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，起转录共调控因子的作用。通过与序列特异性转录因子和CBP/p300相互作用，被引用的蛋白质影响CBP/p300依赖性转录。重要的是，所引用的蛋白的共调节活性表现出显著的基因特异性；与引文相互作用的转录因子与启动子的结合是必要的，但不是充分的，这是引文蛋白质募集到启动子的必要条件。由于目前仅有TGF-a、c-MYC和糖蛋白a亚基基因3个基因被确定为cites调控基因，我们对这些被引用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cites调控基因，并阐明被引蛋白基因特异性协同调控活性的生化基础。在Specific Aim 1.1中，为了深入了解cites调控的基因，将检查缺乏被引用基因（cited1、cited2和cited4）的敲除小鼠的表型。为了确定候选的cites调控基因，将确定受影响组织的mRNA表达谱。在Specific Aim 1.2中，为了获得鉴定新的cites调控启动子的线索，将尝试通过染色质免疫沉淀分离招募被引用蛋白的基因组DNA片段。在Specific Aim 1.3中，我们将研究CITED4在三个候选CITED4调控基因转录中的作用。在人骨肉瘤细胞中，这些候选基因的mRNA转录量随着诱导过表达CITED4而增加。将尝试通过基于细胞培养的分析来证明CITED4直接参与这些基因的转录激活：报告基因测定和染色质免疫沉淀。我们将使用CITED4敲除小鼠来评估CITED4在其调控中的生理作用。最后，在特异性目标2中，将通过染色质免疫沉淀检测培养细胞中已知的引用1调节启动子的蛋白因子的募集和修饰。

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.