Function of a Putative Determinant in Hematopoiesis

描述（由申请人提供）：细胞限制性转录调节剂在造血过程中的选择性基因调控过程中发挥关键作用。本研究对红系Kruppel样因子（EKLF）的分子生物学功能进行了研究。EKLF是一种红细胞限制性转录因子，是完成红细胞程序的重要组成部分。分子和遗传学数据表明，EKLF发挥了重要作用，在发育开关成人β-珠蛋白的表达。它通过在β-珠蛋白启动子处与其同源CACCC元件结合时的强转录激活功能以及通过其与在β-样珠蛋白基因座处产生适当染色质构型的共激活因子相互作用的能力来实现这一点。虽然EKLF如何整合这些信号的细节仍有待完全破译，但最近的证据出人意料地表明，它可以作为转录抑制因子发挥作用，也可能在细胞核外发挥作用。EKLF阻遏和激活之间的转换受到细胞环境及其与辅阻遏物相互作用的影响。此外，它的乙酰化修饰也可能影响这些决定。 该提案旨在揭示这些不同的过程是如何通过识别EKLF蛋白复合物的组成部分和检查这些相互作用中的翻译后修饰的作用来控制的细节。这将通过以下方式实现：（1）检查（a）体外和（B）斜体EKLF蛋白与其活化配偶体的相互作用如何在β-珠蛋白启动子处协调;（2）使用串联亲和纯化来分离衍生自选定细胞类型和亚细胞级分的EKLF蛋白复合物;（3）通过（a）鉴定EKLF阻遏靶点并分析其乙酰化在阻遏中的作用和（B）阐明EKLF的替代作用表征细胞质EKLF复合物。

DESCRIPTION (provided by applicant): Cell-restricted transcriptional modulators play a critical role in the process of selective gene regulation during hematopoiesis. 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. Although the details of how EKLF integrates these signals remain to be fully deciphered, recent evidence unexpectedly suggests that it can function as a transcriptional repressor and may also play a role outside of the nucleus. Shifts between EKLF repression and activation are influenced by the cellular environment and by its interaction with corepressors. In addition, its modification by acetylation may also affect these decisions. This proposal is designed to expose the details of how these varied processes are controlled by identifying components of EKLF protein complexes and examining the role of post-translational modifications in these interactions. This will be accomplished by: (1) examining (a) in vitro and (b) italic how EKLF protein interactions with its activation partners are coordinated at the beta-globin promoter; (2) using tandem affinity purification to isolate EKLF protein complexes derived from selected cell types and subcellular fractions; (3) illuminating alternate roles for EKLF by (a) identifying EKLF repression targets and analyzing the role of its acetylation in repression and (b) characterizing cytoplasmic EKLF complexes.