The roles of nuclear orphan receptor NR2F6 in anti-viral innate immunity.

Proper transcription regulation by key transcription factors, such as IRF3, is critical for anti-viral defense. Dynamics of enhancer activity play important roles in many biological processes, and epigenomic analysis is used to determine the involved enhancers and transcription factors. To determine new transcription factors in anti-DNA-virus response, we have performed H3K27ac ChIP-Seq and identified three transcription factors, NR2F6, MEF2D and MAFF, in promoting HSV-1 replication. NR2F6 promotes HSV-1 replication and gene expression in vitro and in vivo, but not dependent on cGAS/STING pathway. NR2F6 binds to the promoter of MAP3K5 and activates AP-1/c-Jun pathway, which is critical for DNA virus replication. On the other hand, NR2F6 is transcriptionally repressed by c-Jun and forms a negative feedback loop. Meanwhile, cGAS/STING innate immunity signaling represses NR2F6 through STAT3. Taken together, we have identified new transcription factors and revealed the underlying mechanisms involved in the network between DNA viruses and host cells.

Cooperation of MLL1 and Jun in controlling H3K4me3 on enhancers in colorectal cancer.

BACKGROUND: Enhancer dysregulation is one of the important features for cancer cells. Enhancers enriched with H3K4me3 have been implicated to play important roles in cancer. However, their detailed features and regulatory mechanisms have not been well characterized. RESULTS: Here, we profile the landscape of H3K4me3-enriched enhancers (m3Es) in 43 pairs of colorectal cancer (CRC) samples. M3Es are widely distributed in CRC and averagely possess around 10% of total active enhancers. We identify 1322 gain variant m3Es and 367 lost variant m3Es in CRC. The target genes of the gain m3Es are enriched in immune response pathways. We experimentally prove that repression of CBX8 and RPS6KA5 m3Es inhibits target gene expression in CRC. Furthermore, we find histone methyltransferase MLL1 is responsible for depositing H3K4me3 on the identified Vm3Es. We demonstrate that the transcription factor AP1/JUN interacts with MLL1 and regulates m3E activity. Application of a small chemical inhibitor for MLL1 activity, OICR-9429, represses target gene expression of the identified Vm3Es, enhances anti-tumor immunity and inhibits CRC growth in an animal model. CONCLUSIONS: Taken together, our study illustrates the genome-wide landscape and the regulatory mechanisms of m3Es in CRC, and reveals potential novel strategies for cancer treatment.

Removal of epigenetic repressive mark on inflammatory genes in fat liver.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide. The detailed epigenomic changes during fat accumulation in liver are not clear yet. Here, we performed ChIP-Seq analysis in the liver tissues of high-fat diet and regular chow diet mice and investigated the dynamic landscapes of H3K27ac and H3K9me3 marks on chromatin. We find that the activated typical enhancers marked with H3K27ac are enriched on lipid metabolic pathways in fat liver; however, super enhancers do not change much. The regions covered with H3K9me3 repressive mark seem to undergo great changes, and its peak number and intensity both decrease in fat liver. The enhancers located in lost H3K9me3 regions are enriched in lipid metabolism and inflammatory pathways; and motif analysis shows that they are potential targets for transcription factors involved in metabolic and inflammatory processes. Our study has revealed that H3K9me3 may play an important role during the pathogenesis of NAFLD through regulating the accessibility of enhancers.

Regulation of KDM5C stability and enhancer reprogramming in breast cancer.

Abnormality of enhancer regulation has emerged as one of the critical features for cancer cells. KDM5C is a histone H3K4 demethylase and frequently mutated in several types of cancer. It is critical for H3K4me3 and activity of enhancers, but its regulatory mechanisms remain elusive. Here, we identify TRIM11 as one ubiquitin E3 ligase for KDM5C. TRIM11 interacts with KDM5C, catalyzes K48-linked ubiquitin chain on KDM5C, and promotes KDM5C degradation through proteasome. TRIM11 deficiency in an animal model represses the growth of breast tumor and stabilizes KDM5C. In breast cancer patient tissues, TRIM11 is highly expressed and KDM5C is lower expressed, and their expression is negatively correlated. Mechanistically, TRIM11 regulates the enhancer activity of genes involved in cell migration and immune response by targeting KDM5C. TRIM11 and KDM5C regulate MCAM expression and cell migration through targeting H3K4me3 on MCAM enhancer. Taken together, our study reveals novel mechanisms for enhancer regulation during breast cancer tumorigenesis and development.

