Chromatin Topology Research Articles

EPCO-18. HETEROCHROMATIN DYSFUNCTION IMPACTING GENOMIC TOPOLOGY AND SUPERENHANCER LANDSCAPES PROMOTES ATRX-DEFICIENT GLIOMAGENESIS

Abstract Mutational inactivation of the SWI/SNF chromatin regulator ATRX occurs frequently in diffuse gliomas. Whether and how ATRX deficiency promotes oncogenesis remains unclear, despite its implication across a broad array of physiological processes. We sought to delineate the molecular mechanisms by which global epigenomic dysfunction promotes oncogenic phenotypes in ATRX-deficient glioma models. Building on earlier work implicating chromatin accessibility and transcriptional shifts, we employed integrated epigenomic profiling in Atrx-intact and -deficient murine neuroepithelial progenitor cells (mNPCs), validating findings will functional approaches in human glioma stem cells (GSCs) of appropriate genotype. We found that ATRX deficiency dramatically impacted the integrity of large heterochromatin domains genome-wide, with tangible effects on underlying gene expression. This process fundamentally altered chromatin topology, as assessed by Hi-C analysis, and associated superenhancer landscapes, influencing key developmental gene sets like the HOXA cluster. Pharmacological inhibition of HOXA signaling in GSCs selectively reduced proliferation, increased apoptosis, and impaired in vivo growth in the ATRX-deficient context. Finally, our results pointed to derepression of LINE1 endogenous retroviral elements as key mediators of ATRX-deficient heterochromatin dysfunction. Our work implicates novel and targetable molecular mechanisms involving complex epigenomic rewiring and chromatin topology in ATRX-deficient oncogenesis. In doing so, we advance the understanding of a deadly brain tumor along with broader conceptions of epigenetic mechanisms as fundamental drivers in cancer.

Multi-omics comparison of malignant and normal uveal melanocytes reveals molecular features of uveal melanoma

Uveal melanoma (UM) is a rare cancer resulting from the transformation of melanocytes in the uveal tract. Integrative analysis has identified four molecular and clinical subsets of UM. To improve our molecular understanding of UM, we performed extensive multi-omics characterization comparing two aggressive UM patient-derived xenograft models with normal choroidal melanocytes, including DNA optical mapping, specific histone modifications, and DNA topology analysis using Hi-C. Our gene expression and cytogenetic analyses suggest that genomic instability is a hallmark of UM. We also identified a recurrent deletion in the BAP1 promoter resulting in loss of expression and associated with high risk of metastases in UM patients. Hi-C revealed chromatin topology changes associated with the upregulation of PRAME, an independent prognostic biomarker in UM, and a potential therapeutic target. Our findings illustrate how multi-omics approaches can improve our understanding of tumorigenesis and reveal two distinct mechanisms of gene expression dysregulation in UM.

Imaging-based study demonstrates how the DEK nanoscale distribution differentially correlates with epigenetic marks in a breast cancer model

Epigenetic dysregulation of chromatin is one of the hallmarks of cancer development and progression, and it is continuously investigated as a potential general bio-marker of this complex disease. One of the nuclear factors involved in gene regulation is the unique DEK protein—a histone chaperon modulating chromatin topology. DEK expression levels increase significantly from normal to cancer cells, hence raising the possibility of using DEK as a tumor marker. Although DEK is known to be implicated in epigenetic and transcriptional regulation, the details of these interactions and their relevance in cancer development remain largely elusive. In this work, we investigated the spatial correlation between the nuclear distribution of DEK and chromatin patterns—alongside breast cancer progression—leveraging image cross-correlation spectroscopy (ICCS) coupled with Proximity Ligation Assay (PLA) analysis. We performed our study on the model based on three well-established human breast cell lines to consider this tumor's heterogeneity (MCF10A, MCF7, and MDA-MB-231 cells). Our results show that overexpression of DEK correlates with the overall higher level of spatial proximity between DEK and histone marks corresponding to gene promoters regions (H3K9ac, H3K4me3), although it does not correlate with spatial proximity between DEK and gene enhancers (H3K27ac). Additionally, we observed that colocalizing fractions of DEK and histone marks are lower for the non-invasive cell subtype than for the highly invasive cell line (MDA-MB-231). Thus, this study suggests that the role of DEK on transcriptionally active chromatin regions varies depending on the subtype of the breast cancer cell line.

