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The three-dimensional structure of the human genome has been proven to have a significant functional impact on gene expression. The high-order spatial chromatin is organised first by looping mediated by multiple protein factors, and then it is further formed into larger structures of topologically associated domains (TADs) or chromatin contact domains (CCDs), followed by A/B compartments and finally the chromosomal territories (CTs). The genetic variation observed in human population influences the multi-scale structures, posing a question regarding the functional impact of structural variants reflected by the variability of the genes expression patterns. The current methods of evaluating the functional effect include eQTLs analysis which uses statistical testing of influence of variants on spatially close genes. Rarely, non-coding DNA sequence changes are evaluated by their impact on the biomolecular interaction network (BIN) reflecting the cellular interactome that can be analysed by the classical graph-theoretic algorithms. Therefore, in the second part of the review, we introduce the concept of BIN, i.e. a meta-network model of the complete molecular interactome developed by integrating various biological networks. The BIN meta-network model includes DNA-protein binding by the plethora of protein factors as well as chromatin interactions, therefore allowing connection of genomics with the downstream biomolecular processes present in a cell. As an illustration, we scrutinise the chromatin interactions mediated by the CTCF protein detected in a ChIA-PET experiment in the human lymphoblastoid cell line GM12878. In the corresponding BIN meta-network the DNA spatial proximity is represented as a graph model, combined with the Proteins-Interaction Network (PIN) of human proteome using the Gene Association Network (GAN). Furthermore, we enriched the BIN with the signalling and metabolic pathways and Gene Ontology (GO) terms to assert its functional context. Finally, we mapped the Single Nucleotide Polymorphisms (SNPs) from the GWAS studies and identified the chromatin mutational hot-spots associated with a significant enrichment of SNPs related to autoimmune diseases. Afterwards, we mapped Structural Variants (SVs) from healthy individuals of 1000 Genomes Project and identified an interesting example of the missing protein complex associated with protein Q6GYQ0 due to a deletion on chromosome 14. Such an analysis using the meta-network BIN model is therefore helpful in evaluating the influence of genetic variation on spatial organisation of the genome and its functional effect in a cell.

Genome-wide chromosome conformation capture (3C)-based high-throughput sequencing (Hi-C) has enabled identification of genome-wide chromatin loops. Because the Hi-C map with restriction fragment resolution is intrinsically associated with sparsity and stochastic noise, Hi-C data are usually binned at particular intervals; however, the binning method has limited reliability, especially at high resolution. Here, we describe a new method called HiCORE, which provides simple pipelines and algorithms to overcome the limitations of single-layered binning and predict core chromatin regions with three-dimensional physical interactions. In this approach, multiple layers of binning with slightly shifted genome coverage are generated, and interacting bins at each layer are integrated to infer narrower regions of chromatin interactions. HiCORE predicts chromatin looping regions with higher resolution, both in human and Arabidopsis genomes, and contributes to the identification of the precise positions of potential genomic elements in an unbiased manner.

Although previous studies identified numerous single nucleotide polymorphisms (SNPs) and their target genes predisposed to prostate cancer (PrCa) risks, SNP-related splicing associations are rarely reported. In this study, we applied distance-based sQTL analysis (sQTLseekeR) using RNA-seq and SNP genotype data from benign prostate tissue (n = 467) and identified significant associations in 3344 SNP-transcript pairs (P ≤ 0.05) at PrCa risk loci. We characterized a common SNP (rs7247241) and its target gene (PPP1R14A) located in chr19q13, an sQTL with risk allele T associated with upregulation of long isoform (P = 9.99E−7). We confirmed the associations in both TCGA (P = 2.42E−24) and GTEX prostate cohorts (P = 9.08E−78). To functionally characterize this SNP, we performed chromatin immunoprecipitation qPCR and confirmed stronger CTCF and PLAGL2 binding in rs7247241 C than T allele. We found that CTCF binding enrichment was negatively associated with methylation level at the SNP site in human cell lines (r = −0.58). Bisulfite sequencing showed consistent association of rs7247241-T allele with nearby sequence CpG hypermethylation in prostate cell lines and tissues. Moreover, the methylation level at CpG sites nearest to the CTCF binding and first exon splice-in (ψ) of PPP1R14A was significantly associated with aggressive phenotype in the TCGA PrCa cohort. Meanwhile, the long isoform of the gene also promoted cell proliferation. Taken together, with the most updated gene annotations, we reported a set of sQTL associated with multiple traits related to human prostate diseases and revealed a unique role of PrCa risk SNP rs7247241 on PPP1R14A isoform transition.

Deregulations of DNA-methylation-related genes are common in cancers, but frameshift mutation status in colon cancer (CC) is unknown. Our study aims to assess whether CTCF, ZFP57, and ATF7IP genes in this category are mutated in CC. CTCF, ZFP57, and ATF7IP genes have repeat coding sequences, which are frequently deleted or duplicated in CC, harboring the phenotype of unstable or high microsatellite instability (MSI-H). We studied 140 CCs [95 MSI-H CCs and 45 stable MSI (MSS) CCs], and found 7 CCs with MSI-H (6/95: 6.3%) harbored frameshift mutations within the repeats, whereas those with MSS did not. Of note, the CTCF frameshift mutations showed the regional difference in the 2 (12.5%) of 16 MSI-H CCs, indicating there was intratumoral heterogeneity. In the immunohistochemistry for ATF7IP, the MSI-H CC showed low intensity compared to MSS CC. Together, CTCF, ZFP57, and ATF7IP genes, despite the low incidence of the mutations, are altered in several ways (mutation, expression, and intratumoral heterogeneity) and could contribute to MSI-H CC development.

Transcription is regulated and mediated by multiprotein complexes in a chromatin context. Transcription causes changes in DNA topology which is modulated by DNA topoisomerases, enzymes that catalyse changes in DNA topology via transient breaking and re-joining of one or both strands of the phosphodiester backbone. Mammals have six DNA topoisomerases, this review focuses on one, DNA topoisomerase II beta (TOP2B). In the absence of TOP2B transcription of many developmentally regulated genes is altered. Long genes seem particularly susceptible to the lack of TOP2B. Biochemical studies of the role of TOP2B in transcription regulated by ligands such as nuclear hormones, growth factors and insulin has revealed PARP1 associated with TOP2B and also PRKDC, XRCC5 and XRCC6. Analysis of publicly available databases of protein interactions confirms these interactions and illustrates interactions with other key transcriptional regulators including TRIM28. TOP2B has been shown to interact with proteins involved in chromosome organisation including CTCF and RAD21. Comparison of publicly available Chip-seq datasets reveals the location at which these proteins interact with genes. The availability of resources such as large datasets of protein–protein interactions, e.g. BioGrid and IntAct and protein–DNA interactions such as Chip-seq in GEO enables scientists to extend models and propose new hypotheses.