Multifaceted regulation of enhancers in cancer.

Enhancer is one kind of cis-elements regulating gene transcription, whose activity is tightly controlled by epigenetic enzymes and histone modifications. Active enhancers are classified into typical enhancers, super-enhancers and over-active enhancers, according to the enrichment and location of histone modifications. Epigenetic factors control the level of histone modifications on enhancers to determine their activity, such as histone methyltransferases and acetylases. Transcription factors, cofactors and mediators co-operate together and are required for enhancer functions. In turn, abnormalities in these trans-acting factors affect enhancer activity. Recent studies have revealed enhancer dysregulation as one of the important features for cancer. Variations in enhancer regions and mutations of enhancer regulatory genes are frequently observed in cancer cells, and altering the activity of onco-enhancers is able to repress oncogene expression, and suppress tumorigenesis and metastasis. Here we summarize the recent discoveries about enhancer regulation in cancer and discuss their potential application in diagnosis and treatment.

Inhibition of histone methyltransferase SETD8 represses DNA virus replication.

Multiple diseases, such as cancer and neural degeneration diseases, are related with the latent infection of DNA viruses. However, it is still difficult to clean up the latent DNA viruses and new anti-viral strategies are critical for disease treatment. Here, we screen a pool of small chemical molecules and identify UNC0379, an inhibitor for histone H4K20 methyltransferase SETD8, as an effective inhibitor for multiple DNA viruses. UNC0379 not only enhances the expression of anti-viral genes in THP-1 cells, but also repress DNA virus replication in multiple cell lines with defects in cGAS pathway. We prove that SETD8 promotes DNA virus replication in a manner dependent on its enzyme activity. Our results further indicated that SETD8 is required for PCNA stability, one factor critical for viral DNA replication. Viral infection stimulates the interaction between SETD8 and PCNA and thus enhances PCNA stability and viral DNA replication. Taken together, our study reveals a new mechanism for regulating viral DNA replication and provides a potential strategy for treatment of diseases related with DNA viruses.

Genome-wide profiling in colorectal cancer identifies PHF19 and TBC1D16 as oncogenic super enhancers.

Colorectal cancer is one of the most common cancers in the world. Although genomic mutations and single nucleotide polymorphisms have been extensively studied, the epigenomic status in colorectal cancer patient tissues remains elusive. Here, together with genomic and transcriptomic analysis, we use ChIP-Seq to profile active enhancers at the genome wide level in colorectal cancer paired patient tissues (tumor and adjacent tissues from the same patients). In total, we sequence 73 pairs of colorectal cancer tissues and generate 147 H3K27ac ChIP-Seq, 144 RNA-Seq, 147 whole genome sequencing and 86 H3K4me3 ChIP-Seq samples. Our analysis identifies 5590 gain and 1100 lost variant enhancer loci in colorectal cancer, and 334 gain and 121 lost variant super enhancer loci. Multiple key transcription factors in colorectal cancer are predicted with motif analysis and core regulatory circuitry analysis. Further experiments verify the function of the super enhancers governing PHF19 and TBC1D16 in regulating colorectal cancer tumorigenesis, and KLF3 is identified as an oncogenic transcription factor in colorectal cancer. Taken together, our work provides an important epigenomic resource and functional factors for epigenetic studies in colorectal cancer.

Histone demethylase LSD1 promotes RIG-I poly-ubiquitination and anti-viral gene expression.

Under RNA virus infection, retinoic acid-inducible gene I (RIG-I) in host cells recognizes viral RNA and activates the expression of type I IFN. To investigate the roles of protein methyltransferases and demethylases in RIG-I antiviral signaling pathway, we screened all the known related enzymes with a siRNA library and identified LSD1 as a positive regulator for RIG-I signaling. Exogenous expression of LSD1 enhances RIG-I signaling activated by virus stimulation, whereas its deficiency restricts it. LSD1 interacts with RIG-I, promotes its K63-linked polyubiquitination and interaction with VISA/MAVS. Interestingly, LSD1 exerts its function in antiviral response not dependent on its demethylase activity but through enhancing the interaction between RIG-I with E3 ligases, especially TRIM25. Furthermore, we provide in vivo evidence that LSD1 increases antiviral gene expression and inhibits viral replication. Taken together, our findings demonstrate that LSD1 is a positive regulator of signaling pathway triggered by RNA-virus through mediating RIG-I polyubiquitination.

