Inhibition of enhancer-promoter interaction involved in the developmental-stage specific gene regulation

人类3-珠蛋白位点包含5个发育调控的3型珠蛋白基因。这五个基因都依赖于位于基因座5′末端的基因座控制区（locus control region， LCR）进行大量的珠蛋白基因转录。LCR由5个dna酶I超敏位点（HSs）组成，其中至少有一个子集在激活基因座内形成一个完整的复合体。我们之前通过倒置人β-珠蛋白YAC转基因小鼠（TgM）的LCR极性来测试其对适当LCR极性的需求，并观察到所有β型珠蛋白基因的表达减少，无论发育阶段如何。这种表型清楚地证明了LCR的定向依赖性活性，尽管观察到的活性的机制基础尚不清楚。在本研究项目中，我们获得了遗传证据，证明人类HSS包括增强子阻断（绝缘子）活性，这依赖于LCR HSS中完整的ctcf结合基序的存在。奇怪的是，这种ctcf依赖性活性似乎是基因和发育阶段特异性的。这些观察结果表明，在LCR-倒置位点上观察到的表型部分归因于将HSS绝缘子置于LCR HS“增强子（HS1-4）”和β-珠蛋白基因启动子之间，但进一步说，HS5序列还具有原始的红细胞沉默活性，不依赖于ctcf。

The human (3-globin locus contains five developmentally regulated (3-type globin genes. All five genes depend on the locus control region (LCR), located at the 5' end of the locus, for abundant globin gene transcription. The LCR is composed of five DNase I hypersensitive sites (HSs), at least a subset of which appear to cooperate to form a holocomplex in activating genes within the locus. We previously tested the requirement for proper LCR polarity by inverting it in human β-globin YAC transgenic mice (TgM) and observed reduced expression of all the β-type globin genes, regardless of developmental stage. This phenotype clearly demonstrated an orientation-dependent activity of the LCR, although the mechanistic basis for the observed activity was obscure. In this research project, we obtained genetic evidence demonstrating that human HSS includes enhancer-blocking (insulator) activity that was dependent on the presence of an intact CTCF-binding motif within LCR HSS. Curiously, this CTCF-dependent activity appeared to be both gene and developmental-stage specific. These observations demonstrated that the phenotype observed in the LCR-inverted locus was, in part, attributable to placing the HSS insulator between the LCR HS "enhancers (HS1-4)" and the promoter of the β-globin gene, but further that the HS5 sequence also beared a primitive erythroid silencing activity that is CTCF-independent.