CircPVT1 promotes ER-positive breast tumorigenesis and drug resistance by targeting ESR1 and MAVS.

The molecular mechanisms underlying estrogen receptor (ER)-positive breast carcinogenesis and endocrine therapy resistance remain incompletely understood. Here, we report that circPVT1, a circular RNA generated from the lncRNA PVT1, is highly expressed in ERα-positive breast cancer cell lines and tumor samples and is functionally important in promoting ERα-positive breast tumorigenesis and endocrine therapy resistance. CircPVT1 acts as a competing endogenous RNA (ceRNA) to sponge miR-181a-2-3p, promoting the expression of ESR1 and downstream ERα-target genes and breast cancer cell growth. Furthermore, circPVT1 directly interacts with MAVS protein to disrupt the RIGI-MAVS complex formation, inhibiting type I interferon (IFN) signaling pathway and anti-tumor immunity. Anti-sense oligonucleotide (ASO)-targeting circPVT1 inhibits ERα-positive breast cancer cell and tumor growth, re-sensitizing tamoxifen-resistant ERα-positive breast cancer cells to tamoxifen treatment. Taken together, our data demonstrated that circPVT1 can work through both ceRNA and protein scaffolding mechanisms to promote cancer. Thus, circPVT1 may serve as a diagnostic biomarker and therapeutic target for ERα-positive breast cancer in the clinic.

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.

JMJD6 and U2AF65 co-regulate alternative splicing in both JMJD6 enzymatic activity dependent and independent manner.

JMJD6, a jumonji C (Jmj C) domain-containing protein demethylase and hydroxylase, has been implicated in an array of biological processes. It has been shown that JMJD6 interacts with and hydroxylates multiple serine/arginine-rich (SR) proteins and SR related proteins, including U2AF65, all of which are known to function in alternative splicing regulation. However, whether JMJD6 is widely involved in alternative splicing and the molecular mechanism underlying JMJD6-regulated alternative splicing have remained incompletely understood. Here, by using RASL-Seq, we investigated the functional impact of RNA-dependent interaction between JMJD6 and U2AF65, revealing that JMJD6 and U2AF65 co-regulated a large number of alternative splicing events. We further demonstrated the JMJD6 function in alternative splicing in jmjd6 knockout mice. Mechanistically, we showed that the enzymatic activity of JMJD6 was required for a subset of JMJD6-regulated splicing, and JMJD6-mediated lysine hydroxylation of U2AF65 could account for, at least partially, their co-regulated alternative splicing events, suggesting both JMJD6 enzymatic activity-dependent and independent control of alternative splicing. These findings reveal an intimate link between JMJD6 and U2AF65 in alternative splicing regulation, which has important implications in development and disease processes.

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.

Estrogen-induced circRNA, circPGR, functions as a ceRNA to promote estrogen receptor-positive breast cancer cell growth by regulating cell cycle-related genes.