Neutrophil nucleus: shaping the past and the future.

Neutrophils are innate immune cells that are key to protecting the host against infection and maintaining body homeostasis. However, if dysregulated, they can contribute to disease, such as in cancer or chronic autoinflammatory disorders. Recent studies have highlighted the heterogeneity in the neutrophil compartment and identified the presence of immature neutrophils and their precursors in these pathologies. Therefore, understanding neutrophil maturity and the mechanisms through which they contribute to disease is critical. Neutrophils were first characterized morphologically by Ehrlich in 1879 using microscopy, and since then, different technologies have been used to assess neutrophil maturity. The advances in the imaging field, including state-of-the-art microscopy and machine learning algorithms for image analysis, reinforce the use of neutrophil nuclear morphology as a fundamental marker of maturity, applicable for objective classification in clinical diagnostics. New emerging approaches, such as the capture of changes in chromatin topology, will provide mechanistic links between the nuclear shape, chromatin organization, and transcriptional regulation during neutrophil maturation.

Identification of a ΔNp63-Dependent Basal-Like A Subtype-Specific Transcribed Enhancer Program (B-STEP) in Aggressive Pancreatic Ductal Adenocarcinoma.

Subtype-specific enhancer activity analysis via detection of eRNAs on a single-cell level in patient material can be used as a potential tool for treatment stratification.

1440 DNA dioxygenases Tet2/3 regulate gene promoter accessibility and chromatin topology in lineage-specific loci to control keratinocyte differentiation and hair growth

1440 DNA dioxygenases Tet2/3 regulate gene promoter accessibility and chromatin topology in lineage-specific loci to control keratinocyte differentiation and hair growth

Dynamic chromatin conformation and accessibility changes mediate the spatial-specific gene regulatory network in Bombyx mori

Silk gland genes of Bombyx mori can have strict spatial expression patterns, which impact their functions and silk quality. However, our understanding of their regulation mechanisms is currently insufficient. To address this, the middle silk gland (MSG) and posterior silk gland (PSG) of the silkworm were investigated. Gene ontology annotation showed that spatially specific expressed genes were involved in the formation of H3k9me and chromatin topology. Chromatin conformation data generated by Hi-C showed that the topologically associated domain boundaries around FibL and Sericin1 genes were significantly different between MSG and PSG. Changes in chromatin conformation led to changes in chromatin activity, which significantly affected the expression of nearby genes in silkworm. Chromatin accessibility regions of MSG and PSG were analyzed using FAIRE-seq, and 1006 transcription factor motifs were identified in open chromatin regions. Furthermore, the spatial-specific expression patterns of silk gland genes were mainly associated with homeobox-contained transcription factors, such as POU-M2, which was specifically bound and relatively highly expressed in the MSG. The regulatory network mediated by POU-M2 regulated most of the spatial-specific expressed genes in MSG, such as ADH1. These results can aid in improving silk performance, optimizing silkworm breeding, and improving the gene spatial regulatory model research for insects.

Abstract 1404: A functional genetic polymorphism of mitochondrion-associated DNAJA3 in nonalcoholic steatohepatitis-related hepatocellular carcinoma

