Capture Hi-C Research Articles

STAG2 loss in Ewing sarcoma alters enhancer-promoter contacts dependent and independent of EWS::FLI1.

Cohesin complexes carrying STAG1 or STAG2 organize the genome into chromatin loops. STAG2 loss-of-function mutations promote metastasis in Ewing sarcoma, a pediatric cancer driven by the fusion transcription factor EWS::FLI1. We integrated transcriptomic data from patients and cellular models to identify a STAG2-dependent gene signature associated with worse prognosis. Subsequent genomic profiling and high-resolution chromatin interaction data from Capture Hi-C indicated that cohesin-STAG2 facilitates communication between EWS::FLI1-bound long GGAA repeats, presumably acting as neoenhancers, and their target promoters. Changes in CTCF-dependent chromatin contacts involving signature genes, unrelated to EWS::FLI1 binding, were also identified. STAG1 is unable to compensate for STAG2 loss and chromatin-bound cohesin is severely decreased, while levels of the processivity factor NIPBL remain unchanged, likely affecting DNA looping dynamics. These results illuminate how STAG2 loss modifies the chromatin interactome of Ewing sarcoma cells and provide a list of potential biomarkers and therapeutic targets.

Investigating the role of WT1 in Brugada syndrome pathophysiology

Abstract Background Brugada syndrome (BrS) is a heritable arrhythmic disorder presumably caused by conduction slowing in the right ventricular (RV) outflow tract (RVOT). A recent genome-wide association study found an association between common variants on chromosome 11 near WT1, tagged by the SNP rs72905083, and BrS susceptibility. Purpose To study whether WT1 affects cardiac conduction and could therefore represents the underlying association of the chr11 locus with BrS susceptibility. Methods We conducted in silico analyses of omics datasets to identify the likely causal gene at the chr11 BrS locus. Next, we conducted electrophysiological studies in heterozygous young adult (4 months old) Wt1-deficient (Wt1+/-) mice and wild-type littermates to explore the effect of Wt1 on cardiac conduction. To study the potential role of Wt1 in the setting of reduced conduction reserve, we compared aged Scn5a haploinsufficient mice (Scn5a+/-;Wt1+/+, 17 months) with littermates haploinsufficient for both Wt1 and Scn5a (Scn5a+/-;Wt1+/-). Optical mapping was performed in Langendorff-perfused hearts, where conduction was further challenged by acute ajmaline administration or by pacing at the effective refractory period (ERP). Transcriptomic analyses were conducted in Wt1+/- and wild-type mice by RNAseq of PCM1-sorted cardiomyocytes. Results Analysis of omics datasets uncovered WT1 as the likely causal gene since WT1 was the closest gene to, and in fact overlapped, the BrS-associated region; promoter capture Hi-C data in human tissue showed chromatin contact between the promoter of WT1 and the associated region; and H3K27ac ChIP-seq signals (i.e. enhancer activity) of putative cardiac enhancers located within the associated region were correlated with WT1 transcript abundance across multiple tissues (Z-score = 4.32, p=1.57e-05). No differences were observed in ECG parameters or optically mapped epicardial conduction between Wt1+/- mice and wild-type littermates. However, comparison of aged Scn5a+/-;Wt1+/- and Scn5a+/-; Wt1+/+ hearts, in the setting of acute ajmaline challenge or pacing at the ERP, showed faster conduction in RV/RVOT in the presence of Wt1 haploinsufficiency (genotype effect: p=0.057 and p=0.041 for RV and RVOT epicardial conduction, respectively, during ajmaline challenge; p=0.034 for RV epicardial conduction during pacing at ERP). RNAseq of RV/RVOT cardiomyocytes revealed higher Scn5a expression in Wt1+/- vs wild-type (p=0.006). Conclusion We identified WT1 as the gene that most likely underlies the effect of the chr11 BrS susceptibility locus. Functional data revealed protective effects of reduced Wt1 expression in the setting of reduced conduction reserve. Transcriptomic data suggest that this occurs through a transcriptional effect on Scn5a expression.

