The ChAHP Complex Counteracts Chromatin Looping at CTCF Sites that Emerged from SINE Expansions in Mouse.

CCCTC-binding factor (CTCF) and cohesin are key players in three-dimensional chromatin organization. The topologically associating domains (TADs) demarcated by CTCF are remarkably well conserved between species, although genome-wide CTCF binding has diverged substantially following transposon-mediated motif expansions. Therefore, the CTCF consensus motif poorly predicts TADs, and additional factors must modulate CTCF binding and subsequent TAD formation. Here, we demonstrate that the ChAHP complex (CHD4, ADNP, HP1) competes with CTCF for a common set of binding motifs. In Adnp knockout cells, novel insulated regions are formed at sites normally bound by ChAHP, whereas proximal canonical boundaries are weakened. These data reveal that CTCF-mediated loop formation is modulated by a distinct zinc-finger protein complex. Strikingly, ChAHP-bound loci are mainly situated within less diverged SINE B2 transposable elements. This implicates ChAHP in maintenance of evolutionarily conserved spatial chromatin organization by buffering novel CTCF binding sites that emerged through SINE expansions.

The Cohesin Release Factor WAPL Restricts Chromatin Loop Extension.

The spatial organization of chromosomes influences many nuclear processes including gene expression. The cohesin complex shapes the 3D genome by looping together CTCF sites along chromosomes. We show here that chromatin loop size can be increased and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. Cohesin's DNA release factor WAPL restricts this loop extension and also prevents looping between incorrectly oriented CTCF sites. We reveal that the SCC2/SCC4 complex promotes the extension of chromatin loops and the formation of topologically associated domains (TADs). Our data support the model that cohesin structures chromosomes through the processive enlargement of loops and that TADs reflect polyclonal collections of loops in the making. Finally, we find that whereas cohesin promotes chromosomal looping, it rather limits nuclear compartmentalization. We conclude that the balanced activity of SCC2/SCC4 and WAPL enables cohesin to correctly structure chromosomes.

The Pol II preinitiation complex (PIC) influences Mediator binding but not promoter-enhancer looping.

Knowledge of how Mediator and TFIID cross-talk contributes to promoter-enhancer (P-E) communication is important for elucidating the mechanism of enhancer function. We conducted an shRNA knockdown screen in murine embryonic stem cells to identify the functional overlap between Mediator and TFIID subunits on gene expression. Auxin-inducible degrons were constructed for TAF12 and MED4, the subunits eliciting the greatest overlap. Degradation of TAF12 led to a dramatic genome-wide decrease in gene expression accompanied by destruction of TFIID, loss of Pol II preinitiation complex (PIC) at promoters, and significantly decreased Mediator binding to promoters and enhancers. Interestingly, loss of the PIC elicited only a mild effect on P-E looping by promoter capture Hi-C (PCHi-C). Degradation of MED4 had a minor effect on Mediator integrity but led to a consistent twofold loss in gene expression, decreased binding of Pol II to Mediator, and decreased recruitment of Pol II to the promoters, but had no effect on the other PIC components. PCHi-C revealed no consistent effect of MED4 degradation on P-E looping. Collectively, our data show that TAF12 and MED4 contribute mechanistically in different ways to P-E communication but neither factor appears to directly control P-E looping, thereby dissociating P-E communication from physical looping.

TEAD2 initiates ground-state pluripotency by mediating chromatin looping.

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.

Spliceosome component PHD finger 5A is essential for early B lymphopoiesis.

The spliceosome, a multi-megadalton ribonucleoprotein complex, is essential for pre-mRNA splicing in the nucleus and ensuring genomic stability. Its precise and dynamic assembly is pivotal for its function. Spliceosome malfunctions can lead to developmental abnormalities and potentially contribute to tumorigenesis. The specific role of the spliceosome in B cell development is poorly understood. Here, we reveal that the spliceosomal U2 snRNP component PHD finger protein 5A (Phf5a) is vital for early B cell development. Loss of Phf5a results in pronounced defects in B cell development, causing an arrest at the transition from pre-pro-B to early pro-B cell stage in the bone marrow of mutant mice. Phf5a-deficient B cells exhibit impaired immunoglobulin heavy (IgH) chain expression due to defective V-to-DJ gene rearrangement. Mechanistically, our findings suggest that Phf5a facilitates IgH gene rearrangement by regulating the activity of recombination-activating gene endonuclease and influencing chromatin interactions at the Igh locus.

Promoter-Enhancer Communication Occurs Primarily within Insulated Neighborhoods.

