p53 rapidly restructures 3D chromatin organization to trigger a transcriptional response.

Activation of the p53 tumor suppressor triggers a transcriptional program to control cellular response to stress. However, the molecular mechanisms by which p53 controls gene transcription are not completely understood. Here, we uncover the critical role of spatio-temporal genome architecture in this process. We demonstrate that p53 drives direct and indirect changes in genome compartments, topologically associating domains, and DNA loops prior to one hour of its activation, which escort the p53 transcriptional program. Focusing on p53-bound enhancers, we report 340 genes directly regulated by p53 over a median distance of 116 kb, with 74% of these genes not previously identified. Finally, we showcase that p53 controls transcription of distal genes through newly formed and pre-existing enhancer-promoter loops in a cohesin dependent manner. Collectively, our findings demonstrate a previously unappreciated architectural role of p53 as regulator at distinct topological layers and provide a reliable set of new p53 direct target genes that may help designs of cancer therapies.

The genetic regulatory architecture and epigenomic basis for age-related changes in rattlesnake venom.

Developmental phenotypic changes can evolve under selection imposed by age- and size-related ecological differences. Many of these changes occur through programmed alterations to gene expression patterns, but the molecular mechanisms and gene-regulatory networks underlying these adaptive changes remain poorly understood. Many venomous snakes, including the eastern diamondback rattlesnake (Crotalus adamanteus), undergo correlated changes in diet and venom expression as snakes grow larger with age, providing models for identifying mechanisms of timed expression changes that underlie adaptive life history traits. By combining a highly contiguous, chromosome-level genome assembly with measures of expression, chromatin accessibility, and histone modifications, we identified cis-regulatory elements and trans-regulatory factors controlling venom ontogeny in the venom glands of C. adamanteus. Ontogenetic expression changes were significantly correlated with epigenomic changes within genes, immediately adjacent to genes (e.g., promoters), and more distant from genes (e.g., enhancers). We identified 37 candidate transcription factors (TFs), with the vast majority being up-regulated in adults. The ontogenetic change is largely driven by an increase in the expression of TFs associated with growth signaling, transcriptional activation, and circadian rhythm/biological timing systems in adults with corresponding epigenomic changes near the differentially expressed venom genes. However, both expression activation and repression contributed to the composition of both adult and juvenile venoms, demonstrating the complexity and potential evolvability of gene regulation for this trait. Overall, given that age-based trait variation is common across the tree of life, we provide a framework for understanding gene-regulatory-network-driven life-history evolution more broadly.

Cellular reprogramming is driven by widespread rewiring of promoter-enhancer interactions.

BACKGROUND: Long-range interactions between promoters and cis-regulatory elements, such as enhancers, play critical roles in gene regulation. However, the role of three-dimensional (3D) chromatin structure in orchestrating changes in transcriptional regulation during direct cell reprogramming is not fully understood. RESULTS: Here, we performed integrated analyses of chromosomal architecture, epigenetics, and gene expression using Hi-C, promoter Capture Hi-C (PCHi-C), ChIP-seq, and RNA-seq during trans-differentiation of Pre-B cells into macrophages with a ß-estradiol inducible C/EBPαER transgene. Within 1h of ß-estradiol induction, C/EBPα translocated from the cytoplasm to the nucleus, binding to thousands of promoters and putative regulatory elements, resulting in the downregulation of Pre-B cell-specific genes and induction of macrophage-specific genes. Hi-C results were remarkably consistent throughout trans-differentiation, revealing only a small number of TAD boundary location changes, and A/B compartment switches despite significant changes in the expression of thousands of genes. PCHi-C revealed widespread changes in promoter-anchored loops with decreased interactions in parallel with decreased gene expression, and new and increased promoter-anchored interactions in parallel with increased expression of macrophage-specific genes. CONCLUSIONS: Overall, our data demonstrate that C/EBPα-induced trans-differentiation involves few changes in genome architecture at the level of TADs and A/B compartments, in contrast with widespread reorganization of thousands of promoter-anchored loops in association with changes in gene expression and cell identity.