Abstract Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease worldwide and may progress to nonalcoholic steatohepatitis (NASH). Although emerging evidence suggests that NASH would be an important driver of hepatocellular carcinoma (HCC), molecularly what drives the transition of NAFLD to NASH and then HCC has not been clearly defined. Since NASH is a mitochondrial disease, we aimed to determine genetic variants associated with mitochondrial dysfunction linked to NASH and NASH-related HCC. We performed an integrative genomics analysis by analyzing a large-scale genome-wide association study of the body fat distribution-related variants in 344,369 individuals from multiple ancestries. And we linked these results to the top 24 significant variants to NASH and NASH-related HCC in 1,009 individuals from the NCI-UMD cohort. We also performed the expression quantitative trait loci (eQTL) analysis on candidate variants. We analyzed the 3D genome architecture especially chromatin topology based on UCSC Genome Browser that influences the expression of candidate variants. As a result, we identified rs3747579-TT, an eQTL to a mitochondrial chaperone Hsp40, also known as DNAJA3 rather than a coding variant, as a risk allele linked to NASH-related HCC. HCC patients with rs3747579-TT variant had a poor prognosis compared to HCC patients with rs3747579-CT or CC variant. We confirmed that HCC with rs3747579-TT had a reduced expression of DNAJA3, encoding a protein that maintains mitochondrial homeostasis. Consistently, HCC patients with low DNAJA3 expression had a poor prognosis. We found a specific site based on chromatin topology that may influence rs3747579 to regulate the expression of DNAJA3. In summary, we identified rs3747579-TT as a functional polymorphic variant of DNAJA3 that may be responsible for the development of NASH and NASH-related HCC. Citation Format: Yuto Shiode, Ching-Wen Chang, Xin Wei Wang. A functional genetic polymorphism of mitochondrion-associated DNAJA3 in nonalcoholic steatohepatitis-related hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1404.

Abstract 3136: HYENA detects non-coding genes activated by distal enhancers in cancer

Abstract Genome instability is a hallmark of cancer, resulting in the accumulation of mutation. Among different types of mutations, structural variants (SVs) widely spread in cancer genomes and can be important cancer drivers, demonstrated in multiple cancer types. SVs can induce oncogene activation by redirecting enhancers to promoter regions or disrupting chromatin topology. These events are called “enhancer hijacking” and known to upregulate oncogenes in multiple cancers. Enhancers are among the best understood cis-regulatory elements and are essential regulators for precise gene expression in normal tissue. Since SV associated gene deregulation has been shown in multiple cancer types, it is a feasible way to infer putative oncogenes by detecting enhancer hijacking events. Taking advantage of next generation sequencing, bioinformatic tools were developed to study pan-histology protein-coding gene deregulation caused by SVs. However, algorithms that can unbiasedly detect enhancer hijacking in both coding and non-coding genes are yet to be developed. Here we present a computational algorithm “HYENA” to identify putative oncogenes activated by enhancer hijacking using whole-genome sequencing and RNA sequencing data. It deploys rank-based regression model to test whether an SV breakpoint located close to a gene significantly increases the gene expression. Permutation tests were used to generate a false discovery rate for each gene. By benchmarking with existing tools serving similar purposes, we confirmed that HYENA has superior capabilities to infer enhancer hijacking events. We detected 192 oncogene candidates in 1,148 tumor samples from 25 cancer types. While confirming the known cases of enhancer hijacking, we also identified numerous novel oncogene candidates, of which a substantial proportion was non-coding genes. Specifically, we found that a long non-coding RNA TOB1-AS1 was activated by rearranged enhancers in 10% of pancreatic cancer samples. Furthermore, experimental validation with pancreatic cancer cell lines and immunodeficient mice indicated that high expression of TOB1-AS1 is capable to promote cancer cell invasion and tumor metastasis, suggesting it is a novel cancer driver. As the existing treatments for pancreatic cancer are largely inefficacious and patients still have poor prognosis, new targets are urgently needed to improve pancreatic cancer treatments. Although the molecular mechanisms underlying TOB1-AS1 promoted metastasis remain to be unveiled, this non-coding gene can be a promising target in the patients carrying this enhancer hijacking event. In summary, our results demonstrated that HYENA is a sensitive and reliable tool to infer enhancer hijacking activated oncogenes and spot new potential targets to treat cancer. Citation Format: Anqi Yu, Ali E. Yesilkanal, Ashish Thakur, Fan Wang, Xiaoyang Wu, Alexander Muir, Xin He, Francois Spitz, Lixing Yang. HYENA detects non-coding genes activated by distal enhancers in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3136.