Genome-Wide Enhancer Analysis Reveals the Role of AP-1 Transcription Factor in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers in the world, but its epigenomic features have not been determined. Here, we studied the chromatin landscape of active enhancers of HNSCC head tumor tissues by performing H3K27ac and H3K4me1 ChIP-Seq with a Tgfbr1/Pten double conditional knockout HNSCC mouse model. We identified 1,248 gain variant enhancer loci (VELs) and 2,188 lost VELs, as well as 153 gain variant super enhancer loci (VSELs) and 234 lost VSELs. Potentially involved transcription factors were predicted with motif analysis, and we identified AP-1 as one of the critical oncogenic transcription factors in HNSCC and many other types of cancer. Combining transcriptomic and epigenomic data, our analysis also showed that AP-1 and histone modifications coordinately regulate target gene expression in HNSCC. In conclusion, our study provides important epigenomic information for enhancer studies in HNSCC and reveals new mechanism for AP-1 regulating HNSCC.

Epigenetic Dysregulation Induces Translocation of Histone H3 into Cytoplasm.

In eukaryote cells, core components of chromatin, such as histones and DNA, are packaged in nucleus. Leakage of nuclear materials into cytosol will induce pathological effects. However, the underlying mechanisms remain elusive. Here, cytoplasmic localization of nuclear materials induced by chromatin dysregulation (CLIC) in mammalian cells is reported. H3K9me3 inhibition by small chemicals, HP1α knockdown, or knockout of H3K9 methylase SETDB1, induces formation of cytoplasmic puncta containing histones H3.1, H4 and cytosolic DNA, which in turn activates inflammatory genes and autophagic degradation. Autophagy deficiency rescues H3 degradation, and enhances the activation of inflammatory genes. MRE11, a subunit of MRN complex, enters cytoplasm after heterochromatin dysregulation. Deficiency of MRE11 or NBS1, but not RAD50, inhibits CLIC puncta in cytosol. MRE11 depletion represses tumor growth enhanced by HP1α deficiency, suggesting a connection between CLIC and tumorigenesis. This study reveals a novel pathway that heterochromatin dysregulation induces translocation of nuclear materials into cytoplasm, which is important for inflammatory diseases and cancer.

STAT1 epigenetically regulates LCP2 and TNFAIP2 by recruiting EP300 to contribute to the pathogenesis of inflammatory bowel disease.

BACKGROUND: The aetiology of inflammatory bowel disease (IBD) is related to genetics and epigenetics. Epigenetic regulation of the pathogenesis of IBD has not been well defined. Here, we investigated the role of H3K27ac events in the pathogenesis of IBD. Based on previous ChIP-seq and RNA-seq assays, we studied signal transducer and activator of transcription 1 (STAT1) as a transcription factor (TF) and investigated whether the STAT1-EP300-H3K27ac axis contributes to the development of IBD. We performed ChIP-PCR to investigate the interaction between STAT1 and H3K27ac, and co-IP assays were performed to investigate the crosstalk between STAT1 and EP300. RESULTS: Lymphocyte cytosolic protein 2 (LCP2) and TNF-α-inducible protein 2 (TNFAIP2) are target genes of STAT1. p-STAT1 binds to the enhancer loci of the two genes where H3K27ac is enriched, and EP300 subsequently binds to regulate their expression. In mice with dextran sulfate sodium (DSS)-induced acute colitis, an EP300 inhibitor significantly inhibited colitis. CONCLUSIONS: p-STAT1 and EP300 promote TNFAIP2 and LCP2 expression through an increase in H3K27ac enrichment on their enhancers and contribute to the pathogenesis of chronic inflammation.

MLL3 suppresses tumorigenesis through regulating TNS3 enhancer activity.

MLL3 is a histone H3K4 methyltransferase that is frequently mutated in cancer, but the underlying molecular mechanisms remain elusive. Here, we found that MLL3 depletion by CRISPR/sgRNA significantly enhanced cell migration, but did not elevate the proliferation rate of cancer cells. Through RNA-Seq and ChIP-Seq approaches, we identified TNS3 as the potential target gene for MLL3. MLL3 depletion caused downregulation of H3K4me1 and H3K27ac on an enhancer ~ 7 kb ahead of TNS3. 3C assay indicated the identified enhancer interacts with TNS3 promoter and repression of enhancer activity by dCas9-KRAB system impaired TNS3 expression. Exogenous expression of TNS3 in MLL3 deficient cells completely blocked the enhanced cell migration phenotype. Taken together, our study revealed a novel mechanism for MLL3 in suppressing cancer, which may provide novel targets for diagnosis or drug development.