Intra- and interchromosomal contact mapping reveals the Igh locus has extensive conformational heterogeneity and interacts with B-lineage genes.

To produce a diverse antibody repertoire, immunoglobulin heavy-chain (Igh) loci undergo large-scale alterations in structure to facilitate juxtaposition and recombination of spatially separated variable (VH), diversity (DH), and joining (JH) genes. These chromosomal alterations are poorly understood. Uncovering their patterns shows how chromosome dynamics underpins antibody diversity. Using tiled Capture Hi-C, we produce a comprehensive map of chromatin interactions throughout the 2.8-Mb Igh locus in progenitor B cells. We find that the Igh locus folds into semi-rigid subdomains and undergoes flexible looping of the VH genes to its 3' end, reconciling two views of locus organization. Deconvolution of single Igh locus conformations using polymer simulations identifies thousands of different structures. This heterogeneity may underpin the diversity of V(D)J recombination events. All three immunoglobulin loci also participate in a highly specific, developmentally regulated network of interchromosomal interactions with genes encoding B cell-lineage factors. This suggests a model of interchromosomal coordination of B cell development.

Rationally designed chimeric PI3K-BET bromodomain inhibitors elicit curative responses in MYC-driven lymphoma.

Targeted inhibitors of bromodomain and extraterminal (BET)-bromodomains and phosphatidylinositol-3-kinase (PI3K) signaling demonstrate potent but self-limited antilymphoma activity as single agents in the context of cellular Myelocytomatosis (cMYC) oncogene-dysregulation. However, combined PI3K and BET inhibition imparts synergistic anticancer activity with the potential for more sustained disease responses due to the mutual antagonism of compensatory epigenetic and signaling networks. Here, we describe the mechanistic and therapeutic validation of rationally designed dual PI3K/BET bromodomain inhibitors, built by linkage of established PI3K and BET inhibitor pharmacophores. The lead candidate demonstrates high selectivity, nanomolar range cellular potency, and compelling in vivo efficacy, including curative responses in the aggressive Eµ-Myc lymphoma model. These studies further support the therapeutic strategy of combined PI3K and BET inhibition and provide a potential step-change in approach to orthogonal MYC antagonism using optimized chimeric small-molecule technology.

Single cell Hi-C identifies plastic chromosome conformations underlying the gastrulation enhancer landscape.

Embryonic development involves massive proliferation and differentiation of cell lineages. This must be supported by chromosome replication and epigenetic reprogramming, but how proliferation and cell fate acquisition are balanced in this process is not well understood. Here we use single cell Hi-C to map chromosomal conformations in post-gastrulation mouse embryo cells and study their distributions and correlations with matching embryonic transcriptional atlases. We find that embryonic chromosomes show a remarkably strong cell cycle signature. Despite that, replication timing, chromosome compartment structure, topological associated domains (TADs) and promoter-enhancer contacts are shown to be variable between distinct epigenetic states. About 10% of the nuclei are identified as primitive erythrocytes, showing exceptionally compact and organized compartment structure. The remaining cells are broadly associated with ectoderm and mesoderm identities, showing only mild differentiation of TADs and compartment structures, but more specific localized contacts in hundreds of ectoderm and mesoderm promoter-enhancer pairs. The data suggest that while fully committed embryonic lineages can rapidly acquire specific chromosomal conformations, most embryonic cells are showing plastic signatures driven by complex and intermixed enhancer landscapes.

3D genome organization links non-coding disease-associated variants to genes.