Enhlink infers distal and context-specific enhancer–promoter linkages

Enhlink is a computational tool for scATAC-seq data analysis, facilitating precise interrogation of enhancer function at the single-cell level. It employs an ensemble approach incorporating technical and biological covariates to infer condition-specific regulatory DNA linkages. Enhlink can integrate multi-omic data for enhanced specificity, when available. Evaluation with simulated and real data, including multi-omic datasets from the mouse striatum and novel promoter capture Hi-C data, demonstrate that Enhlink outperfoms alternative methods. Coupled with eQTL analysis, it identified a putative super-enhancer in striatal neurons. Overall, Enhlink offers accuracy, power, and potential for revealing novel biological insights in gene regulation.

The long non-coding RNA Meg3 mediates imprinted gene expression during stem cell differentiation.

The imprinted Dlk1-Dio3 domain comprises the developmental genes Dlk1 and Rtl1, which are silenced on the maternal chromosome in different cell types. On this parental chromosome, the domain's imprinting control region activates a polycistron that produces the lncRNA Meg3 and many miRNAs (Mirg) and C/D-box snoRNAs (Rian). Although Meg3 lncRNA is nuclear and associates with the maternal chromosome, it is unknown whether it controls gene repression in cis. We created mouse embryonic stem cells (mESCs) that carry an ectopic poly(A) signal, reducing RNA levels along the polycistron, and generated Rian-/- mESCs as well. Upon ESC differentiation, we found that Meg3 lncRNA (but not Rian) is required for Dlk1 repression on the maternal chromosome. Biallelic Meg3 expression acquired through CRISPR-mediated demethylation of the paternal Meg3 promoter led to biallelic Dlk1 repression, and to loss of Rtl1 expression. lncRNA expression also correlated with DNA hypomethylation and CTCF binding at the 5'-side of Meg3. Using Capture Hi-C, we found that this creates a Topologically Associating Domain (TAD) organization that brings Meg3 close to Dlk1 on the maternal chromosome. The requirement of Meg3 for gene repression and TAD structure may explain how aberrant MEG3 expression at the human DLK1-DIO3 locus associates with imprinting disorders.

Integrative Multi-omics Analysis Identifies Genetic Variants Contributing to Non-syndromic Cleft Lip with or without Cleft Palate.

To provide novel insights into the aetiology of non-syndromic cleft lip with or without cleft palate (NSCL/P) by integrating multi-omics data and exploring susceptibility genes associated with NSCL/P. A two-stage genome-wide association study (GWAS) of NSCL/P was performed, involving a total of 1,069 cases and 1,724 controls. Using promoter capture Hi-C (pCHi-C) datasets in human embryonic stem cells (hESC) and chromatin immunoprecipitation sequencing (ChIP-seq) in craniofacial tissues, we filtered out single nucleotide polymorphisms (SNPs) with active cis-regulation and their target genes. Additionally, we employed expression quantitative trait loci (eQTL) analysis to identify candidate genes. Thirteen SNPs were identified as cis-regulation units associated with the risk of NSCL/P. Five of these were proven to be active in chromatin states in early human craniofacial development (rs7218002: odds ratio [OR] 1.50, P = 8.14E-08; rs835367: OR 0.78, P = 3.48E- 05; rs77022994: OR 0.55, P = 1.05E-04; rs961470: OR 0.73, P = 1.38E-04; rs17314727: OR 0.73, P = 1.85E-04). Additionally, pCHi-C and eQTL analysis prioritised three candidate genes (rs7218002: NTN1, rs835367: FGGY, LINC01135). NTN1 and FGGY were expressed in mouse orofacial development. Deficiencies in NTN1, FGGY and LINC01135 were associated with cleft palate and cleft lip, abnormal facial shape and bifid uvula, and abnormality of the face, respectively. Our study identified five SNPs (rs7218002, rs835367, rs77022994, rs961470 and rs17314727) and three susceptibility genes (NTN1, FGGY and LINC01135) associated with NSCL/P. These findings contribute to a better understanding of the genetic factors involved.

