REGULATION OF HUMAN GLOBIN GENE EXPRESSION

这项资助的长期目标是鉴定和表征调节人类β珠蛋白基因复合物表达的DNA序列和反式作用因子，特别是那些参与胎儿晚期从γ合成到β合成的转换的因子。 已经鉴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β珠蛋白基因复合物，既作用于基因座控制区（LCR），也作用于接近单个珠蛋白基因的区域，所述珠蛋白基因是它们的最佳红细胞特异性或发育阶段特异性调节所需的，或两者。 然而，到目前为止，还没有单一的因素涉及从人类γ合成到β合成的转变。 我们已经描述了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），其结合富含嘧啶的序列，包括位于人δ珠蛋白基因上游1 kb的序列（δ PYR结合位点），其可以在血红蛋白转换中起作用。 最近，我们发现PYR复合物是一种特异的人SWI/SNF样复合物。已知SWI/SNF复合物破坏染色质结构并允许转录因子结合和基因活化。 PYR复合物是第一个具有DNA序列依赖性结合位点的SWI/SNF复合物。 我们还证明了δ PYR结合位点在增强人γ至β转换中的功能作用。 在转基因小鼠的这些研究中，我们已经表明，该序列的缺失导致延迟转换。该基金的具体目标是：（1）通过纯化和测序来表征PYR复合物的蛋白亚基;（2）更精确地确定PYR复合物结合位点的结构和构型;（3）确定PYR复合物对染色质结构的一般功能效应;（4）定位PYR复合物对珠蛋白转换的功能效应所需的最小DNA序列;（5）确定PYR复合物对珠蛋白转换的功能效应。（5）在红系细胞和其它表达该复合物的成人造血细胞中，寻找除人β珠蛋白基因座处的那些以外的PYR复合物作用的基因靶;和（6）确定其它SWI/SNF复合物，特别是人α珠蛋白基因座处的推定复合物。这些研究将为控制血红蛋白转换的机制提供新的见解。 它们可能导致治疗β地中海贫血和镰状细胞病的新方法，因为这些疾病是由于异常β珠蛋白合成引起的，并且理论上可以通过允许最佳γ珠蛋白合成持续到成人生活而治愈。

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.