Evidence that pre-mRNA processing events are temporally and, in some cases, mechanistically coupled to transcription has led to the proposal that RNA polymerase II (Pol II) recruits pre-mRNA splicing factors to active genes. Here we address two key questions raised by this proposal: (i) whether the U1 snRNP, which binds to the 5 splice site of each intron, is recruited cotranscriptionally in vivo and, (ii) if so, where along the length of active genes the U1 snRNP is concentrated. Using chromatin immunoprecipitation (ChIP) in yeast, we show that elevated levels of the U1 snRNP were specifically detected in gene regions containing introns and downstream of introns but not along the length of intronless genes. In contrast to capping enzymes, which bind directly to Pol II, the U1 snRNP was poorly detected in promoter regions, except in genes harboring promoterproximal introns. Detection of the U1 snRNP was dependent on RNA synthesis and was abolished by intron removal. Microarray analysis revealed that intron-containing genes were preferentially selected by ChIP with the U1 snRNP. Thus, U1 snRNP accumulation at genes correlated with the presence and position of introns, indicating that introns are necessary for cotranscriptional U1 snRNP recruitment and/or retention.Pre-mRNA splicing is a two-step transesterification reaction carried out by the spliceosome, a large and dynamic multicomponent RNA-protein complex (52). The first steps in the assembly of the spliceosome on pre-mRNA involve the recognition of the 5Ј and 3Ј ends of each intron (5Ј and 3Ј splice sites) by small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP splicing factors. Regulation of this process determines splice site usage in alternative pre-mRNA splicing (50). A report that 40 to 60% of human genes are alternatively spliced to produce multiple gene products (26) underscores the importance of understanding splice site recognition and subsequent spliceosome assembly. Although much progress has been made in recent years toward understanding the biochemical activities of many splicing regulators, it has been difficult to establish systems for examining the roles of such regulators on endogenous pre-mRNAs in vivo and the mechanisms by which they are recruited.An important clue to understanding how splicing factors might initially assemble on pre-mRNA is provided by observations that splicing begins and is sometimes completed cotranscriptionally (for a review, see reference 39). For a number of genes, intron removal has been detected in nascent RNAs still tethered to the DNA axis by RNA polymerase II (Pol II) (3,5,42,53,54,56). Evidence that transcription rates and promoter identity influence alternative splice site selection is consistent with a cotranscriptional splicing mechanism in humans (9, 21, 45) and yeast (K. J. Howe, C. M. Kane, and M. Ares, unpublished data). The findings that the C-terminal domain (CTD) of RNA Pol II is required for efficient capping, splicing, and polyadenylation of pre-mRNA (33) and specifically stimulates spl...

Abnormal activities of histone lysine demethylases (KDMs) and lysine deacetylases (HDACs) are associated with aberrant gene expression in breast cancer development. However, the precise molecular mechanisms underlying the crosstalk between KDMs and HDACs in chromatin remodeling and regulation of gene transcription are still elusive. In this study, we showed that treatment of human breast cancer cells with inhibitors targeting the zinc cofactor dependent class I/II HDAC, but not NAD+ dependent class III HDAC, led to significant increase of H3K4me2 which is a specific substrate of histone lysine-specific demethylase 1 (LSD1) and a key chromatin mark promoting transcriptional activation. We also demonstrated that inhibition of LSD1 activity by a pharmacological inhibitor, pargyline, or siRNA resulted in increased acetylation of H3K9 (AcH3K9). However, siRNA knockdown of LSD2, a homolog of LSD1, failed to alter the level of AcH3K9, suggesting that LSD2 activity may not be functionally connected with HDAC activity. Combined treatment with LSD1 and HDAC inhibitors resulted in enhanced levels of H3K4me2 and AcH3K9, and exhibited synergistic growth inhibition of breast cancer cells. Finally, microarray screening identified a unique subset of genes whose expression was significantly changed by combination treatment with inhibitors of LSD1 and HDAC. Our study suggests that LSD1 intimately interacts with histone deacetylases in human breast cancer cells. Inhibition of histone demethylation and deacetylation exhibits cooperation and synergy in regulating gene expression and growth inhibition, and may represent a promising and novel approach for epigenetic therapy of breast cancer.

Histone acetylation and methylation are two important epigenetic entities that interact with each other in chromatin remodeling and gene transcription regulation. It has been shown that abnormal activities of histone lysine deacetylases (HDACs) and histone lysine demethylases (KDMs) are associated with aberrant gene expression in cancer development. However, the precise molecular mechanisms underlying the crosstalk between HDACs and KDMs are still elusive. In this study, we showed that exposure of human breast cancer MDA-MB-231 and MDA-MB-468 cells to inhibitors against the zinc cofactor dependent class I or II HDACs, but not NAD+ dependent class III HDAC, led to significant increases of global di or mono-methylation of histone 3 lysine 4 (H3K4me2/me1) which are specific substrates of histone lysine-specific demethylase 1 (LSD1) and key positive chromatin marks promoting transcriptional activation. HDAC inhibitors did not affect in vitro demethylation activity of recombinant LSD1 and did not alter the nuclear presence of the LSD-CoREST-HDAC1/2 complex, indicating the inhibition of LSD1 activity in breast cancer cells by specific HDAC inhibitors likely occurs through disrupting the functional interaction between LSD1 and HDACs rather than direct competition with enzymatic substrate at the active site of LSD1 or suppression of LSD1 protein levels. Inhibition of LSD1 activity by its inhibitor, pargyline, or RNAi in MDA-MB-231 cells caused an increase in acetylation of H3K9 (AcH3K9), suggesting that LSD1 is an important regulator of HDAC activity in breast cancer. Chromatin immunoprecipitation (ChIP) demonstrated that LSD1/HDAC repressive complex is recruited to the promoter of one candidate target gene, estrogen receptor alpha (ERα), in ER negative breast cancer cells and the reactivation of silenced ERα by HDAC inhibitor is associated with increased H3K4me2, acetylH3K9 and acetylH4K16 at ERα promoter. In addition, combined treatment with HDAC inhibitors and the LSD1 inhibitor, pargyline, resulted in enhanced levels of H3K4me2 and AcH3K9, and synergistic growth inhibition of MDA-MB-231 cells. These data suggest a crosstalk between the histone demethylase, LSD1, and histone deacetylases as a novel regulatory mechanism in mediating activity of HDAC inhibitors and may help in designing combinations that could have significant clinical translation in breast cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1993. doi:10.1158/1538-7445.AM2011-1993