Deficiency of Histone Methyltransferase SET Domain-Containing 2 in Liver Leads to Abnormal Lipid Metabolism and HCC.

BACKGROUND AND AIMS: Trimethylation of Lys36 on histone 3 (H3K36me3) catalyzed by histone methyltransferase SET domain-containing 2 (SETD2) is one of the most conserved epigenetic marks from yeast to mammals. SETD2 is frequently mutated in multiple cancers and acts as a tumor suppressor. APPROACH AND RESULTS: Here, using a liver-specific Setd2 depletion model, we found that Setd2 deficiency is sufficient to trigger spontaneous HCC. Meanwhile, Setd2 depletion significantly increased tumor and tumor size of a diethylnitrosamine-induced HCC model. The mechanistic study showed that Setd2 suppresses HCC not only through modulating DNA damage response, but also by regulating lipid metabolism in the liver. Setd2 deficiency down-regulated H3K36me3 enrichment and expression of cholesterol efflux genes and caused lipid accumulation. High-fat diet enhanced lipid accumulation and promoted the development of HCC in Setd2-deficient mice. Chromatin immunoprecipitation sequencing analysis further revealed that Setd2 depletion induced c-Jun/activator protein 1 (AP-1) activation in the liver, which was trigged by accumulated lipid. c-Jun acts as an oncogene in HCC and functions through inhibiting p53 in Setd2-deficient cells. CONCLUSIONS: We revealed the roles of Setd2 in HCC and the underlying mechanisms in regulating cholesterol homeostasis and c-Jun/AP-1 signaling.

Regulation of IL12B Expression in Human Macrophages by TALEN-mediated Epigenome Editing.

Although the exact etiology of inflammatory bowel disease (IBD) remains unclear, exaggerated immune response in genetically predisposed individuals has been reported. Th1 and Th17 cells mediate IBD development. Macrophages produce IL-12 and IL-23 that share p40 subunit encoded by IL12B gene as heteromer partner to drive Th1 and Th17 differentiation. The available animal and human data strongly support the pathogenic role of IL-12/IL-23 in IBD development and suggest that blocking p40 might be the potential strategy for IBD treatment. Furthermore, aberrant alteration of some cytokines expression via epigenetic mechanisms is involved in pathogenesis of IBD. In this study, we analyzed core promoter region of IL12B gene and investigated whether IL12B expression could be regulated through targeted epigenetic modification with gene editing technology. Transcription activator-like effectors (TALEs) are widely used in the field of genome editing and can specifically target DNA sequence in the host genome. We synthesized the TALE DNA-binding domains that target the promoter of human IL12B gene and fused it with the functional catalytic domains of epigenetic enzymes. Transient expression of these engineered enzymes demonstrated that the TALE-DNMT3A targeted the selected IL12B promoter region, induced loci-specific DNA methylation, and down-regulated IL-12B expression in various human cell lines. Collectively, our data suggested that epigenetic editing of IL12B through methylating DNA on its promoter might be developed as a potential therapeutic strategy for IBD treatment.

GLIS2 promotes colorectal cancer through repressing enhancer activation.

Gene transcription is coordinately regulated by multiple transcription factors. However, a systematic approach is still lacking to identify co-regulators for transcription factors. Here, we performed ChIP-Seq analysis and predicted the regulators for p53-mediated transcription process, from which we confirmed the roles of GLIS2, MAZ and MEF2A in regulating p53 target genes. We revealed that GLIS2 selectively regulates the transcription of PUMA but not p21. GLIS2 deficiency caused the elevation of H3K27ac and p53 binding on the PUMA enhancer, and promoted PUMA expression. It increased the rate of apoptosis, but not cell cycle. Moreover, GLIS2 represses H3K27ac level on enhancers, regulates the gene expression related with focal adhesion and promotes cell migration, through inhibiting p300. Big data analysis supports GLIS2 as an oncogene in colon cancer, and perhaps other cancers. Taken together, we have predicted candidates for p53 transcriptional regulators, and provided evidence for GLIS2 as an oncogene through repressing enhancer activation.

The epigenetic landscapes of histone modifications on HSV-1 genome in human THP-1 cells.

