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During the germinal center (GC) reaction, B cells undergo profound transcriptional, epigenetic and genomic architectural changes. How such changes are established remains unknown. Mapping chromatin accessibility during the humoral immune response, we show that OCT2 was the dominant transcription factor linked to differential accessibility of GC regulatory elements. Silent chromatin regions destined to become GC-specific super-enhancers (SEs) contained pre-positioned OCT2-binding sites in naive B cells (NBs). These preloaded SE ‘seeds’ featured spatial clustering of regulatory elements enriched in OCT2 DNA-binding motifs that became heavily loaded with OCT2 and its GC-specific coactivator OCAB in GC B cells (GCBs). SEs with high abundance of pre-positioned OCT2 binding preferentially formed long-range chromatin contacts in GCs, to support expression of GC-specifying factors. Gain in accessibility and architectural interactivity of these regions were dependent on recruitment of OCAB. Pre-positioning key regulators at SEs may represent a broadly used strategy for facilitating rapid cell fate transitions. Elemento, Melnick and colleagues examine the chromatin and transcriptional changes that occur during differentiation of human primary B cells into antibody-secreting cells. In naive B cells, the transcription factor OCT2 is preloaded at high-affinity super-enhancer sites present in repressed ‘silent’ chromatin; upon activation, OCAB is recruited to these regions, where it facilitates arrays of OCT2 binding to lower-affinity octamer motifs, leading to active formation of germinal center B cell-specific super-enhancers.

Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors, as is exemplified by reprogramming somatic cells to pluripotent stem cells through the expression of OCT4, KLF4, SOX2 and cMYC. How transcription factors orchestrate the complex molecular changes around their target gene loci remains incompletely understood. Here, using KLF4 as a paradigm, we provide a transcription-factor-centric view of chromatin reorganization and its association with three-dimensional enhancer rewiring and transcriptional changes during the reprogramming of mouse embryonic fibroblasts to pluripotent stem cells. Inducible depletion of KLF factors in PSCs caused a genome-wide decrease in enhancer connectivity, whereas disruption of individual KLF4 binding sites within pluripotent-stem-cell-specific enhancers was sufficient to impair enhancer–promoter contacts and reduce the expression of associated genes. Our study provides an integrative view of the complex activities of a lineage-specifying transcription factor and offers novel insights into the nature of the molecular events that follow transcription factor binding. Di Giammartino, Kloetgen, Polyzos, Liu et al. probe chromatin organization, enhancer status and transcriptional changes and show that KLF4 acts as a transcriptional regulator and chromatin organizer during induced pluripotent stem cell reprogramming and in pluripotent stem cells.

The revolutionary discovery that somatic cells can be reprogrammed by a defined set transcription factors to induced pluripotent stem cells (iPSCs) changed dramatically the way we perceive cell fate determination. Importantly, iPSCs, similar to embryo-derived stem cells (ESCs), are characterized by a remarkable developmental plasticity and the capacity to self-renew “indefinitely” under appropriate culture conditions, opening new avenues for personalized therapy and disease modeling. Elucidating the molecular mechanisms that maintain, induce, or alter stem cell identity is crucial for a deeper understanding of cell fate determination and potential translational applications. Intense research over the last 10 years exploiting technological advances in epigenomics and genome editing has unraveled many of the mysteries of pluripotent identity enabling novel and efficient ways to manipulate it for biomedical purposes. In this review, we focus on the chromatin and epigenetic characteristics that distinguish stem cells from somatic cells and their dynamic changes during differentiation and reprogramming.

Activation of regulatory elements is thought to be inversely correlated with DNA methylation levels. However, it is difficult to determine whether DNA methylation is compatible with chromatin accessibility or transcription factor (TF) binding if assays are performed separately. We developed a fast, low-input, low sequencing depth method, EpiMethylTag, that combines ATAC-seq or ChIP-seq (M-ATAC or M-ChIP) with bisulfite conversion, to simultaneously examine accessibility/TF binding and methylation on the same DNA. Here we demonstrate that EpiMethylTag can be used to study the functional interplay between chromatin accessibility and TF binding (CTCF and KLF4) at methylated sites.

