REGULATION OF HUMAN GLOBIN GENE EXPRESSION

这项资助的长期目标是鉴定和表征DNA序列和调节人类-珠蛋白基因复合物表达的反式作用因子，特别是那些参与胎儿生命后期从γ合成到β合成转换的因子。已经确定了几种不同的特异性DNA序列和反式作用因子，它们作用于-珠蛋白基因复合物，既在位点控制区（LCR），又靠近单个珠蛋白基因，这些基因是红细胞特异性或发育阶段特异性调节所必需的，或者两者都需要。然而，到目前为止，没有单一的因素涉及到从人类合成到β合成的转变。我们已经描述了一种主要存在于成人造血细胞中的反式作用蛋白复合物（PYR复合物），它与富含嘧啶的序列结合，包括位于人类δ珠蛋白基因上游1kb的一个（δ PYR结合位点），它可能在血红蛋白转换中起作用。近年来，我们发现PYR复合体是一种特殊的人类SWI/ snf样复合体。SWI/SNF复合物已知破坏染色质结构，允许转录因子结合和基因激活。PYR复合物是首个具有DNA序列依赖结合位点的SWI/SNF复合物。我们还证明了δ PYR结合位点在增强人类γ到β转换中的功能作用。在这些转基因小鼠的研究中，我们已经表明，该序列的删除导致延迟开关。该资助的具体目的是：(1)通过纯化和测序来表征PYR复合物的蛋白质亚基；(2)更精确地定义PYR复合体结合位点的结构和构型；(3)确定复合体对染色质结构的一般功能效应；(4)定位复合物对珠蛋白转换的功能作用所需的最小DNA序列；(5)在红细胞和其他表达该复合物的成人造血细胞中寻找除人β -珠蛋白基因位点以外的PYR复合物作用的基因靶点；(6)定义其他SWI/SNF复合物，特别是在人类α -珠蛋白位点的推定复合物。这些研究应该为控制血红蛋白转换的机制提供新的见解。它们可能会带来治疗地中海贫血和镰状细胞病的新方法，因为这些疾病是由于β -珠蛋白合成异常引起的，理论上可以通过使最佳的γ -珠蛋白合成持续到成年而治愈。

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.