Functions of MSG1 Family Transcription Activators

MSG1家族转录激活剂的功能

• 批准号: 6546683
• 负责人: TOSHIHIRO SHIODA
• 金额: $ 32.52万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6546683
• 关键词: DNA; DNA binding protein; biological signal transduction; chromatin; gene expression; gene targeting; genetic promoter element; genetic regulation; genetic transcription; genetically modified animals; immunoprecipitation; intermolecular interaction; laboratory mouse; microarray technology; molecular cloning; nucleic acid sequence; protein structure function; tissue /cell culture; transcription factor; transfection; western blottings

DESCRIPTION (provided by applicant): Members of the CITED family, formerly called the MSG 1 family, function as transcriptional coregulators. By interacting with both sequence-specific transcription factors and CBP/p300, the CITED proteins affect CBP/p300-dependent transcription. Importantly, the coregulating activity of the CITED proteins show remarkable gene-specificity; binding of a CITED-interacting transcription factor to a promoter is necessary - but not sufficient - for recruitment of the CITED proteins to promoters. Since only three genes - TGF-a, c-MYC, and glycoprotein a-subunit gene - have been identified as CITED-regulated genes so far, our knowledge of biological roles and molecular mechanisms of functions of the CITED proteins is still very limited. Therefore, in this project, attempts will be made to identify more CITED-regulated genes and to elucidate biochemical basis of the gene-specific coregulating activity of the CITED proteins. In Specific Aim 1.1, to obtain insights into CITED-regulated genes, phenotypes of knockout mice lacking the cited genes (cited1, cited2, and cited4) will be examined. To identify candidate CITED-regulated genes, mRNA expression profiles of the affected tissues will be determined. In Specific Aim 1.2, to obtain clues for identifying novel CITED-regulated promoters, attempts will be made to isolate genomic DNA fragments that recruit the CITED proteins by chromatin immunoprecipitation. In Specific Aim 1.3, roles of CITED4 in transcription of three candidate CITED4-regulated genes will be examined. Amounts of the mRNA transcripts of those candidates increased along with induced overexpression of CITED4 in human osteosarcoma cells. Attempts will be made to demonstrate direct involvement of CITED4 in transcriptional activation of these genes by cell culture-based analyses: reporter assays and chromatin immunoprecipitation. Physiological roles of CITED4 in their regulation will be evaluated using cited4 knockout mice. Finally, in Specific Aim 2, recruitment and modifications of protein factors to known CITED 1-regulated promoters in cultured cells will be examined by chromatin immunoprecipitation.

描述（由申请人提供）：CITED 家族的成员（以前称为 MSG 1 家族）充当转录共调节子。通过与序列特异性转录因子和 CBP/p300 相互作用，CITED 蛋白影响 CBP/p300 依赖性转录。重要的是，CITED 蛋白的共调节活性表现出显着的基因特异性。 CITED 相互作用转录因子与启动子的结合对于将 CITED 蛋白募集到启动子来说是必要的，但还不够。由于迄今为止只有三个基因——TGF-a、c-MYC和糖蛋白a亚基基因——被确定为CITED调节基因，我们对CITED蛋白的生物学作用和功能分子机制的了解仍然非常有限。因此，本项目将尝试鉴定更多的CITED调控基因，并阐明CITED蛋白基因特异性共调控活性的生化基础。在具体目标 1.1 中，为了深入了解 CITED 调节的基因，将检查缺乏引用基因（cited1、cited2 和 cited4）的基因敲除小鼠的表型。为了确定候选 CITED 调节基因，将确定受影响组织的 mRNA 表达谱。在具体目标 1.2 中，为了获得识别新型 CITED 调控启动子的线索，将尝试通过染色质免疫沉淀来分离招募 CITED 蛋白的基因组 DNA 片段。在具体目标 1.3 中，将检查 CITED4 在三个候选 CITED4 调控基因转录中的作用。这些候选者的 mRNA 转录物数量随着 CITED4 在人骨肉瘤细胞中的诱导过度表达而增加。将尝试通过基于细胞培养的分析（报告基因测定和染色质免疫沉淀）来证明 CITED4 直接参与这些基因的转录激活。 CITED4在其调节中的生理作用将使用cited4敲除小鼠进行评估。最后，在具体目标 2 中，将通过染色质免疫沉淀检查培养细胞中已知 CITED 1 调节启动子的蛋白质因子的募集和修饰。