FUNCTION OF A PUTATIVE DETERMINANT IN HEMATOPOIESIS

造血作用中推定决定因素的功能

• 批准号: 6380812
• 负责人: JAMES J BIEKER
• 金额: $ 28.88万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6380812
• 关键词: DNA binding protein; acyltransferase; affinity chromatography; biological signal transduction; cell differentiation; chromatin; embryonic stem cell; erythroid stem cell; erythropoiesis; gene deletion mutation; gene expression; genetic mapping; genetic promoter element; hemoglobin; laboratory mouse; monoclonal antibody; mutant; phosphorylation; posttranslational modifications; protein protein interaction; protein purification; protein structure function; protein transport; transcription factor; transfection /expression vector

Cell-restricted transcriptional modulators play a critical role in the process of lineage selection during hematopoiesis. In addition, the controls that dictate programming of globin expression during erythroid ontogeny rely on higher-order structures that enable proteins bound at distal (LCR) and proximal (promoter) controlling elements to activate transcription of the selected gene within the context of chromatin. Protein-based interactions between factors bound at these elements must play an important part in establishing and maintaining such a structure. However, the molecular details of how this process is coordinated remains to be established. We have been investigating the molecular and biological function of Erythroid Kruppel-like Factor (EKLF). EKLF is a red cell-restricted transcription factor that is an essential component for completion of the erythroid program. Molecular and genetic data demonstrate that EKLF plays a major role in the developmental switch to adult beta-globin expression. It accomplishes this by means of its strong transcriptional activation function when bound to its cognate CACCC element at the beta- globin promoter, and by its ability to interact with coactivators that generate the proper chromatin configuration at the beta-like globin locus. EKLF is post-translationally modified by both acetylation and phosphorylation, and its subcellular localization may play a role in its function. This proposal is designed to test the hypothesis that these observations are interrelated, and thus that EKLF is an integrator of diverse signals that serve to regulate beta-globin expression. This will be accomplished by: (1) demonstrating that EKLF modification status plays a regulated role in protein-protein interactions, both with its known partners (CBP/p300, BRM1) and with other, yet to be identified, EKLF-associated proteins; (2) testing the functional importance of previous and novel EKLF mutants by means of a biological rescue assay of EKLF-null cells; (3) determining the parameters that control EKLF subcellular localization in primitive and definitive erytbroid cells.

细胞限制的转录调节剂在造血过程中的谱系选择过程中起着至关重要的作用。此外，在红斑个体个体发育过程中决定球蛋白表达的编程的控制依赖于使蛋白质在远端（LCR）和控制元素近端（LCR）和近端（启动子）控制元件的高阶结构中激活染色质背景中所选基因的转录。基于蛋白质在这些元素上的因素之间的相互作用必须在建立和维持这种结构中起重要作用。但是，该过程如何协调的分子细节仍有待确定。我们一直在研究红花样因子（EKLF）的分子和生物学功能。 EKLF是一种红细胞限制的转录因子，是完成红细胞程序的重要组成部分。分子和遗传数据表明，EKLF在发展到成人β-珠蛋白表达的发展中起主要作用。当与β球蛋白启动子处的同源CACCC元素绑定时，它通过其强大的转录激活函数来完成此操作，并通过与共激活因子相互作用的能力，该共激活因子在类似β的球蛋白基因座上产生适当的染色质构型。 EKLF是通过乙酰化和磷酸化在后翻译后修饰的，其亚细胞定位可能在其功能中起作用。该建议旨在检验这些观察结果相互关联的假设，因此EKLF是用于调节β-珠蛋白表达的各种信号的集成子。这将通过：（1）证明EKLF修饰状态在蛋白质 - 蛋白质相互作用中起着调节作用，包括其已知伴侣（CBP/P300，BRM1），并且与其他尚未确定的EKLF相关蛋白质扮演着调节的作用； （2）通过对EKLF无效细胞的生物营救测定法测试以前和新型EKLF突变体的功能重要性； （3）确定控制EKLF亚细胞定位的参数。