Background Cardiotoxicity is a common complication following anthracycline chemotherapy and represents one of the serious adverse reactions affecting life, which severely limits the effective use of anthracyclines in cancer therapy. Although some genes have been investigated by individual studies, the comprehensive analysis of key genes and molecular regulatory network in anthracyclines-induced cardiotoxicity (AIC) is lacking but urgently needed. Methods The present study integrating several transcription profiling datasets aimed to identify key genes associated with AIC by weighted correlation network analysis (WGCNA) and differentially expressed analysis (DEA) and also constructed miRNA-transcription factor-gene regulatory network. A total of three transcription profiling datasets involving 47 samples comprising 41 rat heart tissues and 6 human induced pluripotent stem cell-derived cardiomyocytes (hiPSCMs) samples were enrolled. Results The WGCNA and DEA with E-MTAB-1168 identified 14 common genes affected by doxorubicin administrated by 4 weeks or 6 weeks. Functional and signal enrichment analyses revealed that these genes were mainly enriched in the regulation of heart contraction, muscle contraction, heart process, and oxytocin signaling pathway. Ten (Ryr2, Casq1, Fcgr2b, Postn, Tceal5, Ccn2, Tnfrsf12a, Mybpc2, Ankrd23, Scn3b) of the 14 genes were verified by another gene expression profile GSE154603. Importantly, three key genes (Ryr2, Tnfrsf12a, Scn3b) were further validated in a hiPSCMs-based in-vitro model. Additionally, the miRNA-transcription factor-gene regulatory revealed several top-ranked transcription factors including Tcf12, Ctcf, Spdef, Ebf1, Sp1, Rcor1 and miRNAs including miR-124-3p, miR-195-5p, miR-146a-5p, miR-17-5p, miR-15b-5p, miR-424-5p which may be involved in the regulation of genes associated with AIC. Conclusions Collectively, the current study suggested the important role of the key genes, oxytocin signaling pathway, and the miRNA-transcription factor-gene regulatory network in elucidating the molecular mechanism of AIC.

ObjectiveIn a recent genome-wide association study, a significant genetic association between rs34330 of CDKN1B and risk of systemic lupus erythematosus (SLE) in Han Chinese was identified. This study was undertaken to validate the reported association and elucidate the biochemical mechanisms underlying the effect of the variant.MethodsWe performed an allelic association analysis in patients with SLE, followed by a meta-analysis assessing genome-wide association data across 11 independent cohorts (n = 28,872). In silico bioinformatics analysis and experimental validation in SLE-relevant cell lines were applied to determine the functional consequences of rs34330.ResultsWe replicated a genetic association between SLE and rs34330 (meta-analysis P = 5.29 × 10-22 , odds ratio 0.84 [95% confidence interval 0.81-0.87]). Follow-up bioinformatics and expression quantitative trait locus analysis suggested that rs34330 is located in active chromatin and potentially regulates several target genes. Using luciferase and chromatin immunoprecipitation-real-time quantitative polymerase chain reaction, we demonstrated substantial allele-specific promoter and enhancer activity, and allele-specific binding of 3 histone marks (H3K27ac, H3K4me3, and H3K4me1), RNA polymerase II (Pol II), CCCTC-binding factor, and a critical immune transcription factor (interferon regulatory factor 1 [IRF-1]). Chromosome conformation capture revealed long-range chromatin interactions between rs34330 and the promoters of neighboring genes APOLD1 and DDX47, and effects on CDKN1B and the other target genes were directly validated by clustered regularly interspaced short palindromic repeat (CRISPR)-based genome editing. Finally, CRISPR/dead CRISPR-associated protein 9-based epigenetic activation/silencing confirmed these results. Gene-edited cell lines also showed higher levels of proliferation and apoptosis.ConclusionCollectively, these findings suggest a mechanism whereby the rs34330 risk allele (C) influences the presence of histone marks, RNA Pol II, and IRF-1 transcription factor to regulate expression of several target genes linked to proliferation and apoptosis. This process could potentially underlie the association of rs34330 with SLE.

CCCTC-binding factor (CTCF) plays fundamental roles in transcriptional regulation and chromatin architecture maintenance. CTCF is also a tumour suppressor frequently mutated in cancer, however, the structural and functional impact of mutations have not been examined. We performed molecular and structural characterisation of five cancer-specific CTCF missense zinc finger (ZF) mutations occurring within key intra- and inter-ZF residues. Functional characterisation of CTCF ZF mutations revealed a complete (L309P, R339W, R377H) or intermediate (R339Q) abrogation as well as an enhancement (G420D) of the anti-proliferative effects of CTCF. DNA binding at select sites was disrupted and transcriptional regulatory activities abrogated. Molecular docking and molecular dynamics confirmed that mutations in residues specifically contacting DNA bases or backbone exhibited loss of DNA binding. However, R339Q and G420D were stabilised by the formation of new primary DNA bonds, contributing to gain-of-function. Our data confirm that a spectrum of loss-, change- and gain-of-function impacts on CTCF zinc fingers are observed in cell growth regulation and gene regulatory activities. Hence, diverse cellular phenotypes of mutant CTCF are clearly explained by examining structure–function relationships. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-021-03946-z.

ETO2 is a nuclear co-repressor, which plays a critical role in the regulation of the cell cycle, self-renewal capacity, and differentiation of hematopoietic progenitor cells. We identified novel fusion transcripts involving ETO2 and CTCF by RNA-seq in a multiple relapsed AML case. The CTCF-ETO2 and ETO2-CTCF chimeric genes were validated by RT-PCR and Sanger sequencing. In addition, both transcripts apparently promoted cell proliferation via JAK/STAT3 pathway that is sensitive to STAT3 inhibitors. The novel fusions may have prognostic value and pathogenic mechanisms in acute myeloid leukemia.