Metazoan chromosomes are sequentially partitioned into topologically associating domains (TADs) and then into smaller sub-domains. One class of sub-domains, insulated neighborhoods, are proposed to spatially sequester and insulate the enclosed genes through self-association and chromatin looping. However, it has not been determined functionally whether promoter-enhancer interactions and gene regulation are broadly restricted to within these loops. Here, we employed published datasets from murine embryonic stem cells (mESCs) to identify insulated neighborhoods that confine promoter-enhancer interactions and demarcate gene regulatory regions. To directly address the functionality of these regions, we depleted estrogen-related receptor ß (Esrrb), which binds the Mediator co-activator complex, to impair enhancers of genes within 222 insulated neighborhoods without causing mESC differentiation. Esrrb depletion reduces Mediator binding, promoter-enhancer looping, and expression of both nascent RNA and mRNA within the insulated neighborhoods without significantly affecting the flanking genes. Our data indicate that insulated neighborhoods represent functional regulons in mammalian genomes.

Decreased Enhancer-Promoter Proximity Accompanying Enhancer Activation.

Enhancers can regulate the promoters of their target genes over very large genomic distances. It is widely assumed that mechanisms of enhancer action involve the reorganization of three-dimensional chromatin architecture, but this is poorly understood. The predominant model involves physical enhancer-promoter interaction by looping out the intervening chromatin. However, studying the enhancer-driven activation of the Sonic hedgehog gene (Shh), we have identified a change in chromosome conformation that is incompatible with this simple looping model. Using super-resolution 3D-FISH and chromosome conformation capture, we observe a decreased spatial proximity between Shh and its enhancers during the differentiation of embryonic stem cells to neural progenitors. We show that this can be recapitulated by synthetic enhancer activation, is impeded by chromatin-bound proteins located between the enhancer and the promoter, and appears to involve the catalytic activity of poly (ADP-ribose) polymerase. Our data suggest that models of enhancer-promoter communication need to encompass chromatin conformations other than looping.

Impaired DNA demethylation of C/EBP sites causes premature aging.

Changes in DNA methylation are among the best-documented epigenetic alterations accompanying organismal aging. However, whether and how altered DNA methylation is causally involved in aging have remained elusive. GADD45α (growth arrest and DNA damage protein 45A) and ING1 (inhibitor of growth family member 1) are adapter proteins for site-specific demethylation by TET (ten-eleven translocation) methylcytosine dioxygenases. Here we show that Gadd45a/Ing1 double-knockout mice display segmental progeria and phenocopy impaired energy homeostasis and lipodystrophy characteristic of Cebp (CCAAT/enhancer-binding protein) mutants. Correspondingly, GADD45α occupies C/EBPß/δ-dependent superenhancers and, cooperatively with ING1, promotes local DNA demethylation via long-range chromatin loops to permit C/EBPß recruitment. The results indicate that enhancer methylation can affect aging and imply that C/EBP proteins play an unexpected role in this process. Our study suggests a causal nexus between DNA demethylation, metabolism, and organismal aging.

Cell type-specific chromatin topology and gene regulation.

Chromatin structure is critically involved in gene regulation and cell fate determination. How this structure is established and maintained in distinct, terminally differentiated cells remains elusive. Winick-Ng et al. address this puzzle by applying immunoGAM in different brain cell types and reveal cell type-specific chromatin topologies, long gene decompaction, and the involvement of transcription factors (TFs).

Dynamic Rewiring of Promoter-Anchored Chromatin Loops during Adipocyte Differentiation.

Interactions between transcriptional promoters and their distal regulatory elements play an important role in transcriptional regulation; however, the extent to which these interactions are subject to rapid modulations in response to signals is unknown. Here, we use promoter capture Hi-C to demonstrate a rapid reorganization of promoter-anchored chromatin loops within 4 hr after inducing differentiation of 3T3-L1 preadipocytes. The establishment of new promoter-enhancer loops is tightly coupled to activation of poised (histone H3 lysine 4 mono- and dimethylated) enhancers, as evidenced by the acquisition of histone H3 lysine 27 acetylation and the binding of MED1, SMC1, and P300 proteins to these regions, as well as to activation of target genes. Intriguingly, formation of loops connecting activated enhancers and promoters is also associated with extensive recruitment of corepressors such as NCoR and HDACs, indicating that this class of coregulators may play a previously unrecognized role during enhancer activation.

Enhancer-activated RET confers protection against oxidative stress to KMT2A-rearranged acute myeloid leukemia.

