DIFFERENTIAL GENE EXPRESSION IN INTESTINAL CELL LINES

哺乳动物的肠上皮是一个不断发展的 系统 每个肠隐窝包含一个单一的，多能干 快速增殖的细胞。 这个词干的后代 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。 这种偶联的细胞增殖和分化受到 严格的监管。 许多肠道基因的表达遵循 沿着直肠-结肠（水平）轴的分布梯度， 隐窝绒毛和隐窝表面上皮（垂直轴 小肠和大肠。 独特的区域- 基因调控的特异性、细胞特异性和空间特异性 使得肠上皮对于研究 控制细胞增殖和分化的机制。 我们 以前分离出一个基因，命名为A4，这是转录 在人结肠上皮细胞分化时被激活 HT 29 -18行。A4的表达在肠道中富集， 上皮细胞，并遵循增加，水平梯度从 小肠到大肠。 此外，A4的表达是 大量存在于隐窝中， 沿着小泡的隐窝绒毛和隐窝表面上皮轴 和大肠。 这些显著特征表明， A4可以作为一个很好的隐窝细胞分化的标志物， 了解A4表达的调控机制， 体外培养可能有助于阐明控制肠道的机制 上皮分化 在本项目期间， 计划项目，我们已经取得了重大进展， 了解A4启动子在HT 29过程中的调控 分化 具体地说，我们确定了一个阳性顺式元件， 与DNA结合酶相互作用的近端A4启动子 蛋白质（假定的转录因子）与分化- 依赖性活动。 此外，我们还证明了A4是 蛋白脂质家族的蛋白质，是膜蛋白的一种。 内质网与推定的离子通道活动。 两 在竞争性续期申请中提出具体目标 项目5：（1）分离和表征差异- 依赖性DNA结合因子（称为GATG结合蛋白）， 与A4启动子相互作用，并研究其对上皮细胞的影响。 分化，和（2）进一步表征A4的功能， 离子通道和分化的潜在介质。 这些 研究应该有助于了解控制肠道的机制， 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.