Glucocorticoid receptor and nuclear factor kappa-b affect three-dimensional chromatin organization

Long-range chromatin interactions are important for transcriptional regulation of genes, many of which are related to complex agronomics traits. However, the pattern of three-dimensional chromatin interactions remains unclear in plants. Here we report the generation of chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) data and the construction of extensive H3K4me3- and H3K27ac-centered chromatin interaction maps in maize. Results show that the interacting patterns between proximal and distal regulatory regions of genes are highly complex and dynamic. Genes with chromatin interactions have higher expression levels than those without interactions. Genes with proximal-proximal interactions prefer to be transcriptionally coordinated. Tissue-specific proximal–distal interactions are associated with tissue-specific expression of genes. Interactions between proximal and distal regulatory regions further interweave into organized network communities that are enriched in specific biological functions. The high-resolution chromatin interaction maps will help to understand the transcription regulation of genes associated with complex agronomic traits of maize.

Oestrogen receptor α (ERα) is key player in the progression of breast cancer. Recently, the cistrome and interactome of ERα were mapped in breast cancer cells, revealing the importance of spatial organization in oestrogen-mediated transcription. However, the underlying mechanism of this process is unclear. Here, we show that ERα binding sites (ERBS) identified from the Chromatin Interaction Analysis-Paired End DiTag of ERα are enriched for AP-2 motifs. We demonstrate the transcription factor, AP-2γ, which has been implicated in breast cancer oncogenesis, binds to ERBS in a ligand-independent manner. Furthermore, perturbation of AP-2γ expression impaired ERα DNA binding, long-range chromatin interactions, and gene transcription. In genome-wide analyses, we show that a large number of AP-2γ and ERα binding events converge together across the genome. The majority of these shared regions are also occupied by the pioneer factor, FoxA1. Molecular studies indicate there is functional interplay between AP-2γ and FoxA1. Finally, we show that most ERBS associated with long-range chromatin interactions are colocalized with AP-2γ and FoxA1. Together, our results suggest AP-2γ is a novel collaborative factor in ERα-mediated transcription.

<div>Abstract<p>Cancer-specific <i>TERT</i> promoter mutations (-146C>T and -124C>T) have been linked to reactivation of the epigenetically silenced telomerase reverse transcriptase gene (<i>TERT</i>). Understanding how these single-nucleotide alterations drive <i>TERT</i> reactivation is a fundamental unanswered question and is key for making successful therapeutics. We show that unlike wild-type promoters, recruitment of the transcription factor GABPA specifically to mutant <i>TERT</i> promoters mediates long-range chromatin interaction and enrichment of active histone marks, and hence drives <i>TERT</i> transcription. CRISPR-mediated reversal of mutant <i>TERT</i> promoters, or deletion of its long-range interacting chromatin, abrogates GABPA binding and long-range interactions, leading to depletion of active histone marks, loss of POL2 recruitment, and suppression of <i>TERT</i> transcription. In contrast, <i>de novo</i> introduction of a <i>TERT</i> promoter mutation enables GABPA binding and upregulation of <i>TERT</i> via long-range interactions, acquisition of active histone marks, and subsequent POL2 recruitment. This study provides a unifying mechanistic insight into activation of mutant <i>TERT</i> promoters across various human cancers.</p><p><b>Significance:</b> This study identifies a key mechanism by which cancer-specific mutant <i>TERT</i> promoters cause reactivation of <i>TERT</i>. Because the mechanism uncovered here is not utilized by promoters that drive <i>TERT</i> in normal cells, this mechanism could be exploited to make inhibitors which have the potential to block telomerase function and hence the progression of up to 90% of human cancers. <i>Cancer Discov; 6(11); 1276–91. ©2016 AACR.</i></p><p><i>See related commentary by Min and Shay, p. 1212</i>.</p><p><i>This article is highlighted in the In This Issue feature, p. 1197</i></p></div>

