Di Li-Jun, Wang Li, Zhou Guo-Ling, Wu Xue-Song, Guo Zhi-Chen, Ke Xi-Song, Liu De-Pei, Liang Chih-Chuan
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, P.R. China.
J Cell Biochem. 2008 Sep 1;105(1):301-12. doi: 10.1002/jcb.21826.
Chromatin from different regions of the genome frequently forms steady associations that play important roles in regulating gene expression. The widely used chromatin conformation capture (3C) assay allows determination of the in vivo structural organization of an active endogenous locus. However, unpredicted chromatin associations within a given genomic locus can not be identified by 3C. Here, we describe a new strategy, quantitative associated chromatin trap (QACT), which incorporates a modified 3C method and a quantitative assay tool, to capture and quantitatively analyzes all possible associated chromatin partners (ACPs) of a given chromatin fragment. Using QACT, we have analyzed the chromatin conformation of the mouse alpha-globin gene cluster and proved the extensive interaction between HS26 and alpha-globin genes. In addition, we have identified a candidate alpha1-globin gene specific silencer 475A8 which shows the differentiation-stage specific DNase I hypersensitivity. Functional analysis suggests that 475A8 may regulate the alpha1-globin gene during terminal differentiation of committed erythroid progenitor cells. ChIP (chromatin immunoprecipitation) and cotransfection assays demonstrate that GATA-1, a hemopoietic specific transcriptional factor, may increase alpha1-globin gene expression by suppressing the function of 475A8 in terminally differentiated erythroid cells.
基因组不同区域的染色质经常形成稳定的关联，这些关联在调节基因表达中发挥重要作用。广泛使用的染色质构象捕获（3C）分析能够确定活性内源性基因座的体内结构组织。然而，给定基因组基因座内无法预测的染色质关联无法通过3C识别。在此，我们描述了一种新策略，定量关联染色质捕获（QACT），它结合了改良的3C方法和定量分析工具，以捕获并定量分析给定染色质片段的所有可能的关联染色质伙伴（ACP）。使用QACT，我们分析了小鼠α-珠蛋白基因簇的染色质构象，并证明了HS26与α-珠蛋白基因之间存在广泛的相互作用。此外，我们鉴定出一个候选的α1-珠蛋白基因特异性沉默子475A8，它显示出分化阶段特异性的DNase I超敏反应。功能分析表明，475A8可能在定向红系祖细胞的终末分化过程中调节α1-珠蛋白基因。染色质免疫沉淀（ChIP）和共转染分析表明，造血特异性转录因子GATA-1可能通过抑制475A8在终末分化红系细胞中的功能来增加α1-珠蛋白基因的表达。
