FUNCTION OF A PUTATIVE DETERMINANT IN HEMATOPOIESIS

细胞限制性转录调节因子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，在红系个体发育过程中，控制珠蛋白表达的调控依赖于更高级别的结构，使远端(LCR)和近端(启动子)控制元件结合的蛋白质能够在染色质背景下激活所选基因的转录。结合在这些元素上的因子之间基于蛋白质的相互作用必须在建立和维持这样的结构中发挥重要作用。然而，这一过程如何协调的分子细节仍有待确定。我们一直在研究红系Kruppel样因子(EKLF)的分子生物学功能。EKLF是一种红细胞限制性转录因子，是完成红系程序的重要组成部分。分子和遗传学数据表明，EKLF在发育向成年β-珠蛋白表达的转变中起着重要作用。它是通过与其在β-珠蛋白启动子上的同源CACCC元件结合时具有强大的转录激活功能，以及通过与在类β珠蛋白基因座产生适当染色质配置的共激活子相互作用来实现这一点的。EKLF在翻译后被乙酰化和磷酸化修饰，其亚细胞定位可能在其功能中起作用。这一提议旨在检验这样一种假设，即这些观察结果是相互关联的，因此EKLF是多种信号的集成者，这些信号有助于调节β-珠蛋白的表达。这将通过以下方式完成：(1)证明EKLF修饰状态在与其已知伙伴(CBP/p300，BRM1)和其他尚未确定的EKLF相关蛋白的蛋白质相互作用中起调节作用；(2)通过对EKLF缺失细胞的生物拯救试验来测试先前和新的EKLF突变体的功能重要性；(3)确定控制EKLF在原始和最终的红斑细胞中定位的参数。

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.