Binding sites of the chromatin regulator protein CTCF function as important landmarks in the human genome. The recently characterized CTCF-binding sites at LINE-1 repeats depend on another repeat-regulatory protein CGGBP1. These CGGBP1-dependent CTCF-binding sites serve as potential barrier elements for epigenetic marks such as H3K9me3. Such CTCF-binding sites are associated with asymmetric H3K9me3 levels as well as RNA levels in their flanks. The functions of these CGGBP1-dependent CTCF-binding sites remain unknown. By performing targeted studies on candidate CGGBP1-dependent CTCF-binding sites cloned in an SV40 promoter-enhancer episomal system we show that these regions act as inhibitors of ectopic transcription from the SV40 promoter. CGGBP1-dependent CTCF-binding sites that recapitulate their genomic function of loss of CTCF binding upon CGGBP1 depletion and H3K9me3 asymmetry in immediate flanks are also the ones that show the strongest inhibition of ectopic transcription. By performing a series of strand-specific reverse transcription PCRs we demonstrate that this ectopic transcription results in the synthesis of RNA from the SV40 promoter in a direction opposite to the downstream reporter gene in a strand-specific manner. The unleashing of the bidirectionality of the SV40 promoter activity and a breach of the transcription barrier seems to depend on depletion of CGGBP1 and loss of CTCF binding proximal to the SV40 promoter. RNA-sequencing reveals that CGGBP1-regulated CTCF-binding sites act as barriers to transcription at multiple locations genome-wide. These findings suggest a role of CGGBP1-dependent binding sites in restricting ectopic transcription.

BackgroundThe transcription factor CTCF appears indispensable in defining topologically associated domain boundaries and maintaining chromatin loop structures within these domains, supported by numerous functional studies. However, acute depletion of CTCF globally reduces chromatin interactions but does not significantly alter transcription.ResultsHere, we systematically integrate multi-omics data including ATAC-seq, RNA-seq, WGBS, Hi-C, Cut&Run, and CRISPR-Cas9 survival dropout screens, and time-solved deep proteomic and phosphoproteomic analyses in cells carrying auxin-induced degron at endogenous CTCF locus. Acute CTCF protein degradation markedly rewires genome-wide chromatin accessibility. Increased accessible chromatin regions are frequently located adjacent to CTCF-binding sites at promoter regions and insulator sites associated with enhanced transcription of nearby genes. In addition, we use CTCF-associated multi-omics data to establish a combinatorial data analysis pipeline to discover CTCF co-regulatory partners. We successfully identify 40 candidates, including multiple established partners. Interestingly, many CTCF co-regulators that have alterations of their respective downstream gene expression do not show changes of their own expression levels across the multi-omics measurements upon acute CTCF loss, highlighting the strength of our system to discover hidden co-regulatory partners associated with CTCF-mediated transcription.ConclusionsThis study highlights that CTCF loss rewires genome-wide chromatin accessibility, which plays a critical role in transcriptional regulation.

Chromatin interactions play important roles in regulating gene expression. However, the availability of genome-wide chromatin interaction data is limited. We develop a computational method, chromatin interaction neural network (ChINN), to predict chromatin interactions between open chromatin regions using only DNA sequences. ChINN predicts CTCF- and RNA polymerase II-associated and Hi-C chromatin interactions. ChINN shows good across-sample performances and captures various sequence features for chromatin interaction prediction. We apply ChINN to 6 chronic lymphocytic leukemia (CLL) patient samples and a published cohort of 84 CLL open chromatin samples. Our results demonstrate extensive heterogeneity in chromatin interactions among CLL patient samples. Ministry of Education (MOE) National Research Foundation (NRF) Published version This research is supported by the National Research Foundation (NRF) Singapore through an NRF Fellowship awarded to M.J.F (NRF-NRFF2012-054) and NTU start-up funds awarded to M.J.F. This research is supported by the RNA Biology Center at the Cancer Science Institute of Singapore, NUS, as part of funding under the Singapore Ministry of Education Academic Research Fund Tier 3 awarded to Daniel Tenen as lead PI with M.J.F as co-investigator (MOE2014-T3-1-006). This research is supported by a National Research Foundation Competitive Research Programme grant awarded to V.T. as lead PI and M.J.F. as co-PI (NRF-CRP17-2017-02). This research is supported by the National Research Foundation Singapore and the Singapore Ministry of Education under its Research Centres of Excellence initiative. This research is supported by a Ministry of Education Tier II grant awarded to M.J.F (T2EP30120-0020).

Cohesin is a key organizer of chromatin folding in eukaryotic cells. The two main activities of this ring-shaped protein complex are the maintenance of sister chromatid cohesion and the establishment of long-range DNA–DNA interactions through the process of loop extrusion. Although the basic principles of both cohesion and loop extrusion have been described, we still do not understand several crucial mechanistic details. One of such unresolved issues is the question of whether a cohesin ring topologically embraces DNA string(s) during loop extrusion. Here, we show that cohesin complexes residing on CTCF-occupied genomic sites in mammalian cells do not interact with DNA topologically. We assessed the stability of cohesin-dependent loops and cohesin association with chromatin in high-ionic-strength conditions in G1-synchronized HeLa cells. We found that increased salt concentration completely displaces cohesin from those genomic regions that correspond to CTCF-defined loop anchors. Unsurprisingly, CTCF-anchored cohesin loops also dissipate in these conditions. Because topologically engaged cohesin is considered to be salt resistant, our data corroborate a non-topological model of loop extrusion. We also propose a model of cohesin activity throughout the interphase, which essentially equates the termination of non-topological loop extrusion with topological loading of cohesin. This theoretical framework enables a parsimonious explanation of various seemingly contradictory experimental findings. Supplementary Information The online version contains supplementary material available at 10.1186/s13072-021-00411-w.

