A CARM1 Inhibitor Potently Suppresses Breast Cancer Both In Vitro and In Vivo.

CARM1, belonging to the protein arginine methyltransferase (PRMT) family, is intricately associated with the progression of cancer and is viewed as a promising target for both cancer diagnosis and therapy. However, the number of specific and potent CARM1 inhibitors is limited. We herein discovered a CARM1 inhibitor, iCARM1, that showed better specificity and activity toward CARM1 compared to the known CARM1 inhibitors, EZM2302 and TP-064. Similar to CARM1 knockdown, iCARM1 suppressed the expression of oncogenic estrogen/ERα-target genes, whereas activated type I interferon (IFN) and IFN-induced genes (ISGs) in breast cancer cells. Consequently, iCARM1 potently suppressed breast cancer cell growth both in vitro and in vivo. The combination of iCARM1 with either endocrine therapy drugs or etoposide demonstrated synergistic effects in inhibiting the growth of breast tumors. In summary, targeting CARM1 by iCARM1 effectively suppresses breast tumor growth, offering a promising therapeutic approach for managing breast cancers in clinical settings.

CARM1 hypermethylates the NuRD chromatin remodeling complex to promote cell cycle gene expression and breast cancer development.

Protein arginine methyltransferase CARM1 has been shown to methylate a large number of non-histone proteins, and play important roles in gene transcriptional activation, cell cycle progress, and tumorigenesis. However, the critical substrates through which CARM1 exerts its functions remain to be fully characterized. Here, we reported that CARM1 directly interacts with the GATAD2A/2B subunit in the nucleosome remodeling and deacetylase (NuRD) complex, expanding the activities of NuRD to include protein arginine methylation. CARM1 and NuRD bind and activate a large cohort of genes with implications in cell cycle control to facilitate the G1 to S phase transition. This gene activation process requires CARM1 to hypermethylate GATAD2A/2B at a cluster of arginines, which is critical for the recruitment of the NuRD complex. The clinical significance of this gene activation mechanism is underscored by the high expression of CARM1 and NuRD in breast cancers, and the fact that knockdown CARM1 and NuRD inhibits cancer cell growth in vitro and tumorigenesis in vivo. Targeting CARM1-mediated GATAD2A/2B methylation with CARM1 specific inhibitors potently inhibit breast cancer cell growth in vitro and tumorigenesis in vivo. These findings reveal a gene activation program that requires arginine methylation established by CARM1 on a key chromatin remodeler, and targeting such methylation might represent a promising therapeutic avenue in the clinic.

The Dual Function of KDM5C in Both Gene Transcriptional Activation and Repression Promotes Breast Cancer Cell Growth and Tumorigenesis.

Emerging evidence suggested that epigenetic regulators can exhibit both activator and repressor activities in gene transcriptional regulation and disease development, such as cancer. However, how these dual activities are regulated and coordinated in specific cellular contexts remains elusive. Here, it is reported that KDM5C, a repressive histone demethylase, unexpectedly activates estrogen receptor alpha (ERα)-target genes, and meanwhile suppresses type I interferons (IFNs) and IFN-stimulated genes (ISGs) to promote ERα-positive breast cancer cell growth and tumorigenesis. KDM5C-interacting protein, ZMYND8, is found to be involved in both processes. Mechanistically, KDM5C binds to active enhancers and recruits the P-TEFb complex to activate ERα-target genes, while inhibits TBK1 phosphorylation in the cytosol to repress type I IFNs and ISGs. Pharmacological inhibition of both ERα and KDM5C is effective in inhibiting cell growth and tumorigenesis. Taken together, it is revealed that the dual activator and repressor nature of an epigenetic regulator together contributes to cancer development.

Profiling PRMT methylome reveals roles of hnRNPA1 arginine methylation in RNA splicing and cell growth.

Numerous substrates have been identified for Type I and II arginine methyltransferases (PRMTs). However, the full substrate spectrum of the only type III PRMT, PRMT7, and its connection to type I and II PRMT substrates remains unknown. Here, we use mass spectrometry to reveal features of PRMT7-regulated methylation. We find that PRMT7 predominantly methylates a glycine and arginine motif; multiple PRMT7-regulated arginine methylation sites are close to phosphorylations sites; methylation sites and proximal sequences are vulnerable to cancer mutations; and methylation is enriched in proteins associated with spliceosome and RNA-related pathways. We show that PRMT4/5/7-mediated arginine methylation regulates hnRNPA1 binding to RNA and several alternative splicing events. In breast, colorectal and prostate cancer cells, PRMT4/5/7 are upregulated and associated with high levels of hnRNPA1 arginine methylation and aberrant alternative splicing. Pharmacological inhibition of PRMT4/5/7 suppresses cancer cell growth and their co-inhibition shows synergistic effects, suggesting them as targets for cancer therapy.

A hypermethylation strategy utilized by enhancer-bound CARM1 to promote estrogen receptor α-dependent transcriptional activation and breast carcinogenesis.

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.

JMJD6 Licenses ERα-Dependent Enhancer and Coding Gene Activation by Modulating the Recruitment of the CARM1/MED12 Co-activator Complex.

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.

Regulation of Transcription Factor Yin Yang 1 by SET7/9-mediated Lysine Methylation.

Yin Yang 1 (YY1) is a multifunctional transcription factor shown to be critical in a variety of biological processes. Although it is regulated by multiple types of post-translational modifications (PTMs), whether YY1 is methylated, which enzyme methylates YY1, and hence the functional significance of YY1 methylation remains completely unknown. Here we reported the first methyltransferase, SET7/9 (KMT7), capable of methylating YY1 at two highly conserved lysine (K) residues, K173 and K411, located in two distinct domains, one in the central glycine-rich region and the other in the very carboxyl-terminus. Functional studies revealed that SET7/9-mediated YY1 methylation regulated YY1 DNA-binding activity both in vitro and at specific genomic loci in cultured cells. Consistently, SET7/9-mediated YY1 methylation was shown to involve in YY1-regulated gene transcription and cell proliferation. Our findings revealed a novel regulatory strategy, methylation by lysine methyltransferase, imposed on YY1 protein, and linked YY1 methylation with its biological functions.

Arginine methylation of HSP70 regulates retinoid acid-mediated RARß2 gene activation.

Although "histone" methyltransferases and demethylases are well established to regulate transcriptional programs and to use nonhistone proteins as substrates, their possible roles in regulation of heat-shock proteins in the nucleus have not been investigated. Here, we report that a highly conserved arginine residue, R469, in HSP70 (heat-shock protein of 70 kDa) proteins, an evolutionarily conserved protein family of ATP-dependent molecular chaperone, was monomethylated (me1), at least partially, by coactivator-associated arginine methyltransferase 1/protein arginine methyltransferase 4 (CARM1/PRMT4) and demethylated by jumonji-domain-containing 6 (JMJD6), both in vitro and in cultured cells. Functional studies revealed that HSP70 could directly regulate retinoid acid (RA)-induced retinoid acid receptor ß2 (RARß2) gene transcription through its binding to chromatin, with R469me1 being essential in this process. HSP70's function in gene transcriptional regulation appears to be distinct from its protein chaperon activity. R469me1 was shown to mediate the interaction between HSP70 and TFIIH, which involves in RNA polymerase II phosphorylation and thus transcriptional initiation. Our findings expand the repertoire of nonhistone substrates targeted by PRMT4 and JMJD6, and reveal a new function of HSP70 proteins in gene transcription at the chromatin level aside from its classic role in protein folding and quality control.