DIFFERENTIAL GENE EXPRESSION IN INTESTINAL CELL LINES

肠细胞系中的差异基因表达

• 批准号: 6500423
• 负责人: Vincent W Yang
• 金额: $ 10.37万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6500423
• 关键词: DNA binding protein; DNA footprinting; Xenopus oocyte; animal genetic material tag; cell differentiation; cell proliferation; clone cells; gastrointestinal epithelium; gel mobility shift assay; gene expression; genetic regulation; human genetic material tag; human tissue; in situ hybridization; laboratory rat; membrane channels; microinjections; molecular cloning; northern blottings; pluripotent stem cells; protein structure function; site directed mutagenesis; southern blotting; transcription factor; transfection; western blottings

The mammalian intestinal epithelium is a continuously developing system. Each intestinal crypt contains a single, multipotent stem cell which undergoes rapid proliferation. Descendants of this stem cell become the four principal cell types that constitute the gut epithelium: absorptive, goblet, enteroendocrine, and Paneth cells. This coupled cell proliferation and differentiation is subject to stringent regulation. Expression of many intestinal genes follow a gradient of distribution along the duodenal-colon (horizontal) axis, and the crypt-villus and crypt-surface epithelium (vertical axes of the small and large intestines, respectively. The unique region- specific, cell-specific, and spatial-specific nature of gene regulation renders the intestinal epithelium attractive for studying the mechanism controlling cell proliferation and differentiation. We previously isolated a gene, named A4, that is transcriptionally activated upon differentiation of the human colonic epithelial cell line HT29-18. Expression of A4 is enriched in the intestinal epithelium and follows an increasing, horizontal gradient from the small to the large intestine. In addition, expression of A4 is abundant in the crypt and follows a diminishing, vertical gradient along the crypt-villus and crypt-surface epithelium axIs of the small and large intestine, respectively. These salient features suggest that A4 may serve as an excellent marker of crypt cell differentiation, and that understanding the mechanism regulating A4 expression in vitro may shed some light on the mechanism controlling gut epithelial differentiation. Within the project period of the current Program Project, we have made significant progress in understanding the regulation of the A4 promoter during HT29 differentiation. Specifically, we identified a positive cis-element in the proximal A4 promoter that interacts with a DNA-binding protein (putative transcription factor) with a differentiation- dependent activity. In addition, we showed that A4 is a member of the proteolipid family of proteins and is a membrane protein of the endoplasmic reticulum with putative ion channel activity. Two Specific Aims are proposed in the competitive renewal application for Project 5: (1) to isolate and characterize the differentiation- dependent DNA-binding factor (named GATG-binding protein) that interacts with the A4 promoter and to study its effect on epithelial differentiation, and (2) to further characterize the function of A4 as an ion channel and as a potential mediator of differentiation. These studies should help understand the mechanism controlling intestinal epithelial differentiation.

哺乳动物肠上皮是一种不断发展的 系统。 每个肠道隐窝都包含一个单一的多能茎 经历快速增殖的细胞。 这个茎的后代 细胞成为构成肠道的四种主要细胞类型 上皮：吸收性，杯状，肠内分泌和泛细胞。 这种耦合的细胞增殖和分化受 严格的法规。 许多肠基因的表达遵循 沿十二指肠形（水平）轴的分布梯度， 以及地下村和地下表面上皮（垂直轴的垂直轴 小肠和大肠。 独特的地区 - 基因调控的特异性，细胞特异性和空间特异性 使肠上皮在研究 控制细胞增殖和分化的机制。 我们 以前孤立的一个名为A4的基因是转录 随着人类结肠上皮细胞的分化而激活 线HT29-18。 A4的表达富含肠道 上皮并遵循从越来越多的水平梯度 小肠到小。 此外，A4的表达是 地下室丰富，遵循垂直梯度减少 沿着小村庄和地下表面上皮轴 和大肠。 这些显着特征表明 A4可以作为隐窝细胞分化的绝佳标志， 并理解调节A4表达的机制 体外可能会阐明控制肠道的机制 上皮分化。 在当前的项目期间 计划项目，我们在 了解HT29期间A4启动子的调节 分化。 具体而言，我们确定了一个阳性的顺式元素 与DNA结合相互作用的近端A4启动子 蛋白质（推定转录因子）具有分化 - 依赖活动。 此外，我们表明A4是 蛋白质的蛋白质家族，是 内质网具有推定的离子通道活性。 二 在竞争性更新申请中提出了具体目标 对于项目5：（1）分离和表征分化 - 依赖的DNA结合因子（称为GATG结合蛋白） 与A4启动子相互作用并研究其对上皮的影响 分化，（2）进一步表征A4的功能为 离子通道和作为分化的潜在介体。 这些 研究应有助于了解控制肠道的机制 上皮分化。