Genome sequencing has revealed over 300 million genetic variations in human populations. Over 90% of variants are single nucleotide polymorphisms (SNPs), the remainder include short deletions or insertions, and small numbers of structural variants. Hundreds of thousands of these variants have been associated with specific phenotypic traits and diseases through genome wide association studies which link significant differences in variant frequencies with specific phenotypes among large groups of individuals. Only 5% of disease-associated SNPs are located in gene coding sequences, with the potential to disrupt gene expression or alter of the function of encoded proteins. The remaining 95% of disease-associated SNPs are located in non-coding DNA sequences which make up 98% of the genome. The role of non-coding, disease-associated SNPs, many of which are located at considerable distances from any gene, was at first a mystery until the discovery that gene promoters regularly interact with distal regulatory elements to control gene expression. Disease-associated SNPs are enriched at the millions of gene regulatory elements that are dispersed throughout the non-coding sequences of the genome, suggesting they function as gene regulation variants. Assigning specific regulatory elements to the genes they control is not straightforward since they can be millions of base pairs apart. In this review we describe how understanding 3D genome organization can identify specific interactions between gene promoters and distal regulatory elements and how 3D genomics can link disease-associated SNPs to their target genes. Understanding which gene or genes contribute to a specific disease is the first step in designing rational therapeutic interventions.

Inhibition of mutant IDH1 promotes cycling of acute myeloid leukemia stem cells.

Approximately 20% of acute myeloid leukemia (AML) patients carry mutations in IDH1 or IDH2 that result in over-production of the oncometabolite D-2-hydroxyglutarate (2-HG). Small molecule inhibitors that block 2-HG synthesis can induce complete morphological remission; however, almost all patients eventually acquire drug resistance and relapse. Using a multi-allelic mouse model of IDH1-mutant AML, we demonstrate that the clinical IDH1 inhibitor AG-120 (ivosidenib) exerts cell-type-dependent effects on leukemic cells, promoting delayed disease regression. Although single-agent AG-120 treatment does not fully eradicate the disease, it increases cycling of rare leukemia stem cells and triggers transcriptional upregulation of the pyrimidine salvage pathway. Accordingly, AG-120 sensitizes IDH1-mutant AML to azacitidine, with the combination of AG-120 and azacitidine showing vastly improved efficacy in vivo. Our data highlight the impact of non-genetic heterogeneity on treatment response and provide a mechanistic rationale for the observed combinatorial effect of AG-120 and azacitidine in patients.

DeepLoop robustly maps chromatin interactions from sparse allele-resolved or single-cell Hi-C data at kilobase resolution.

Mapping chromatin loops from noisy Hi-C heatmaps remains a major challenge. Here we present DeepLoop, which performs rigorous bias correction followed by deep-learning-based signal enhancement for robust chromatin interaction mapping from low-depth Hi-C data. DeepLoop enables loop-resolution, single-cell Hi-C analysis. It also achieves a cross-platform convergence between different Hi-C protocols and micrococcal nuclease (micro-C). DeepLoop allowed us to map the genetic and epigenetic determinants of allele-specific chromatin interactions in the human genome. We nominate new loci with allele-specific interactions governed by imprinting or allelic DNA methylation. We also discovered that, in the inactivated X chromosome (Xi), local loops at the DXZ4 'megadomain' boundary escape X-inactivation but the FIRRE 'superloop' locus does not. Importantly, DeepLoop can pinpoint heterozygous single-nucleotide polymorphisms and large structure variants that cause allelic chromatin loops, many of which rewire enhancers with transcription consequences. Taken together, DeepLoop expands the use of Hi-C to provide loop-resolution insights into the genetics of the three-dimensional genome.

Chromosome organization through the cell cycle at a glance.

Genome organization and the three-dimensional folding of chromosomes are now seen as major contributors to nearly all nuclear functions including gene regulation, replication and repair. Recent studies have shown that in addition to the dramatic metamorphoses in chromosome conformation associated with entry to, and exit from mitosis, chromosomes undergo continual conformational changes throughout interphase with differential dynamics in loop structure, topological domains, compartments and lamina-associated domains. Understanding and accounting for these cell-cycle-dependent conformational changes is essential for the interpretation of data from a growing array of powerful molecular techniques to investigate genome conformation function, and to identify the molecules and mechanisms that drive chromosome conformational changes. In this Cell Science at a Glance article and the accompanying poster, we review Hi-C and microscopy studies describing cell-cycle-dependent conformational changes in chromosome structure.

