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

描述(改编自申请人的摘要)：I型胶原蛋白， 由两个A1(I)和一个A2(I)多肽组成的杂三聚体是最多的 在皮肤、骨骼和肌腱中发现丰富的蛋白质，并解除对 I型胶原在许多病理情况下都会发生。这个 CCAAT结合因子(CBF)是一种关键的反式作用因子，它激活 A1(I)和A2(I)胶原启动子的体外转录 就像其他推动者一样。CBF由CBF-A、CBF-B和CBF-C三个亚基组成， 它们都是DNA结合所需的。负责编队的部分 每个亚基中CBF-DNA复合体的含量从酵母到人都是保守的，并且 其中两个CBF-A和CBF-C与组蛋白折叠基序同源。在……里面 显性负性CBF突变体对CBF功能的体内抑制作用 小鼠成纤维细胞抑制细胞生长并减少其表达 胶原蛋白基因，以及其他几个在细胞过程中受到调控的基因 成长。在拟议的研究中，将使用 差异显示和基因表达阵列技术筛选基因 由于抑制CBF结合而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环境中进行研究 使用显性负CBF突变体的哺乳动物细胞系。抄写 将在体外重组系统中分析CBF的激活功能 确定与CBF相关的核因子并介导 激活CBF依赖的转录。研究转录本 重组CBF亚基体内激活，改变特异性CBF 与突变的CBF结合位点结合的突变体将被创建以测试 小鼠成纤维细胞中a1(I)和a2(I)胶原启动子的活性。

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.