The long noncoding RNA HOTAIRM1 reportedly plays important roles in acute myeloid leukemia, gastric cancer and colorectal cancer. Here, we analyzed potential function of HOTAIRM1 in glioma and asked whether it participates in long-range chromatin interactions. We monitored expression of HOTAIRM1 in glioma tissues and correlated levels with patient survival using the TCGA dataset. HOTAIRM1 was highly expressed in glioma tissue, with high levels associated with shortened patient survival time. We then suppressed HOTAIRM1 activity in the human glioblastoma U251 line using CRISPR-cas9 to knock in a truncating polyA fragment. Reporter analysis of these and control cells confirmed that the HOTAIRM1 locus serves as an active enhancer. We then performed Capture-C analysis to identify target genes of that locus and applied RNA antisense purification to assess chromatin interactions between the HOTAIRM1 locus and HOXA cluster genes. HOTAIRM1 knockdown in glioma cells decreased proliferation and reduced expression of HOXA cluster genes. HOTAIRM1 regulates long-range interactions between the HOTAIRM1 locus and HOXA genes. Our work suggests a new mechanism by which HOTAIRM1 regulates glioma progression by regulating high-order chromatin structure and could suggest novel therapeutic targets to treat an intractable cancer.

Genome-wide association studies have identified more than 100 common single nucleotide polymorphisms (SNPs) that are associated with prostate cancer risk. However, the vast majority of these SNPs lie in noncoding regions of the genome. To test whether these risk SNPs regulate their target genes through long-range chromatin interactions, we applied capture-based 3C sequencing technology to investigate possible cis-interactions at ten prostate cancer risk loci in six cell lines. We identified significant physical interactions between risk regions and their potential target genes including CAPG at 2p11.2, C2orf43 at 2p24.1, RFX6 at 6q22.1, NFASC at 1q32.1, MYC at 8q24.1 and AGAP7P at 10q11.23. Most of the interaction peaks were co-localized to regions of active histone modification and transcription factor binding sites. Expression quantitative trait locus (eQTL) analysis showed suggestive eQTL signals at rs1446669, rs699664 and rs1078004 for CAPG (p < 0.004), rs13394027 for C2orf43 (p = 2.25E-27), rs10993994 and rs4631830 for AGAP7P (p < 8.02E-5). Further analysis revealed an enhancer activity at genomic region surrounding rs4631830 which was expected to disrupt HOXB-like DNA binding affinity. This study identifies a set of candidate genes and their potential regulatory variants, and provides additional evidence showing the role of long-range chromatin interactions in prostate cancer etiology.

Coordinated regulation of IFITM1, 2 and 3 genes by an IFN-responsive enhancer through long-range chromatin interactions

Deciphering long-range chromatin interactions is critical for understanding temporal and tissue-specific gene expression regulated by cis- and trans-acting factors. By combining the chromosome conformation capture (3C) and biotinylated dCas9 system, we previously established a method CAPTURE-3C-seq to unbiasedly identify high-resolution and locus-specific long-range DNA interactions. Here we present the statistical model and a flexible pipeline, C3S, for analysing CAPTURE-3C-seq or similar experimental data from raw sequencing reads to significantly interacting chromatin loci. C3S provides all steps for data processing, quality control and result illustration. It can automatically define the bin size based on the binding peak of the dCas9-targeted regions. Furthermore, it supports the analysis of intra- and inter-chromosomal interactions for different mammalian cell types. We successfully applied C3S across multiple datasets in human K562 cells and mouse embryonic stem cells (mESC) for detecting known and new chromatin interactions at multiple scales. Integrative and topological analysis of the interacted loci at the human β-globin gene cluster provides new insights into mechanisms in developmental gene regulation and network structure in local chromosomal architecture. Furthermore, computational results in mESCs reveal a role for chromatin interacting loops between enhancers and promoters in regulating alternative transcripts of the pluripotency gene OCT4.