Haplotype mapping of H3K27me3-associated chromatin interactions defines topological regulation of gene silencing in rice

Histone modification H3K27me3 is an important chromatin mark that plays vital roles in repressing expression of developmental genes. Here, we construct high-resolution 3D genome maps using long-read chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) and characterize H3K27me3-associated chromatin interactions in an elite rice hybrid, Shanyou 63. We find that many H3K27me3-marked regions may function as silencer-like regulatory elements. The silencer-like elements can come into proximity with distal target genes via forming chromatin loops in 3D space of the nuclei, regulating gene silencing and plant traits. Natural and induced deletion of silencers upregulate expression of distal connected genes. Furthermore, we identify extensive allele-specific chromatin loops. We find that genetic variations alter allelic chromatin topology, thus modulating allelic gene imprinting in rice hybrids. In conclusion, the characterization of silencer-like regulatory elements and haplotype-resolved chromatin interaction maps provide insights into the understanding of molecular mechanisms underlying allelic gene silencing and plant trait controlling.

SATB1 regulates 3D genome architecture in T cells by constraining chromatin interactions surrounding CTCF-binding sites

Special AT-rich sequence binding protein 1 (SATB1) has long been proposed to act as a global chromatin loop organizer in Tcells. However, the exact functions of SATB1 in spatial genome organization remain elusive. Here we show that the depletion of SATB1 in human and murine Tcells leads to transcriptional dysregulation for genes involved in Tcell activation, as well as alterations of 3D genome architecture at multiple levels, including compartments, topologically associating domains, and loops. Importantly, SATB1 extensively colocalizes with CTCF throughout the genome. Depletion of SATB1 leads to increased chromatin contacts among and across the SATB1/CTCF co-occupied sites, thereby affecting the transcription of critical regulators of Tcell activation. The loss of SATB1 does not affect CTCF occupancy but significantly reduces the retention of CTCF in the nuclear matrix. Collectively, our data show that SATB1 contributes to 3D genome organization by constraining chromatin topology surrounding CTCF-binding sites.

Determining chromatin architecture with Micro Capture-C.

Micro Capture-C (MCC) is a chromatin conformation capture (3C) method for visualizing reproducible three-dimensional contacts of specified regions of the genome at base pair resolution. These methods are an established family of techniques that use proximity ligation to assay the topology of chromatin. MCC can generate data at substantially higher resolution than previous techniques through multiple refinements of the 3C method. Using a sequence agnostic nuclease, the maintenance of cellular integrity and full sequencing of the ligation junctions, MCC achieves subnucleosomal levels of resolution, which can be used to reveal transcription factor binding sites analogous to DNAse I footprinting. Gene dense regions, close-range enhancer-promoter contacts, individual enhancers within super-enhancers and multiple other types of loci or regulatory regions that were previously challenging to assay with conventional 3C techniques, are readily observed using MCC. MCC requires training in common molecular biology techniques and bioinformatics to perform the experiment and analyze the data. The protocol can be expected to be completed in a 3 week timeframe for experienced molecular biologists.

Structural variation cooperates with permissive chromatin to control enhancer hijacking–mediated oncogenic transcription

AbstractStructural variants (SVs) involving enhancer hijacking can rewire chromatin topologies to cause oncogene activation in human cancers, including hematologic malignancies; however, because of the lack of tools to assess their effects on gene regulation and chromatin organization, the molecular determinants for the functional output of enhancer hijacking remain poorly understood. Here, we developed a multimodal approach to integrate genome sequencing, chromosome conformation, chromatin state, and transcriptomic alteration for quantitative analysis of transcriptional effects and structural reorganization imposed by SVs in leukemic genomes. We identified known and new pathogenic SVs, including recurrent t(5;14) translocations that cause the hijacking of BCL11B enhancers for the allele-specific activation of TLX3 in a subtype of pediatric leukemia. Epigenetic perturbation of SV-hijacked BCL11B enhancers impairs TLX3 transcription, which are required for the growth of t(5;14) leukemia cells. By CRISPR engineering of patient-derived t(5;14) in isogenic leukemia cells, we uncovered a new mechanism whereby the transcriptional output of SV-induced BCL11B enhancer hijacking is dependent on the loss of DNA hypermethylation at the TLX3 promoter. Our results highlight the importance of the cooperation between genetic alteration and permissive chromatin as a critical determinant of SV-mediated oncogene activation, with implications for understanding aberrant gene transcription after epigenetic therapies in patients with leukemia. Hence, leveraging the interdependency of genetic alteration on chromatin variation may provide new opportunities to reprogram gene regulation as targeted interventions in human disease.

