A low-input high resolution sequential chromatin immunoprecipitation method captures genome-wide dynamics of bivalent chromatin.

BACKGROUND: Bivalent chromatin is an exemplar of epigenetic plasticity. This co-occurrence of active-associated H3K4me3 and inactive-associated H3K27me3 histone modifications on opposite tails of the same nucleosome occurs predominantly at promoters that are poised for future transcriptional upregulation or terminal silencing. We know little of the dynamics, resolution, and regulation of this chromatin state outside of embryonic stem cells where it was first described. This is partly due to the technical challenges distinguishing bone-fide bivalent chromatin, where both marks are on the same nucleosome, from allelic or sample heterogeneity where there is a mix of H3K4me3-only and H3K27me3-only mononucleosomes. RESULTS: Here, we present a robust and sensitive method to accurately map bivalent chromatin genome-wide, along with controls, from as little as 2 million cells. We optimized and refined the sequential ChIP protocol which uses two sequential overnight immunoprecipitation reactions to robustly purify nucleosomes that are truly bivalent and contain both H3K4me3 and H3K27me3 modifications. Our method generates high quality genome-wide maps with strong peak enrichment and low background, which can be analyzed using standard bioinformatic packages. Using this method, we detect 8,789 bivalent regions in mouse embryonic stem cells corresponding to 3,918 predominantly CpG rich and developmentally regulated gene promoters. Furthermore, profiling Dppa2/4 knockout mouse embryonic stem cells, which lose both H3K4me3 and H3K27me3 at approximately 10% of bivalent promoters, demonstrated the ability of our method to capture bivalent chromatin dynamics. CONCLUSIONS: Our optimized sequential reChIP method enables high-resolution genome-wide assessment of bivalent chromatin together with all required controls in as little as 2 million cells. We share a detailed protocol and guidelines that will enable bivalent chromatin landscapes to be generated in a range of cellular contexts, greatly enhancing our understanding of bivalent chromatin and epigenetic plasticity beyond embryonic stem cells.

Bivalent chromatin: a developmental balancing act tipped in cancer.

Bivalent chromatin is defined by the co-occurrence of otherwise opposing H3K4me3 and H3K27me3 modifications and is typically located at unmethylated promoters of lowly transcribed genes. In embryonic stem cells, bivalent chromatin has been proposed to poise developmental genes for future activation, silencing or stable repression upon lineage commitment. Normally, bivalent chromatin is kept in tight balance in cells, in part through the activity of the MLL/COMPASS-like and Polycomb repressive complexes that deposit the H3K4me3 and H3K27me3 modifications, respectively, but also emerging novel regulators including DPPA2/4, QSER1, BEND3, TET1 and METTL14. In cancers, both the deregulation of existing domains and the creation of de novo bivalent states is associated with either the activation or silencing of transcriptional programmes. This may facilitate diverse aspects of cancer pathology including epithelial-to-mesenchymal plasticity, chemoresistance and immune evasion. Here, we review current methods for detecting bivalent chromatin and discuss the factors involved in the formation and fine-tuning of bivalent domains. Finally, we examine how the deregulation of chromatin bivalency in the context of cancer could facilitate and/or reflect cancer cell adaptation. We propose a model in which bivalent chromatin represents a dynamic balance between otherwise opposing states, where the underlying DNA sequence is primed for the future activation or repression. Shifting this balance in any direction disrupts the tight equilibrium and tips cells into an altered epigenetic and phenotypic space, facilitating both developmental and cancer processes.

Targeting Menin disrupts the KMT2A/B and polycomb balance to paradoxically activate bivalent genes.

Precise control of activating H3K4me3 and repressive H3K27me3 histone modifications at bivalent promoters is essential for normal development and frequently corrupted in cancer. By coupling a cell surface readout of bivalent MHC class I gene expression with whole-genome CRISPR-Cas9 screens, we identify specific roles for MTF2-PRC2.1, PCGF1-PRC1.1 and Menin-KMT2A/B complexes in maintaining bivalency. Genetic loss or pharmacological inhibition of Menin unexpectedly phenocopies the effects of polycomb disruption, resulting in derepression of bivalent genes in both cancer cells and pluripotent stem cells. While Menin and KMT2A/B contribute to H3K4me3 at active genes, a separate Menin-independent function of KMT2A/B maintains H3K4me3 and opposes polycomb-mediated repression at bivalent genes. Release of KMT2A from active genes following Menin targeting alters the balance of polycomb and KMT2A at bivalent genes, facilitating gene activation. This functional partitioning of Menin-KMT2A/B complex components reveals therapeutic opportunities that can be leveraged through inhibition of Menin.