Ectopic activation of rearranged during transfection (RET) has been reported to facilitate lineage differentiation and cell proliferation in different cytogenetic subtypes of acute myeloid leukemia (AML). Herein, we demonstrate that RET is significantly (p < 0.01) upregulated in AML subtypes containing rearrangements of the lysine methyltransferase 2A gene (KMT2A), commonly referred to as KMT2A-rearranged (KMT2A-r) AML. Integrating multi-epigenomics data, we show that the KMT2A-MLLT3 fusion induces the development of CCCTC-binding (CTCF)-guided de novo extrusion enhancer loop to upregulate RET expression in KMT2A-r AML. Based on the finding that RET expression is tightly correlated with the selective chromatin remodeler and mediator (MED) proteins, we used a small-molecule inhibitor having dual inhibition against RET and MED12-associated cyclin-dependent kinase 8 (CDK8) in KMT2A-r AML cells. Dual inhibition of RET and CDK8 restricted cell proliferation by producing multimodal oxidative stress responses in treated cells. Our data suggest that epigenetically enhanced RET protects KMT2A-r AML cells from oxidative stresses, which could be exploited as a potential therapeutic strategy.

Transcriptional Regulation by (Super)Enhancers: From Discovery to Mechanisms.

Accurate control of gene expression in the right cell at the right moment is of fundamental importance to animal development and homeostasis. At the heart of gene regulation lie the enhancers, a class of gene regulatory elements that ensures precise spatiotemporal activation of gene transcription. Mammalian genomes are littered with enhancers, which are frequently organized in cooperative clusters such as locus control regions and superenhancers. Here, we discuss our current knowledge of enhancer biology, including an overview of the discovery of the various enhancer subsets and the mechanistic models used to explain their gene regulatory function.

A spatiotemporally regulated transcriptional complex underlies heteroblastic development of leaf hairs in Arabidopsis thaliana.

Heteroblasty refers to a phenomenon that a plant produces morphologically or functionally different lateral organs in an age-dependent manner. In the model plant Arabidopsis thaliana, the production of trichomes (epidermal leaf hairs) on the abaxial (lower) side of leaves is a heteroblastic mark for the juvenile-to-adult transition. Here, we show that the heteroblastic development of abaxial trichomes is regulated by a spatiotemporally regulated complex comprising the leaf abaxial fate determinant (KAN1) and the developmental timer (miR172-targeted AP2-like proteins). We provide evidence that a short-distance chromatin loop brings the downstream enhancer element into close association with the promoter elements of GL1, which encodes a MYB transcription factor essential for trichome initiation. During juvenile phase, the KAN1-AP2 repressive complex binds to the downstream sequence of GL1 and represses its expression through chromatin looping. As plants age, the gradual reduction in AP2-like protein levels leads to decreased amount of the KAN1-AP2 complex, thereby licensing GL1 expression and the abaxial trichome initiation. Our results thus reveal a novel molecular mechanism by which a heteroblastic trait is governed by integrating age and leaf polarity cue in plants.

Gene Positioning Effects on Expression in Eukaryotes.

The packaging and organization of the genome within the eukaryotic interphase nucleus directly influence how the genes are expressed. An underappreciated aspect of genome structure is that it is highly dynamic and that the physical positioning of a gene can impart control over its transcriptional status. In this review, we assess the current knowledge of how gene positioning at different levels of genome organization can directly influence gene expression during interphase. The levels of organization discussed include chromatin looping, topologically associated domains, chromosome territories, and nuclear compartments. We discuss specific studies demonstrating that gene positioning is a dynamic and highly regulated feature of the eukaryotic genome that allows for the essential spatiotemporal regulation of genes.

Estrogen receptor α (ERα)-binding super-enhancers drive key mediators that control uterine estrogen responses in mice.

Estrogen receptor α (ERα) modulates gene expression by interacting with chromatin regions that are frequently distal from the promoters of estrogen-regulated genes. Active chromatin-enriched "super-enhancer" (SE) regions, mainly observed in in vitro culture systems, often control production of key cell type-determining transcription factors. Here, we defined super-enhancers that bind to ERα in vivo within hormone-responsive uterine tissue in mice. We found that SEs are already formed prior to estrogen exposure at the onset of puberty. The genes at SEs encoded critical developmental factors, including retinoic acid receptor α (RARA) and homeobox D (HOXD). Using high-throughput chromosome conformation capture (Hi-C) along with DNA sequence analysis, we demonstrate that most SEs are located at a chromatin loop end and that most uterine genes in loop ends associated with these SEs are regulated by estrogen. Although the SEs were formed before puberty, SE-associated genes acquired optimal ERα-dependent expression after reproductive maturity, indicating that pubertal processes that occur after SE assembly and ERα binding are needed for gene responses. Genes associated with these SEs affected key estrogen-mediated uterine functions, including transforming growth factor ß (TGFß) and LIF interleukin-6 family cytokine (LIF) signaling pathways. To the best of our knowledge, this is the first identification of SE interactions that underlie hormonal regulation of genes in uterine tissue and optimal development of estrogen responses in this tissue.

