Paul Thankam, Li SiDe, Khurana Sanjeev, Leleiko Neal S, Walsh Martin J
Department of Pediatrics, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA.
Biochem J. 2007 Dec 15;408(3):317-26. doi: 10.1042/BJ20070282.
The CFTR (cystic fibrosis transmembrane conductance regulator) gene is a tightly regulated and differentially expressed transcript in many mucosal epithelial cell types. It appears that DNA sequence variations alone do not explain CFTR-related gastrointestinal disease patterns and that epigenetic modifiers influence CFTR expression. Our aim was to characterize the native chromatin environment in cultured cells for intestinal CFTR expression by determining the relationship between histone acetylation and occupation of CFTR by multiple transcription factors, through a common regulatory element. We used HDAC (histone deacetylase) inhibition and ChIP (chromatin immunoprecipitation) analyses to define regions associated with acute acetylation of histone at the CFTR locus. We identified a region within the first intron associated with acute acetylation of histone H4 as an epigenetic signature corresponding to an intestine-specific enhancer element for CFTR. DHS (DNase I-hypersensitivity) assays and ChIP were used to specify control elements and occupation by regulatory factors. Quantitative ChIP procedures indicate that HNF1alpha (hepatic nuclear factor 1alpha) and Cdx2 (caudal homeobox protein 2) occupy and regulate through a novel intronic enhancer element of CFTR and that Tcf4 (T-cell factor 4) overlaps the same DNA element. RNAi (RNA interference) of Tcf4 and HNF1alpha decreased intestinal cell CFTR expression, identifying these as positive regulatory factors and CFTR as a target for Wnt signalling. We have linked the acetylation signature of nucleosomal histones to active intestinal CFTR expression and occupation by transcription factors HNF1alpha, Cdx2 and Tcf4 which converge to modify chromatin architecture. These studies suggest the therapeutic potential of histone modification strategies, such as inhibition of HDAC activity, to treat CFTR-associated disease by selectively enhancing CFTR expression.
CFTR（囊性纤维化跨膜传导调节因子）基因在许多黏膜上皮细胞类型中是一个受到严格调控且差异表达的转录本。似乎仅DNA序列变异并不能解释与CFTR相关的胃肠道疾病模式，表观遗传修饰因子会影响CFTR的表达。我们的目的是通过确定组蛋白乙酰化与多种转录因子通过一个共同调控元件对CFTR的占据之间的关系，来表征培养细胞中肠道CFTR表达的天然染色质环境。我们使用组蛋白去乙酰化酶（HDAC）抑制和染色质免疫沉淀（ChIP）分析来确定与CFTR基因座处组蛋白急性乙酰化相关的区域。我们在第一个内含子中鉴定出一个与组蛋白H4急性乙酰化相关的区域，作为对应于CFTR肠道特异性增强子元件的表观遗传特征。使用DNase I超敏反应（DHS）分析和ChIP来确定调控元件以及调控因子的占据情况。定量ChIP程序表明，肝细胞核因子1α（HNF1α）和尾型同源盒蛋白2（Cdx2）通过CFTR的一个新的内含子增强子元件占据并发挥调控作用，并且T细胞因子4（Tcf4）与相同的DNA元件重叠。对Tcf4和HNF1α进行RNA干扰（RNAi）可降低肠道细胞CFTR的表达，表明它们是正调控因子，且CFTR是Wnt信号通路的一个靶点。我们已将核小体组蛋白的乙酰化特征与活跃的肠道CFTR表达以及转录因子HNF1α、Cdx2和Tcf4的占据联系起来，这些转录因子共同作用以改变染色质结构。这些研究表明，诸如抑制HDAC活性等组蛋白修饰策略具有通过选择性增强CFTR表达来治疗CFTR相关疾病的治疗潜力。