Dysregulation of enhancer-promoter communication in the context of the three-dimensional (3D) nucleus is increasingly recognized as a potential driver of oncogenic programs. Here, we profiled the 3D enhancer-promoter networks of primary patient-derived glioblastoma stem cells (GSCs) in comparison with neuronal stem cells (NSCs) to identify potential central nodes and vulnerabilities in the regulatory logic of this devastating cancer. Specifically, we focused on hyperconnected 3D regulatory hubs and demonstrated that hub-interacting genes exhibit high and coordinated expression at the single-cell level and strong association with oncogenic programs that distinguish IDH-wt glioblastoma patients from low-grade glioma. Epigenetic silencing of a recurrent 3D enhancer hub-with an uncharacterized role in glioblastoma-was sufficient to cause concordant downregulation of multiple hub-connected genes along with significant shifts in transcriptional states and reduced clonogenicity. By integrating published datasets from other cancer types, we also identified both universal and cancer type-specific 3D regulatory hubs which enrich for varying oncogenic programs and nominate specific factors associated with worse outcomes. Genetic alterations, such as focal duplications, could explain only a small fraction of the detected hyperconnected hubs and their increased activity. Overall, our study provides computational and experimental support for the potential central role of 3D regulatory hubs in controlling oncogenic programs and properties.

Mammalian embryogenesis commences with two pivotal and binary cell fate decisions that give rise to three essential lineages: the trophectoderm, the epiblast and the primitive endoderm. Although key signaling pathways and transcription factors that control these early embryonic decisions have been identified, the non-coding regulatory elements through which transcriptional regulators enact these fates remain understudied. Here, we characterize, at a genome-wide scale, enhancer activity and 3D connectivity in embryo-derived stem cell lines that represent each of the early developmental fates. We observe extensive enhancer remodeling and fine-scale 3D chromatin rewiring among the three lineages, which strongly associate with transcriptional changes, although distinct groups of genes are irresponsive to topological changes. In each lineage, a high degree of connectivity, or ‘hubness’, positively correlates with levels of gene expression and enriches for cell-type specific and essential genes. Genes within 3D hubs also show a significantly stronger probability of coregulation across lineages compared to genes in linear proximity or within the same contact domains. By incorporating 3D chromatin features, we build a predictive model for transcriptional regulation (3D-HiChAT) that outperforms models using only 1D promoter or proximal variables to predict levels and cell-type specificity of gene expression. Using 3D-HiChAT, we identify, in silico, candidate functional enhancers and hubs in each cell lineage, and with CRISPRi experiments, we validate several enhancers that control gene expression in their respective lineages. Our study identifies 3D regulatory hubs associated with the earliest mammalian lineages and describes their relationship to gene expression and cell identity, providing a framework to comprehensively understand lineage-specific transcriptional behaviors. Here, the authors describe 3D hubs as regulatory subunits of gene expression in the three essential lineages of embryogenesis. They develop a computational model that can predict novel enhancers and they validate such enhancers in the context of specific lineages.

Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors (TFs), as is exemplified by reprogramming somatic cells to pluripotent stem cells (PSCs) via expression of OCT4, KLF4, SOX2 and cMYC. How TFs orchestrate the complex molecular changes around their target gene loci remains incompletely understood. Here, using KLF4 as a paradigm, we provide a TF-centric view of chromatin reorganization and its association to 3D enhancer rewiring and transcriptional changes during reprogramming of mouse embryonic fibroblasts to PSCs. Inducible depletion of KLF factors in PSCs caused a genome-wide decrease in enhancer connectivity, while disruption of individual KLF4 binding sites within PSC-specific enhancers was sufficient to impair enhancer-promoter contacts and reduce expression of associated genes. Our study provides an integrative view of the complex activities of a lineage-specifying TF and offers novel insights into the nature of molecular events that follow TF binding.