Abstract 6154: Targeted CRISPRi screen identifies functional variants and novel target genes at multiple renal cell carcinoma (RCC) susceptibility loci

Abstract Genome-wide association studies (GWAS) have identified 13 renal cell carcinoma (RCC) risk regions, as well as 7 regions with nominal significance, but detailed investigation of how these regions function is required to reveal the underlying biological bases of disease susceptibility. While detailed study of four loci have implicated critical pathways in RCC, causal genes and pathways for most loci remain unidentified. To identify such causative variants and target genes, we evaluated a set of variants based on Massively Parallel Reporter Assays (MPRA), eQTL analysis, capture Hi-C, and an arrayed CRISPR inhibition (CRISPRi) screen. The MPRA identified 196 variants across 19 regions with significant allele-specific effects, indicating cis-regulatory activity. Of these, 39 variants across 10 RCC loci displayed chromatin loops allowing physical interaction with the promoter of 24 nearby putative gene targets, as well as a significant cis-eQTL with the same gene. To confirm the presence of a cis-regulatory relationship between these 39 candidate variants and 24 putative target genes, we performed an arrayed CRISPRi screen in ACHN (RCC) and HEK293T (embryonic renal) cells covering each of the 10 nominated regions. Cells were stably transduced with inactive Cas9 fused to the ZIM3 transcriptional repressor domain and cis-regulatory relationships were assessed by TaqMan qPCR for the target genes. The screen identified multiple novel relevant target genes of RCC predisposition variants, including UCK2 at 1q24, ABL2 and TDRD5 at 1q25, GTDC1 at 2q22, GPR37 at 7q31, AGBL3 and CALD1 at 7q33, and MAP2K1 at 15q22. At some regions, evidence indicates cis-regulatory relationships occur between a target gene and all variants predicted to be ‘causative’ at the region (e.g., UCK2 at 1q24 and ABL2 at 1q25). For other regions, cis-regulatory relationships occur between a gene and only one variant tested (e.g., GPR37 at 7q31 and TDRD5 at 1q25).Previously, target genes of only four RCC susceptibility regions had been identified and published. This study identifies eight novel target genes across six additional RCC susceptibility regions, representing a significant advance in understanding of the underlying biology of RCC susceptibility variants. Furthermore, ongoing screening using the orthologous method, CRISPR activation (CRISPRa), and integration of both normoxic and hypoxic conditions continues to provide further insight into the underlying biology at these regions. Citation Format: Timothy Winter, Timothy Myers, Leandro Colli, Lea Jessop, Jiyeon Choi, Mitchell J. Machiela, Mark P. Purdue, Kevin Brown, Stephen Chanock. Targeted CRISPRi screen identifies functional variants and novel target genes at multiple renal cell carcinoma (RCC) susceptibility loci [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6154.

Abstract 2850: Noncoding regulatory mutations as driving event for the oncogenic core regulatory circuitries of neuroblastoma