Three decades of research have established the CCCTC-binding factor (CTCF) as a ubiquitously expressed chromatin organizing factor and master regulator of gene expression. A new role for CTCF as a regulator of alternative splicing (AS) has now emerged. CTCF has been directly and indirectly linked to the modulation of AS at the individual transcript and at the transcriptome-wide level. The emerging role of CTCF-mediated regulation of AS involves diverse mechanisms; including transcriptional elongation, DNA methylation, chromatin architecture, histone modifications, and regulation of splicing factor expression and assembly. CTCF thereby appears to not only co-ordinate gene expression regulation but contributes to the modulation of transcriptomic complexity. In this review, we highlight previous discoveries regarding the role of CTCF in AS. In addition, we summarize detailed mechanisms by which CTCF mediates AS regulation. We propose opportunities for further research designed to examine the possible fate of CTCF-mediated alternatively spliced genes and associated biological consequences. CTCF has been widely acknowledged as the ‘master weaver of the genome’. Given its multiple connections, further characterization of CTCF’s emerging role in splicing regulation might extend its functional repertoire towards a ‘conductor of the splicing orchestra’.

DNA methylation, chromatin-binding proteins, and DNA looping are common components regulating genomic imprinting which leads to parent-specific monoallelic gene expression. Loss of methylation (LOM) at the human imprinting center 2 (IC2) on chromosome 11p15 is the most common cause of the imprinting overgrowth disorder Beckwith-Wiedemann Syndrome (BWS). Here, we report a familial transmission of a 7.6 kB deletion that ablates the core promoter of KCNQ1. This structural alteration leads to IC2 LOM and causes recurrent BWS. We find that occupancy of the chromatin organizer CTCF is disrupted proximal to the deletion, which causes chromatin architecture changes both in cis and in trans. We also profile the chromatin architecture of IC2 in patients with sporadic BWS caused by isolated LOM to identify conserved features of IC2 regulatory disruption. A strong interaction between CTCF sites around KCNQ1 and CDKN1C likely drive their expression on the maternal allele, while a weaker interaction involving the imprinting control region element may impede this connection and mediate gene silencing on the paternal allele. We present an imprinting model in which KCNQ1 transcription is necessary for appropriate CTCF binding and a novel chromatin conformation to drive allele-specific gene expression.

Studies of nuclear architecture using chromosome conformation capture methods have provided a detailed view of how chromatin folds in the 3D nuclear space. New variants of this technology now afford unprecedented resolution and allow the identification of ever smaller folding domains that offer new insights into the mechanisms by which this organization is established and maintained. Here we review recent results in this rapidly evolving field with an emphasis on CTCF function, with the goal of gaining a mechanistic understanding of the principles by which chromatin is folded in the eukaryotic nucleus.

While constitutive CCCTC-binding factor (CTCF)–binding sites are needed to maintain relatively invariant chromatin structures, such as topologically associating domains, the precise roles of CTCF to control cell-type–specific transcriptional regulation remain poorly explored. We examined CTCF occupancy in different types of primary blood cells derived from the same donor to elucidate a new role for CTCF in gene regulation during blood cell development. We identified dynamic, cell-type–specific binding sites for CTCF that colocalize with lineage-specific transcription factors. These dynamic sites are enriched for single-nucleotide polymorphisms that are associated with blood cell traits in different linages, and they coincide with the key regulatory elements governing hematopoiesis. CRISPR-Cas9–based perturbation experiments demonstrated that these dynamic CTCF-binding sites play a critical role in red blood cell development. Furthermore, precise deletion of CTCF-binding motifs in dynamic sites abolished interactions of erythroid genes, such as RBM38, with their associated enhancers and led to abnormal erythropoiesis. These results suggest a novel, cell-type–specific function for CTCF in which it may serve to facilitate interaction of distal regulatory emblements with target promoters. Our study of the dynamic, cell-type–specific binding and function of CTCF provides new insights into transcriptional regulation during hematopoiesis.

Numerous studies of relationship between epigenomic features have focused on their strong correlation across the genome, likely because such relationship can be easily identified by many established methods for correlation analysis. However, two features with little correlation may still colocalize at many genomic sites to implement important functions. There is no bioinformatic tool for researchers to specifically identify such feature pairs. Here, we develop a method to identify feature pairs in which two features have maximal colocalization minimal correlation (MACMIC) across the genome. By MACMIC analysis of 3306 feature pairs in 16 human cell types, we reveal a dual role of CCCTC-binding factor (CTCF) in epigenetic regulation of cell identity genes. Although super-enhancers are associated with activation of target genes, only a subset of super-enhancers colocalized with CTCF regulate cell identity genes. At super-enhancers colocalized with CTCF, CTCF is required for the active marker H3K27ac in cell types requiring the activation, and also required for the repressive marker H3K27me3 in other cell types requiring repression. Our work demonstrates the biological utility of the MACMIC analysis and reveals a key role for CTCF in epigenetic regulation of cell identity. The code for MACMIC is available at https://github.com/bxia888/MACMIC.

Switch/sucrose-nonfermentable (SWI/SNF) chromatin-remodeling complexes are critical regulators of chromatin dynamics during transcription, DNA replication, and DNA repair. A recently identified SWI/SNF subcomplex termed GLTSCR1/1L-BAF (GBAF; or “noncanonical BAF”, ncBAF) uniquely contains bromodomain-containing protein BRD9 and glioma tumor suppressor candidate region 1 (GLTSCR1) or its paralog GLTSCR1-like (GLTSCR1L). Recent studies have identified a unique dependency on GBAF (ncBAF) complexes in synovial sarcoma and malignant rhabdoid tumors, both of which possess aberrations in canonical BAF (cBAF) and Polybromo-BAF (PBAF) complexes. Dependencies on GBAF in malignancies without SWI/SNF aberrations, however, are less defined. Here, we show that GBAF, particularly its BRD9 subunit, is required for the viability of prostate cancer cell lines in vitro and for optimal xenograft tumor growth in vivo. BRD9 interacts with androgen receptor (AR) and CCCTC-binding factor (CTCF), and modulates AR-dependent gene expression. The GBAF complex exhibits overlapping genome localization and transcriptional targets as bromodomain and extraterminal domain–containing (BET) proteins, which are established AR coregulators. Our results demonstrate that GBAF is critical for coordinating SWI/SNF–BET cooperation and uncover a new druggable target for AR-positive prostate cancers, including those resistant to androgen deprivation or antiandrogen therapies. Significance: Advanced prostate cancers resistant to androgen receptor antagonists are still susceptible to nontoxic BRD9 inhibitors, making them a promising alternative for halting AR signaling in progressed disease.

