The mechanism of the human β-globin gene switching from an aspect of genomic organization.

本研究旨在从人β-珠蛋白基因的基因组组织角度探讨其表达的开关机制。5个活性基因(ε-, g -, a -， δ-和（3-珠蛋白基因）在发育过程中表现出时间和组织特异性表达。为了从DNA结构的基础上研究β-珠蛋白基因家族的表达调控，我们重点研究了一类非b DNA结构，即固有弯曲DNA。利用圆形排列法，我们绘制了β-家族珠蛋白位点整个区域的弯曲DNA位点，并对弯曲DNA位点区域进行了详细的分析。基于上述结果，本研究的主要目的是在体外和体内分析基因座控制区（Locus Control region， LCR）中dna酶I超敏感位点(HS)-2增强子区域的核小体相。HS-2区两侧有两个DNA弯曲位点，四个核小体被安置在位点之间。在体外和体内测定了含有弯曲核核序列的核体相。另一方面，在红系K562细胞中，中间的核小体（核小体与HS-2的确切位置相对应，包含了红系特异性转录因子NF-E2的结合位点）呈现出多个期相。而该区域非红系HeLa细胞仅采用一个相。当染色质结构处于抑制状态时，转录效率是通过在染色质重塑之前控制NF-E2与其染色体上同源基序的相互作用来调节的。根据结果，我们假设周期性弯曲DNA的功能是关键核小体对核小体排列的基本因素。我们目前通过使用二核体DNA文库筛选和表征关键核小体来证实这一假设。

The purpose of the study is to investigate the switching mechanism of the human β-globin gene expression from an aspect of its genomic organization. Five active genes (ε-, Gγ-,Aγ-, δ-and (3-globin genes) exhibit temporal and tissue-specific expression during development. In an attempt to examine the regulation of expression of the β-globin gene family from the basis of DNA structure, we focused on a type of non-B DNA structures, intrinsically bent DNA. Using the circular permutation assay, we mapped the bent DNA sites in the entire region of the β-family globin locus and carried out the detail analyses on the region of bent DNA sites. Based on the previous results, the main object of the study supported by a Grant-in-Aid for Scientific Research (C) was to analyze nucleosomal phases in vitro and in vivo in the DNase I hypersensitive sites (HS)-2 enhancer region of the Locus Control Region (LCR).The HS-2 region was flanked by two DNA bend sites and four nucleosomes were accommodated between the sites. The nucleosomal phases that contained the bend core sequences were determined in vitro and in vivo. On the other hand, the nucleosome in the middle which corresponded to the exact location of HS-2 and included the binding site for the erythroid-specific transcription factor NF-E2 showed several phases in erythroid K562 cells. In contrast, only one phase was adopted in non-erythroid HeLa cells in this region. When the chromatin structure is in a repressive state, the transcriptional efficiency is regulated by controlling the interaction of NF-E2 to its cognate motif on the chromosome before chromatin remodeling. From the results, we hypothesized the function of the periodic bent DNA is a basic factor of key nucleosomes on nucleosome alignment. We currently confirmed the hypothesis by screening and characterizing key nucleosomes using a dinucleosome DNA library.