Abstract Background and Aim: Neuroblastoma (NB) is the most aggressive solid tumor of infancy and arises from neural-crest-derived progenitor cells. It is composed by cell types reflecting developmental stages of the adrenergic lineage: mesenchymal (MES) and adrenergic (ADRN) cell types. These cell identities result from the activity of NB specific Core Regulatory Circuitries (CRCs), transcription factor (TF) sets that co-occupy regulatory regions together impacting the expression of their own genes and those of lineage-specific ones governing cell state transitions and cellular identities. Our hypothesis is that noncoding somatic single nucleotide variants (SNVs) located in NB CRCs TF binding sites (TFBSs) underlie NB CRCs activity, affecting cellular differentiation and promoting tumor onset. We aim to investigate such pattern of regulatory elements and identify putative driver SNVs, exploring their role and characterizing their action mechanism. Methods: Transcriptionally active TFBSs (aTFBSs) bound by ADRN CRC TFs (GATA3, HAND2, ISL1, TBX2, MYCN, PHOX2B, ASCL1 and LMO1) were identified integrating 27 ChIP-seq and 11 ATAC-seq experiments in 6 NB ADRN cell lines (SKNBE2C, Kelly, NGP, COGN415, LAN5 and NB1643). In particular, aTFBSs were defined as ChIP-seq peaks overlapping ATAC-seq signals in the same cell line. SNVs from 317 NB whole genomes were mapped on aTFBSs and the enrichment of mutations in core regions respect to flanking ones was tested using Fisher test. Observed mutation rate in aTFBSs was compared with background mutation rate due to local sequence context. SNVs in mutated aTFBSs were selected and their impact on TF binding affinity was evaluated with FABIAN-variant and motifbreakR tools. aTFBSs target-genes were identified through promoter capture HiC (CHiC) in ADRN NB cells (SKNBE2C and SHSY5Y). Results: We found a significant (FDR < 0.05) mutation enrichment in aTFBSs of ISL1, PHOX2B, TBX2, GATA3 and MYCN, suggesting a common somatic mutation burden for a subset of ADRN CRC TFBSs in ADRN NB cells. Starting from 839 SNVs falling in significantly mutated aTFBSs, we selected 269 mutations impacting the binding of ADRN CRC TFs. This mutations subset mapped in aTFBSs interacting with genes that significantly enriched pathways involved in neuronal development and migration (FDR < 0.05), thus suggesting their possible role in affecting cell identity definition. As an example, chr6:137753475:G>T mapped in aTFBSs interacting with the promoter of OLIG3, which takes part in neuronal differentiation. Interestingly, chr6:137753475:G>T was predicted to break the binding to ISL1 (FABIAN score: -0.24), a key regulator of ADRN CRC. Conclusions: These results demonstrate that somatic noncoding SNVs can act synergistically to promote NB onset, affecting CRCs activity and, in turn, the normal cell identity definition. Citation Format: Mario Capasso, Vincenzo Aievola, Vito Alessandro Lasorsa, Annalaura Montella, Ferdinando Bonfiglio, Marianna Avitabile, Teresa Maiorino, Matilde Tirelli, Giuseppe D'Alterio, Matthias Fischer, Frank Westermann, Achille Iolascon. Noncoding regulatory mutations as driving event for the oncogenic core regulatory circuitries of neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2850.

Abstract 143: Molecular profiling of tumor-initiation events in rhabdomyosarcoma uncovers mitochondrial vulnerabilities

Abstract Background: Alveolar Rhabdomyosarcoma (ARMS) is a very aggressive soft tissue tumor affecting children and adolescents. It is found that pediatric patients with RMS harboring chimeric PAX fusion transcription factors (TFs) exhibit a greater incidence of tumor relapse, metastasis, and poor survival outcome, thereby underscoring the urgent need to develop effective therapies to treat this subtype of childhood cancer. Methods: We generated a novel ARMS model by stable transduction of myogenic progenitor cells with a cassette encoding the exact PAX3/7-FOXO1 fusion protein found in ARMS tumors. We analyzed genome-wide expression levels with RNA-seq and utilized Cut&Run to map the binding sites of PAX3/7-FOXO1, active histone marks, enhancers, and super-enhancers in our novel model. Hi-C and promoter capture HiC (pCHiC) were also performed to generate 3D chromatin conformation maps in cells expressing the oncogenic TFs. We compared these data with analogous datasets from orthotopic patient-derived xenografts (O-PDXs) of PAX3/7-FOXO1 fusion-positive ARMS tumors from St. Jude and established ARMS cell lines to validate whether our model system recapitulates ARMS tumors. CRISPRi was performed to silence key targets in two representative ARMS cell lines and proliferation was scored after silencing. Next, we found that ARMS tumors displayed anomalies in mitochondrial mass and numbers, metabolic parameters, and membrane potential by performing TEM, Seahorse assays, and FACS-based TMRM and mitotracker Red dye uptake in a panel of ARMS cells and PDX samples. We performed cell viability and colony forming assays in ARMS cells treated with single drugs and combinations of drugs targeting FGFR4 and mitochondrial metabolism. Results: Our data provide evidence to support muscle progenitors as potential cells-of-origin for ARMS. We showed that rewiring of chromatin architecture mediated by PAX3/7-FOXO1 TFs is evident across established cell lines, primary tumors as well as O-PDXs. By integrating PAX3/7-FOXO1 binding, RNA-seq, and pCHiC data, we identified key promoter-enhancer (P-En) interactions that are erased or established de novo at multiple loci of master TFs like MYOD, MYOG and MYCN. We also identified key regulators of proliferation, including FGFR4, and mitochondrial function that are essential for tumor growth and invasiveness. Our data indicated aberrant and enhanced mitochondrial metabolism in ARMS as well as selective sensitivity to mitochondrial inhibitors in RMS cells. Administration of these inhibitors preferentially inhibited cell viability and clonogenicity in ARMS, and prompted a pre-clinical phase II study to test these inhibitors at St. Jude Research Hospital. Conclusion: We present a novel treatment strategy leveraging the mitochondrial dependencies of fusion positive PAX-driven tumors which can be exploited for precision therapy in high-risk ARMS. Citation Format: Bhargab Kalita, Gerard Martinez-Cebrian, Justina McEvoy, Brittney Gordon, Kaley Blankenship, Asa Karlstorm, Elizabeth Stewart, Michael Dyer, Brian Dynlacht. Molecular profiling of tumor-initiation events in rhabdomyosarcoma uncovers mitochondrial vulnerabilities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 143.