Estrogen and estrogen receptor (ER)-regulated gene transcriptional events have been well known to be involved in ER-positive breast carcinogenesis. Meanwhile, circular RNAs (circRNAs) are emerging as a new family of functional non-coding RNAs (ncRNAs) with implications in a variety of pathological processes, such as cancer. However, the estrogen-regulated circRNA program and the function of such program remain uncharacterized. Methods: CircRNA sequencing (circRNA-seq) was performed to identify circRNAs induced by estrogen, and cell proliferation, colony formation, wound healing, transwell and tumor xenograft experiments were applied to examine the function of estrogen-induced circRNA, circPGR. RNA sequencing (RNA-seq) and ceRNA network analysis wereperformed to identify circPGR's target genes and the microRNA (miRNA) bound to circPGR. Anti-sense oligonucleotide (ASO) was used to assess circPGR's effects on ER-positive breast cancer cell growth. Results: Genome-wide circRNA profiling by circRNA sequencing (circRNA-seq) revealed that a large number of circRNAs were induced by estrogen, and further functional screening for the several circRNAs originated from PGR revealed that one of them, which we named as circPGR, was required for ER-positive breast cancer cell growth and tumorigenesis. CircPGR was found to be localized in the cytosol of cells and functioned as a competing endogenous RNA (ceRNA) to sponge miR-301a-5p to regulate the expression of multiple cell cycle genes. The clinical relevance of circPGR was underscored by its high and specific expression in ER-positive breast cancer cell lines and clinical breast cancer tissue samples. Accordingly, anti-sense oligonucleotide (ASO) targeting circPGR was proven to be effective in suppressing ER-positive breast cancer cell growth. Conclusions: These findings reveled that, besides the well-known messenger RNA (mRNA), microRNA (miRNA), long non-coding RNA (lncRNA) and enhancer RNA (eRNA) programs, estrogen also induced a circRNA program, and exemplified by circPGR, these estrogen-induced circRNAs were required for ER-positive breast cancer cell growth, providing a new class of therapeutic targets for ER-positive breast cancer.

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.

DDB1 binds histone reader BRWD3 to activate the transcriptional cascade in adipogenesis and promote onset of obesity.

Obesity has become a global pandemic. Identification of key factors in adipogenesis helps to tackle obesity and related metabolic diseases. Here, we show that DDB1 binds the histone reader BRWD3 to promote adipogenesis and diet-induced obesity. Although typically recognized as a component of the CUL4-RING E3 ubiquitin ligase complex, DDB1 stimulates adipogenesis independently of CUL4. A DDB1 mutant that does not bind CUL4A or CUL4B fully restores adipogenesis in DDB1-deficient cells. Ddb1+/- mice show delayed postnatal development of white adipose tissues and are protected from diet-induced obesity. Mechanistically, by interacting with BRWD3, DDB1 is recruited to acetylated histones in the proximal promoters of ELK1 downstream immediate early response genes and facilitates the release of paused RNA polymerase II, thereby activating the transcriptional cascade in adipogenesis. Our findings have uncovered a CUL4-independent function of DDB1 in promoting the transcriptional cascade of adipogenesis, development of adipose tissues, and onset of obesity.

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.

Methylation of transcription factor YY2 regulates its transcriptional activity and cell proliferation.

Yin Yang 1 (YY1) is a multifunctional DNA-binding transcription factor shown to be critical in a variety of biological processes, and its activity and function have been shown to be regulated by multitude of mechanisms, which include but are not limited to post-translational modifications (PTMs), its associated proteins and cellular localization. YY2, the paralog of YY1 in mouse and human, has been proposed to function redundantly or oppositely in a context-specific manner compared with YY1. Despite its functional importance, how YY2's DNA-binding activity and function are regulated, particularly by PTMs, remains completely unknown. Here we report the first PTM with functional characterization on YY2, namely lysine 247 monomethylation (K247me1), which was found to be dynamically regulated by SET7/9 and LSD1 both in vitro and in cultured cells. Functional study revealed that SET7/9-mediated YY2 methylation regulated its DNA-binding activity in vitro and in association with chromatin examined by chromatin immunoprecipitation coupled with sequencing (ChIP-seq) in cultured cells. Knockout of YY2, SET7/9 or LSD1 by CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9-mediated gene editing followed by RNA sequencing (RNA-seq) revealed that a subset of genes was positively regulated by YY2 and SET7/9, but negatively regulated by LSD1, which were enriched with genes involved in cell proliferation regulation. Importantly, YY2-regulated gene transcription, cell proliferation and tumor growth were dependent, at least partially, on YY2 K247 methylation. Finally, somatic mutations on YY2 found in cancer, which are in close proximity to K247, altered its methylation, DNA-binding activity and gene transcription it controls. Our findings revealed the first PTM with functional implications imposed on YY2 protein, and linked YY2 methylation with its biological functions.

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.