The candidate anticancer drug curaxins can insert into DNA base pairs and efficiently inhibit the growth of various cancers. However, how curaxins alter the genomic DNA structure and affect the DNA binding property of key proteins remains to be clarified. Here, we first showed that curaxin CBL0137 strongly stabilizes the interaction between the double strands of DNA and reduces DNA bending and twist rigidity simultaneously, by single-molecule magnetic tweezers. More importantly, we found that CBL0137 greatly impairs the binding of CTCF but facilitates trapping FACT on DNA. We revealed that CBL0137 clamps the DNA double helix that may induce a huge barrier for DNA unzipping during replication and transcription and causes the distinct binding response of CTCF and FACT on DNA. Our work provides a novel mechanical insight into CBL0137's anticancer mechanisms at the nucleic acid level.

Recent technological advancements in the field of chromatin biology have rewritten the textbook on nuclear organization. We now appreciate that the folding of chromatin in the three-dimensional space (i.e. its 3D "architecture") is non-random, hierarchical, and highly complex. While 3D chromatin structure is partially encoded in the primary sequence and thereby broadly conserved across cell types and states, a substantial portion of the genome seems to be dynamic during development or in disease. Moreover, there is growing evidence that at least some of the 3D structure of chromatin is functionally linked to gene regulation, both being modulated by and impacting on multiple nuclear processes (including DNA replication, transcription, and RNA splicing). In recent years, these new concepts have nourished several investigations about the functional role of 3D chromatin topology dynamics in the heart during development and disease. This review aims to provide a comprehensive overview of our current understanding in this field, and to discuss how this knowledge can inform further research as well as clinical practice.

The three-dimensional (3D) organization of the human genome is of crucial importance for gene regulation, and the CCCTC-binding factor (CTCF) plays an important role in chromatin interactions. However, it is still unclear what sequence patterns in addition to CTCF motif pairs determine chromatin loop formation. To discover the underlying sequence patterns, we have developed a deep learning model, called DeepCTCFLoop, to predict whether a chromatin loop can be formed between a pair of convergent or tandem CTCF motifs using only the DNA sequences of the motifs and their flanking regions. Our results suggest that DeepCTCFLoop can accurately distinguish the CTCF motif pairs forming chromatin loops from the ones not forming loops. It significantly outperforms CTCF-MP, a machine learning model based on word2vec and boosted trees, when using DNA sequences only. Furthermore, we show that DNA motifs binding to several transcription factors, including ZNF384, ZNF263, ASCL1, SP1, and ZEB1, may constitute the complex sequence patterns for CTCF-mediated chromatin loop formation. DeepCTCFLoop has also been applied to disease-associated sequence variants to identify candidates that may disrupt chromatin loop formation. Therefore, our results provide useful information for understanding the mechanism of 3D genome organization and may also help annotate and prioritize the noncoding sequence variants associated with human diseases.

During human spermatogenesis, germ cells undergo dynamic changes in chromatin organization/re-packaging and in transcriptomes. In order to better understand the underlying mechanism(s), scATAC-Seq of 5376 testicular cells from 3 normal men were performed. Data were analyzed in parallel with the scRNA-Seq data of human testicular cells. In all, 10 germ cell types associated with spermatogenesis and 6 testicular somatic cell types were identified, along with 142 024 peaks located in promoter, genebody and CpG Island. We had examined chromatin accessibility of all chromosomes, with chromosomes 19 and 17 emerged as the leading chromosomes that displayed high chromatin accessibility. In accessible chromatin regions, transcription factor-binding sites were identified and specific motifs with high frequencies at different spermatogenesis stages were detected, including CTCF, BORIS, NFY, DMRT6, EN1, ISL1 and GLI3. Two most remarkable observations were noted. First, TLE3 was specifically expressed in differentiating spermatogonia. Second, PFN4 was found to be involved in actin cytoskeletal organization during meiosis. More important, unique regions upstream of PFN4 and TLE3 were shown to display high accessibility, illustrating their significance in supporting human spermatogenesis.

Tumor metastasis remains an obstacle in cancer treatment and is responsible for most cancer-related deaths. Nm23-H1 is one of the first metastasis suppressor proteins discovered with the ability to inhibit metastasis of many cancers including breast, colon, and liver cancer. Although loss of Nm23-H1 is observed in aggressive cancers and correlated with metastatic potential, little is known regarding the mechanisms that regulate its cellular level. Here, we examined the mechanisms that control Nm23-H1 expression in breast cancer cells. Initial studies in aggressive MDA-MB-231 cells (expressing low Nm23-H1) and less invasive MCF-7 cells (expressing high Nm23-H1) revealed that mRNA levels correlated with protein expression, suggesting that transcriptional mechanisms may control Nm23-H1 expression. Truncational analysis of the Nm23-H1 promoter revealed a proximal and minimal promoter that harbor putative binding sites for transcription factors including CTCF and EGR1. CTCF and EGR1 induced Nm23-H1 expression and reduced cell migration of MDA-MB-231 cells. Moreover, CTCF and EGR1 were recruited to the Nm23-H1 promoter in MCF-7 cells and their expression correlated with Nm23-H1 levels. This study indicates that loss of Nm23-H1 in aggressive breast cancer is apparently caused by downregulation of CTCF and EGR1, which potentially drive Nm23-H1 expression to promote a less invasive phenotype.

The human genome is partitioned into a collection of genomic features, inclusive of genes, transposable elements, lamina interacting regions, early replicating control elements and cis-regulatory elements, such as promoters, enhancers, and anchors of chromatin interactions. Uneven distribution of these features within chromosomes gives rise to clusters, such as topologically associating domains (TADs), lamina-associated domains, clusters of cis-regulatory elements or large organized chromatin lysine (K) domains (LOCKs). Here we show that LOCKs from diverse histone modifications discriminate primitive from differentiated cell types. Active LOCKs (H3K4me1, H3K4me3 and H3K27ac) cover a higher fraction of the genome in primitive compared to differentiated cell types while repressive LOCKs (H3K9me3, H3K27me3 and H3K36me3) do not. Active LOCKs in differentiated cells lie proximal to highly expressed genes while active LOCKs in primitive cells tend to be bivalent. Genes proximal to bivalent LOCKs are minimally expressed in primitive cells. Furthermore, bivalent LOCKs populate TAD boundaries and are preferentially bound by regulators of chromatin interactions, including CTCF, RAD21 and ZNF143. Together, our results argue that LOCKs discriminate primitive from differentiated cell populations., Large organized chromatin lysine (K) domains (LOCKs) are a genomic feature derived from the cluster of modified nucleosomes. Here the authors provide evidence that LOCKs discriminate primitive from differentiated cell populations and that they relate to transcription regulating events.