Maternal Dppa2 and Dppa4 are dispensable for zygotic genome activation but important for offspring survival.

Zygotic genome activation (ZGA) represents the initiation of transcription following fertilisation. Despite its importance, we know little of the molecular events that initiate mammalian ZGA in vivo. Recent in vitro studies in mouse embryonic stem cells have revealed developmental pluripotency associated 2 and 4 (Dppa2/4) as key regulators of ZGA-associated transcription. However, their roles in initiating ZGA in vivo remain unexplored. We reveal that Dppa2/4 proteins are present in the nucleus at all stages of preimplantation development and associate with mitotic chromatin. We generated conditional single and double maternal knockout mouse models to deplete maternal stores of Dppa2/4. Importantly, Dppa2/4 maternal knockout mice were fertile when mated with wild-type males. Immunofluorescence and transcriptome analyses of two-cell embryos revealed that, although ZGA took place, there were subtle defects in embryos that lacked maternal Dppa2/4. Strikingly, heterozygous offspring that inherited the null allele maternally had higher preweaning lethality than those that inherited the null allele paternally. Together, our results show that although Dppa2/4 are dispensable for ZGA transcription, maternal stores have an important role in offspring survival, potentially via epigenetic priming of developmental genes.

Pooled CRISPR-activation screening coupled with single-cell RNA-seq in mouse embryonic stem cells.

CRISPR/Cas9 screens are a powerful approach to identify key regulators of biological processes. By combining pooled CRISPR/Cas9 screening with single-cell RNA-sequencing readout, individual perturbations can be assessed in parallel both comprehensively and at scale. Importantly, this allows gene function and regulation to be interrogated at a cellular level in an unbiased manner. Here, we present a protocol to perform pooled CRISPR-activation screens in mouse embryonic stem cells using 10× Genomics scRNA-seq as a readout. For complete information on the generation and use of this protocol, please refer to Alda-Catalinas et al. (2020).

Keeping your options open: insights from Dppa2/4 into how epigenetic priming factors promote cell plasticity.

The concept of cellular plasticity is particularly apt in early embryonic development, where there is a tug-of-war between the stability and flexibility of cell identity. This balance is controlled in part through epigenetic mechanisms. Epigenetic plasticity dictates how malleable cells are to change by adjusting the potential to initiate new transcriptional programmes. The higher the plasticity of a cell, the more readily it can adapt and change its identity in response to external stimuli such as differentiation cues. Epigenetic plasticity is regulated in part through the action of epigenetic priming factors which establish this permissive epigenetic landscape at genomic regulatory elements to enable future transcriptional changes. Recent studies on the DNA binding proteins Developmental Pluripotency Associated 2 and 4 (Dppa2/4) support their roles as epigenetic priming factors in facilitating cell fate transitions. Here, using Dppa2/4 as a case study, the concept of epigenetic plasticity and molecular mechanism of epigenetic priming factors will be explored. Understanding how epigenetic priming factors function is key not only to improve our understanding of the tight control of development, but also to give insights into how this goes awry in diseases of cell identity, such as cancer.

A Single-Cell Transcriptomics CRISPR-Activation Screen Identifies Epigenetic Regulators of the Zygotic Genome Activation Program.