PAN RNA: transcriptional exhaust from a viral engine.

Kaposi's sarcoma-associated herpesvirus (KSHV), also designated human herpesvirus 8 (HHV-8), has been linked to Kaposi's sarcoma, as well as to primary effusion lymphoma (PEL), and a subset of multicentric Castleman's disease. KSHV genomes are maintained as episomes within infected cells and the virus exhibits a biphasic life cycle consisting of a life-long latent phase during which only a few viral genes are expressed and no viral progeny are produced and a transient lytic reactivation phase, in which a full repertoire of ~ 80 lytic genes are activated in a temporally regulated manner culminating in the release of new virions. Lytic replication is initiated by a single viral protein, K-Rta (ORF50), which activates more than 80 viral genes from multiple resident viral episomes (i.e., viral chromosomes). One of the major targets of K-Rta is a long non-coding nuclear RNA, PAN RNA (polyadenylated nuclear RNA), a lncRNA that accumulates to exceedingly high levels in the nucleus during viral reactivation. K-Rta directly binds to the PAN RNA promoter and robustly activates PAN RNA expression. Although PAN RNA has been known for over 20 years, its role in viral replication is still incompletely understood. In this perspective, we will briefly review the current understanding of PAN RNA and then describe our current working model of this RNA. The model is based on our observations concerning events that occur during KSHV lytic reactivation including (i) a marked accumulation of RNA Pol II at the PAN promoter, (ii) genomic looping emanating from the PAN locus, (iii) interaction of a second viral lytic protein (ORF57) with K-Rta, PAN RNA and RNA Pol II, (iv) the essential requirement for PAN RNA expression in cis for optimal transcriptional execution needed for the entire lytic program, and (v) ORF57 recruitment of RNA Pol II to the PAN genomic locus. Together our results generate a model in which the PAN locus serves as a hub for sequestration/trapping of the cellular transcriptional machinery proximal to viral episomes. Sequestration at the PAN locus facilitates high levels of viral transcription throughout the viral genome during lytic replication. ORF57 acts as a transcription-dependent transactivator at the PAN locus by binding to both Rta and PAN to locally trap RNA Pol II. The resulting accumulation of high levels of nuclear PAN RNA created by this process is an inducible enhancer-derived (eRNA) by-product that litters the infected cell nucleus.

Enhancer RNAs in cancer: regulation, mechanisms and therapeutic potential.

Enhancers are distal genomic elements critical for gene regulation and cell identify control during development and diseases. Many human cancers were found to associate with enhancer malfunction, due to genetic and epigenetic alterations, which in some cases directly drive tumour growth. Conventionally, enhancers are known to provide DNA binding motifs to recruit transcription factors (TFs) and to control target genes. However, recent progress found that most, if not all, active enhancers pervasively transcribe noncoding RNAs that are referred to as enhancer RNAs (eRNAs). Increasing evidence points to functional roles of at least a subset of eRNAs in gene regulation in both normal and cancer cells, adding new insights into the action mechanisms of enhancers. eRNA expression was observed to be widespread but also specific to tumour types and individual patients, serving as opportunities to exploit them as potential diagnosis markers or therapeutic targets. In this review, we discuss the brief history of eRNA research, their functional mechanisms and importance in cancer gene regulation, as well as their therapeutic and diagnostic values in cancer. We propose that further studies of eRNAs in cancer will offer a promising 'eRNA targeted therapy' for human cancer intervention.

BRCA1 mutations attenuate super-enhancer function and chromatin looping in haploinsufficient human breast epithelial cells.

