Zhan Jun, Johnson Irudayam Maria, Wielgosz Matthew, Nienhuis Arthur W
Department of Hematology, Division of Experimental Hematology , St. Jude Children's Research Hospital , Memphis, Tennessee, USA.
Mol Ther Methods Clin Dev. 2016 Dec 14;3:16077. doi: 10.1038/mtm.2016.77. eCollection 2016.
Chromosome Conformation Capture (3C) technology was used to identify physical interactions between the proximal Wiskott-Aldrich Syndrome protein () promoter and its distant DNA segments in Jurkat-T cells. We found that two hematopoietic specific DNase I hypersensitive (DHS) sites (proximal DHS-A, and distal DHS-B) which had high interaction frequencies with the proximal WASp promoter indicating potential regulatory activity for these DHS sites. Subsequently, we cloned several DNA fragments around the proximal DHS-A site into a luciferase reporter vector. Interestingly, no fragments showed enhancer activity, but two fragments exhibited strong silencing activity in Jurkat-T cells. After aligning the chromatin state profiling for hematopoietic and nonhematopoietic cells using the human genome browser (UCSC), we found a 5 kb putative hematopoietic specific enhancer region located 250 kb downstream of the gene. This putative enhancer region contains two hematopoietic cell specific DHS sites. Subsequently, the hematopoietic specific DHS sites enhanced luciferase expression from the proximal WASp promoter in all hematopoietic cells we tested. Finally, using a lentiviral vector stable expression system, the hematopoietic specific-enhancer(s) increased GFP reporter gene expression in hematopoietic cells, and increased gene expression in WASp deficient cells. This enhancer may have the potential to be used in gene therapy for hematological diseases.
染色体构象捕获（3C）技术被用于鉴定Jurkat - T细胞中威斯科特-奥尔德里奇综合征蛋白（WASp）近端启动子与其远端DNA片段之间的物理相互作用。我们发现两个造血特异性脱氧核糖核酸酶I超敏（DHS）位点（近端DHS - A和远端DHS - B）与近端WASp启动子具有高相互作用频率，表明这些DHS位点具有潜在的调控活性。随后，我们将近端DHS - A位点周围的几个DNA片段克隆到荧光素酶报告载体中。有趣的是，没有片段显示出增强子活性，但有两个片段在Jurkat - T细胞中表现出强烈的沉默活性。使用人类基因组浏览器（UCSC）对造血细胞和非造血细胞的染色质状态图谱进行比对后，我们在WASp基因下游250 kb处发现了一个5 kb的假定造血特异性增强子区域。这个假定的增强子区域包含两个造血细胞特异性DHS位点。随后，造血特异性DHS位点增强了我们测试的所有造血细胞中近端WASp启动子的荧光素酶表达。最后，使用慢病毒载体稳定表达系统，造血特异性增强子在造血细胞中增加了绿色荧光蛋白（GFP）报告基因的表达，并在WASp缺陷细胞中增加了WASp基因的表达。这种增强子可能具有用于血液系统疾病基因治疗的潜力。