LINE-1 repression in Epstein-Barr virus-associated gastric cancer through viral-host genome interaction.

Long INterspersed Element 1 (LINE-1 or L1) acts as a major remodeling force in genome regulation and evolution. Accumulating evidence shows that virus infection impacts L1 expression, potentially impacting host antiviral response and diseases. The underlying regulation mechanism is unclear. Epstein-Barr virus (EBV), a double-stranded DNA virus linked to B-cell and epithelial malignancies, is known to have viral-host genome interaction, resulting in transcriptional rewiring in EBV-associated gastric cancer (EBVaGC). By analyzing publicly available datasets from the Gene Expression Omnibus (GEO), we found that EBVaGC has L1 transcriptional repression compared with EBV-negative gastric cancer (EBVnGC). More specifically, retrotransposition-associated young and full-length L1s (FL-L1s) were among the most repressed L1s. Epigenetic alterations, especially increased H3K9me3, were observed on FL-L1s. H3K9me3 deposition was potentially attributed to increased TASOR expression, a key component of the human silencing hub (HUSH) complex for H3K9 trimethylation. The 4C- and HiC-seq data indicated that the viral DNA interacted in the proximity of the TASOR enhancer, strengthening the loop formation between the TASOR enhancer and its promoter. These results indicated that EBV infection is associated with increased H3K9me3 deposition, leading to L1 repression. This study uncovers a regulation mechanism of L1 expression by chromatin topology remodeling associated with viral-host genome interaction in EBVaGC.

Interactive enhancer hubs (iHUBs) mediate transcriptional reprogramming and adaptive resistance in pancreatic cancer

ObjectivePancreatic ductal adenocarcinoma (PDAC) displays a remarkable propensity towards therapy resistance. However, molecular epigenetic and transcriptional mechanisms enabling this are poorly understood. In this study, we aimed to identify novel...

Function and Evolution of the Loop Extrusion Machinery in Animals.

Structural maintenance of chromosomes (SMC) complexes are essential proteins found in genomes of all cellular organisms. Essential functions of these proteins, such as mitotic chromosome formation and sister chromatid cohesion, were discovered a long time ago. Recent advances in chromatin biology showed that SMC proteins are involved in many other genomic processes, acting as active motors extruding DNA, which leads to the formation of chromatin loops. Some loops formed by SMC proteins are highly cell type and developmental stage specific, such as SMC-mediated DNA loops required for VDJ recombination in B-cell progenitors, or dosage compensation in Caenorhabditis elegans and X-chromosome inactivation in mice. In this review, we focus on the extrusion-based mechanisms that are common for multiple cell types and species. We will first describe an anatomy of SMC complexes and their accessory proteins. Next, we provide biochemical details of the extrusion process. We follow this by the sections describing the role of SMC complexes in gene regulation, DNA repair, and chromatin topology.

SLFN5-mediated chromatin dynamics sculpt higher-order DNA repair topology

Repair of DNA double-strand breaks (DSBs) elicits three-dimensional (3D) chromatin topological changes. A recent finding reveals that 53BP1 assembles into a 3D chromatin topology pattern around DSBs. How this formation of a higher-order structure is configured and regulated remains enigmatic. Here, we report that SLFN5 is a critical factor for 53BP1 topological arrangement at DSBs. Using super-resolution imaging, we find that SLFN5 binds to 53BP1 chromatin domains to assemble a higher-order microdomain architecture by driving damaged chromatin dynamics at both DSBs and deprotected telomeres. Mechanistically, we proposethat 53BP1 topology is shaped by two processes: (1) chromatin mobility driven by the SLFN5-LINC-microtubule axis and (2) the assembly of 53BP1 oligomers mediated by SLFN5. In mammals, SLFN5 deficiency disrupts the DSB repair topology and impairs non-homologous end joining, telomere fusions, class switch recombination, and sensitivity to poly (ADP-ribose) polymerase inhibitor. We establish a molecular mechanism that shapes higher-order chromatin topologies to safeguard genomic stability.