Single-cell multi-ome regression models identify functional and disease-associated enhancers and enable chromatin potential analysis

We present a gene-level regulatory model, single-cell ATAC + RNA linking (SCARlink), which predicts single-cell gene expression and links enhancers to target genes using multi-ome (scRNA-seq and scATAC–seq co-assay) sequencing data. The approach uses regularized Poisson regression on tile-level accessibility data to jointly model all regulatory effects at a gene locus, avoiding the limitations of pairwise gene–peak correlations and dependence on peak calling. SCARlink outperformed existing gene scoring methods for imputing gene expression from chromatin accessibility across high-coverage multi-ome datasets while giving comparable to improved performance on low-coverage datasets. Shapley value analysis on trained models identified cell-type-specific gene enhancers that are validated by promoter capture Hi-C and are 11× to 15× and 5× to 12× enriched in fine-mapped eQTLs and fine-mapped genome-wide association study (GWAS) variants, respectively. We further show that SCARlink-predicted and observed gene expression vectors provide a robust way to compute a chromatin potential vector field to enable developmental trajectory analysis.

Abstract B057: Oncogenic Foxl2 directs enhancer reprogramming and changes in 3D genome structure in ovarian granulosa cell tumors

Abstract Background: Adult-type granulosa cell tumors (aGCTs) are a rare monogenic disease. Nearly all aGCTs carry a missense point mutation in the Forkhead domain containing FOXL2 (Foxl2 p.C134W) transcription factor, but the oncogenic mechanism of this mutation is unknown. Other Forkhead family transcription factors have well-described “pioneer” activity, binding to compacted, nucleosome-bound DNA and increasing accessibility for other regulatory proteins. Objectives: To investigate if oncogenic Foxl2-C134W has pioneering activity and if mutant Foxl2 operates its oncogenic activity through enhancer reprogramming. Methods: CRISPR/Cas9 editing was used to generate isogenic granulosa cell tumor cells lacking either the FOXL2 wild-type allele (single knock-out; SKO) or both the mutant and wild-type FOXL2 alleles (double knock-out; DKO). ATAC-Seq and endogenous Foxl2 ChIP-Seq were performed on these isogenic lines to determine the differential chromatin accessibility at Foxl2-bound regulatory regions across genotypes. Promoter capture HiC was performed using a bespoke genome-wide hybrid capture probe set, CHiCAGO was used to identify significant HiC interactions, and an activity-by-contact model was constructed to identify enhancer elements bound by endogenous Foxl2-C134W and determine their target promoter contact frequency (promoter capture HiC) and enhancer activity (geometric mean of H3K27ac and ATACseq signal. Results: Endogenous ChIP-seq of Foxl2-C134W from the SKO cell line identified 1147 high-confidence peaks. De novo motif discovery identified the canonical Foxl2 binding motif (TGTTTACATT, P = 1.4 × 10−7) in 44.9% of peaks, as well as a novel variant motif (TGTTTTGTCT, P = 6.7 × 10−15) in 68.5% of peaks. Median chromatin accessibility at Foxl2-C134W peak regions, as measured by ATAC-seq, was significantly decreased in the DKO cells compared to either SKO or parental cell lines (P < 2 × 10-16 for both comparisons). Decreased chromatin accessibility at Foxl2-C134W ChIP-seq peaks in the DKO cells was driven by peaks containing the novel variant Foxl2 binding motif. Promoter capture HiC through activity-by-contact model identified promoter-enhancer interactions lost in the DKO cell line involving several genes, including ADAMTS5, FOXF2, and PRICKLE1 (adjusted P < 0.05). Conclusion: Foxl2-C134W exhibits “pioneering” activity, increasing chromatin accessibility at key gene regulatory elements with associated re-programming of promoter-enhancer interactions. The oncogenic mechanism of Foxl2-C134W in granulosa cell tumors may involve changes in DNA binding specificity, re-directing this pioneering function to sites containing a novel variant Foxl2 binding motif. Citation Format: Veena K. Vuttaradhi, Eleonora Khlebus, Thomas Welte, Barrett Lawson, Robert Tyler Hillman. Oncogenic Foxl2 directs enhancer reprogramming and changes in 3D genome structure in ovarian granulosa cell tumors [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B057.

