found that DNA shape features varied significantly at regions with shifted nucleosomes. CONCLUSION: Altogether, our data support a role for KDM5B in regulating nucleosome positioning in ES cells.

KDM5B is a master regulator of the H3K4-methylome in stem cells, development and cancer.

Epigenetic regulation of chromatin plays a critical role in controlling stem cell function and tumorigenesis. The histone lysine demethylase, KDM5B, which catalyzes the demethylation of histone 3 lysine 4 (H3K4), is important for embryonic stem (ES) cell differentiation, and is a critical regulator of the H3K4-methylome during early mouse embryonic pre-implantation stage development. KDM5B is also overexpressed, amplified, or mutated in many cancer types. In cancer cells, KDM5B regulates expression of oncogenes and tumor suppressors by modulating H3K4 methylation levels. In this review, we examine how KDM5B regulates gene expression and cellular fates of stem cells and cancer cells by temporally and spatially controlling H3K4 methylation levels.

Strategy for Tumor-Selective Disruption of Androgen Receptor Function in the Spectrum of Prostate Cancer.

PURPOSE: Testosterone suppression in prostate cancer is limited by serious side effects and resistance via restoration of androgen receptor (AR) functionality. ELK1 is required for AR-dependent growth in various hormone-dependent and castration-resistant prostate cancer models. The amino-terminal domain of AR docks at two sites on ELK1 to coactivate essential growth genes. This study explores the ability of small molecules to disrupt the ELK1-AR interaction in the spectrum of prostate cancer, inhibiting AR activity in a manner that would predict functional tumor selectivity. EXPERIMENTAL DESIGN: Small-molecule drug discovery and extensive biological characterization of a lead compound. RESULTS: We have discovered a lead molecule (KCI807) that selectively disrupts ELK1-dependent promoter activation by wild-type and variant ARs without interfering with ELK1 activation by ERK. KCI807 has an obligatory flavone scaffold and functional hydroxyl groups on C5 and C3'. KCI807 binds to AR, blocking ELK1 binding, and selectively blocks recruitment of AR to chromatin by ELK1. KCI807 primarily affects a subset of AR target growth genes selectively suppressing AR-dependent growth of prostate cancer cell lines with a better inhibitory profile than enzalutamide. KCI807 also inhibits in vivo growth of castration/enzalutamide-resistant cell line-derived and patient-derived tumor xenografts. In the rodent model, KCI807 has a plasma half-life of 6 hours, and maintenance of its antitumor effect is limited by self-induced metabolism at its 3'-hydroxyl. CONCLUSIONS: The results offer a mechanism-based therapeutic paradigm for disrupting the AR growth-promoting axis in the spectrum of prostate tumors while reducing global suppression of testosterone actions. KCI807 offers a good lead molecule for drug development.

Identification of H4K20me3- and H3K4me3-associated RNAs using CARIP-Seq expands the transcriptional and epigenetic networks of embryonic stem cells.

RNA has been shown to interact with various proteins to regulate chromatin dynamics and gene expression. However, it is unknown whether RNAs associate with epigenetic marks such as post-translational modifications of histones, including histone 4 lysine 20 trimethylation (H4K20me3) or trimethylated histone 3 lysine 4 (H3K4me3), to regulate chromatin and gene expression. Here, we used chromatin-associated RNA immunoprecipitation (CARIP) followed by next-generation sequencing (CARIP-Seq) to survey RNAs associated with H4K20me3- and H3K4me3-marked chromatin on a global scale in embryonic stem (ES) cells. We identified thousands of mRNAs and noncoding RNAs that associate with H4K20me3- and H3K4me3-marked chromatin. H4K20me3- and H3K4me3-interacting RNAs are involved in chromatin organization and modification and RNA processing, whereas H4K20me3-only RNAs are involved in cell motility and differentiation, and H3K4me3-only RNAs are involved in metabolic processes and RNA processing. Expression of H3K4me3-associated RNAs is enriched in ES cells, whereas expression of H4K20me3-associated RNAs is enriched in ES cells and differentiated cells. H4K20me3- and H3K4me3-interacting RNAs originate from genes that co-localize with features of active chromatin, including transcriptional machinery and active promoter regions, and the histone modification H3K36me3 in gene body regions. We also found that H4K20me3 and H3K4me3 are associated with distinct gene features including transcripts of greater length and exon number relative to unoccupied transcripts. H4K20me3- and H3K4me3-marked chromatin is also associated with processed RNAs (exon transcripts) relative to unspliced pre-mRNA and ncRNA transcripts. In summary, our results provide evidence that H4K20me3- and H3K4me3-associated RNAs represent a distinct subnetwork of the ES cell transcriptional repertoire.

H4K20me3 co-localizes with activating histone modifications at transcriptionally dynamic regions in embryonic stem cells.

