JMJD6 licenses estrogen receptor alpha-dependent enhancer RNA and coding gene activation by modulating CARM1/MED12 co-activator complex in breast cancer
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE101562 | GEO | 2018/04/16
REPOSITORIES: GEO
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Project description:Whereas the actions of enhancers in gene transcriptional regulation are well established, roles of JmjC-domain-containing proteins in mediating enhancer activation remain poorly understood. Here, we report that recruitment of the JmjC-domain-containing protein 6 (JMJD6) to estrogen receptor alpha (ERα)-bound active enhancers is required for RNA polymerase II recruitment and enhancer RNA production on enhancers, resulting in transcriptional pause release of cognate estrogen target genes. JMJD6 is found to interact with MED12 in the mediator complex to regulate its recruitment. Unexpectedly, JMJD6 is necessary for MED12 to interact with CARM1, which methylates MED12 at multiple arginine sites and regulates its chromatin binding. Consistent with its role in transcriptional activation, JMJD6 is required for estrogen/ERα-induced breast cancer cell growth and tumorigenesis. Our data have uncovered a critical regulator of estrogen/ERα-induced enhancer coding gene activation and breast cancer cell potency, providing a potential therapeutic target of ER-positive breast cancers.
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Project description:Enhancers are genomic regulatory elements shown to play key roles in controlling cell type-specific gene expression, regulated by signal-dependent transcription factors and co-factors. Distinct classes of enhancers can specify distinct gene expression profiles and biological outcomes. Recent studies suggested that bidirectional non-coding RNAs (ncRNAs), referred as enhancer RNA (eRNAs), are transcribed on enhancers, which are tightly associated with enhancer activity. However, the protein factors which govern the activation of enhancers remain poorly characterized. Here we reported that a Jumonji C (JmjC) domain-containing protein demethylase, JMJD6, is a determinant for estrogen/estrogen receptor (ER)-induced enhancer activation, coding gene transcription, breast cancer cell growth and tumorigenesis. Mechanistically, JMJD6 was found to be specifically recruited onto ER-bound active enhancers in response to estrogen stimulation, which was required for RNA Pol II recruitment and eRNA production of enhancers, leading to transcriptional activation of cognate estrogen target genes. Furthermore, JMJD6 was found to be physically and functionally associated with MED12 in the mediator complex to regulate estrogen-induced transcriptional activation, with JMJD6 was required for MED12 recruitment onto active enhancers. Specifically, JMJD6 was essential for MED12 to interact with CARM1, an arginine methyltransferase well known to be implicated in estrogen-induced transcriptional activation, which methylated MED12 at its c-terminus at multiple arginine methylation sites and was required for MED12 binding with chromatin. Collectively, our data uncovered a critical regulator of estrogen/estrogen receptor-induced enhancer activation and coding gene transcription, providing a potential drug candidate for developing novel strategies targeting ER-positive breast cancers.
2018-04-16 | GSE101559 | GEO
Project description:Enhancers are genomic regulatory elements shown to play key roles in controlling cell type-specific gene expression, regulated by signal-dependent transcription factors and co-factors. Distinct classes of enhancers can specify distinct gene expression profiles and biological outcomes. Recent studies suggested that bidirectional non-coding RNAs (ncRNAs), referred as enhancer RNA (eRNAs), are transcribed on enhancers, which are tightly associated with enhancer activity. However, the protein factors which govern the activation of enhancers remain poorly characterized. Here we reported that a Jumonji C (JmjC) domain-containing protein demethylase, JMJD6, is a determinant for estrogen/estrogen receptor (ER)-induced enhancer activation, coding gene transcription, breast cancer cell growth and tumorigenesis. Mechanistically, JMJD6 was found to be specifically recruited onto ER-bound active enhancers in response to estrogen stimulation, which was required for RNA Pol II recruitment and eRNA production of enhancers, leading to transcriptional activation of cognate estrogen target genes. Furthermore, JMJD6 was found to be physically and functionally associated with MED12 in the mediator complex to regulate estrogen-induced transcriptional activation, with JMJD6 was required for MED12 recruitment onto active enhancers. Specifically, JMJD6 was essential for MED12 to interact with CARM1, an arginine methyltransferase well known to be implicated in estrogen-induced transcriptional activation, which methylated MED12 at its c-terminus at multiple arginine methylation sites and was required for MED12 binding with chromatin. Collectively, our data uncovered a critical regulator of estrogen/estrogen receptor-induced enhancer activation and coding gene transcription, providing a potential drug candidate for developing novel strategies targeting ER-positive breast cancers.
