ROLE OF CBF/NF-Y IN TRANSCRIPTION: TYPE I COLLAGEN GENES

CBF/NF-Y 在转录中的作用：I 型胶原蛋白基因

• 批准号: 6375246
• 负责人: SANKAR N MAITY
• 金额: $ 26.25万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6375246
• 关键词: binding sites; cell growth regulation; cell line; collagen; differential display technique; fibroblasts; gene induction /repression; genetic promoter element; immunoprecipitation; mutant; transcription factor

DESCRIPTION (Adapted from applicant's abstract): Type I collagen, a heterotrimer consisting of two a1(I) and one a2(I) polypeptides, is the most abundant protein found in skin, bone, and tendons and deregulated synthesis of type I collagen occurs in a number of pathological conditions. The CCAAT-binding factor (CBF) is a critical trans-acting factor that activates the in vitro transcription of both the a1(I) and a2(I) collagen promoters, as well as other promoters. CBF consists of three subunits, CBF-A, CBF-B, and CBF-C, which are all needed for DNA binding. The segment responsible for the formation of the CBF-DNA complex in each subunits is conserved from yeast to human, and in two of them, CBF-A and CBF-C, is homologous to the histone-fold motif. In vivo inhibition of CBF function by a dominant negative CBF mutant expressed in mouse fibroblasts inhibited cell growth and decreased expression of the collagen gene, as well as several other genes that are regulated during cell growth. In the proposed study, a comprehensive analysis will be done using the differential display and cDNA expression array to identify genes whose expression is decreased or increased as a result of inhibition of CBF binding in fibroblasts. The in vivo function of CBF will be studied in different mammalian cell lines using the dominant negative CBF mutant. The transcription activation function of CBF will be analyzed in an in vitro reconstituted system to identify the nuclear factor that associates with CBF and mediates the activation of CBF-dependent transcription. To study the transcriptional activation by recombinant CBF subunits in vivo, altered-specificity CBF mutants, that bind to a mutant CBF binding site will be created to test activity of the a1(I) and a2(I) collagen promoters in mouse fibroblasts.

描述（改编自申请人的摘要）：I 型胶原蛋白，一种 由两个 a1(I) 和一个 a2(I) 多肽组成的异源三聚体是最常见的 皮肤、骨骼和肌腱中含有丰富的蛋白质，并且蛋白质的合成失调 I 型胶原蛋白出现在多种病理状况中。这 CCAAT 结合因子 (CBF) 是一种关键的反式作用因子，可激活 a1(I) 和 a2(I) 胶原蛋白启动子的体外转录 和其他发起人一样。 CBF由三个亚基组成：CBF-A、CBF-B和CBF-C， 这些都是DNA结合所需要的。负责形成的部分 每个亚基中的 CBF-DNA 复合物从酵母到人类都是保守的，并且 其中两个 CBF-A 和 CBF-C 与组蛋白折叠基序同源。在 显性失活 CBF 突变体对 CBF 功能的体内抑制 小鼠成纤维细胞抑制细胞生长并降低 胶原蛋白基因以及其他几个在细胞生长过程中受到调节的基因 生长。在拟议的研究中，将利用 差异显示和 cDNA 表达阵列来鉴定其基因 抑制 CBF 结合导致表达减少或增加 在成纤维细胞中。 CBF的体内功能将在不同的方面进行研究 使用显性失活 CBF 突变体的哺乳动物细胞系。转录 CBF 的激活功能将在体外重构系统中进行分析 确定与 CBF 相关并介导的核因子 CBF 依赖性转录的激活。研究转录 体内重组 CBF 亚基的激活，改变特异性 CBF 突变体，结合到突变 CBF 结合位点将被创建来测试 小鼠成纤维细胞中 a1(I) 和 a2(I) 胶原蛋白启动子的活性。