Histone positioning and modifications on viral genomes are important factors regulating virus replication. To investigate the dynamics of modified histones on the viral genome and their potential roles in antiviral response, we studied the dynamic changes of histone modifications across the HSV-1 genome in THP-1 cells. Histone modifications were detected on the HSV-1 genome soon after infection, including H3K9me3, H3K27me3, H3K4me3 and H3K27ac. These modifications emerged on the viral genome soon after infection and changed rapidly along with virus life cycle progression. The transcription repression marks, H3K9me3 and H3K27me3, decreased on the viral genome during the infection process; the transcription activation mark H3K27ac increased. Treatment with C646, an inhibitor of H3K27ac transferase p300, significantly repressed virus replication and viral gene expression. Our study reveals the relationship between histone modifications and viral gene expression and provides potential novel strategies for antiviral treatment.

Epigenetic plasticity of enhancers in cancer.

Enhancers are cis-acting elements with many sites bound by transcription factors and activate transcription over long distance. Histone modifications are critical for enhancer activity and utilized as hallmarks for the identification of putative enhancers. Monomethylation of histone H3 lysine 4 (H3K4me1) is the mark for enhancer priming; acetylation of histone H3 lysine 27 (H3K27ac) for active enhancers and trimethylation of histone H3 lysine 27 (H3K27me3) for silent enhancers. Recent studies from multiple groups have provided evidence that enhancer reprogramming, especially gain of enhancer activity, is closely related to tumorigenesis and cancer development. In this review, we will summarize the recent discoveries about enhancer regulation and the mechanisms of enhancer reprogramming in tumorigenesis, and discuss the potential application of enhancer manipulation in precision medicine.

The hyper-activation of transcriptional enhancers in breast cancer.

BACKGROUND: Activation of transcription enhancers, especially super-enhancers, is one of the critical epigenetic features of tumorigenesis. However, very few studies have systematically identified the enhancers specific in cancer tissues. METHODS: Here, we studied the change of histone modifications in MMTV-PyVT breast cancer model, combining mass spectrometry-based proteomics and ChIP-seq-based epigenomics approaches. Some of the proteomic results were confirmed with western blotting and IHC staining. An inhibitor of H3K27ac was applied to study its effect on cancer development. RESULTS: H3K27ac and H4K8ac are elevated in cancer, which was confirmed in patient tissue chips. ChIP-seq revealed that H4K8ac is co-localized with H3K27ac on chromatin, especially on distal enhancers. Epigenomic studies further identified a subgroup of super-enhancers marked by H3K4me3 peaks in the intergenic regions. The H3K4me3-enriched regions enhancers are associated with higher level of H3K27ac and H4K8ac compared with the average level of conventional super-enhancers and are associated with higher transcription level of their adjacent genes. We identified 148 H3K4me3-enriched super-enhancers with higher gene expression in tumor, which may be critical for breast cancer. One inhibitor for p300 and H3K27ac, C646, repressed tumor formation probably through inhibiting Vegfa and other genes. CONCLUSIONS: Taken together, our work identifies novel regulators and provides important resource to the genome-wide enhancer studies in breast cancer and raises the possibility of cancer treatment through modulating enhancer activity.

Histone demethylase KDM3A is required for enhancer activation of hippo target genes in colorectal cancer.

Hippo pathway is involved in tumorigenesis, and its regulation in cytosol has been extensively studied, but its regulatory mechanisms in the nuclear are not clear. In the current study, using a FBS-inducing model following serum starvation, we identified KDM3A, a demethylase of histone H3K9me1/2, as a positive regulator for hippo target genes. KDM3A promotes gene expression through two mechanisms, one is to upregulate YAP1 expression, and the other is to facilitate H3K27ac on the enhancers of hippo target genes. H3K27ac upregulation is more relevant with gene activation, but not H3K4me3; and KDM3A depletion caused H3K9me2 upregulation mainly on TEAD1-binding enhancers rather than gene bodies, further resulting in H3K27ac decrease, less TEAD1 binding on enhancers and impaired transcription. Moreover, KDM3A is associated with p300 and required for p300 recruitment to enhancers. KDM3A deficiency delayed cancer cell growth and migration, which was rescued by YAP1 expression. KDM3A expression is correlated with YAP1 and hippo target genes in colorectal cancer patient tissues, and may serve as a potential prognosis mark. Taken together, our study reveals novel mechanisms for hippo signaling and enhancer activation, which is critical for tumorigenesis of colorectal cancer.