Zygotic genome activation (ZGA) is an essential transcriptional event in embryonic development that coincides with extensive epigenetic reprogramming. Complex manipulation techniques and maternal stores of proteins preclude large-scale functional screens for ZGA regulators within early embryos. Here, we combined pooled CRISPR activation (CRISPRa) with single-cell transcriptomics to identify regulators of ZGA-like transcription in mouse embryonic stem cells, which serve as a tractable, in vitro proxy of early mouse embryos. Using multi-omics factor analysis (MOFA+) applied to ∼200,000 single-cell transcriptomes comprising 230 CRISPRa perturbations, we characterized molecular signatures of ZGA and uncovered 24 factors that promote a ZGA-like response. Follow-up assays validated top screen hits, including the DNA-binding protein Dppa2, the chromatin remodeler Smarca5, and the transcription factor Patz1, and functional experiments revealed that Smarca5's regulation of ZGA-like transcription is dependent on Dppa2. Together, our single-cell transcriptomic profiling of CRISPRa-perturbed cells provides both system-level and molecular insights into the mechanisms that orchestrate ZGA.

Epigenetic priming by Dppa2 and 4 in pluripotency facilitates multi-lineage commitment.

How the epigenetic landscape is established in development is still being elucidated. Here, we uncover developmental pluripotency associated 2 and 4 (DPPA2/4) as epigenetic priming factors that establish a permissive epigenetic landscape at a subset of developmentally important bivalent promoters characterized by low expression and poised RNA-polymerase. Differentiation assays reveal that Dppa2/4 double knockout mouse embryonic stem cells fail to exit pluripotency and differentiate efficiently. DPPA2/4 bind both H3K4me3-marked and bivalent gene promoters and associate with COMPASS- and Polycomb-bound chromatin. Comparing knockout and inducible knockdown systems, we find that acute depletion of DPPA2/4 results in rapid loss of H3K4me3 from key bivalent genes, while H3K27me3 is initially more stable but lost following extended culture. Consequently, upon DPPA2/4 depletion, these promoters gain DNA methylation and are unable to be activated upon differentiation. Our findings uncover a novel epigenetic priming mechanism at developmental promoters, poising them for future lineage-specific activation.

Establishment of porcine and human expanded potential stem cells.

We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro. Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine.

Dynamics of the epigenetic landscape during the maternal-to-zygotic transition.

A remarkable epigenetic remodelling process occurs shortly after fertilization, which restores totipotency to the zygote. This involves global DNA demethylation, chromatin remodelling, genome spatial reorganization and substantial transcriptional changes. Key to these changes is the transition from the maternal environment of the oocyte to an embryonic-driven developmental expression programme, a process termed the maternal-to-zygotic transition (MZT). Zygotic genome activation occurs predominantly at the two-cell stage in mice and the eight-cell stage in humans, yet the dynamics of its control are still mostly obscure. In recent years, partly due to single-cell and low-cell number epigenomic studies, our understanding of the epigenetic and chromatin landscape of preimplantation development has improved considerably. In this Review, we discuss the latest advances in the study of the MZT, focusing on DNA methylation, histone post-translational modifications, local chromatin structure and higher-order genome organization. We also discuss key mechanistic studies that investigate the mode of action of chromatin regulators, transcription factors and non-coding RNAs during preimplantation development. Finally, we highlight areas requiring additional research, as well as new technological advances that could assist in eventually completing our understanding of the MZT.

Establishment of mouse expanded potential stem cells.

Mouse embryonic stem cells derived from the epiblast contribute to the somatic lineages and the germline but are excluded from the extra-embryonic tissues that are derived from the trophectoderm and the primitive endoderm upon reintroduction to the blastocyst. Here we report that cultures of expanded potential stem cells can be established from individual eight-cell blastomeres, and by direct conversion of mouse embryonic stem cells and induced pluripotent stem cells. Remarkably, a single expanded potential stem cell can contribute both to the embryo proper and to the trophectoderm lineages in a chimaera assay. Bona fide trophoblast stem cell lines and extra-embryonic endoderm stem cells can be directly derived from expanded potential stem cells in vitro. Molecular analyses of the epigenome and single-cell transcriptome reveal enrichment for blastomere-specific signature and a dynamic DNA methylome in expanded potential stem cells. The generation of mouse expanded potential stem cells highlights the feasibility of establishing expanded potential stem cells for other mammalian species.

MERVL/Zscan4 Network Activation Results in Transient Genome-wide DNA Demethylation of mESCs.

