OCT2 pre-positioning facilitates cell fate transition and chromatin architecture changes in humoral immunity.

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.

H3K27ac bookmarking promotes rapid post-mitotic activation of the pluripotent stem cell program without impacting 3D chromatin reorganization.

During self-renewal, cell-type-defining features are drastically perturbed in mitosis and must be faithfully reestablished upon G1 entry, a process that remains largely elusive. Here, we characterized at a genome-wide scale the dynamic transcriptional and architectural resetting of mouse pluripotent stem cells (PSCs) upon mitotic exit. We captured distinct waves of transcriptional reactivation with rapid induction of stem cell genes and transient activation of lineage-specific genes. Topological reorganization at different hierarchical levels also occurred in an asynchronous manner and showed partial coordination with transcriptional resetting. Globally, rapid transcriptional and architectural resetting associated with mitotic retention of H3K27 acetylation, supporting a bookmarking function. Indeed, mitotic depletion of H3K27ac impaired the early reactivation of bookmarked, stem-cell-associated genes. However, 3D chromatin reorganization remained largely unaffected, suggesting that these processes are driven by distinct forces upon mitotic exit. This study uncovers principles and mediators of PSC molecular resetting during self-renewal.

RBBP6 isoforms regulate the human polyadenylation machinery and modulate expression of mRNAs with AU-rich 3' UTRs.

Polyadenylation of mRNA precursors is mediated by a large multisubunit protein complex. Here we show that RBBP6 (retinoblastoma-binding protein 6), identified initially as an Rb- and p53-binding protein, is a component of this complex and functions in 3' processing in vitro and in vivo. RBBP6 associates with other core factors, and this interaction is mediated by an unusual ubiquitin-like domain, DWNN ("domain with no name"), that is required for 3' processing activity. The DWNN is also expressed, via alternative RNA processing, as a small single-domain protein (isoform 3 [iso3]). Importantly, we show that iso3, known to be down-regulated in several cancers, competes with RBBP6 for binding to the core machinery, thereby inhibiting 3' processing. Genome-wide analyses following RBBP6 knockdown revealed decreased transcript levels, especially of mRNAs with AU-rich 3' untranslated regions (UTRs) such as c-Fos and c-Jun, and increased usage of distal poly(A) sites. Our results implicate RBBP6 and iso3 as novel regulators of 3' processing, especially of RNAs with AU-rich 3' UTRs.

PARP1 represses PAP and inhibits polyadenylation during heat shock.

The 3' ends of most eukaryotic mRNAs are produced by an endonucleolytic cleavage followed by synthesis of a poly(A) tail. Poly(A) polymerase (PAP), the enzyme that catalyzes the formation of the tail, is subject to tight regulation involving several posttranslational modifications. Here we show that the enzyme poly(ADP-ribose) polymerase 1 (PARP1) modifies PAP and regulates its activity both in vitro and in vivo. PARP1 binds to and modifies PAP by poly(ADP-ribosyl)ation (PARylation) in vitro, which inhibits PAP activity. In vivo we show that PAP is PARylated during heat shock, leading to inhibition of polyadenylation in a PARP1-dependent manner. The observed inhibition reflects reduced RNA binding affinity of PARylated PAP in vitro and decreased PAP association with non-heat shock protein-encoding genes in vivo. Our results provide direct evidence that PARylation can control processing of mRNA precursors, and also identify PARP1 as a regulator of polyadenylation during thermal stress.

Mechanisms and consequences of alternative polyadenylation.

Alternative polyadenylation (APA) is emerging as a widespread mechanism used to control gene expression. Like alternative splicing, usage of alternative poly(A) sites allows a single gene to encode multiple mRNA transcripts. In some cases, this changes the mRNA coding potential; in other cases, the code remains unchanged but the 3' UTR length is altered, influencing the fate of mRNAs in several ways, for example, by altering the availability of RNA binding protein sites and microRNA binding sites. The mechanisms governing both global and gene-specific APA are only starting to be deciphered. Here we review what is known about these mechanisms and the functional consequences of alternative polyadenylation.

Molecular architecture of the human pre-mRNA 3' processing complex.

Pre-mRNA 3' end formation is an essential step in eukaryotic gene expression. Over half of human genes produce alternatively polyadenylated mRNAs, suggesting that regulated polyadenylation is an important mechanism for posttranscriptional gene control. Although a number of mammalian mRNA 3' processing factors have been identified, the full protein composition of the 3' processing machinery has not been determined, and its structure is unknown. Here we report the purification and subsequent proteomic and structural characterization of human mRNA 3' processing complexes. Remarkably, the purified 3' processing complex contains approximately 85 proteins, including known and new core 3' processing factors and over 50 proteins that may mediate crosstalk with other processes. Electron microscopic analyses show that the core 3' processing complex has a distinct "kidney" shape and is approximately 250 A in length. Together, our data has revealed the complexity and molecular architecture of the pre-mRNA 3' processing complex.

3D Enhancer-promoter networks provide predictive features for gene expression and coregulation in early embryonic lineages.

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.

Systematic mapping and modeling of 3D enhancer-promoter interactions in early mouse embryonic lineages reveal regulatory principles that determine the levels and cell-type specificity of gene expression.

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 in silico 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.

Assessing Specific Networks of Chromatin Interactions with HiChIP.

