Regulation of a dual functional nuclear receptor coactivator CARM1

双功能核受体共激活因子 CARM1 的调节

• 批准号: 7389549
• 负责人: Qin Feng
• 金额: $ 5.29万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7389549
• 关键词: Arginine; Attenuated; Binding; Biochemical; Biological Assay; Breast; Cells; Cellular biology; Chimeric Proteins; Chromatin; Co-Immunoprecipitations; Code; Complex; Estrogen Receptor alpha; Estrogen Receptors; Estrogens; Family; Female; Gene Targeting; Genes; Genetic Transcription; Hela Cells; Histone H3; Histones; Hormones; Immunoprecipitation; In Vitro; Laboratories; Light; MCF7 cell; Mammary Neoplasms; Mediating; Methods; Methylation; Methyltransferase; Names; Nuclear; Nuclear Receptors; Oncogenes; Pattern; Phosphorylation; Phosphorylation Site; Phosphotransferases; Post-Translational Protein Processing; Proteins; Recombinants; Regulation; Reporter; Reporting; Role; Signal Pathway; Signal Transduction; Site; Site-Directed Mutagenesis; Steroid Receptors; Substrate Specificity; System; Testing; Training; Transcript; Transcription Initiation; Transcriptional Activation; Transfection; base; coactivator-associated arginine methyltransferase 1; design; expression cloning; in vivo; malignant breast neoplasm; mutant; novel; polypeptide; protein degradation; receptor; reproductive function; response; tumorigenesis

DESCRIPTION (provided by applicant): The estrogen signaling pathway is pivotal for maintaining female reproductive function and contributes extensively to breast tumorigenesis. Estrogen exerts its effect mainly through binding to its cognate receptors, estrogen receptor (ER) alpha and beta, resulting in recruitment of coactivators and subsequent transcriptional activation of estrogen-dependent genes. Studies over last decade have established the critical roles of nuclear receptor coactivators (including the SRC family of coactivators and CARM1) in estrogen receptor-mediated transcription. Our ongoing studies indicate that CARM1 is a dual-functional nuclear receptor coactivator, as it not only activates transcription by methylating core histone, but also terminates hormone induced transcription by methylating non-histone coactivators and resulting in disassembly of the coactivator complex. One critical question arises about how CARM1 selectively methylates histone and non-histone coactivators in an appropriate order during transcription initiation. Although intensive studies from different laboratories have been focused on identification of CARM1 substrates, very little is known about how the functions of CARM1 are regulated by diverse signals in the cells. We hypothesize that enzymatic activity and substrate specificity of CARM1 are regulated by posttranslational modifications such as phosphorylation and/or CARM1 interacting proteins. The specific aims of this proposal are to: 1. Determine if SRC-3 needs a phosphorylation code to be methylated by CARM1 with in vitro methylation assay. 2. Identify the phosphorylation sites of CARM1 by mass spectrometric analysis. 3. Determine the role of phosphorylation on CARM1 enzymatic activity and substrate specificity. Various assays including in vitro methylation and cell-free chromatin-based in vitro transcription will be used. 4. Investigate the roles of the CARM1 interacting proteins on CARM1 functions by different biochemical and cell biology methods. The SRC-3/AIB1 is an authentic oncogene and its transcript was reported to be over-expressed in 64% of primary breast cancers. In our current study, we found that SRC-3/AIB1 is methylated by CARM1, and methylation of SRC-3 attenuates its coactivator activity. Since CARM1 is the sole methyltransferase responsible for SRC-3 arginine methylation, understanding how CARM1 activity is regulated will not only shed more light on the mechanisms by which nuclear receptors guide transcription, but also will provide further information for design of novel and effective therapy to treat breast tumors.

描述（由申请人提供）：雌激素信号通路对于维持女性生殖功能至关重要，并广泛促进乳腺肿瘤发生。雌激素主要通过与其同源受体雌激素受体（ER）α和β结合发挥其作用，导致辅激活因子的募集和随后雌激素依赖性基因的转录激活。过去十年的研究已经确定了核受体辅激活因子（包括SRC家族的辅激活因子和CARM 1）在雌激素受体介导的转录中的关键作用。我们正在进行的研究表明，CARM 1是一种双功能核受体共激活因子，因为它不仅通过甲基化核心组蛋白激活转录，而且通过甲基化非组蛋白共激活因子终止激素诱导的转录，并导致共激活因子复合物的分解。一个关键的问题是，在转录起始过程中，CARM 1如何以适当的顺序选择性地甲基化组蛋白和非组蛋白共激活因子。虽然来自不同实验室的深入研究一直集中在识别CARM 1底物上，但关于CARM 1的功能如何受到细胞中不同信号的调节知之甚少。我们推测，酶活性和底物特异性的CARM 1的调节翻译后修饰，如磷酸化和/或CARM 1相互作用蛋白。这项建议的具体目标是：1.通过体外甲基化试验确定SRC-3是否需要磷酸化密码被CARM 1甲基化。2.通过质谱分析鉴定CARM 1的磷酸化位点。3.确定磷酸化对CARM 1酶活性和底物特异性的作用。将使用各种测定，包括体外甲基化和基于无细胞染色质的体外转录。4.通过不同的生物化学和细胞生物学方法研究CARM 1相互作用蛋白对CARM 1功能的作用。SRC-3/AIB 1是一种真正的癌基因，据报道其转录本在64%的原发性乳腺癌中过表达。在我们目前的研究中，我们发现SRC-3/AIB 1被CARM 1甲基化，SRC-3的甲基化减弱了其共激活因子活性。由于CARM 1是负责SRC-3精氨酸甲基化的唯一甲基转移酶，因此了解CARM 1活性是如何调节的，不仅将进一步阐明核受体引导转录的机制，而且还将为设计治疗乳腺肿瘤的新型有效疗法提供进一步的信息。