Epigenetic Activation of Plasmacytoid DCs Drives IFNAR-Dependent Therapeutic Differentiation of AML.

Pharmacologic inhibition of epigenetic enzymes can have therapeutic benefit against hematologic malignancies. In addition to affecting tumor cell growth and proliferation, these epigenetic agents may induce antitumor immunity. Here, we discovered a novel immunoregulatory mechanism through inhibition of histone deacetylases (HDAC). In models of acute myeloid leukemia (AML), leukemia cell differentiation and therapeutic benefit mediated by the HDAC inhibitor (HDACi) panobinostat required activation of the type I interferon (IFN) pathway. Plasmacytoid dendritic cells (pDC) produced type I IFN after panobinostat treatment, through transcriptional activation of IFN genes concomitant with increased H3K27 acetylation at these loci. Depletion of pDCs abrogated panobinostat-mediated induction of type I IFN signaling in leukemia cells and impaired therapeutic efficacy, whereas combined treatment with panobinostat and IFNα improved outcomes in preclinical models. These discoveries offer a new therapeutic approach for AML and demonstrate that epigenetic rewiring of pDCs enhances antitumor immunity, opening the possibility of exploiting this approach for immunotherapies. SIGNIFICANCE: We demonstrate that HDACis induce terminal differentiation of AML through epigenetic remodeling of pDCs, resulting in production of type I IFN that is important for the therapeutic effects of HDACis. The study demonstrates the important functional interplay between the immune system and leukemias in response to HDAC inhibition. This article is highlighted in the In This Issue feature, p. 1397.

Locus-specific induction of gene expression from heterochromatin loci during cellular senescence.

Senescence is a fate-determined state, accompanied by reorganization of heterochromatin. Although lineage-appropriate genes can be temporarily repressed through facultative heterochromatin, stable silencing of lineage-inappropriate genes often involves the constitutive heterochromatic mark, histone H3 lysine 9 trimethylation (H3K9me3). The fate of these heterochromatic genes during senescence is unclear. In the present study, we show that a small number of lineage-inappropriate genes, exemplified by the LCE2 skin genes, are derepressed during senescence from H3K9me3 regions in fibroblasts. DNA FISH experiments reveal that these gene loci, which are condensed at the nuclear periphery in proliferative cells, are decompacted during senescence. Decompaction of the locus is not sufficient for LCE2 expression, which requires p53 and C/EBPß signaling. NLRP3, which is predominantly expressed in macrophages from an open topologically associated domain (TAD), is also derepressed in senescent fibroblasts due to the local disruption of the H3K9me3-rich TAD that contains it. NLRP3 has been implicated in the amplification of inflammatory cytokine signaling in senescence and aging, highlighting the functional relevance of gene induction from 'permissive' H3K9me3 regions in senescent cells.

Short- and long-range cis interactions between integrated HPV genomes and cellular chromatin dysregulate host gene expression in early cervical carcinogenesis.

Development of cervical cancer is directly associated with integration of human papillomavirus (HPV) genomes into host chromosomes and subsequent modulation of HPV oncogene expression, which correlates with multi-layered epigenetic changes at the integrated HPV genomes. However, the process of integration itself and dysregulation of host gene expression at sites of integration in our model of HPV16 integrant clone natural selection has remained enigmatic. We now show, using a state-of-the-art 'HPV integrated site capture' (HISC) technique, that integration likely occurs through microhomology-mediated repair (MHMR) mechanisms via either a direct process, resulting in host sequence deletion (in our case, partially homozygously) or via a 'looping' mechanism by which flanking host regions become amplified. Furthermore, using our 'HPV16-specific Region Capture Hi-C' technique, we have determined that chromatin interactions between the integrated virus genome and host chromosomes, both at short- (<500 kbp) and long-range (>500 kbp), appear to drive local host gene dysregulation through the disruption of host:host interactions within (but not exceeding) host structures known as topologically associating domains (TADs). This mechanism of HPV-induced host gene expression modulation indicates that integration of virus genomes near to or within a 'cancer-causing gene' is not essential to influence their expression and that these modifications to genome interactions could have a major role in selection of HPV integrants at the early stage of cervical neoplastic progression.