BACKGROUND: Bivalent chromatin domains consisting of the activating histone 3 lysine 4 trimethylation (H3K4me3) and repressive histone 3 lysine 27 trimethylation (H3K27me3) histone modifications are enriched at developmental genes that are repressed in embryonic stem cells but active during differentiation. However, it is unknown whether another repressive histone modification, histone 4 lysine 20 trimethylation (H4K20me3), co-localizes with activating histone marks in ES cells. RESULTS: Here, we describe the previously uncharacterized coupling of the repressive H4K20me3 heterochromatin mark with the activating histone modifications H3K4me3 and histone 3 lysine 36 trimethylation (H3K36me3), and transcriptional machinery (RNA polymerase II; RNAPII), in ES cells. These newly described bivalent domains consisting of H3K4me3/H4K20me3 are predominantly located in intergenic regions and near transcriptional start sites of active genes, while H3K36me3/H4K20me3 are located in intergenic regions and within gene body regions of active genes. Global sequential ChIP, also termed reChIP-Seq, confirmed the simultaneous presence of H3K4me3 and H4K20me3 at the same genomic regions in ES cells. Genes containing H3K4me3/H4K20me3 exhibit decreased RNAPII pausing and are poised for deactivation of RNAPII binding during differentiation relative to H3K4me3 marked genes. An evaluation of transcription factor (TF) binding motif enrichment revealed that DNA sequence may play a role in shaping the landscape of these novel bivalent domains. Moreover, H3K4me3/H4K20me3 and H3K36me3/H4K20me3 bound regions are enriched with repetitive LINE and LTR elements. CONCLUSIONS: Overall, these findings highlight a previously undescribed subnetwork of ES cell transcriptional circuitry that utilizes dual marking of the repressive H4K20me3 mark with activating histone modifications.

Culture of haploid blastocysts in FGF4 favors the derivation of epiblast stem cells with a primed epigenetic and transcriptional landscape.

Pluripotent stem cells within the inner cell mass and epiblast of mammalian embryos have the capacity to form all lineages in the adult organism, while multipotent trophoblast stem (TS) cells derived from the trophectoderm are capable of differentiating into fetal lineages of the placenta. While mouse embryonic stem (ES) cells and epiblast stem cells (EpiSCs) exhibit distinct expression patterns and utilize distinct external signaling pathways for self-renewal, because mouse EpiSCs resemble human ES cells they are a useful model to investigate mechanisms of human ES cell self-renewal and differentiation. Recent studies have shown that haploid embryos and ES cells can be generated from chemically-activated unfertilized mouse oocytes. However, it is unclear whether EpiSCs or TS cells can be derived from haploid embryos. Here, we describe the derivation of EpiSCs from haploid blastocyst-stage embryos using culture conditions that promote TS cell self-renewal. Maternal (parthenogenetic/gynogenetic) EpiSCs (maEpiSCs) functionally and morphologically resemble conventional EpiSCs. Established maEpiSCs and conventional EpiSCs are diploid and exhibit a normal number of chromosomes. Moreover, global expression analyses and epigenomic profiling revealed that maEpiSCs and conventional EpiSCs exhibit similarly primed transcriptional programs and epigenetic profiles, respectively. Altogether, our results describe a useful experimental model to generate EpiSCs from haploid embryos, provide insight into self-renewal mechanisms of EpiSCs, and suggest that FGF4 is not sufficient to derive TS cells from haploid blastocyst-stage embryos.

CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells.

Embryonic stem (ES) cell self-renewal and differentiation is governed by extrinsic signals and intrinsic networks of transcription factors, epigenetic regulators, and post-translation modifications of histones that combinatorially influence the gene expression state of nearby genes. RNA has also been shown to interact with various proteins to regulate chromatin dynamics and gene expression. Chromatin-associated RNA immunoprecipitation (CARIP) followed by next-generation sequencing (CARIP-Seq) is a novel method to survey RNAs associated with chromatin proteins, while chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq) is a powerful genomics technique to map the location of post-translational modification of histones, transcription factors, and epigenetic modifiers on a global-scale in ES cells. Here, we describe methods to perform CARIP-Seq and ChIP-Seq, including library construction for next-generation sequencing, to generate global chromatin-associated RNA and epigenomic maps in ES cells.

KDM5B decommissions the H3K4 methylation landscape of self-renewal genes during trophoblast stem cell differentiation.

Trophoblast stem (TS) cells derived from the trophectoderm (TE) of mammalian embryos have the ability to self-renew indefinitely or differentiate into fetal lineages of the placenta. Epigenetic control of gene expression plays an instrumental role in dictating the fate of TS cell self-renewal and differentiation. However, the roles of histone demethylases and activating histone modifications such as methylation of histone 3 lysine 4 (H3K4me3/me2) in regulating TS cell expression programs, and in priming the epigenetic landscape for trophoblast differentiation, are largely unknown. Here, we demonstrate that the H3K4 demethylase, KDM5B, regulates the H3K4 methylome and expression landscapes of TS cells. Depletion of KDM5B resulted in downregulation of TS cell self-renewal genes and upregulation of trophoblast-lineage genes, which was accompanied by altered H3K4 methylation. Moreover, we found that KDM5B resets the H3K4 methylation landscape during differentiation in the absence of the external self-renewal signal, FGF4, by removing H3K4 methylation from promoters of self-renewal genes, and of genes whose expression is enriched in TS cells. Altogether, our data indicate an epigenetic role for KDM5B in regulating H3K4 methylation in TS cells and during trophoblast differentiation.