2018-04-16 | GSE101560 | GEO
Project description:JMJD6 licenses estrogen receptor alpha-dependent enhancer RNA and coding gene activation by modulating CARM1/MED12 co-activator complex in breast cancer
| PRJNA394829 | ENA
Project description:The coactivator-associated arginine methyltransferase (CARM1) functions as a regulator of transcription by methylating a diverse array of substrates. To broaden our understanding of CARM1's mechanistic actions, we sought to identify additional substrates for this enzyme. To do this, we generated CARM1 substrate motif antibodies, and used immunoprecipitation coupled with mass spectrometry to identify cellular targets of CARM1, including mediator complex subunit 12 (MED12) and the lysine methyltransferase KMT2D. Both of these proteins are implicated in enhancer function. We identified the major CARM1-mediated MED12 methylation site as arginine 1899 (R1899), which interacts with the Tudor domain-containing effector molecule, TDRD3. Chromatin immunoprecipitation-seq studies revealed that CARM1 and the methyl mark it deposits are tightly associated with ERα-specific enhancers and positively modulate transcription of estrogen-regulated genes. In addition, we showed that the methylation of MED12, at the R1899 site, and the recruitment of TDRD3 by this methylated motif are critical for the ability of MED12 to interact with activating noncoding RNAs.
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Project description:Estrogen and its cognate receptor, ERα, regulate cell proliferation, differentiation, and carcinogenesis in the endometrium by controlling gene transcription. ERα requires co-activators to mediate transcription via mechanisms that are largely uncharacterized. Herein, using growth-regulating estrogen receptor binding 1 (GREB1) as an ERα target gene in Ishikawa cells, we demonstrate that nuclear receptor co-activator 6 (NCOA6) is essential for estradiol (E2)/ERα-activated GREB1 transcription. We found that NCOA6 associates with the GREB1 promoter and enhancer in an E2-independent manner and that NCOA6 knockout reduces chromatin looping, enhancer-promoter interactions, and basal GREB1 expression in the absence of E2. In the presence of E2, ERα bound the GREB1 enhancer and also associated with its promoter, and p300, myeloid/lymphoid or mixed-lineage leukemia protein 4 (MLL4), and RNA polymerase II were recruited to the GREB1 enhancer and promoter. Consequently, the levels of the histone modifications H3K4me1/3, H3K9ac, and H3K27ac were significantly increased; enhancer and promoter regions were transcribed; and GREB1 mRNA was robustly transcribed. NCOA6 knockout reduced ERα recruitment and abolished all of the aforementioned E2-induced events, making GREB1 completely insensitive to E2 induction. We also found that GREB1-deficient Ishikawa cells are much more resistant to chemotherapy and that human endometrial cancers with low GREB1 expression predict poor overall survival. These results indicate that NCOA6 has an essential role in ERα-mediated transcription by increasing enhancer-promoter interactions through chromatin looping and by recruiting RNA polymerase II and the histone-code modifiers p300 and MLL4. Moreover, GREB1 loss may predict chemoresistance of endometrial cancer.