BACKGROUND: BRCA1-associated breast cancer originates from luminal progenitor cells. BRCA1 functions in multiple biological processes, including double-strand break repair, replication stress suppression, transcriptional regulation, and chromatin reorganization. While non-malignant cells carrying cancer-predisposing BRCA1 mutations exhibit increased genomic instability, it remains unclear whether BRCA1 haploinsufficiency affects transcription and chromatin dynamics in breast epithelial cells. METHODS: H3K27ac-associated super-enhancers were compared in primary breast epithelial cells from BRCA1 mutation carriers (BRCA1mut/+) and non-carriers (BRCA1+/+). Non-tumorigenic MCF10A breast epithelial cells with engineered BRCA1 haploinsufficiency were used to confirm the H3K27ac changes. The impact of BRCA1 mutations on enhancer function and enhancer-promoter looping was assessed in MCF10A cells. RESULTS: Here, we show that primary mammary epithelial cells from women with BRCA1 mutations display significant loss of H3K27ac-associated super-enhancers. These BRCA1-dependent super-enhancers are enriched with binding motifs for the GATA family. Non-tumorigenic BRCA1mut/+ MCF10A cells recapitulate the H3K27ac loss. Attenuated histone mark and enhancer activity in these BRCA1mut/+ MCF10A cells can be partially restored with wild-type BRCA1. Furthermore, chromatin conformation analysis demonstrates impaired enhancer-promoter looping in BRCA1mut/+ MCF10A cells. CONCLUSIONS: H3K27ac-associated super-enhancer loss is a previously unappreciated functional deficiency in ostensibly normal BRCA1 mutation-carrying breast epithelium. Our findings offer new mechanistic insights into BRCA1 mutation-associated transcriptional and epigenetic abnormality in breast epithelial cells and tissue/cell lineage-specific tumorigenesis.

Invariant TAD Boundaries Constrain Cell-Type-Specific Looping Interactions between Promoters and Distal Elements around the CFTR Locus.

Three-dimensional genome structure plays an important role in gene regulation. Globally, chromosomes are organized into active and inactive compartments while, at the gene level, looping interactions connect promoters to regulatory elements. Topologically associating domains (TADs), typically several hundred kilobases in size, form an intermediate level of organization. Major questions include how TADs are formed and how they are related to looping interactions between genes and regulatory elements. Here we performed a focused 5C analysis of a 2.8 Mb chromosome 7 region surrounding CFTR in a panel of cell types. We find that the same TAD boundaries are present in all cell types, indicating that TADs represent a universal chromosome architecture. Furthermore, we find that these TAD boundaries are present irrespective of the expression and looping of genes located between them. In contrast, looping interactions between promoters and regulatory elements are cell-type specific and occur mostly within TADs. This is exemplified by the CFTR promoter that in different cell types interacts with distinct sets of distal cell-type-specific regulatory elements that are all located within the same TAD. Finally, we find that long-range associations between loci located in different TADs are also detected, but these display much lower interaction frequencies than looping interactions within TADs. Interestingly, interactions between TADs are also highly cell-type-specific and often involve loci clustered around TAD boundaries. These data point to key roles of invariant TAD boundaries in constraining as well as mediating cell-type-specific long-range interactions and gene regulation.

Takayasu arteritis risk locus in IL6 represses the anti-inflammatory gene GPNMB through chromatin looping and recruiting MEF2-HDAC complex.

OBJECTIVE: Previous work has revealed a genetic association between Takayasu arteritis and a non-coding genetic variant in an enhancer region within IL6 (rs2069837 A/G). The risk allele in this variant (allele A) has a protective effect against chronic viral infection and cancer. The goal of this study was to characterise the functional consequences of this disease-associated risk locus. METHODS: A combination of experimental and bioinformatics tools were used to mechanistically understand the effects of the disease-associated genetic locus in IL6. These included electrophoretic mobility shift assay, DNA affinity precipitation assays followed by mass spectrometry and western blotting, luciferase reporter assays and chromosome conformation capture (3C) to identify chromatin looping in the IL6 locus. Both cell lines and peripheral blood primary monocyte-derived macrophages were used. RESULTS: We identified the monocyte/macrophage anti-inflammatory gene GPNMB,~520 kb from IL6, as a target gene regulated by rs2069837. We revealed preferential recruitment of myocyte enhancer factor 2-histone deacetylase (MEF2-HDAC) repressive complex to the Takayasu arteritis risk allele. Further, we demonstrated suppression of GPNMB expression in monocyte-derived macrophages from healthy individuals with AA compared with AG genotype, which was reversed by histone deacetylase inhibition. Our data show that the risk allele in rs2069837 represses the expression of GPNMB by recruiting MEF2-HDAC complex, enabled through a long-range intrachromatin looping. Suppression of this anti-inflammatory gene might mediate increased susceptibility in Takayasu arteritis and enhance protective immune responses in chronic infection and cancer. CONCLUSIONS: Takayasu arteritis risk locus in IL6 might increase disease susceptibility by suppression of the anti-inflammatory gene GPNMB through chromatin looping and recruitment of MEF2-HDAC epigenetic repressive complex. Our data highlight long-range chromatin interactions in functional genomic and epigenomic studies in autoimmunity.