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.

Psip1/p52 regulates posterior Hoxa genes through activation of lncRNA Hottip.

Long noncoding RNAs (lncRNAs) have been implicated in various biological functions including the regulation of gene expression, however, the functionality of lncRNAs is not clearly understood and conflicting conclusions have often been reached when comparing different methods to investigate them. Moreover, little is known about the upstream regulation of lncRNAs. Here we show that the short isoform (p52) of a transcriptional co-activator-PC4 and SF2 interacting protein (Psip1), which is known to be involved in linking transcription to RNA processing, specifically regulates the expression of the lncRNA Hottip-located at the 5' end of the Hoxa locus. Using both knockdown and knockout approaches we show that Hottip expression is required for activation of the 5' Hoxa genes (Hoxa13 and Hoxa10/11) and for retaining Mll1 at the 5' end of Hoxa. Moreover, we demonstrate that artificially inducing Hottip expression is sufficient to activate the 5' Hoxa genes and that Hottip RNA binds to the 5' end of Hoxa. By engineering premature transcription termination, we show that it is the Hottip lncRNA molecule itself, not just Hottip transcription that is required to maintains active expression of posterior Hox genes. Our data show a direct role for a lncRNA molecule in regulating the expression of developmentally-regulated mRNA genes in cis.

Ring1B compacts chromatin structure and represses gene expression independent of histone ubiquitination.

How polycomb group proteins repress gene expression in vivo is not known. While histone-modifying activities of the polycomb repressive complexes (PRCs) have been studied extensively, in vitro data have suggested a direct activity of the PRC1 complex in compacting chromatin. Here, we investigate higher-order chromatin compaction of polycomb targets in vivo. We show that PRCs are required to maintain a compact chromatin state at Hox loci in embryonic stem cells (ESCs). There is specific decompaction in the absence of PRC2 or PRC1. This is due to a PRC1-like complex, since decompaction occurs in Ring1B null cells that still have PRC2-mediated H3K27 methylation. Moreover, we show that the ability of Ring1B to restore a compact chromatin state and to repress Hox gene expression is not dependent on its histone ubiquitination activity. We suggest that Ring1B-mediated chromatin compaction acts to directly limit transcription in vivo.

Psip1/Ledgf p75 restrains Hox gene expression by recruiting both trithorax and polycomb group proteins.

Trithorax and polycomb group proteins are generally thought to antagonize one another. The trithorax family member MLL (myeloid/lymphoid or mixed-lineage leukemia) is presumed to activate Hox expression, counteracting polycomb-mediated repression. PC4 and SF2 interacting protein 1 (PSIP1)/p75, also known as LEDGF, whose PWWP domain binds to H3K36me3, interacts with MLL and tethers MLL fusion proteins to HOXA9 in leukaemias. Here we show, unexpectedly, that Psip1/p75 regulates homeotic genes by recruiting not only MLL complexes, but also the polycomb group protein Bmi1. In Psip1(-/-) cells binding of Mll1/2, Bmi1 and the co-repressor Ctbp1 at Hox loci are all abrogated and Hoxa and Hoxd mRNA expression increased. Our data not only reveal a potential mechanism of action for Psip1 in the regulation of Hox genes but also suggest an unexpected interplay between proteins usually considered as transcriptional activators and repressors.

Anterior-posterior differences in HoxD chromatin topology in limb development.

A late phase of HoxD activation is crucial for the patterning and growth of distal structures across the anterior-posterior (A-P) limb axis of mammals. Polycomb complexes and chromatin compaction have been shown to regulate Hox loci along the main body axis in embryonic development, but the extent to which they have a role in limb-specific HoxD expression, an evolutionary adaptation defined by the activity of distal enhancer elements that drive expression of 5' Hoxd genes, has yet to be fully elucidated. We reveal two levels of chromatin topology that differentiate distal limb A-P HoxD activity. Using both immortalised cell lines derived from posterior and anterior regions of distal E10.5 mouse limb buds, and analysis in E10.5 dissected limb buds themselves, we show that there is a loss of polycomb-catalysed H3K27me3 histone modification and a chromatin decompaction over HoxD in the distal posterior limb compared with anterior. Moreover, we show that the global control region (GCR) long-range enhancer spatially colocalises with the 5' HoxD genomic region specifically in the distal posterior limb. This is consistent with the formation of a chromatin loop between 5' HoxD and the GCR regulatory module at the time and place of distal limb bud development when the GCR participates in initiating Hoxd gene quantitative collinearity and Hoxd13 expression. This is the first example of A-P differences in chromatin compaction and chromatin looping in the development of the mammalian secondary body axis (limb).

Psip1/Ledgf p52 binds methylated histone H3K36 and splicing factors and contributes to the regulation of alternative splicing.