The introduction of chromosome conformation capture (3C)-based technologies coupled with next-generation sequencing have significantly advanced our understanding of how the genetic material is organized within the eukaryotic nucleus. Three-dimensional (3D) genomic organization occurs at hierarchical levels, ranging from chromosome territories and subnuclear compartments to smaller self-associated domains and fine-scale chromatin interactions. The latter can be further categorized into different subtypes, such as structural or regulatory, based either on their presumed functionality and/or the factors that mediate their formation. Various enrichment strategies coupled with 3C-based technologies have been developed to prospectively isolate and quantify chromatin interactions around regions occupied by specific proteins or marks of interest. These approaches not only enable high-resolution characterization of the selected chromatin contacts at a cost-effective manner, but also offer important biological insights into their organizational principles and regulatory function. In this chapter, we will focus on the recently developed HiChIP technology with an emphasis on the discovery of putative active enhancers and promoter interactions in cell types of interest. We will describe the specific steps for designing, performing and analyzing successful HiChIP experiments as well as important limitations and considerations.

A Knock-In Mouse Model of Thymoma With the GTF2I L424H Mutation.

INTRODUCTION: The pathogenesis of thymic epithelial tumors remains largely unknown. We previously identified GTF2I L424H as the most frequently recurrent mutation in thymic epithelial tumors. Nevertheless, the precise role of this mutation in tumorigenesis of thymic epithelial cells is unclear. METHODS: To investigate the role of GTF2I L424H mutation in thymic epithelial cells in vivo, we generated and characterized a mouse model in which the Gtf2i L424H mutation was conditionally knocked-in in the Foxn1+ thymic epithelial cells. Digital spatial profiling was performed on thymomas and normal thymic tissues with GeoMx-mouse whole transcriptome atlas. Immunohistochemistry staining was performed using both mouse tissues and human thymic epithelial tumors. RESULTS: We observed that the Gtf2i mutation impairs development of the thymic medulla and maturation of medullary thymic epithelial cells in young mice and causes tumor formation in the thymus of aged mice. Cell cycle-related pathways, such as E2F targets and MYC targets, are enriched in the tumor epithelial cells. Results of gene set variation assay analysis revealed that gene signatures of cortical thymic epithelial cells and thymic epithelial progenitor cells are also enriched in the thymomas of the knock-in mice, which mirrors the human counterparts in The Cancer Genome Atlas database. Immunohistochemistry results revealed similar expression pattern of epithelial cell markers between mouse and human thymomas. CONCLUSIONS: We have developed and characterized a novel thymoma mouse model. This study improves knowledge of the molecular drivers in thymic epithelial cells and provides a tool for further study of the biology of thymic epithelial tumors and for development of novel therapies.

Transcription factors: building hubs in the 3D space.

The hierarchical three-dimensional folding of the mammalian genome constitutes an important regulatory layer of gene expression and cell fate control during processes such as development and tumorigenesis. Accumulating evidence supports the existence of complex topological assemblies in which multiple genes and regulatory elements are frequently interacting with each other in the 3D nucleus. Here, we will discuss the nature, organizational principles, and potential function of such assemblies, including the recently reported enhancer "hubs," "cliques," and FIREs (frequently interacting regions) as well as multi-contact hubs. We will also review recent studies that investigate the role of transcription factors (TFs) in driving the topological genome reorganization and hub formation in the context of cell fate transitions and cancer. Finally, we will highlight technological advances that enabled these studies, current limitations, and future directions necessary to advance our understating in the field.

KLF4 is involved in the organization and regulation of pluripotency-associated three-dimensional enhancer networks.

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.

EpiMethylTag: simultaneous detection of ATAC-seq or ChIP-seq signals with DNA methylation.

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.

Widespread Mitotic Bookmarking by Histone Marks and Transcription Factors in Pluripotent Stem Cells.

During mitosis, transcription is halted and many chromatin features are lost, posing a challenge for the continuity of cell identity, particularly in fast cycling stem cells, which constantly balance self-renewal with differentiation. Here we show that, in pluripotent stem cells, certain histone marks and stem cell regulators remain associated with specific genomic regions of mitotic chromatin, a phenomenon known as mitotic bookmarking. Enhancers of stem cell-related genes are bookmarked by both H3K27ac and the master regulators OCT4, SOX2, and KLF4, while promoters of housekeeping genes retain high levels of mitotic H3K27ac in a cell-type invariant manner. Temporal degradation of OCT4 during mitotic exit compromises its ability both to maintain and induce pluripotency, suggesting that its regulatory function partly depends on its bookmarking activity. Together, our data document a widespread yet specific bookmarking by histone modifications and transcription factors promoting faithful and efficient propagation of stemness after cell division.

The Chromatin Signature of Pluripotency: Establishment and Maintenance.

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.

New links between mRNA polyadenylation and diverse nuclear pathways.

The 3' ends of most eukaryotic messenger RNAs must undergo a maturation step that includes an endonuc-leolytic cleavage followed by addition of a polyadenylate tail. While this reaction is catalyzed by the action of only two enzymes it is supported by an unexpectedly large number of proteins. This complexity reflects the necessity of coordinating this process with other nuclear events, and growing evidence indicates that even more factors than previously thought are necessary to connect 3' processing to additional cellular pathways. In this review we summarize the current understanding of the molecular machinery involved in this step of mRNA maturation, focusing on new core and auxiliary proteins that connect polyadenylation to splicing, DNA damage, transcription and cancer.