REGULATION OF HUMAN GLOBIN GENE EXPRESSION

这笔赠款的长期目标是识别和表征调节人类β珠蛋白基因复合体表达的DNA序列和反式作用因子，特别是那些参与晚期胎儿从伽马合成到β合成的DNA序列和反式作用因子。已经确定了几种不同的特异DNA序列和反式作用因子，它们作用于β珠蛋白基因复合体，既位于位点控制区(LCR)，也接近单个珠蛋白基因，这是其最佳红系特异性调控或发育阶段特异性调控所必需的，或者两者兼而有之。然而，到目前为止，还没有任何单一因素涉及到从人类伽马合成到贝塔合成的转变。我们已经描述了一个主要存在于成人造血细胞中的反式作用蛋白复合体(PYR复合体)，它与富含嘧啶的序列结合，包括位于人类增量珠蛋白基因上游1kb的一个可能在血红蛋白开关中发挥功能的蛋白质复合体(DeltaPYR结合位点)。最近，我们发现PYR复合体是一种特化的人类SWI/SNF样复合体。已知SWI/SNF复合体可以破坏染色质结构，允许转录因子结合和基因激活。PYR复合体是第一个具有DNA序列依赖结合位点的SWI/SNF复合体。我们还证明了deltaPYR结合位点在促进人类伽马向β转换方面的功能作用。在转基因小鼠的这些研究中，我们已经证明了该序列的缺失会导致延迟切换。这项资助的具体目的是：(1)通过纯化和测序来表征PYR复合体的蛋白质亚单位；(2)更精确地定义PYR复合体结合部位的结构和构型；(3)确定该复合体对染色质结构的一般功能影响；(4)定位该复合体对珠蛋白开关的功能影响所需的最小DNA序列；(5)在红系细胞和其他表达该复合体的成人造血细胞中寻找PYR复合体作用的基因靶点，而不是人β珠蛋白基因位点上的那些基因靶点；以及(6)定义其他SWI/SNF复合体，特别是人类α珠蛋白基因座上的一个假定复合体。这些研究应该为控制血红蛋白转换的机制提供新的见解。它们可能会导致治疗贝塔地中海贫血和镰状细胞疾病的新方法，因为这些疾病是由于贝塔珠蛋白合成异常造成的，理论上可以通过让最佳伽马珠蛋白合成持续到成年生活来治愈。

The long-term goals of this grant are to identify and characterize DNA sequences and trans-acting factors that regulate the expression of the human beta globin gene complex, especially those involved in the switch from gamma to beta synthesis in late fetal life. Several different specific DNA sequences and trans- acting factors have been identified that act at the beta globin gene complex, both at the locus-control region (LCR) and close to individual globin genes which are required for either their optimal erythroid-specific or developmental stage-specific regulation, or both. However, to date, no single factor has been implicated in the switch from human gamma to beta synthesis. We have described a trans-acting protein complex (PYR complex) present primarily in adult hematopoietic cells which binds to pyrimidine-rich sequences, including one located 1 kb upstream of the human delta globin gene (delta PYR binding site) which may function in hemoglobin switching. Recently, we have discovered that PYR complex is a specialized human SWI/SNF-like complex. SWI/SNF complexes are known to disrupt chromatin structure and permit transcription factor binding and gene activation. PYR complex is the first SWI/SNF complex with a DNA sequence- dependent binding site. We have also demonstrated a functional role for the delta PYR binding site in enhancing human gamma to beta switching. In these studies in transgenic mice, we have shown that deletion of this sequence leads to delayed switching. The specific aims of this grant are to: (1) characterize the protein subunits of PYR complex by purification and sequencing; (2) define the structure and configuration of the PYR complex binding site more precisely; (3) determine the general function effects of the complex on chromatin structure; (4) localize the minimal DNA sequence required for the functional effects of the complex on globin switching; (5) search for gene targets of PYR complex action other than those at the human beta globin gene locus in erythroid cells, and in other adult hematopoietic cells that express the complex; and (6) define other SWI/SNF complexes, particularly a putative one at the human alpha globin locus. These studies should provide new insights into the mechanisms controlling the hemoglobin switching. They may result in new approaches to the treatment of the beta thalassemias and sickle cell disease since these diseases are due to abnormal beta globin synthesis and could theoretically be cured by allowing optimal gamma globin synthesis to persist into adult life.