Gene expression and regulation with or without alternative splicing are crucial for tissues and cells to function correctly. They have been studied from three almost independent perspectives at the genome level: 1- Recognition of splicing quantitative trait loci (sQTLs), 2- Expression quantitative trait loci (eQTLs) recognition, and 3- Recognition of longer-range physical chromatin interactions between genome segments which model 3D dynamics of cells and tissues. Even though the associations between eQTLs and longer-range chromatin interactions have been previously studied, a similar relationship between sQTLs and chromatin interactions has yet to be analyzed. In this case, examining whether sQTLs control the alternative splicing of their target genes’ mRNA via physically interacting genome segments is crucial. We have jointly analyzed high-throughput chromatin conformation capture (Hi-C) and sQTL datasets over eight human cancer tissues. We have discovered a positive association between the number of genes having sQTLs and chromatin interaction frequency. Such a positive association still exists when we also control for eQTLs. Additionally, sQTLs and their target genes generally exist inside identical topologically associating domains (TADs). These findings are observed over the whole set of analyzed cancer types and functional subsets of the sQTL dataset, such as survival-related sQTLs. Furthermore, tissue-specific sQTLs are statistically enriched in tissue-specific frequently interacting regions (FIREs) in 6 out of 8 human cancer tissues (Chronic Myeloid Leukemia, Colon Adenocarcinoma, Acute Myeloid Leukemia, Lung Adenocarcinoma, Prostate Cancer, Sarcoma). Our sQTL and Hi-C datasets have shown the existence of closer spatial distance between sQTLs and their target genes with possible alternative splicing across several different cancer types in humans. Such a closer spatial distance also exists, independent of whether we integrate eQTLs into the analysis. We found that sQTLs regulate alternative splicing through chromatin interactions. Source code of the analysis in this research is available on https://github.com/seferlab/sqtlhic .

In cellular genomes, CTCF insulators impact transcription over small distances in a one-dimensional manner and over much longer distances in a three-dimensional manner by maintaining chromatin loops. We have previously shown that the latent HSV-1 genome contains CTCF insulators that function to regulate lytic transcription of adjacent genes in a one-dimensional manner. Here, we test the hypothesis that HSV-1 CTCF insulators nucleate chromatin loops to regulate the expression of distance separated gene regions through three-dimensional organization of viral genomes. We used 4C- seq methods to identify multiple long-range cis interactions in HSV-1 genomes that generate viral chromatin domains, including those nucleated by the viral CTCF insulator CTRL2. Deletion of the CTRL2 insulator disrupted these viral chromatin domains. Loop-nucleating interactions were quantitated with a novel approach (UMI-4C- seq ) that utilizes unique molecular identifiers to label and count chromatin interactions associated with specific viewpoint primers. Cis -interaction peaks across four different viewpoints were quantified. Viral genomes lacking CTRL2 displayed more cis -interaction peaks and wider ranges of interaction lengths compared to wt virus, suggesting altered chromatin organization. Furthermore, differential looping analysis showed viral genomes lacking CTRL2 displayed a more transcriptionally permissive chromatin environment. Thus, the CTRL2 insulator functions as a critical regulator of long-range chromatin interactions and its deletion reshapes the viral chromatin landscape, leading to a more accessible and dynamic regulatory environment that may influence HSV-1 transcriptional programs and latency-associated chromatin states.