Increasing evidence suggests that chromatin modifications have important roles in modulating constitutive or alternative splicing. Here we demonstrate that the PWWP domain of the chromatin-associated protein Psip1/Ledgf can specifically recognize tri-methylated H3K36 and that, like this histone modification, the Psip1 short (p52) isoform is enriched at active genes. We show that the p52, but not the long (p75), isoform of Psip1 co-localizes and interacts with Srsf1 and other proteins involved in mRNA processing. The level of H3K36me3 associated Srsf1 is reduced in Psip1 mutant cells and alternative splicing of specific genes is affected. Moreover, we show altered Srsf1 distribution around the alternatively spliced exons of these genes in Psip1 null cells. We propose that Psip1/p52, through its binding to both chromatin and splicing factors, might act to modulate splicing.

Dispersal of PRC1 condensates disrupts polycomb chromatin domains and loops.

Polycomb repressive complex 1 (PRC1) strongly influences 3D genome organization, mediating local chromatin compaction and clustering of target loci. Several PRC1 subunits have the capacity to form biomolecular condensates through liquid-liquid phase separation in vitro and when tagged and over-expressed in cells. Here, we use 1,6-hexanediol, which can disrupt liquid-like condensates, to examine the role of endogenous PRC1 biomolecular condensates on local and chromosome-wide clustering of PRC1-bound loci. Using imaging and chromatin immunoprecipitation, we show that PRC1-mediated chromatin compaction and clustering of targeted genomic loci-at different length scales-can be reversibly disrupted by the addition and subsequent removal of 1,6-hexanediol to mouse embryonic stem cells. Decompaction and dispersal of polycomb domains and clusters cannot be solely attributable to reduced PRC1 occupancy detected by chromatin immunoprecipitation following 1,6-hexanediol treatment as the addition of 2,5-hexanediol has similar effects on binding despite this alcohol not perturbing PRC1-mediated 3D clustering, at least at the sub-megabase and megabase scales. These results suggest that weak hydrophobic interactions between PRC1 molecules may have a role in polycomb-mediated genome organization.

Human ß-defensin 3 affects the activity of pro-inflammatory pathways associated with MyD88 and TRIF.

ß-Defensins are cationic host defense peptides that form an amphipathic structure stabilized by three intramolecular disulfide bonds. They are key players in innate and adaptive immunity and have recently been shown to limit the production of pro-inflammatory cytokines in TLR4-stimulated macrophages. In the present study, we investigate the mechanism underlying the anti-inflammatory effect of human ß-defensin 3 (hBD3). We show that the canonical structure of hBD3 is required for this immunosuppressive effect and that hBD3 rapidly associates with and enters macrophages. Examination of the global effect of hBD3 on transcription in TLR4-stimulated macrophages shows that hBD3 inhibits the transcription of pro-inflammatory genes. Among the altered genes there is significant enrichment of groups involved in the positive regulation of NF-κB including components of Toll-like receptor signaling pathways. We confirm these observations by showing corresponding decreases in protein levels of pro-inflammatory cytokines and cell surface molecules. In addition, we show that hBD3 reduces NF-κB signaling in cells transfected with MyD88 or TRIF and that hBD3 inhibits the TLR4 response in both MyD88- and TRIF-deficient macrophages. Taken together these findings suggest that the mechanism of hBD3 anti-inflammatory activity involves specific targeting of TLR signaling pathways resulting in transcriptional repression of pro-inflammatory genes.

Human centromere repositioning activates transcription and opens chromatin fibre structure.

Human centromeres appear as constrictions on mitotic chromosomes and form a platform for kinetochore assembly in mitosis. Biophysical experiments led to a suggestion that repetitive DNA at centromeric regions form a compact scaffold necessary for function, but this was revised when neocentromeres were discovered on non-repetitive DNA. To test whether centromeres have a special chromatin structure we have analysed the architecture of a neocentromere. Centromere repositioning is accompanied by RNA polymerase II recruitment and active transcription to form a decompacted, negatively supercoiled domain enriched in 'open' chromatin fibres. In contrast, centromerisation causes a spreading of repressive epigenetic marks to surrounding regions, delimited by H3K27me3 polycomb boundaries and divergent genes. This flanking domain is transcriptionally silent and partially remodelled to form 'compact' chromatin, similar to satellite-containing DNA sequences, and exhibits genomic instability. We suggest transcription disrupts chromatin to provide a foundation for kinetochore formation whilst compact pericentromeric heterochromatin generates mechanical rigidity.

Characterisation of a nucleo-adhesome.

