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.

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.

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.

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.

SPOP suppresses prostate cancer through regulation of CYCLIN E1 stability.

SPOP is one of the important subunits for CUL3/SPOP/RBX1 complex tightly connected with tumorigenesis. However, its exact roles in different cancers remain debatable. Here, we identify CYCLIN E1, as a novel substrate for SPOP. SPOP directly interacts with CYCLIN E1 and specific regulates its stability in prostate cancer cell lines. SPOP/CUL3/RBX1 complex regulates CYCLIN E1 stability through poly-ubiquitination. CDK2 competes with SPOP for CYCLIN E1 interaction, suggesting that SPOP probably regulates the stability of CDK2-free CYCLIN E1. CYCLIN E1 expression rescued proliferation, migration, and tumor formation of prostate cancer cell suppressed by SPOP. Furthermore, we found SPOP selectively regulates the substrates' stability and signaling pathways in prostate cancer and CCRC cell lines, suggesting that complicated mechanisms exist for SPOP to regulate substrate specificity. Altogether, we have revealed a novel mechanism for SPOP in suppressing prostate cancer and provided evidence to show SPOP has dual functions in prostate cancer and CCRC.

Characterization of WDR20: A new regulator of the ERAD machinery.

ERAD is an important process of protein quality control that eliminates misfolded or unassembled proteins from ER. Before undergoing proteasome degradation, the misfolded proteins are dislocated from ER membrane into cytosol, which requires the AAA ATPase p97/VCP and its cofactor, the NPL4-UFD1 dimer. Here, we performed a CRISPR-based screen and identify many candidates for ERAD regulation. We further confirmed four proteins, FBOX2, TRIM6, UFL1 and WDR20, are novel regulators for ERAD. Then the molecular mechanism for WDR20 in ERAD is further characterized. Depletion of WDR20 inhibits the degradation of TCRα, a typical ERAD substrate, while WDR20 overexpression reduces TCRα protein level. WDR20 associates with TCRα and central regulators of the ERAD system, p97, GP78 and HRD1. A portion of WDR20 localizes to the ER-containing microsomal membrane. WDR20 expression increases TCRα ubiquitination, and HRD1 E3 ligase is essential for the process. WDR20 seems to serve as an adaptor protein to mediate the interaction between p97 and TCRα. Our study provides novel candidates and reveals an unexpected role of WDR20 in ERAD regulation.

TTLL12 Inhibits the Activation of Cellular Antiviral Signaling through Interaction with VISA/MAVS.

Upon virus infection, host cells use retinoic-acid-inducible geneI I (RIG-I)-like receptors to recognize viral RNA and activate type I IFN expression. To investigate the role of protein methylation in the antiviral signaling pathway, we screened all the SET domain-containing proteins and identified TTLL12 as a negative regulator of RIG-I signaling. TTLL12 contains SET and TTL domains, which are predicted to have lysine methyltransferase and tubulin tyrosine ligase activities, respectively. Exogenous expression of TTLL12 represses IFN-ß expression induced by Sendai virus. TTLL12 deficiency by RNA interference and CRISPR-gRNA techniques increases the induced IFN-ß expression and inhibits virus replication in the cell. The global gene expression profiling indicated that TTLL12 specifically inhibits the expression of the downstream genes of innate immunity pathways. Cell fractionation and fluorescent staining indicated that TTLL12 is localized in the cytosol. The mutagenesis study suggested that TTLL12's ability to repress the RIG-I pathway is probably not dependent on protein modifications. Instead, TTLL12 directly interacts with virus-induced signaling adaptor (VISA), TBK1, and IKKε, and inhibits the interactions of VISA with other signaling molecules. Taken together, our findings demonstrate TTLL12 as a negative regulator of RNA-virus-induced type I IFN expression by inhibiting the interaction of VISA with other proteins.

SPOP-containing complex regulates SETD2 stability and H3K36me3-coupled alternative splicing.

Trimethylation of histone H3K36 is a chromatin mark associated with active gene expression, which has been implicated in coupling transcription with mRNA splicing and DNA damage response. SETD2 is a major H3K36 trimethyltransferase, which has been implicated as a tumor suppressor in mammals. Here, we report the regulation of SETD2 protein stability by the proteasome system, and the identification of SPOP, a key subunit of the CUL3 ubiquitin E3 ligase complex, as a SETD2-interacting protein. We demonstrate that SPOP is critically involved in SETD2 stability control and that the SPOP/CUL3 complex is responsible for SETD2 polyubiquitination both in vivo and in vitro ChIP-Seq analysis and biochemical experiments demonstrate that modulation of SPOP expression confers differential H3K36me3 on SETD2 target genes, and induce H3K36me3-coupled alternative splicing events. Together, these findings establish a functional connection between oncogenic SPOP and tumor suppressive SETD2 in the dynamic regulation of gene expression on chromatin.

WDR82 Negatively Regulates Cellular Antiviral Response by Mediating TRAF3 Polyubiquitination in Multiple Cell Lines.

Upon virus infection, retinoic acid-inducible gene I-like receptors in host cells recognize viral RNA and activate type I IFN expression. Previously, we identified WD repeat domain (WDR) 5 as one positive regulator for pathway activation. In this study, we report that WDR82, a homolog protein of WDR5, acts opposite to WDR5 and inhibits the activation of the retinoic acid-inducible gene I signaling pathway. WDR82 overexpression inhibits virus-triggered pathway activation, whereas its knockdown enhances induced IFN-ß expression. WDR82 is localized on the mitochondria, and its first N-terminal WD40 domain is critical for localization. WDR82 interacts with TNFR-associated factor (TRAF) 3, and its overexpression promotes K48-linked, but not K63-linked, polyubiquitination on TRAF3. Furthermore, WDR82 knockdown inhibits viral replication in the cell, whereas its overexpression has the opposite effect. Interestingly, WDR82 regulates Sendai virus-induced IFNB1 expression in a cell type-specific manner. Taken together, our findings demonstrate that WDR82 is a negative regulator of virus-triggered type I IFNs pathway through mediating TRAF3 polyubiquitination status and stability on mitochondria.