Estrogen receptor alpha (ER/ESR1) is frequently mutated in endocrine resistant ER-positive (ER+) breast cancer and linked to ligand-independent growth and metastasis. Despite the distinct clinical features of ESR1 mutations, their role in intrinsic subtype switching remains largely unknown. Here we find that ESR1 mutant cells and clinical samples show a significant enrichment of basal subtype markers, and six basal cytokeratins (BCKs) are the most enriched genes. Induction of BCKs is independent of ER binding and instead associated with chromatin reprogramming centered around a progesterone receptor-orchestrated insulated neighborhood. BCK-high ER+ primary breast tumors exhibit a number of enriched immune pathways, shared with ESR1 mutant tumors. S100A8 and S100A9 are among the most induced immune mediators and involve in tumor-stroma paracrine crosstalk inferred by single-cell RNA-seq from metastatic tumors. Collectively, these observations demonstrate that ESR1 mutant tumors gain basal features associated with increased immune activation, encouraging additional studies of immune therapeutic vulnerabilities.

Genome-wide association studies have identified 3p21.31 as the main risk locus for severe COVID-19, although underlying mechanisms remain elusive. We perform an epigenomic dissection of 3p21.31, identifying a CTCF-dependent tissue-specific 3D regulatory chromatin hub that controls the activity of several chemokine receptor genes. Risk SNPs colocalize with regulatory elements and are linked to increased expression of CCR1, CCR2 and CCR5 in monocytes and macrophages. As excessive organ infiltration of inflammatory monocytes and macrophages is a hallmark of severe COVID-19, our findings provide a rationale for the genetic association of 3p21.31 variants with elevated risk of hospitalization upon SARS-CoV-2 infection. B.S. and R.W.H. are supported by Dutch Lung Foundation grant 4.1.18.226. G.S. was supported by the ‘Fundación Científica de la Asociación Española Contra el Cáncer’. R.S. is supported by an Erasmus MC Fellowship, a Dutch Lung Foundation Junior Investigator grant (4.2.19.041JO) and a VIDI grant (09150172010068) from the Dutch Research Council (NWO). A.F.v.O. was supported by a postdoctoral fellowship from the Foundation Recherche Médicale. S.S. was supported by an Agence National pour la Recherche” grant (ANR-18-CE12-0019)

Eukaryotic genomes are structurally organized via the formation of multiple loops that create gene expression regulatory units called topologically associating domains (TADs). Here we revealed the KSHV TAD structure at 500 base pair resolution and constructed a 3D KSHV genomic structural model with 2kb binning. The latent KSHV genome formed very similar genomic architectures in three different naturally infected PEL cell lines and in an experimentally infected epithelial cell line. The majority of the TAD boundaries were occupied by CTCF and SMC1, and the KSHV transactivator was recruited to these sites during reactivation. Triggering KSHV gene expression decreased pre-wired genomic loops within the regulatory unit, while contacts extending outside of regulatory borders increased, leading to formation of a larger regulatory unit with a shift from repressive to active compartments (B to A). The 3D genomic structural model proposes that the immediate-early promoter region is localized on the periphery of the 3D viral genome during latency, while highly inducible non-coding RNA regions moved toward the inner space of the structure, resembling the configuration of a “bird cage” during reactivation. The compartment-like properties of viral episomal chromatin structure and its reorganization during the transition from latency may help coordinate viral gene transcription.ImportanceThe 3D architecture of chromatin allows for efficient arrangement, expression, and replication of genetic material. The genomes of all organisms studied to date have been found to be organized through some form of tiered domain structures. However, the architectural framework of the genomes of large double-stranded DNA viruses such as the herpesvirus family has not been reported. Prior studies with Kaposi’s sarcoma-associated herpesvirus (KSHV) have indicated that the viral chromatin shares many biological properties exhibited by the host cell genome, essentially behaving as a mini human chromosome. Thus, we hypothesized that the KSHV genome may be organized in a similar manner. In this report, we describe the domain structure of the latent and lytic KSHV genome at 500 base pair resolution and present a 3D genomic structural model for KSHV under each condition. These results add new insights into the complex regulation of the viral lifecycle.

Gene transcription is regulated by distant regulatory elements via combinatorial binding of transcription factors. It is increasingly recognized that alterations in chromatin state and transcription factor binding in these distant regulatory elements may have key roles in cancer development. Here we focused on the first stages of oncogene-induced carcinogenic transformation, and characterized the regulatory network underlying transcriptional changes associated with this process. Using Hi-C data, we observe spatial coupling between differentially expressed genes and their differentially accessible regulatory elements and reveal two candidate transcription factors, p53 and CTCF, as determinants of transcriptional alterations at the early stages of oncogenic HRas-induced transformation in human mammary epithelial cells. Strikingly, the malignant transcriptional reprograming is promoted by redistribution of chromatin binding of these factors without major variation in their expression level. Our results demonstrate that alterations in the regulatory landscape have a major role in driving oncogene-induced transcriptional reprogramming., By integrating transcriptome, regulatory element and chromosome topology profiles, Schwartz et al identify redistribution of p53 and CTCF genomic binding to be associated with transcriptional and phenotypical changes during Ras-induced transformation of mammary epithelial cells.

Testicular germ cell tumours (TGCTs) respond well to cisplatin-based therapy. However, cisplatin resistance and poor outcomes do occur. It has been suggested that a shift towards DNA hypermethylation mediates cisplatin resistance in TGCT cells, although there is little direct evidence to support this claim. Here we utilized a series of isogenic cisplatin-resistant cell models and observed a strong association between cisplatin resistance in TGCT cells and a net increase in global CpG and non-CpG DNA methylation spanning regulatory, intergenic, genic and repeat elements. Hypermethylated loci were significantly enriched for repressive DNA segments, CTCF and RAD21 sites and lamina associated domains, suggesting that global nuclear reorganization of chromatin structure occurred in resistant cells. Hypomethylated CpG loci were significantly enriched for EZH2 and SUZ12 binding and H3K27me3 sites. Integrative transcriptome and methylome analyses showed a strong negative correlation between gene promoter and CpG island methylation and gene expression in resistant cells and a weaker positive correlation between gene body methylation and gene expression. A bidirectional shift between gene promoter and gene body DNA methylation occurred within multiple genes that was associated with upregulation of polycomb targets and downregulation of tumour suppressor genes. These data support the hypothesis that global remodelling of DNA methylation is a key factor in mediating cisplatin hypersensitivity and chemoresistance of TGCTs and furthers the rationale for hypomethylation therapy for refractory TGCT patients.