Hi-C techniques: from genome assemblies to transcription regulation.

The invention of chromosome conformation capture (3C) techniques, in particular the key method Hi-C providing genome-wide information about chromatin contacts, revolutionized the way we study the three-dimensional organization of the nuclear genome and how it affects transcription, replication, and DNA repair. Because the frequency of chromatin contacts between pairs of genomic segments predictably relates to the distance in the linear genome, the information obtained by Hi-C has also proved useful for scaffolding genomic sequences. Here, we review recent improvements in experimental procedures of Hi-C and its various derivatives, such as Micro-C, HiChIP, and Capture Hi-C. We assess the advantages and limitations of the techniques, and present examples of their use in recent plant studies. We also report on progress in the development of computational tools used in assembling genome sequences.

Identification of a distal enhancer regulating hedgehog interacting protein gene in human lung epithelial cells

An intergenic region at chromosome 4q31 is one of the most significant regions associated with COPD susceptibility and lung function in GWAS. In this region, the implicated causal gene HHIP has a unique epithelial expression pattern in adult human lungs, in contrast to dominant expression in fibroblasts in murine lungs. However, the mechanism underlying the species-dependent cell type-specific regulation of HHIP remains largely unknown. We employed snATAC-seq analysis to identify open chromatin regions within the COPD GWAS region in various human lung cell types. ChIP-quantitative PCR, reporter assays, chromatin conformation capture assays and Hi-C assays were conducted to characterize the regulatory element in this region. CRISPR/Cas9-editing was performed in BEAS-2B cells to generate single colonies with stable knockout of the regulatory element. RT-PCR and Western blot assays were used to evaluate expression of HHIP and epithelial-mesenchymal transition (EMT)-related marker genes. We identified a distal enhancer within the COPD 4q31 GWAS locus that regulates HHIP transcription at baseline and after TGFβ treatment in a SMAD3-dependent, but Hedgehog-independent manner in human bronchial epithelial cells. The distal enhancer also maintains chromatin topological domains near 4q31 locus and HHIP gene. Reduced HHIP expression led to increased EMT induced by TGFβ in human bronchial epithelial cells. A distal enhancer regulates HHIP expression both under homeostatic condition and upon TGFβ treatment in human bronchial epithelial cells. The interaction between HHIP and TGFβ signalling possibly contributes to COPD pathogenesis. Supported by NIH grants R01HL127200, R01HL148667 and R01HL162783 (to X. Z).

Transcription induces context-dependent remodeling of chromatin architecture during differentiation.

Metazoan chromosomes are organized into discrete spatial domains (TADs), believed to contribute to the regulation of transcriptional programs. Despite extensive correlation between domain organization and gene activity, a direct mechanistic link is unclear, with perturbation studies often showing little effect. To follow chromatin architecture changes during development, we used Capture Hi-C to interrogate the domains around key differentially expressed genes during mouse thymocyte maturation, uncovering specific remodeling events. Notably, one TAD boundary was broadened to accommodate RNA polymerase elongation past the border, and subdomains were formed around some activated genes without changes in CTCF binding. The ectopic induction of some genes was sufficient to recapitulate domain formation in embryonic stem cells, providing strong evidence that transcription can directly remodel chromatin structure. These results suggest that transcriptional processes drive complex chromosome folding patterns that can be important in certain genomic contexts.