Disruption of polyhomeotic polymerization decreases nucleosome occupancy and alters genome accessibility.

Chromatin attains its three-dimensional (3D) conformation by establishing contacts between different noncontiguous regions. Sterile Alpha Motif (SAM)-mediated polymerization of the polyhomeotic (PH) protein regulates subnuclear clustering of Polycomb Repressive Complex 1 (PRC1) and chromatin topology. The mutations that perturb the ability of the PH to polymerize, disrupt long-range chromatin contacts, alter Hox gene expression, and lead to developmental defects. To understand the underlying mechanism, we combined the experiments and theory to investigate the effect of this SAM domain mutation on nucleosome occupancy and accessibility on a genome wide scale. Our data show that disruption of PH polymerization because of SAM domain mutation decreases nucleosome occupancy and alters accessibility. Polymer simulations investigating the interplay between distant chromatin contacts and nucleosome occupancy, both of which are regulated by PH polymerization, suggest that nucleosome density increases when contacts between different regions of chromatin are established. Taken together, it appears that SAM domain-mediated PH polymerization biomechanically regulates the organization of chromatin at multiple scales from nucleosomes to chromosomes and we suggest that higher order organization can have a top-down causation effect on nucleosome occupancy.

On the Advent of Super-Resolution Microscopy in the Realm of Polycomb Proteins.

The genomes of metazoans are organized at multiple spatial scales, ranging from the double helix of DNA to whole chromosomes. The intermediate genomic scale of kilobases to megabases, which corresponds to the 50-300 nm spatial scale, is particularly interesting, as the 3D arrangement of chromatin is implicated in multiple regulatory mechanisms. In this context, polycomb group (PcG) proteins stand as major epigenetic modulators of chromatin function, acting prevalently as repressors of gene transcription by combining chemical modifications of target histones with physical crosslinking of distal genomic regions and phase separation. The recent development of super-resolution microscopy (SRM) has strongly contributed to improving our comprehension of several aspects of nano-/mesoscale (10-200 nm) chromatin domains. Here, we review the current state-of-the-art SRM applied to PcG proteins, showing that the application of SRM to PcG activity and organization is still quite limited and mainly focused on the 3D assembly of PcG-controlled genomic loci. In this context, SRM approaches have mostly been applied to multilabel fluorescence in situ hybridization (FISH). However, SRM data have complemented the maps obtained from chromosome capture experiments and have opened a new window to observe how 3D chromatin topology is modulated by PcGs.

The "Superoncogene" Myc at the Crossroad between Metabolism and Gene Expression in Glioblastoma Multiforme.

The concept of the Myc (c-myc, n-myc, l-myc) oncogene as a canonical, DNA-bound transcription factor has consistently changed over the past few years. Indeed, Myc controls gene expression programs at multiple levels: directly binding chromatin and recruiting transcriptional coregulators; modulating the activity of RNA polymerases (RNAPs); and drawing chromatin topology. Therefore, it is evident that Myc deregulation in cancer is a dramatic event. Glioblastoma multiforme (GBM) is the most lethal, still incurable, brain cancer in adults, and it is characterized in most cases by Myc deregulation. Metabolic rewiring typically occurs in cancer cells, and GBM undergoes profound metabolic changes to supply increased energy demand. In nontransformed cells, Myc tightly controls metabolic pathways to maintain cellular homeostasis. Consistently, in Myc-overexpressing cancer cells, including GBM cells, these highly controlled metabolic routes are affected by enhanced Myc activity and show substantial alterations. On the other hand, deregulated cancer metabolism impacts Myc expression and function, placing Myc at the intersection between metabolic pathway activation and gene expression. In this review paper, we summarize the available information on GBM metabolism with a specific focus on the control of the Myc oncogene that, in turn, rules the activation of metabolic signals, ensuring GBM growth.