Both HOX gene expression and CTCF regulation have been well demonstrated to play a critical role in regulating maintenance of leukemic stem cells (LSCs) that are known to be resistant to BET inhibitor (BETi). To investigate the regulatory role of CTCF boundary in aberrant HOX gene expression and the therapeutic sensitivity of BETi in AML, we employed CRISPR-Cas9 genome editing technology to delete 47 base pairs of the CTCF binding motif which is located between HOXA7 and HOXA9 genes (CBS7/9) in different subtypes of AML with either MLL-rearrangement or NPM1 mutation. Our results revealed that HOXA9 is significantly downregulated in response to the CBS7/9 deletion. Moreover, CBS7/9 boundary deletion sensitized the BETi treatment reaction in both MOLM-13 and OCI-AML3 cells. To further examine whether BETi therapeutic sensitivity in AML is depended on the expression level of the HOXA9 gene, we overexpressed the HOXA9 in the CBS7/9 deleted AML cell lines, which can rescue and restore the resistance to BETi treatment of the CBS7/9 KO cells by activating MAPK signaling pathway. Deletion of CBS7/9 specifically decreased the recruitment of BRD4 and RNA pol II to the posterior HOXA genes, in which, a transcription elongation factor ELL3 is the key factor in regulating HOXA gene transcription monitored by CBS7/9 chromatin boundary. Thus, disruption of CBS7/9 boundary perturbs HOXA9 transcription and regulates BETi sensitivity in AML treatment. Moreover, alteration of CTCF boundaries in the oncogene loci may provide a novel strategy to overcome the drug resistance of LSCs. Graphical abstract.

CCCTC-binding factor (CTCF) sites interact with each other in the chromatin environment, establishing chromatin domains. Our previous study showed that interaction between CTCF sites is cell type-specific around the β-globin locus and is dependent on erythroid-specific activator GATA-1. To find out molecular mechanisms of the cell type-specific interaction, we directly inhibited GATA-1 binding to the β-globin enhancers by deleting its binding motifs and found that histone H3K27 acetylation (H3K27ac) was decreased at CTCF sites surrounding the β-globin locus, even though CTCF binding itself was maintained at the sites. Forced H3K27ac by Trichostatin A treatment or CBP/p300 KD affected the interactions between CTCF sites around the β-globin locus without changes in CTCF binding. Analysis of public ChIA-PET data revealed that H3K27ac is higher at CTCF sites forming short interactions than long interactions. GATA-1 was identified as a representative transcription factor that relates with genes present inside the short interactions in erythroid K562 cells. Depletion of GATA-1-reduced H3K27ac at CTCF sites near erythroid-specific enhancers. These results indicate that H3K27ac at CTCF sites is required for cell type-specific chromatin interactions between them. Tissue-specific activator GATA-1 appears to play a role in H3K27ac at CTCF sites in erythroid cells.

Background: The cohesin complex plays an essential role in genome organisation and cell division. A full complement of the cohesin complex and its regulators is important for normal development, since heterozygous mutations in genes encoding these components can be sufficient to produce a disease phenotype. The implication that genes encoding the cohesin subunits or cohesin regulators must be tightly controlled and resistant to variability in expression has not yet been formally tested.Methods: Here, we identify spatial-regulatory connections with potential to regulate expression of cohesin loci (Mitotic: SMC1A, SMC3, STAG1, STAG2, RAD21/RAD21-AS; Meiotic: SMC1B, STAG3, REC8, RAD21L1), cohesin-ring support genes (NIPBL, MAU2, WAPL, PDS5A, PDS5B) and CTCF, including linking their expression to that of other genes. We searched the genome-wide association studies (GWAS) catalogue for SNPs mapped or attributed to cohesin genes by GWAS (GWAS-attributed) and the GTEx catalogue for SNPs mapped to cohesin genes by cis-regulatory variants in one or more of 44 tissues across the human body (expression quantitative trail locus-attributed).Results: Connections that centre on the cohesin ring subunits provide evidence of coordinated regulation that has little tolerance for perturbation. We used the CoDeS3D SNP-gene attribution methodology to identify transcriptional changes across a set of genes coregulated with the cohesin loci that include biological pathways such as extracellular matrix production and proteasome-mediated protein degradation. Remarkably, many of the genes that are coregulated with cohesin loci are themselves intolerant to loss-of-function.Conclusions: The results highlight the importance of robust regulation of cohesin genes and implicate novel pathways that may be important in the human cohesinopathy disorders.

CTCF plays a pivotal role in mediating chromatin interactions, but it does not do so alone. A number of factors have been reported to co-localize with CTCF and regulate CTCF loops, but no comprehensive analysis of binding partners has been performed. This prompted us to identify CTCF loop participants and regulators by co-localization analysis with CTCF. We screened all factors that had ChIP-seq data in humans by co-localization analysis with human super conserved CTCF (hscCTCF) binding sites, and identified many new factors that overlapped with hscCTCF binding sites. Combined with CTCF loop information, we observed that clustered factors could promote CTCF loops. After in-depth mining of each factor, we found that many factors might have the potential to promote CTCF loops. Our data further demonstrated that BHLHE40 affected CTCF loops by regulating CTCF binding. Together, this study revealed that many factors have the potential to participate in or regulate CTCF loops, and discovered a new role for BHLHE40 in modulating CTCF loop formation.

Colorectal cancer (CRC) mostly arises from progressive accumulation of somatic mutations within cells. Most commonly mutated genes like TP53, APC and KRAS can promote survival and proliferation of cancer cells. Although the molecular alterations and landscape of some specific mutations in CRC are well known, the presence of a somatic mutation signature related to genomic regions and epigenetic markers remain unclear. To find the signatures from a random distribution of somatic mutations in CRCs, we carried out enrichment analysis in different genomic regions and identified peaks of epigenetic markers. We validated that the mutation frequency in miRNA is dramatically higher than in flanking genomic regions. Moreover, we observed that somatic mutations in CRC and colon cancer cell lines are significantly enriched in CTCF binding sites. We also found these mutations are enriched for H3K27me3 in both normal sigmoid colon and colon cancer cell lines. Taken together, our findings suggest that there are some common somatic mutations signatures which provide new directions to study CRC.