BackgroundOne of the greatest challenges in cancer genomics is to distinguish driver mutations from passenger mutations. Whereas recurrence is a hallmark of driver mutations, it is difficult to observe recurring noncoding mutations owing to a limited amount of whole-genome sequenced samples. Hence, it is required to develop a method to predict potentially recurrent mutations.ResultsIn this work, we developed a random forest classifier that predicts regulatory mutations that may recur based on the features of the mutations repeatedly appearing in a given cohort. With breast cancer as a model, we profiled 35 quantitative features describing genetic and epigenetic signals at the mutation site, transcription factors whose binding motif was disrupted by the mutation, and genes targeted by long-range chromatin interactions. A true set of mutations for machine learning was generated by interrogating publicly available pan-cancer genomes based on our statistical model of mutation recurrence. The performance of our random forest classifier was evaluated by cross validations. The variable importance of each feature in the classification of mutations was investigated. Our statistical recurrence model for the random forest classifier showed an area under the curve (AUC) of ~0.78 in predicting recurrent mutations. Chromatin accessibility at the mutation sites, the distance from the mutations to known cancer risk loci, and the role of the target genes in the regulatory or protein interaction network were among the most important variables.ConclusionsOur methods enable to characterize recurrent regulatory mutations using a limited number of whole-genome samples, and based on the characterization, to predict potential driver mutations whose recurrence is not found in the given samples but likely to be observed with additional samples.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1385-y) contains supplementary material, which is available to authorized users.

Long-range chromatin interactions control metazoan gene transcription. However, the involvement of intra- and interchromosomal interactions in development and oncogenesis remains unclear. TAL1/SCL is a critical transcription factor required for the development of all hematopoietic lineages; yet, aberrant TAL1 transcription often occurs in T-cell acute lymphoblastic leukemia (T-ALL). Here, we report that oncogenic TAL1 expression is regulated by different intra- and interchromosomal loops in normal hematopoietic and leukemic cells, respectively. These intra- and interchromosomal loops alter the cell-type-specific enhancers that interact with the TAL1 promoter. We show that human SET1 (hSET1)-mediated H3K4 methylations promote a long-range chromatin loop, which brings the +51 enhancer in close proximity to TAL1 promoter 1 in erythroid cells. The CCCTC-binding factor (CTCF) facilitates this long-range enhancer/promoter interaction of the TAL1 locus in erythroid cells while blocking the same enhancer/promoter interaction of the TAL1 locus in human T-cell leukemia. In human T-ALL, a T-cell-specific transcription factor c-Maf-mediated interchromosomal interaction brings the TAL1 promoter into close proximity with a T-cell-specific regulatory element located on chromosome 16, activating aberrant TAL1 oncogene expression. Thus, our study reveals a novel molecular mechanism involving changes in three-dimensional chromatin interactions that activate the TAL1 oncogene in human T-cell leukemia.

The activation of transcription factors such as nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1) plays an important role in regulating the expression of target genes, including those for cytokines involved in pathogenesis of minimal change nephrotic syndrome (MCNS). The therapeutic effects of glucocorticoids depend on the glucocorticoid receptor (GR) acting on gene transcription and interacting with certain transcription factors. To explore the role of transcription factors in the pathogenesis of MCNS and the therapeutic effects of glucocorticoids, we examined the DNA-binding abilities of NF-kappaB, AP-1, and GR in peripheral blood mononuclear cells (PBMC) from 6 children with MCNS and 6 healthy controls by electrophoretic mobility shift assay (EMSA). NF-kappaB and AP-1 DNA-binding abilities were significantly increased both at baseline and after stimulation by phorbol 12-myristate 13-acetate (TPA) in PBMC from MCNS patients compared with controls, but declined to normal levels after treatment with dexamethasone (DEX). GR DNA-binding abilities were significantly reduced at baseline and after treatment with TPA, but were enhanced markedly by DEX. There were strong correlations between urinary protein and the baseline DNA binding ability of NF-kappaB or AP-1, or GR. These results suggested that the abnormal activation of NF-kappaB and AP-1 and the reduction of GR DNA-binding abilities may be involved in the pathogenesis of MCNS. Inhibition of NF-kappaB and AP-1 and enhancement of GR DNA-binding abilities by DEX may form the molecular basis of the effects of glucocorticoids in MCNS.