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Project description:Genome-wide RNA sequencing has shown that only a small fraction of the human genome is transcribed into protein-coding mRNAs. While once thought to be "junk" DNA, recent findings indicate that the rest of the genome encodes many types of non-coding RNA molecules with a myriad of functions still being determined. Among the non-coding RNAs, long non-coding RNAs (lncRNA) and enhancer RNAs (eRNA) are found to be most copious. While their exact biological functions and mechanisms of action are currently unknown, technologies such as next-generation RNA sequencing (RNA-seq) and global nuclear run-on sequencing (GRO-seq) have begun deciphering their expression patterns and biological significance. In addition to their identification, it has been shown that the expression of long non-coding RNAs and enhancer RNAs can vary due to spatial, temporal, developmental, or hormonal variations. In this review, we explore newly reported information on estrogen-regulated eRNAs and lncRNAs and their associated biological functions to help outline their markedly prominent roles in estrogen-dependent signaling.
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Project description:While protein arginine methyltransferases (PRMTs) and PRMT-catalyzed protein methylation have been well-known to be involved in a myriad of biological processes, their functions and the underlying molecular mechanisms in cancers, particularly in estrogen receptor alpha (ERα)-positive breast cancers, remain incompletely understood. Here we focused on investigating PRMT4 (also called coactivator associated arginine methyltransferase 1, CARM1) in ERα-positive breast cancers due to its high expression and the associated poor prognosis. Methods: ChIP-seq and RNA-seq were employed to identify the chromatin-binding landscape and transcriptional targets of CARM1, respectively, in the presence of estrogen in ERα-positive MCF7 breast cancer cells. High-resolution mass spectrometry analysis of enriched peptides from anti-monomethyl- and anti-asymmetric dimethyl-arginine antibodies in SILAC labeled wild-type and CARM1 knockout cells were performed to globally map CARM1 methylation substrates. Cell viability was measured by MTS and colony formation assay, and cell cycle was measured by FACS analysis. Cell migration and invasion capacities were examined by wound-healing and trans-well assay, respectively. Xenograft assay was used to analyze tumor growth in vivo. Results: CARM1 was found to be predominantly and specifically recruited to ERα-bound active enhancers and essential for the transcriptional activation of cognate estrogen-induced genes in response to estrogen treatment. Global mapping of CARM1 substrates revealed that CARM1 methylated a large cohort of proteins with diverse biological functions, including regulation of intracellular estrogen receptor-mediated signaling, chromatin organization and chromatin remodeling. A large number of CARM1 substrates were found to be exclusively hypermethylated by CARM1 on a cluster of arginine residues. Exemplified by MED12, hypermethylation of these proteins by CARM1 served as a molecular beacon for recruiting coactivator protein, tudor-domain-containing protein 3 (TDRD3), to CARM1-bound active enhancers to activate estrogen/ERα-target genes. In consistent with its critical role in estrogen/ERα-induced gene transcriptional activation, CARM1 was found to promote cell proliferation of ERα-positive breast cancer cells in vitro and tumor growth in mice. Conclusions: our study uncovered a "hypermethylation" strategy utilized by enhancer-bound CARM1 in gene transcriptional regulation, and suggested that CARM1 can server as a therapeutic target for breast cancer treatment.
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Project description:A crucial feature of differentiated cells is the rapid activation of enhancer-driven transcriptional programs in response to signals. The potential contributions of physicochemical properties of enhancer assembly in signaling events remain poorly understood. Here we report that in human breast cancer cells, the acute 17β-estradiol-dependent activation of functional enhancers requires assembly of an enhancer RNA-dependent ribonucleoprotein (eRNP) complex exhibiting properties of phase-separated condensates. Unexpectedly, while acute ligand-dependent assembly of eRNPs resulted in enhancer activation sensitive to chemical disruption of phase separation, chronically activated enhancers proved resistant to such disruption, with progressive maturation of eRNPs to a more gel-like state. Acute, but not chronic, stimulation resulted in ligand-induced, condensin-dependent changes in spatial chromatin conformation based on homotypic enhancer association, resulting in cooperative enhancer-activation events. Thus, distinct physicochemical properties of eRNP condensates on enhancers serve as determinants of rapid ligand-dependent alterations in chromosomal architecture and cooperative enhancer activation.
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