The global and promoter-centric 3D genome organization temporally resolved during a circadian cycle.

BACKGROUND: Circadian gene expression is essential for organisms to adjust their physiology and anticipate daily changes in the environment. The molecular mechanisms controlling circadian gene transcription are still under investigation. In particular, how chromatin conformation at different genomic scales and regulatory elements impact rhythmic gene expression has been poorly characterized. RESULTS: Here we measure changes in the spatial chromatin conformation in mouse liver using genome-wide and promoter-capture Hi-C alongside daily oscillations in gene transcription. We find topologically associating domains harboring circadian genes that switch assignments between the transcriptionally active and inactive compartment at different hours of the day, while their boundaries stably maintain their structure over time. To study chromatin contacts of promoters at high resolution over time, we apply promoter capture Hi-C. We find circadian gene promoters displayed a maximal number of chromatin contacts at the time of their peak transcriptional output. Furthermore, circadian genes, as well as contacted and transcribed regulatory elements, reach maximal expression at the same timepoints. Anchor sites of circadian gene promoter loops are enriched in DNA binding sites for liver nuclear receptors and other transcription factors, some exclusively present in either rhythmic or stable contacts. Finally, by comparing the interaction profiles between core clock and output circadian genes, we show that core clock interactomes are more dynamic compared to output circadian genes. CONCLUSION: Our results identify chromatin conformation dynamics at different scales that parallel oscillatory gene expression and characterize the repertoire of regulatory elements that control circadian gene transcription through rhythmic or stable chromatin configurations.

Genetic perturbation of PU.1 binding and chromatin looping at neutrophil enhancers associates with autoimmune disease.

Neutrophils play fundamental roles in innate immune response, shape adaptive immunity, and are a potentially causal cell type underpinning genetic associations with immune system traits and diseases. Here, we profile the binding of myeloid master regulator PU.1 in primary neutrophils across nearly a hundred volunteers. We show that variants associated with differential PU.1 binding underlie genetically-driven differences in cell count and susceptibility to autoimmune and inflammatory diseases. We integrate these results with other multi-individual genomic readouts, revealing coordinated effects of PU.1 binding variants on the local chromatin state, enhancer-promoter contacts and downstream gene expression, and providing a functional interpretation for 27 genes underlying immune traits. Collectively, these results demonstrate the functional role of PU.1 and its target enhancers in neutrophil transcriptional control and immune disease susceptibility.

High-resolution three-dimensional chromatin profiling of the Chinese hamster ovary cell genome.

Chinese hamster ovary (CHO) cell lines are the pillars of a multibillion-dollar biopharmaceutical industry producing recombinant therapeutic proteins. The effects of local chromatin organization and epigenetic repression within these cell lines result in unpredictable and unstable transgene expression following random integration. Limited knowledge of the CHO genome and its higher order chromatin organization has thus far impeded functional genomics approaches required to tackle these issues. Here, we present an integrative three-dimensional (3D) map of genome organization within the CHOK1SV® 10E9 cell line in conjunction with an improved, less fragmented CHOK1SV 10E9 genome assembly. Using our high-resolution chromatin conformation datasets, we have assigned ≈90% of sequence to a chromosome-scale genome assembly. Our genome-wide 3D map identifies higher order chromatin structures such as topologically associated domains, incorporates our chromatin accessibility data to enhance the identification of active cis-regulatory elements, and importantly links these cis-regulatory elements to target promoters in a 3D promoter interactome. We demonstrate the power of our improved functional annotation by evaluating the 3D landscape of a transgene integration site and two phenotypically different cell lines. Our work opens up further novel genome engineering targets, has the potential to inform vital improvements for industrial biotherapeutic production, and represents a significant advancement for CHO cell line development.