In addition to central functions in cell adhesion signalling, integrin-associated proteins have wider roles at sites distal to adhesion receptors. In experimentally defined adhesomes, we noticed that there is clear enrichment of proteins that localise to the nucleus, and conversely, we now report that nuclear proteomes contain a class of adhesome components that localise to the nucleus. We here define a nucleo-adhesome, providing experimental evidence for a remarkable scale of nuclear localisation of adhesion proteins, establishing a framework for interrogating nuclear adhesion protein functions. Adding to nuclear FAK's known roles in regulating transcription, we now show that nuclear FAK regulates expression of many adhesion-related proteins that localise to the nucleus and that nuclear FAK binds to the adhesome component and nuclear protein Hic-5. FAK and Hic-5 work together in the nucleus, co-regulating a subset of genes transcriptionally. We demonstrate the principle that there are subcomplexes of nuclear adhesion proteins that cooperate to control transcription.

In Vivo Modeling of Patient Genetic Heterogeneity Identifies New Ways to Target Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy of the bile ducts within the liver characterized by high levels of genetic heterogeneity. In the context of such genetic variability, determining which oncogenic mutations drive ICC growth has been difficult, and developing modes of patient stratification and targeted therapies remains challenging. Here we model the interactions between rare mutations with more common driver genes and combine in silico analysis of patient data with highly multiplexed in vivo CRISPR-spCas9 screens to perform a functional in vivo study into the role genetic heterogeneity plays in driving ICC. Novel tumor suppressors were uncovered, which, when lost, cooperate with the RAS oncoprotein to drive ICC growth. Focusing on a set of driver mutations that interact with KRAS to initiate aggressive, sarcomatoid-type ICC revealed that tumor growth relies on Wnt and PI3K signaling. Pharmacologic coinhibition of Wnt and PI3K in vivo impeded ICC growth regardless of mutational profile. Therefore, Wnt and PI3K activity should be considered as a signature by which patients can be stratified for treatment independent of tumor genotype, and inhibitors of these pathways should be levied to treat ICC. SIGNIFICANCE: This work shows that, despite significant genetic heterogeneity, intrahepatic cholangiocarcinoma relies on a limited number of signaling pathways to grow, suggesting common therapeutic vulnerabilities across patients.

Monoallelic variants resulting in substitutions of MAB21L1 Arg51 Cause Aniridia and microphthalmia.

Classical aniridia is a congenital and progressive panocular disorder almost exclusively caused by heterozygous loss-of-function variants at the PAX6 locus. We report nine individuals from five families with severe aniridia and/or microphthalmia (with no detectable PAX6 mutation) with ultrarare monoallelic missense variants altering the Arg51 codon of MAB21L1. These mutations occurred de novo in 3/5 families, with the remaining families being compatible with autosomal dominant inheritance. Mice engineered to carry the p.Arg51Leu change showed a highly-penetrant optic disc anomaly in heterozygous animals with severe microphthalmia in homozygotes. Substitutions of the same codon (Arg51) in MAB21L2, a close homolog of MAB21L1, cause severe ocular and skeletal malformations in humans and mice. The predicted nucleotidyltransferase function of MAB21L1 could not be demonstrated using purified protein with a variety of nucleotide substrates and oligonucleotide activators. Induced expression of GFP-tagged wildtype and mutant MAB21L1 in human cells caused only modest transcriptional changes. Mass spectrometry of immunoprecipitated protein revealed that both mutant and wildtype MAB21L1 associate with transcription factors that are known regulators of PAX6 (MEIS1, MEIS2 and PBX1) and with poly(A) RNA binding proteins. Arg51 substitutions reduce the association of wild-type MAB21L1 with TBL1XR1, a component of the NCoR complex. We found limited evidence for mutation-specific interactions with MSI2/Musashi-2, an RNA-binding proteins with effects on many different developmental pathways. Given that biallelic loss-of-function variants in MAB21L1 result in a milder eye phenotype we suggest that Arg51-altering monoallelic variants most plausibly perturb eye development via a gain-of-function mechanism.

FAK regulates IL-33 expression by controlling chromatin accessibility at c-Jun motifs.

Focal adhesion kinase (FAK) localizes to focal adhesions and is overexpressed in many cancers. FAK can also translocate to the nucleus, where it binds to, and regulates, several transcription factors, including MBD2, p53 and IL-33, to control gene expression by unknown mechanisms. We have used ATAC-seq to reveal that FAK controls chromatin accessibility at a subset of regulated genes. Integration of ATAC-seq and RNA-seq data showed that FAK-dependent chromatin accessibility is linked to differential gene expression, including of the FAK-regulated cytokine and transcriptional regulator interleukin-33 (Il33), which controls anti-tumor immunity. Analysis of the accessibility peaks on the Il33 gene promoter/enhancer regions revealed sequences for several transcription factors, including ETS and AP-1 motifs, and we show that c-Jun, a component of AP-1, regulates Il33 gene expression by binding to its enhancer in a FAK kinase-dependent manner. This work provides the first demonstration that FAK controls transcription via chromatin accessibility, identifying a novel mechanism by which nuclear FAK regulates biologically important gene expression.