The mature 3' ends of most mRNAs are created by a two-step reaction that involves an endonucleolytic cleavage of the pre-mRNA followed by polyadenylation of the upstream product. The 3' processing machinery is composed of four multisubunit complexes, which, together with a few other proteins, constitute the core components required for cleavage and polyadenylation. A proteomic analysis led to the identification of approximately 80 proteins that associate with the human pre-mRNA 3' processing complex, including new core 3' factors and other proteins that might mediate crosstalk between 3' processing and other nuclear pathways. This thesis focuses on two of the newly identified proteins, which we found particularly intriguing: PARP1 and RBBP6. PARP1 is an enzyme that, when activated, catalyzes the polymerization of ADP-ribose units from donor NAD molecules to acceptor proteins, a reaction known as PARylation. This post-translational modification has been shown to modulate critical events such as DNA damage response and transcription. We found that PARP1 binds PAP, the enzyme responsible for polyadenylating the 3' ends of mRNAs, and modifies it by PARylation. In vivo PAP is PARylated during heat shock, leading to inhibition of polyadenylation in a PARP1-dependent manner. Finally, we show that the observed inhibition reflects decreased PAP association with 3' end of genes. These results identify PARP1 as a regulator of polyadenylation during thermal stress and show for the first time that PARylation can control gene expression by modulating processing of mRNA. The second project involves RBBP6, a large multidomain protein that is known to interact with p53 and Rb. The N-terminal part of the human RBBP6 includes a DWNN domain, which is particularly interesting because it adopts a ubiquitin-like fold and, in addition to forming part of the full-length RBBP6 protein, is also expressed as a small protein (RBBP6 isoform3) which has been shown to be downregulated in several human cancers. We found that RBBP6 is essential for the cleavage activity of the 3' processing complex and that an N-terminal derivative of RBBP6 (RBBP6-N), containing only the DWNN, Zinc and Ring domains, is enough to rescue cleavage activity. The RBBP6 and RBBP6 isoform3 can compete with each other in binding to Cstf64 (an interaction mediated by the DWNN domain). In addition, overexpression of isoform3 inhibits cleavage raising intriguing possibilities of modulation of 3' processing by fine-tuning the levels of the two RBBP6 isoforms. To better characterize the function of RBBP6 globally, we also performed genome-wide analysis, both by microarray and deep sequencing. Following RBBP6 knockdown we observed a general lengthening of 3' UTRs accompanied by an overall downregulation in gene expression, especially of RNAs with AU-rich 3'UTRs. We show that this is the result of a defect in their 3' cleavage and subsequent degradation by the exosome. All together our results point to a role for RBBP6 as a new core 3' processing factor able to regulate the expression of AU-rich mRNAs.

Abstract Activation of regulatory elements is thought to be inversely correlated with DNA methylation levels. However, it is difficult to determine whether DNA methylation is compatible with chromatin accessibility or transcription factor (TF) binding if assays are performed separately. We developed a fast, low input, low sequencing depth method, EpiMethylTag that combines ATAC-seq or ChIP-seq (M-ATAC or M-ChIP) with bisulfite conversion, to simultaneously examine accessibility/TF binding and methylation on the same DNA. Here we demonstrate that EpiMethylTag can be used to study the functional interplay between chromatin accessibility and TF binding (CTCF and KLF4) at methylated sites. Footnotes * Email addresses: Priscillia.Lhoumaud{at}nyumc.org, Gunjan.Sethia{at}nyumc.org, fri2002{at}med.cornell.edu, Theodoros.Sakellaropoulos{at}nyulangone.org, snetkv01{at}nyu.edu, simon.vidal{at}gmail.com, Sana.Badri{at}nyumc.org, MacIntosh.Cornwell{at}nyulangone.org, dac2051{at}med.cornell.edu, thinktank.q{at}gmail.com, efa2001{at}med.cornell.edu, mas4011{at}med.cornell.edu, dlandau{at}nygenome.org * authors and new data were added. author affiliations updated; Supplemental files updated.

Mammalian embryogenesis commences with two pivotal and binary cell fate decisions that give rise to three essential lineages, the trophectoderm (TE), the epiblast (EPI) and the primitive endoderm (PrE). Although key signaling pathways and transcription factors that control these early embryonic decisions have been identified, the non-coding regulatory elements via which transcriptional regulators enact these fates remain understudied. To address this gap, we have characterized, at a genome-wide scale, enhancer activity and 3D connectivity in embryo-derived stem cell lines that represent each of the early developmental fates. We observed extensive enhancer remodeling and fine-scale 3D chromatin rewiring among the three lineages, which strongly associate with transcriptional changes, although there are distinct groups of genes that are irresponsive to topological changes. In each lineage, a high degree of connectivity or \"hubness\" positively correlates with levels of gene expression and enriches for cell-type specific and essential genes. Genes within 3D hubs also show a significantly stronger probability of coregulation across lineages, compared to genes in linear proximity or within the same contact domains. By incorporating 3D chromatin features, we build a novel predictive model for transcriptional regulation (3D-HiChAT), which outperformed models that use only 1D promoter or proximal variables in predicting levels and cell-type specificity of gene expression. Using 3D-HiChAT, we performed genome-wide perturbations to nominate candidate functional enhancers and hubs in each cell lineage, and with CRISPRi experiments we validated several novel enhancers that control expression of one or more genes in their respective lineages. Our study comprehensively identifies 3D regulatory hubs associated with the earliest mammalian lineages and describes their relationship to gene expression and cell identity, providing a framework to understand lineage-specific transcriptional behaviors.