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

描述（改编自申请人摘要）：I型胶原蛋白，a 由两个α 1（I）和一个α 2（I）多肽组成的异源三聚体是最常见的 在皮肤、骨骼和肌腱中发现丰富的蛋白质， I型胶原蛋白存在于许多病理状态中。的 CCAAT结合因子（CBF）是一种关键的反式作用因子， α 1（I）和α 2（I）胶原启动子的体外转录，以及 作为其他推动者。CBF由CBF-A、CBF-B和CBF-C三个亚基组成， 这些都是DNA结合所必需的负责形成的部分 CBF-DNA复合物在每个亚基中的比例从酵母到人是保守的， 其中CBF-A和CBF-C与组蛋白折叠基序同源。在 通过表达于细胞中的显性阴性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 胶原基因，以及其他几个在细胞生长过程中受到调控的基因， 增长在拟议的研究中，将使用 差异显示和cDNA表达阵列，以确定基因， 由于CBF结合的抑制， 在成纤维细胞中。将在不同的条件下研究CBF的体内功能。 使用显性负CBF突变体的哺乳动物细胞系。转录 将在体外重建系统中分析CBF的激活功能 确定与CBF相关并介导 CBF依赖性转录的激活。研究转录 重组CBF亚基体内激活，特异性改变的CBF 将产生结合突变CBF结合位点的突变体，以测试 小鼠成纤维细胞中的α 1（I）和α 2（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.