Cornelia de Lange syndrome-associated mutations cause a DNA damage signalling and repair defect.

Cornelia de Lange syndrome is a multisystem developmental disorder typically caused by mutations in the gene encoding the cohesin loader NIPBL. The associated phenotype is generally assumed to be the consequence of aberrant transcriptional regulation. Recently, we identified a missense mutation in BRD4 associated with a Cornelia de Lange-like syndrome that reduces BRD4 binding to acetylated histones. Here we show that, although this mutation reduces BRD4-occupancy at enhancers it does not affect transcription of the pluripotency network in mouse embryonic stem cells. Rather, it delays the cell cycle, increases DNA damage signalling, and perturbs regulation of DNA repair in mutant cells. This uncovers a role for BRD4 in DNA repair pathway choice. Furthermore, we find evidence of a similar increase in DNA damage signalling in cells derived from NIPBL-deficient individuals, suggesting that defective DNA damage signalling and repair is also a feature of typical Cornelia de Lange syndrome.

Structural Variants at the BRCA1/2 Loci are a Common Source of Homologous Repair Deficiency in High-grade Serous Ovarian Carcinoma.

PURPOSE: The abundance and effects of structural variation at BRCA1/2 in tumors are not well understood. In particular, the impact of these events on homologous recombination repair deficiency (HRD) has yet to be demonstrated. EXPERIMENTAL DESIGN: Exploiting a large collection of whole-genome sequencing data from high-grade serous ovarian carcinoma (N = 205) together with matched RNA sequencing for the majority of tumors (N = 150), we have comprehensively characterized mutation and expression at BRCA1/2. RESULTS: In addition to the known spectrum of short somatic mutations (SSM), we discovered that multi-megabase structural variants (SV) were a frequent, unappreciated source of BRCA1/2 disruption in these tumors, and we found a genome-wide enrichment for large deletions at the BRCA1/2 loci across the cohort. These SVs independently affected a substantial proportion of patients (16%) in addition to those affected by SSMs (24%), conferring HRD and impacting patient survival. We also detail compound deficiencies involving SSMs and SVs at both loci, demonstrating that the strongest risk of HRD emerges from combined SVs at both BRCA1 and BRCA2 in the absence of SSMs. Furthermore, these SVs are abundant and disruptive in other cancer types. CONCLUSIONS: These results extend our understanding of the mutational landscape underlying HRD, increase the number of patients predicted to benefit from therapies exploiting HRD, and suggest there is currently untapped potential in SV detection for patient stratification.

DNA Methylation Directs Polycomb-Dependent 3D Genome Re-organization in Naive Pluripotency.

The DNA hypomethylation that occurs when embryonic stem cells (ESCs) are directed to the ground state of naive pluripotency by culturing in two small molecule inhibitors (2i) results in redistribution of polycomb (H3K27me3) away from its target loci. Here, we demonstrate that 3D genome organization is also altered in 2i, with chromatin decompaction at polycomb target loci and a loss of long-range polycomb interactions. By preventing DNA hypomethylation during the transition to the ground state, we are able to restore to ESC in 2i the H3K27me3 distribution, as well as polycomb-mediated 3D genome organization that is characteristic of primed ESCs grown in serum. However, these cells retain the functional characteristics of 2i ground-state ESCs. Our findings demonstrate the central role of DNA methylation in shaping major aspects of 3D genome organization but caution against assuming causal roles for the epigenome and 3D genome in gene regulation and function in ESCs.

Publisher Correction: BRD4 interacts with NIPBL and BRD4 is mutated in a Cornelia de Lange-like syndrome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: BRD4 interacts with NIPBL and BRD4 is mutated in a Cornelia de Lange-like syndrome.

In the version of this article initially published, Wendy Bickmore and Madapura Pradeepa were incorrectly not indicated as corresponding authors. The error has been corrected in the HTML and PDF versions of the paper.

BRD4 interacts with NIPBL and BRD4 is mutated in a Cornelia de Lange-like syndrome.

We found that the clinical phenotype associated with BRD4 haploinsufficiency overlapped with that of Cornelia de Lange syndrome (CdLS), which is most often caused by mutation of NIPBL. More typical CdLS was observed with a de novo BRD4 missense variant, which retained the ability to coimmunoprecipitate with NIPBL, but bound poorly to acetylated histones. BRD4 and NIPBL displayed correlated binding at super-enhancers and appeared to co-regulate developmental gene expression.