Mouse embryonic stem cells are dynamic and heterogeneous. For example, rare cells cycle through a state characterized by decondensed chromatin and expression of transcripts, including the Zscan4 cluster and MERVL endogenous retrovirus, which are usually restricted to preimplantation embryos. Here, we further characterize the dynamics and consequences of this transient cell state. Single-cell transcriptomics identified the earliest upregulated transcripts as cells enter the MERVL/Zscan4 state. The MERVL/Zscan4 transcriptional network was also upregulated during induced pluripotent stem cell reprogramming. Genome-wide DNA methylation and chromatin analyses revealed global DNA hypomethylation accompanying increased chromatin accessibility. This transient DNA demethylation was driven by a loss of DNA methyltransferase proteins in the cells and occurred genome-wide. While methylation levels were restored once cells exit this state, genomic imprints remained hypomethylated, demonstrating a potential global and enduring influence of endogenous retroviral activation on the epigenome.

Quantitative analysis of chromatin interaction changes upon a 4.3 Mb deletion at mouse 4E2.

BACKGROUND: Circular chromosome conformation capture (4C) has provided important insights into three dimensional (3D) genome organization and its critical impact on the regulation of gene expression. We developed a new quantitative framework based on polymer physics for the analysis of paired-end sequencing 4C (PE-4Cseq) data. We applied this strategy to the study of chromatin interaction changes upon a 4.3 Mb DNA deletion in mouse region 4E2. RESULTS: A significant number of differentially interacting regions (DIRs) and chromatin compaction changes were detected in the deletion chromosome compared to a wild-type (WT) control. Selected DIRs were validated by 3D DNA FISH experiments, demonstrating the robustness of our pipeline. Interestingly, significant overlaps of DIRs with CTCF/Smc1 binding sites and differentially expressed genes were observed. CONCLUSIONS: Altogether, our PE-4Cseq analysis pipeline provides a comprehensive characterization of DNA deletion effects on chromatin structure and function.

Regulation of the ESC transcriptome by nuclear long noncoding RNAs.

Long noncoding (lnc)RNAs have recently emerged as key regulators of gene expression. Here, we performed high-depth poly(A)(+) RNA sequencing across multiple clonal populations of mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs) to comprehensively identify differentially regulated lncRNAs. We establish a biologically robust profile of lncRNA expression in these two cell types and further confirm that the majority of these lncRNAs are enriched in the nucleus. Applying weighted gene coexpression network analysis, we define a group of lncRNAs that are tightly associated with the pluripotent state of ESCs. Among these, we show that acute depletion of Platr14 using antisense oligonucleotides impacts the differentiation- and development-associated gene expression program of ESCs. Furthermore, we demonstrate that Firre, a lncRNA highly enriched in the nucleoplasm and previously reported to mediate chromosomal contacts in ESCs, controls a network of genes related to RNA processing. Together, we provide a comprehensive, up-to-date, and high resolution compilation of lncRNA expression in ESCs and NPCs and show that nuclear lncRNAs are tightly integrated into the regulation of ESC gene expression.

Random monoallelic expression: regulating gene expression one allele at a time.

Monoallelic gene expression is a remarkable process in which transcription occurs from only one of two homologous alleles in a diploid cell. Interestingly, between 0.5% and 15% of autosomal genes exhibit random monoallelic gene expression, in which different cells express only one allele independently of the underlying genomic sequence, in a cell type-specific manner. Recently, genome-wide studies have increased our understanding of the cell type-specific incidence of random monoallelic gene expression, and how the imbalance in allelic expression is distinguished within the cell and potentially maintained across cell generations. Monoallelic gene expression is likely generated through stochastic independent regulation of the two alleles upon differentiation, and has varied implications for the cell and organism, in particular with respect to disease.

Random monoallelic gene expression increases upon embryonic stem cell differentiation.

Random autosomal monoallelic gene expression refers to the transcription of a gene from one of two homologous alleles. We assessed the dynamics of monoallelic expression during development through an allele-specific RNA-sequencing screen in clonal populations of hybrid mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs). We identified 67 and 376 inheritable autosomal random monoallelically expressed genes in ESCs and NPCs, respectively, a 5.6-fold increase upon differentiation. Although DNA methylation and nuclear positioning did not distinguish the active and inactive alleles, specific histone modifications were differentially enriched between the two alleles. Interestingly, expression levels of 8% of the monoallelically expressed genes remained similar between monoallelic and biallelic clones. These results support a model in which random monoallelic expression occurs stochastically during differentiation and, for some genes, is compensated for by the cell to maintain the required transcriptional output of these genes.

