Regulation of the human epsilon-globin gene transcription in the switching mechanism

人ε-珠蛋白基因的上游侧翼区不仅含有沉默子，而且含有几个重要的顺式作用因子，它们调节珠蛋白基因的阶段特异性开关。以前，我们通过体外转录测定来表征人ε-珠蛋白基因沉默子。我们首先检测了S1核酸酶超敏感位点（S1 HS），发现两个S1 HS区域，该区域中的三链DNA或滑动DNA的异常结构很可能是引起S1核酸酶敏感性的结构。我们还研究了弯曲的DNA，这是序列固有的不寻常的结构之一。环形排列实验表明，在整个区域内平均每680 bp出现一个DNA弯曲位点。为了了解其他珠蛋白基因中弯曲位点的周期性，我们接下来定位了β-珠蛋白基因区域中的位点。这些位点与帽位点的相对位置与ε-珠蛋白基因区域中的那些相同，表明弯曲位点在两个基因的基因组序列的分子进化过程中是保守的。这一结果也加强了它们在基因组组织中的重要性，例如核小体定相的信号。最后，我们在13.3kb的G γ-A γ-β-珠蛋白基因区域定位了弯曲位点。弯曲位点以大约700 bp的间隔存在，它们将该区域划分为单元。此外，他们是保守的启动子区的几乎所有的β-家族珠蛋白基因，虽然该网站的周期性局部干扰的交界处的重复γ-和γ-珠蛋白基因和它们的第二内含子。这表明弯曲位点的周期性在基因组DNA组织的层次中的排名低于基因组重排和基因表达。

The human epsilon-globin gene contains not only silencer but also several important cis-acting factors in the upstream flanking region, which regulate stage-specific switching of globin genes. Previously we characterized the human epsilon-globin gene silencer by in vitro transcription assay. Here we first examined the S1 nuclease hypersensitive sites (S1 HS) in the upstream flanking region and found that two regions were S1 HS.The unusual feature in the region, triplex DNA or slipped DNA,was most likely to be the structure that causes S1 nuclease sensitivity. We also examined bent DNA which is one of the unusual structures intrinsically adopted by the sequence. The circular permutation experiment revealed that the DNA bend sites appeared every 680 bp on average in the entire region. In order to know the periodicity of the bend sites in other globin genes, we next mapped the sites in the beta-globin gene region. The relative positions of the sites to the cap site were identical to those in the epsilon-globin gene region, suggesting that the bend sites have been conserved during molecular evolution of the genomic sequence among two genes. The result also strengthens their significance in the genomic organization such as a signal for nucleosome phasing. Finally, we mapped the bend sites in the region spanning 13.3kb containing the Ggamma-Agamma-psibeta-globin genes. The bend sites were present at an interval of roughly 700bp and they divide the region into units. Furthermore, they were conserved in the promoter region of nearly all beta-family globin genes, although the periodicity of the sites was locally disturbed at the junctions of the duplicated Ggamma-and Agamma-globin genes and their second introns. This suggested that the periodicity of the bend sites is ranked lower in the hierarchy of genomic DNA organization than genome rearrangement and gene expression.