GENE REGULATION BY A NOVEL PROTEIN CLONED FROM PITUITARY

从垂体克隆的新型蛋白质的基因调控

• 批准号: 2562575
• 负责人: F C BANCROFT
• 金额: $ 24.47万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2562575
• 关键词: DNA binding protein; RNA splicing; X ray crystallography; chromosomes; embryo /fetus cell /tissue; gel mobility shift assay; genetic mapping; genetic promoter element; genetic regulatory element; human genetic material tag; laboratory mouse; molecular cloning; phenotype; pituitary gland; prolactin; protein binding; protein isoforms; protein kinase A; protein structure function; recombinant proteins; tissue /cell culture; transcription factor; transfection

Recent studies have implied that an additional transcription factor(s) beside the pituitary-specific factor Pit-1 is involved in homeostatic regulation of prolactin (PRL) promoter activity via Pit-1 binding cis active elements. We have employed one of these elements, site 1P, to clone from a pituitary cDNA expression library a protein, termed PBLF, that is a novel member of the WD repeat family. PBLF contains a PQ-rich potential transactivation domain, but no apparent DNA-binding motif. PBLF exhibits sequence-specific binding to site 1P, and is as active as Pit-1 in transactivating expression of PRL promoter constructs containing site 1P. In addition, PBLF, but not Pit-1, can mediate the transcriptional action of protein kinase A. In mouse embryo on day E12.5, PBLF is expressed specifically in Rathke's pouch, suggesting a role in pituitary development. PBLF accumulates in the nuclei of GH3 rat pituitary cells, and its mRNA is present in these cells at levels comparable to Pit-1 mRNA. PBLF transcripts are expressed in all adult human tissues examined. However, most tissues express multiple PBLF mRNA species that display tissue-specific patterns, suggesting that alternative splicing of the initial PBLF transcript may yield tissue- specific synthesis of isoforms of PBLF. These observations imply that PBLF will prove to play a significant transcriptional regulatory role, in the pituitary as well as in other organs; and thus provide the conceptual basis for our further investigations of the biochemical and functional properties of this novel protein. Biochemical studies will involve first production of soluble recombinant PBLF, followed by determination of the PBLF domain structure, characterization of its DNA- binding properties, and studies of the binding of PBLF to site 1P in the presence of Pit-1. Functional studies will involve investigations of the role of PBLF as a transcription factor in pituitary cells; of alternative splicing of the PBLF transcript in the brain; and of the PBLF phenotype, beginning with chromosomal localization of the PBLF gene in human, followed by attempts to identify a known organism (human or mouse) mutant for PBLF. These studies should provide further insight into the biochemical and physiological roles of PBLF.

最近的研究表明，另一种转录因子 除了垂体特异性因子坑外，pit与稳态有关 通过PIT-1结合顺式调节催乳素（PRL）启动子活性 活动元素。 我们已经采用了这些元素之一，即站点1p 来自垂体cDNA表达式文库A蛋白的克隆，称为PBLF， 那是WD重复家庭的新成员。 PBLF包含一个富含PQ的 潜在的反式激活结构域，但没有明显的DNA结合基序。 PBLF表现出序列特异性与位点1p的结合，并且与 PRL启动子构建体反式激活表达中的PIT-1 包含站点1p。 另外，PBLF（但不是PIT-1）可以介导 蛋白激酶A的转录作用。 e12.5，PBLF专门用Rathke的小袋表达 在垂体发展中的作用。 PBLF积聚在GH3的核中 大鼠垂体细胞及其mRNA存在于这些细胞中的水平 与PIT-1 mRNA相当。 PBLF成绩单在所有成年人中均表示 人体组织检查了。 但是，大多数组织表达多个PBLF 显示组织特异性模式的mRNA物种，表明 初始PBLF转录本的替代剪接可能会产生组织 - PBLF同工型的特异性合成。 这些观察表明 PBLF将证明发挥重要的转录调节作用， 在垂体和其他器官中；因此提供了 我们对生化和进一步研究的概念基础 这种新蛋白的功能特性。 生化研究将 涉及首次生产可溶性重组PBLF，其次是 PBLF结构结构的确定，其DNA-的表征 结合特性，以及PBLF与位点1p结合的研究 Pit-1的存在。 功能研究将涉及对 PBLF作为垂体细胞中转录因子的作用；的 PBLF转录本在大脑中的替代剪接；和 PBLF表型，从PBLF基因的染色体定位开始 在人类中，然后尝试识别已知生物（人类或 鼠标）PBLF的突变体。 这些研究应提供进一步的见识 进入PBLF的生化和生理作用。