DIFFERENTIAL GENE EXPRESSION IN INTESTINAL CELL LINES

哺乳动物的肠上皮是一种不断发育的 系统。每个肠道隐窝包含一个单一的、多能的干细胞 经历快速增殖的细胞。这根茎的后代 细胞成为构成肠道的四种主要细胞类型 上皮：吸收细胞、杯状细胞、肠内分泌细胞和潘氏细胞。 这种耦合的细胞增殖和分化受到 严格的监管。许多肠道基因的表达伴随着 沿十二指肠-结肠(水平)轴的分布梯度， 和隐窝绒毛和隐窝表面上皮(垂直轴 分别是小肠和大肠。独特的地区- 基因调控的特异性、细胞特异性和空间特异性 使肠上皮具有研究的吸引力 控制细胞增殖和分化的机制。我们 之前分离到了一种基因，命名为A4，它在转录上 人结肠上皮细胞分化后被激活 HT29-18线。A4在肠道中的表达丰富 上皮，并遵循一个递增的水平梯度从 从小肠到大肠。此外，A4的表达是 在地穴中含量丰富，并沿着一个递减的垂直坡度 沿隐窝绒毛和隐窝表面上皮轴的小 和大肠。这些显著的特征表明 A4可能是隐窝细胞分化的一个很好的标志， 而对A4基因表达调控机制的理解 体外实验可能为肠道控制机制提供一些线索 上皮分化。在当前的项目期内 计划项目，我们在以下方面取得了重大进展 理解A4启动子在HT29过程中的调控 差异化。具体来说，我们确定了一个积极的顺式元素在 近端A4启动子与DNA结合的相互作用 具有分化的蛋白质(假定的转录因子)- 从属活动。此外，我们还证明了A4是 蛋白脂家族的蛋白质，是一种膜蛋白 内质网具有可能的离子通道活性。二 在竞争性续签申请中提出了具体目标 对于项目5：(1)分离和表征差异- 依赖DNA结合因子(称为GATG结合蛋白) 与A4启动子的相互作用及其对上皮细胞的影响 分化，以及(2)进一步鉴定A4AS的功能 作为一种离子通道，并作为一种潜在的分化介质。这些 研究应该有助于了解肠道的控制机制 上皮分化。

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.