Histone H3 lysine-9 methyltransferase G9a/EHMT2/KMT1C is a key corepressor of gene expression. However, activation of a limited number of genes by G9a (independent of its catalytic activity) has also been observed, although the precise molecular mechanisms are unknown. By using RNAi in combination with gene expression microarray analysis, we found that G9a functions as a positive and a negative transcriptional coregulator for discrete subsets of genes that are regulated by the hormone-activated Glucocorticoid Receptor (GR). G9a was recruited to GR-binding sites (but not to the gene body) of its target genes and interacted with GR, suggesting recruitment of G9a by GR. In contrast to its corepressor function, positive regulation of gene expression by G9a involved G9a-mediated enhanced recruitment of coactivators CARM1 and p300 to GR target genes. Further supporting a role for G9a as a molecular scaffold for its coactivator function, the G9a-specific methyltransferase inhibitor UNC0646 did not affect G9a coactivator function but selectively decreased G9a corepressor function for endogenous target genes. Overall, G9a functioned as a coactivator for hormone-activated genes and as a corepressor in support of hormone-induced gene repression, suggesting that the positive or negative actions of G9a are determined by the gene-specific regulatory environment and chromatin architecture. These findings indicate distinct mechanisms of G9a coactivator vs. corepressor functions in transcriptional regulation and provide insight into the molecular mechanisms of G9a coactivator function. Our results also suggest a physiological role of G9a in fine tuning the set of genes that respond to glucocorticoids.
Pre-mRNA splicing and polyadenylation are tightly connected to transcription, and transcriptional stimuli and elongation dynamics can affect mRNA maturation. However, whether this regulatory mechanism has a physio/pathological impact is not known. In cancer, where splice variant expression is often deregulated, many mutated oncogenes are transcriptional regulators. In particular, the Ewing sarcoma (EwSa) oncogene, resulting from a fusion of the EWS and FLI1 genes, encodes a well characterized transcription factor. EWS-FLI1 directly stimulates transcription of the CCND1 protooncogene encoding cyclin D1a and a less abundant but more oncogenic splice isoform, D1b. We show that, although both EWS and EWS-FLI1 enhance cyclin D1 gene expression, they regulate the D1b/D1a transcript ratio in an opposite manner. Detailed analyses of RNA polymerase dynamics along the gene and of the effects of an inhibitor of elongation show that EWS-FLI1 favors D1b isoform expression by decreasing the elongation rate, whereas EWS has opposite effects. As a result, the D1b/D1a ratio is elevated in EwSa cell lines and tumors. The endogenous D1b protein is enriched in nuclei, where the oncogenic activity of cyclin D1 is known to occur, and depleting D1b in addition to D1a results in a stronger reduction of EwSa cell growth than depleting D1a only. These data show that elevated expression of a splice isoform in cancer can be due to an alteration of the transcription process by a mutated transcriptional regulator and provide evidence for a physio/pathological impact of the coupling between transcription and mRNA maturation.coregulator ͉ Ewing sarcoma ͉ EWS-FLI1 ͉ polyadenylation ͉ splicing G ene expression in cancer cells is altered at the transcriptional level by many mutated oncogenes acting as transcriptional regulators. A second level of gene expression that is often altered in cancer cells is pre-mRNA splicing. Indeed, most human genes give rise to several transcripts with different exon content because of alternative splicing and alternative cleavage/ polyadenylation sites (1). Genes involved in major cellular programs often give rise to splice isoforms with distinct biological activities and deregulated expression in cancer (2, 3). In some cases, cancer-associated deregulation of alternative splicing arises from mutations within splicing regulatory sequences or from alterations of the expression of splicing factors involved in splicing regulation (2, 3). However, only few splicing factors have been found to be altered in cancer. Moreover, the role of another level of splicing regulation that involves transcriptional regulators has not been investigated yet.It is now widely accepted that pre-mRNA splicing and 3Ј-end maturation are tightly connected to transcription in Metazoans and that transcription impacts RNA processing (4, 5). It has been shown that the recruitment of processing factors and the maturation of pre-mRNAs occur at least in part cotranscriptionally and are enhanced by RNA polymerase II (Pol II) and its phosphorylation (...
Background:The transcriptional corepressor G9a also activates genes by an unknown mechanism. Results: The N-terminal region of G9a binds estrogen receptor ␣ and is necessary and sufficient for enhancing estrogen-induced gene activation and for occupancy of G9a on target genes. Conclusion: The domains of G9a responsible for activation and inhibition of transcription are different. Significance: G9a has inherent coactivator as well as corepressor activity.
Like many transcription regulators, histone methyltransferases G9a and G9a-like protein (GLP) can act gene-specifically as coregulators, but mechanisms controlling this specificity are mostly unknown. We show that adjacent post-translational methylation and phosphorylation regulate binding of G9a and GLP to heterochromatin protein 1 gamma (HP1γ), formation of a ternary complex with the glucocorticoid receptor (GR) on chromatin, and function of G9a and GLP as coactivators for a subset of GR target genes. HP1γ is recruited by G9a and GLP to GR binding sites associated with genes that require G9a, GLP, and HP1γ for glucocorticoid-stimulated transcription. At the physiological level, G9a and GLP coactivator function is required for glucocorticoid activation of genes that repress cell migration in A549 lung cancer cells. Thus, regulated methylation and phosphorylation serve as a switch controlling G9a and GLP coactivator function, suggesting that this mechanism may be a general paradigm for directing specific transcription factor and coregulator actions on different genes.
Progesterone receptor (PgR) controls the menstrual cycle, pregnancy, embryonic development, and homeostasis, and it plays important roles in breast cancer development and progression. However, the requirement of coregulators for estrogen-induced expression of the PgR gene has not been fully explored. Here we used RNA interference to demonstrate dramatic differences in requirements of 10 different coregulators for estrogen-regulated expression of six different genes, including PgR and the well-studied TFF1 (or pS2) gene in MCF-7 breast cancer cells. Full estrogen-induced expression of TFF1 required all ten coregulators, but PgR induction required only four of the 10 coregulators. Chromatin immunoprecipitation studies demonstrated several mechanisms responsible for the differential coregulator requirements. Actin-binding coregulator Flightless-I, required for TFF1 expression and recruited to that gene by estrogen receptor-α (ERα), is not required for PgR expression and not recruited to that gene. Protein acetyltransferase tat-interactive protein 60 and ATP-dependent chromatin remodeler Brahma Related Gene 1 are recruited to both genes but are required only for TFF1 expression. Histone methyltransferase G9a is recruited to both genes and required for estrogen-induced expression of TFF1 but negatively regulates estrogen-induced expression of PgR. In contrast, histone methyltransferase myeloid/lymphoid or mixed-lineage leukemia 1 (MLL1), pioneer factor Forkhead box A1, and p160 coregulator steroid receptor coactivator-3 are required for expression of and are recruited to both genes. Depletion of MLL1 decreased ERα binding to the PgR and TFF1 genes. In contrast, depletion of G9a enhanced ERα binding to the PgR gene but had no effect on ERα binding to the TFF1 gene. These studies suggest that differential promoter architecture is responsible for promoter-specific mechanisms of gene regulation.
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