Functions of MSG1 Family Transcription Activators

描述（由申请人提供）：CITED家族的成员，以前称为MSG 1家族，作为转录辅助调节因子发挥作用。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蛋白的共调节活性显示出显著的基因特异性; CITED相互作用转录因子与启动子的结合对于CITED蛋白向启动子的募集是必要的，但不是足够的。由于迄今为止只有三个基因-TGF-α、c-MYC和糖蛋白α亚基基因-被鉴定为CITED调节基因，因此我们对CITED蛋白的生物学作用和功能的分子机制的了解仍然非常有限。因此，在本项目中，将尝试鉴定更多的CITED调节基因，并阐明CITED蛋白的基因特异性共调节活性的生化基础。在Specific Aim 1.1中，为了深入了解CITED调控的基因，将检查缺乏引用基因（cited 1，cited 2和cited 4）的敲除小鼠的表型。为了鉴定候选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，为了获得用于鉴定新的CITED调节的启动子的线索，将尝试通过染色质免疫沉淀分离募集CITED蛋白的基因组DNA片段。在具体目标1.3中，将检查CITED 4在三个候选CITED 4调节基因的转录中的作用。在人骨肉瘤细胞中，这些候选物的mRNA转录物的量随着CITED 4的诱导过表达而沿着增加。将尝试证明直接参与CITED 4在这些基因的转录激活细胞培养为基础的分析：报告分析和染色质免疫沉淀。将使用cited 4基因敲除小鼠评估CITED 4在其调节中的生理作用。最后，在特定目标2中，将通过染色质免疫沉淀来检查蛋白因子对培养细胞中已知的CITED 1调节的启动子的募集和修饰。

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.