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

BackgroundLong-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.ResultsHere, 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 β-estradiol inducible C/EBPαER transgene. Within 1h of β-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.ConclusionsOverall, 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.

Integration of genetic fine-mapping and multi-omics data reveals candidate effector genes for hypertension

Genome-wide association studies of blood pressure (BP) have identified >1,000 loci, but the effector genes and biological pathways at these loci are mostly unknown. Using published association summary statistics, we conducted annotation-informed fine-mapping incorporating tissue-specific chromatin segmentation and colocalization to identify causal variants and candidate effector genes for systolic BP, diastolic BP, and pulse pressure. We observed 532 distinct signals associated with ≥2 BP traits and 84 with all three. For >20% of signals, a single variant accounted for >75% posterior probability, 65 were missense variants in known (SLC39A8, ADRB2, and DBH) and previously unreported BP candidate genes (NRIP1 and MMP14). In disease-relevant tissues, we colocalized >80 and >400 distinct signals for each BP trait with cis-eQTLs and regulatory regions from promoter capture Hi-C, respectively. Integrating mouse, human disorder, gene expression and tissue abundance data, and literature review, we provide consolidated evidence for 436 BP candidate genes for future functional validation and discover several potential drug targets.

Allelic chromatin structure precedes imprinted expression of Kcnk9 during neurogenesis.

Differences in chromatin state inherited from the parental gametes influence the regulation of maternal and paternal alleles in offspring. This phenomenon, known as genomic imprinting, results in genes preferentially transcribed from one parental allele. While local epigenetic factors such as DNA methylation are known to be important for the establishment of imprinted gene expression, less is known about the mechanisms by which differentially methylated regions (DMRs) lead to differences in allelic expression across broad stretches of chromatin. Allele-specific higher-order chromatin structure has been observed at multiple imprinted loci, consistent with the observation of allelic binding of the chromatin-organizing factor CTCF at multiple DMRs. However, whether allelic chromatin structure impacts allelic gene expression is not known for most imprinted loci. Here we characterize the mechanisms underlying brain-specific imprinted expression of the Peg13-Kcnk9 locus, an imprinted region associated with intellectual disability. We performed region capture Hi-C on mouse brains from reciprocal hybrid crosses and found imprinted higher-order chromatin structure caused by the allelic binding of CTCF to the Peg13 DMR. Using an in vitro neuron differentiation system, we showed that imprinted chromatin structure precedes imprinted expression at the locus. Additionally, activation of a distal enhancer induced imprinted expression of Kcnk9 in an allelic chromatin structure-dependent manner. This work provides a high-resolution map of imprinted chromatin structure and demonstrates that chromatin state established in early development can promote imprinted expression upon differentiation.

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.

A deep learning method for replicate-based analysis of chromosome conformation contacts using Siamese neural networks

The organisation of the genome in nuclear space is an important frontier of biology. Chromosome conformation capture methods such as Hi-C and Micro-C produce genome-wide chromatin contact maps that provide rich data containing quantitative and qualitative information about genome architecture. Most conventional approaches to genome-wide chromosome conformation capture data are limited to the analysis of pre-defined features, and may therefore miss important biological information. One constraint is that biologically important features can be masked by high levels of technical noise in the data. Here we introduce a replicate-based method for deep learning from chromatin conformation contact maps. Using a Siamese network configuration our approach learns to distinguish technical noise from biological variation and outperforms image similarity metrics across a range of biological systems. The features extracted from Hi-C maps after perturbation of cohesin and CTCF reflect the distinct biological functions of cohesin and CTCF in the formation of domains and boundaries, respectively. The learnt distance metrics are biologically meaningful, as they mirror the density of cohesin and CTCF binding. These properties make our method a powerful tool for the exploration of chromosome conformation capture data, such as Hi-C capture Hi-C, and Micro-C.

