Hinnebusch Brian F, Henderson J Welles, Siddique Aleem, Malo Madhu S, Zhang Wenying, Abedrapo Mario A, Hodin Richard A
Department of Surgery, Massachusetts General Hospital/Harvard Medical School, Grey-Bigelow 504, 55 Fruit Street, Boston, MA 02114, USA.
J Gastrointest Surg. 2003 Feb;7(2):237-44; discussion 244-5. doi: 10.1016/s1091-255x(02)00140-3.
Enterocyte differentiation is thought to occur through the transcriptional regulation of a small subset of specific genes. A recent growing body of evidence indicates that post-translational modifications of chromatin proteins (histones) play an important role in the control of gene transcription. Previous work has demonstrated that one such modification, histone acetylation, occurs in an in vitro model of enterocyte differentiation, butyrate-treated HT-29 cells. In the present work, we sought to determine if the epigenetic signal of histone acetylation occurs in an identifiable pattern in association with the transcriptional activation of the enterocyte differentiation marker gene intestinal alkaline phosphatase (IAP). HT-29 cells were maintained under standard culture conditions and differentiated with sodium butyrate. The chromatin immunoprecipitation (ChIP) assay was used to compare the acetylation state of histones associated with specific regions of the IAP promoter in the two cell populations (undifferentiated vs. differentiated). Chromatin was extracted from cells and cleaved by sonication or enzymatic digestion to obtain fragments of approximately 200 to 600 base-pairs, as confirmed by polymerase chain reaction using primers designed to amplify the IAP segments of interest. The ChIP assay selects DNA sequences that are associated with acetylated histones by immunoprecipitation. Unbound segments represent DNA sequences whose histones are not acetylated. After immunoprecipitation, sequences were detected by radiolabeled polymerase chain reaction, and the relative intensity of the bands was quantified by densitometry. The relative acetylation state of histones at specific sites was determined by comparing the ratios of bound/unbound segments. We determined that in a segment of the IAP promoter between -378 and -303 base-pairs upstream from the transcriptional start site, the acetylation state of histone H3 increased twofold in the differentiated, IAP expressing cells, whereas that of histone H4 remained essentially constant. Additionally, at a distant site, between -1378 and -1303 base-pairs, the acetylation state of H3 and H4 did not change appreciably between the undifferentiated and differentiated cells. We conclude that butyrate-induced differentiation is associated with specific and localized changes in the histone acetylation state within the IAP promoter. These changes within the endogenous IAP gene may underlie its transcriptional activation in the context of the enterocyte differentiation program.
肠上皮细胞分化被认为是通过一小部分特定基因的转录调控来发生的。最近越来越多的证据表明，染色质蛋白（组蛋白）的翻译后修饰在基因转录控制中起重要作用。先前的研究表明，一种这样的修饰，即组蛋白乙酰化，发生在肠上皮细胞分化的体外模型，即丁酸盐处理的HT-29细胞中。在本研究中，我们试图确定组蛋白乙酰化的表观遗传信号是否以与肠上皮细胞分化标志物基因肠碱性磷酸酶（IAP）转录激活相关的可识别模式出现。HT-29细胞在标准培养条件下培养并用丁酸钠诱导分化。染色质免疫沉淀（ChIP）分析用于比较两个细胞群体（未分化与分化）中与IAP启动子特定区域相关的组蛋白的乙酰化状态。从细胞中提取染色质并通过超声处理或酶消化进行切割，以获得约200至600个碱基对的片段，这通过使用设计用于扩增感兴趣的IAP片段的引物进行聚合酶链反应得到证实。ChIP分析通过免疫沉淀选择与乙酰化组蛋白相关的DNA序列。未结合的片段代表其组蛋白未乙酰化的DNA序列。免疫沉淀后，通过放射性标记的聚合酶链反应检测序列，并通过光密度测定法定量条带的相对强度。通过比较结合/未结合片段的比率来确定特定位点处组蛋白的相对乙酰化状态。我们确定，在转录起始位点上游-378至-303碱基对的IAP启动子片段中，在分化的、表达IAP的细胞中组蛋白H3的乙酰化状态增加了两倍，而组蛋白H4的乙酰化状态基本保持不变。此外，在一个较远的位点，即-1378至-1303碱基对之间，未分化细胞和分化细胞之间H3和H4的乙酰化状态没有明显变化。我们得出结论，丁酸盐诱导的分化与IAP启动子内组蛋白乙酰化状态的特定和局部变化相关。内源性IAP基因内的这些变化可能是其在肠上皮细胞分化程序背景下转录激活的基础。