Whole-exome and whole-genome sequencing have revealed millions of somatic mutations associated with different human cancers, and the vast majority of them are located outside of coding sequences, making it challenging to directly interpret their functional effects. With the rapid advances in high-throughput sequencing technologies, genome-scale long-range chromatin interactions were detected, and distal target genes of regulatory elements were determined using three-dimensional (3D) chromatin looping. Herein, we present OncoBase (http://www.oncobase.biols.ac.cn/), an integrated database for annotating 81 385 242 somatic mutations in 68 cancer types from more than 120 cancer projects by exploring their roles in distal interactions between target genes and regulatory elements. OncoBase integrates local chromatin signatures, 3D chromatin interactions in different cell types and reconstruction of enhancer-target networks using state-of-the-art algorithms. It employs informative visualization tools to display the integrated local and 3D chromatin signatures and effects of somatic mutations on regulatory elements. Enhancer-promoter interactions estimated from chromatin interactions are integrated into a network diffusion system that quantitatively prioritizes somatic mutations and target genes from a large pool. Thus, OncoBase is a useful resource for the functional annotation of regulatory noncoding regions and systematically benchmarking the regulatory effects of embedded noncoding somatic mutations in human carcinogenesis.

Cortisol, the central stress hormone in humans, activates the glucocorticoid receptor (GR). Anti-inflammatory effects are the most important pharmaceutical effects mediated by the GR. Inasmuch as electrophilic cyclopentenone prostaglandin 15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2) has potent anti-inflammatory properties and activates the SUMOylation pathway, we have investigated the effect of 15d-PGJ2 on glucocorticoid signaling and receptor SUMOylation. To this end, we studied isogenic HEK293 cells expressing either wild-type GR or SUMOylation-defective GR. Interestingly, 15d-PGJ2 triggered SUMO-2 and -3 (SUMO-2/3) modification in the primary SUMOylation sites of the GR. Gene expression profiling and pathway analyses indicate that 15d-PGJ2 inhibits GR signaling in a genome-wide fashion that is significantly dependent on the GR SUMOylation sites. Chromatin immunoprecipitation assays showed that the repressive effect of 15d-PGJ2 on GR target gene expression occurs in parallel with the inhibition of receptor binding to the target gene chromatin. Furthermore, depletion of UBC9, the sole SUMO E2 conjugase, from HEK293 cells confirmed the involvement of active SUMOylation in the regulatory process. Taken together, our data indicate that GR SUMOylation modulates the glucocorticoid signaling during acute cell stress. Our data also suggest that GR SUMOylation modulates cross talk of the glucocorticoid signaling with other transcription factors that are responsive to cell stress.

Background/Aims: Prolonged physical exercise induces adaptive alterations in the hypothalamic-pituitary axis, increasing cortisol metabolism, and reducing cortisol synthesis and glucocorticoid sensitivity. The mechanisms responsible for this relative glucocorticoid resistance remain unknown but may involve expression of genes encoding glucocorticoid receptor (GR) and/or inflammatory molecules of nuclear factor kappa B1 (NFkB1) signaling pathway and cytokines. This study aimed to determine the impact of prolonged physical training on the expression of genes involved in glucocorticoid action and inflammatory response. Methods: Normal sedentary male cadets of the Brazilian Air Force Academy were submitted to 6 weeks of standardized physical training. Eighteen of 29 initially selected cadets were able to fully complete the training program. Fasting glucose, insulin and cortisol levels, cytokine concentration and the expression of genes encoding GR, NFkB1, inhibitor of NFkB1 and IkB kinase A were determined before and after the training period. Results: Prolonged physical exercise reduced the basal cortisol levels and the percent cortisol reduction after dexamethasone. These findings were associated with a significant reduction in the mRNA levels of GR (6.3%), NFkB1 (63%), inhibitor of NFkB1 (25%) and IkB kinase A (46%) with concomitant reduction in cytokine concentrations (ELISA). Conclusions: Prolonged physical training decreases the glucocorticoid sensitivity and the mRNA levels of the GR gene combined with decreased mRNA of genes related to the NFkB pathway.