Role of SWI/SNF in acute leukemia maintenance and enhancer-mediated Myc regulation.

Cancer cells frequently depend on chromatin regulatory activities to maintain a malignant phenotype. Here, we show that leukemia cells require the mammalian SWI/SNF chromatin remodeling complex for their survival and aberrant self-renewal potential. While Brg1, an ATPase subunit of SWI/SNF, is known to suppress tumor formation in several cell types, we found that leukemia cells instead rely on Brg1 to support their oncogenic transcriptional program, which includes Myc as one of its key targets. To account for this context-specific function, we identify a cluster of lineage-specific enhancers located 1.7 Mb downstream from Myc that are occupied by SWI/SNF as well as the BET protein Brd4. Brg1 is required at these distal elements to maintain transcription factor occupancy and for long-range chromatin looping interactions with the Myc promoter. Notably, these distal Myc enhancers coincide with a region that is focally amplified in ∼3% of acute myeloid leukemias. Together, these findings define a leukemia maintenance function for SWI/SNF that is linked to enhancer-mediated gene regulation, providing general insights into how cancer cells exploit transcriptional coactivators to maintain oncogenic gene expression programs.

Lamin A/C is expressed in pluripotent mouse embryonic stem cells.

The pluripotent nature of embryonic stem cells (ESC) is associated with a dynamic open chromatin state and an irregular nuclear shape. It has been postulated that the absence of Lamin A/C contributes to these features. However, we show that mouse ESCs express low, yet readily detectable, amounts of Lamin A/C at both the RNA and protein levels. Full-length transcripts of both isoforms were readily detected by q-PCR and deep RNA sequencing. Additionally, protein expression was validated in multiple primary and established ESC lines by immunoblotting using several independent antibodies. Immunofluorescence labeling showed localization of Lamin A/C at the nuclear periphery of all Oct4/Nanog double-positive ESC lines examined, as well as in the inner cell mass of blastocysts. Our results demonstrate ESCs do express low levels of Lamin A/C, thus models linking pluripotency and nuclear dynamics with the absence of Lamin A/C need to be revisited.

Chromatin organization and transcriptional regulation.

Cell type specific transcriptional regulation must be adhered to in order to maintain cell identity throughout the lifetime of an organism, yet it must be flexible enough to allow for responses to endogenous and exogenous stimuli. This regulation is mediated not only by molecular factors (e.g. cell type specific transcription factors, histone and DNA modifications), but also on the level of chromatin and genome organization. In this review we focus on recent findings that have contributed to our understanding of higher order chromatin structure and genome organization within the nucleus. We highlight new findings on the dynamic positioning of genes relative to each other, as well as to their chromosome territory and the nuclear lamina, and how the position of genes correlates with their transcriptional activity.

Bone marrow stem cells and the liver: are they relevant?

The contribution of bone marrow stem cell responses to liver homeostasis, injury and malignancy is discussed in this review. Pluripotent stem cells or their more committed progenitor progeny are essential to tissue development, regeneration and repair and are widely implicated in the pathogenesis of malignancy. Stem cell responses to injury are the focus of intense research efforts in the hope of future therapeutic manipulation. Stem cells occur within tissues, such as the liver, or arise from extrahepatic sites, in particular, the bone marrow. As the largest reservoir of stem cells in the adult, the bone marrow has been implicated in the stem cell response associated with liver injury. However, in liver injury, the relative contribution of bone marrow stem cells compared to intrahepatic progenitor responses is poorly characterized. Intrahepatic progenitor responses have been recently reviewed elsewhere. In this review, we have summarized liver-specific extrahepatic stem cell responses originating from the bone marrow. The physiological relevance of bone marrow stem cell responses to adult liver homeostasis, injury and malignancy is discussed with emphasis on mechanisms of bone marrow stem cell recruitment to sites of liver injury and its contribution to intrahepatic malignancy.