Transcription-dependent cohesin repositioning rewires chromatin loops in cellular senescence.

Senescence is a state of stable proliferative arrest, generally accompanied by the senescence-associated secretory phenotype, which modulates tissue homeostasis. Enhancer-promoter interactions, facilitated by chromatin loops, play a key role in gene regulation but their relevance in senescence remains elusive. Here, we use Hi-C to show that oncogenic RAS-induced senescence in human diploid fibroblasts is accompanied by extensive enhancer-promoter rewiring, which is closely connected with dynamic cohesin binding to the genome. We find de novo cohesin peaks often at the 3' end of a subset of active genes. RAS-induced de novo cohesin peaks are transcription-dependent and enriched for senescence-associated genes, exemplified by IL1B, where de novo cohesin binding is involved in new loop formation. Similar IL1B induction with de novo cohesin appearance and new loop formation are observed in terminally differentiated macrophages, but not TNFα-treated cells. These results suggest that RAS-induced senescence represents a cell fate determination-like process characterised by a unique gene expression profile and 3D genome folding signature, mediated in part through cohesin redistribution on chromatin.

Highly interconnected enhancer communities control lineage-determining genes in human mesenchymal stem cells.

Adipocyte differentiation is driven by waves of transcriptional regulators that reprogram the enhancer landscape and change the wiring of the promoter interactome. Here, we use high-throughput chromosome conformation enhancer capture to interrogate the role of enhancer-to-enhancer interactions during differentiation of human mesenchymal stem cells. We find that enhancers form an elaborate network that is dynamic during differentiation and coupled with changes in enhancer activity. Transcription factors (TFs) at baited enhancers amplify TF binding at target enhancers, a phenomenon we term cross-interaction stabilization of TFs. Moreover, highly interconnected enhancers (HICE) act as integration hubs orchestrating differentiation by the formation of three-dimensional enhancer communities, inside which, HICE, and other enhancers, converge on phenotypically important gene promoters. Collectively, these results indicate that enhancer interactions play a key role in the regulation of enhancer function, and that HICE are important for both signal integration and compartmentalization of the genome.

Analysis of chromatin organization and gene expression in T cells identifies functional genes for rheumatoid arthritis.

Genome-wide association studies have identified genetic variation contributing to complex disease risk. However, assigning causal genes and mechanisms has been more challenging because disease-associated variants are often found in distal regulatory regions with cell-type specific behaviours. Here, we collect ATAC-seq, Hi-C, Capture Hi-C and nuclear RNA-seq data in stimulated CD4+ T cells over 24 h, to identify functional enhancers regulating gene expression. We characterise changes in DNA interaction and activity dynamics that correlate with changes in gene expression, and find that the strongest correlations are observed within 200 kb of promoters. Using rheumatoid arthritis as an example of T cell mediated disease, we demonstrate interactions of expression quantitative trait loci with target genes, and confirm assigned genes or show complex interactions for 20% of disease associated loci, including FOXO1, which we confirm using CRISPR/Cas9.

Cohesin-Dependent and -Independent Mechanisms Mediate Chromosomal Contacts between Promoters and Enhancers.

It is currently assumed that 3D chromosomal organization plays a central role in transcriptional control. However, depletion of cohesin and CTCF affects the steady-state levels of only a minority of transcripts. Here, we use high-resolution Capture Hi-C to interrogate the dynamics of chromosomal contacts of all annotated human gene promoters upon degradation of cohesin and CTCF. We show that a majority of promoter-anchored contacts are lost in these conditions, but many contacts with distinct properties are maintained, and some new ones are gained. The rewiring of contacts between promoters and active enhancers upon cohesin degradation associates with rapid changes in target gene transcription as detected by SLAM sequencing (SLAM-seq). These results provide a mechanistic explanation for the limited, but consistent, effects of cohesin and CTCF depletion on steady-state transcription and suggest the existence of both cohesin-dependent and -independent mechanisms of enhancer-promoter pairing.