Diabetic kidney disease (DKD) is a major complication of diabetes and the leading cause of end-stage renal failure. Epigenetics has been associated with metabolic memory in which prior periods of hyperglycemia enhance the future risk of developing DKD despite subsequent glycemic control. To understand the mechanistic role of such epigenetic memory in human DKD and to identify new therapeutic targets, we profiled gene expression, DNA methylation, and chromatin accessibility in kidney proximal tubule epithelial cells (PTECs) derived from subjects with and without type 2 diabetes (T2D). T2D-PTECs displayed persistent gene expression and epigenetic changes with and without transforming growth factor-β1 treatment, even after culturing in vitro under similar conditions as nondiabetic PTECs, signified by deregulation of fibrotic and transport-associated genes (TAGs). Motif analysis of differential DNA methylation and chromatin accessibility regions associated with genes differentially regulated in T2D revealed enrichment for SMAD3, HNF4A, and CTCF transcription factor binding sites. Furthermore, the downregulation of several TAGs in T2D (including CLDN10, CLDN14, CLDN16, SLC16A2, and SLC16A5) was associated with promoter hypermethylation, decreased chromatin accessibility, and reduced enrichment of HNF4A, histone H3-lysine-27-acetylation, and CTCF. Together, these integrative analyses reveal epigenetic memory underlying the deregulation of key target genes in T2D-PTECs that may contribute to sustained renal dysfunction in DKD.

The tumor microenvironment (TME) plays critical roles in tumor growth and progression, however key regulators of gene expression in the TME of cutaneous malignant peripheral nerve sheath tumor (C-MPNST) and spindle cell melanoma (SCM) have not been well elucidated. Herein, we investigate the epigenetic regulation of promoters and gene bodies and their effect on the TME composition of C-MPNSTs and SCMs. A cohort of 30 patients was analyzed using differential gene expression (DGE) and gene set enrichment analysis (GSEA) using the Nanostring platform. Methylation analysis was carried out utilizing an Infinium Methylation EPIC array targeting 866,562 methylation site (CpG) islands. DGE revealed overexpression of genes related to mast cells in the TME of SCMs, and a predominance of exhausted CD8+ T cells and macrophages in the TME of C-MPNSTs. Interestingly, we further observed promoter hypermethylation in key overexpressed genes and corresponding gene body hypomethylation. Analysis using ENCODE ChIP-sequencing data identified CTCF as the common transcription factor at the site of the hypomethylated probe. These findings support that the TME composition of C-MPNSTs and SCMs is at least partially independent on promoter methylation status, suggesting a possible relationship between gene body enhancers and expression of key TME genes in both entities.

Background The three-dimensional organization of the genome in the nucleus plays an integral role in many biological processes, including gene expression. The genome is folded into DNA loops that bring together distal regulatory elements and genes. Cohesin, a ring-shaped protein complex, is a major player in the formation of DNA loops. Cohesin is composed of a core trimer and one of two variant STAG subunits, STAG1 or STAG2. It is not understood whether variant STAG proteins give rise to cohesin complexes with distinct functions. Recent studies have begun to characterize the roles of STAG1 and STAG2, with partially contradictory results. Results Here, we generate stable single-knockout embryonic stem cell lines to investigate the individual contributions of STAG1 and STAG2 in regulating cohesin chromosomal localization and function. We report both overlapping roles for STAG1 and STAG2 in cohesin localization and somewhat distinct roles in gene expression. STAG1 and STAG2 occupy the same sites across the genome, yet do not exist together in a higher order complex. Despite their shared localization, STAG1 and STAG2 have both distinct and redundant effects on gene expression. Loss of both STAG1 and STAG2 causes widespread transcriptome dysregulation, altered cohesin DNA occupancy, and reduced cell proliferation. Conclusions Together, this work reveals the requirement of at least one STAG protein for proper cohesin function. STAG1 and STAG2 have independent roles in cohesin localization and both overlapping and distinct roles in gene expression. The roles of STAG1 and STAG2 in mouse embryonic stem cells may be somewhat different than in other cell types, due to their relative expression levels. These results advance our understanding of the link between mammalian genome organization and gene expression during development and disease contexts.

The RAG endonuclease initiates Igh locus V(D)J recombination in progenitor (pro)-B cells1. Upon binding a recombination centre-based JH, RAG scans upstream chromatin via loop extrusion, potentially mediated by cohesin, to locate Ds and assemble a DJH-based recombination centre2. CTCF looping factor-bound elements (CBEs) within IGCR1 upstream of Ds impede RAG scanning3–5; however, their inactivation allows scanning to proximal VHs, where additional CBEs activate rearrangement and impede scanning any further upstream5. Distal VH utilization is thought to involve diffusional access to the recombination centre following large-scale Igh locus contraction6–8. Here we test the potential of linear RAG scanning to mediate distal VH usage in G1-arrested v-Abl pro-B cell lines9, which undergo robust D-to-JH but little VH-to-DJH rearrangements, presumably owing to lack of locus contraction2,5. Through an auxin-inducible approach10, we degraded the cohesin component RAD2110–12 or CTCF12,13 in these G1-arrested lines. Degradation of RAD21 eliminated all V(D)J recombination and interactions associated with RAG scanning, except for reecombination centre-located DQ52-to-JH joining, in which synapsis occurs by diffusion2. Remarkably, while degradation of CTCF suppressed most CBE-based chromatin interactions, it promoted robust recombination centre interactions with, and robust VH-to-DJH joining of, distal VHs, with patterns similar to those of ‘locus-contracted’ primary pro-B cells. Thus, downmodulation of CTCF-bound scanning-impediment activity promotes cohesin-driven RAG scanning across the 2.7-Mb Igh locus. Using an auxin-inducible approach, the authors show that downmodulation of CTCF activity promotes cohesin-driven RAG endonuclease scanning, and thus V(D)J recombination, across the Igh locus.