High-throughput identification of regulatory elements and functional assays to uncover susceptibility genes for nasopharyngeal carcinoma

Large-scale genetic association studies have identified multiple susceptibility loci for nasopharyngeal carcinoma (NPC), but the underlying biological mechanisms remain to be explored. To gain insights into the genetic etiology of NPC, we conducted a follow-up study encompassing 6,907 cases and 10,472 controls and identified two additional NPC susceptibility loci, 9q22.33 (rs1867277; OR= 0.74, 95% CI= 0.68-0.81, p= 3.08×10-11) and 17q12 (rs226241; OR= 1.42, 95% CI= 1.26-1.60, p= 1.62×10-8). The two additional loci, together with two previously reported genome-wide significant loci, 5p15.33 and 9p21.3, were investigated by high-throughput sequencing for chromatin accessibility, histone modification, and promoter capture Hi-C (PCHi-C) profiling. Using luciferase reporter assays and CRISPR interference (CRISPRi) to validate the functional profiling, we identified PHF2 at locus 9q22.33 as a susceptibility gene. PHF2 encodes a histone demethylase and acts as a tumor suppressor. The risk alleles of the functional SNPs reduced the expression of the target gene PHF2 by inhibiting the enhancer activity of its long-range (4.3 Mb) cis-regulatory element, which promoted proliferation of NPC cells. In addition, we identified CDKN2B-AS1 as a susceptibility gene at locus 9p21.3, and the NPC risk allele of the functional SNP rs2069418 promoted the expression of CDKN2B-AS1 by increasing its enhancer activity. The overexpression of CDKN2B-AS1 facilitated proliferation of NPC cells. In summary, we identified functional SNPs and NPC susceptibility genes, which provides additional explanations for the genetic association signals and helps to uncover the underlying genetic etiology of NPC development.

RELA governs a network of islet-specific metabolic genes necessary for beta cell function

Aims/hypothesisNF-κB activation unites metabolic and inflammatory responses in many diseases yet less is known about the role that NF-κB plays in normal metabolism. In this study we investigated how RELA impacts the beta cell transcriptional landscape and provides network control over glucoregulation.MethodsWe generated novel mouse lines harbouring beta cell-specific deletion of either the Rela gene, encoding the canonical NF-κB transcription factor p65 (βp65KO mice), or the Ikbkg gene, encoding the NF-κB essential modulator NEMO (βNEMOKO mice), as well as βA20Tg mice that carry beta cell-specific and forced transgenic expression of the NF-κB-negative regulator gene Tnfaip3, which encodes the A20 protein. Mouse studies were complemented by bioinformatics analysis of human islet chromatin accessibility (assay for transposase-accessible chromatin with sequencing [ATAC-seq]), promoter capture Hi-C (pcHi-C) and p65 binding (chromatin immunoprecipitation–sequencing [ChIP-seq]) data to investigate genome-wide control of the human beta cell metabolic programme.ResultsRela deficiency resulted in complete loss of stimulus-dependent inflammatory gene upregulation, consistent with its known role in governing inflammation. However, Rela deletion also rendered mice glucose intolerant because of functional loss of insulin secretion. Glucose intolerance was intrinsic to beta cells as βp65KO islets failed to secrete insulin ex vivo in response to a glucose challenge and were unable to restore metabolic control when transplanted into secondary chemical-induced hyperglycaemic recipients. Maintenance of glucose tolerance required Rela but was independent of classical NF-κB inflammatory cascades, as blocking NF-κB signalling in vivo by beta cell knockout of Ikbkg (NEMO), or beta cell overexpression of Tnfaip3 (A20), did not cause severe glucose intolerance. Thus, basal p65 activity has an essential and islet-intrinsic role in maintaining normal glucose homeostasis. Genome-wide bioinformatic mapping revealed the presence of p65 binding sites in the promoter regions of specific metabolic genes and in the majority of islet enhancer hubs (~70% of ~1300 hubs), which are responsible for shaping beta cell type-specific gene expression programmes. Indeed, the islet-specific metabolic genes Slc2a2, Capn9 and Pfkm identified within the large network of islet enhancer hub genes showed dysregulated expression in βp65KO islets.Conclusions/interpretationThese data demonstrate an unappreciated role for RELA as a regulator of islet-specific transcriptional programmes necessary for the maintenance of healthy glucose metabolism. These findings have clinical implications for the use of anti-inflammatories, which influence NF-κB activation and are associated with diabetes.Graphical