Spatiotemporal Regulation of Intestinal Gene Expression

描述（由申请人提供）：乳糖酶不持续性（成人型乳酸缺乏症）导致消化系统对乳糖（牛奶中存在的碳水化合物常量营养素）吸收不良。最近发现了与肠道乳糖酶持续性和非持续性相关的人类DNA序列变异。本研究项目侧重于人类肠道乳糖酶持久性的遗传决定因素的表征。我们之前已经在细胞培养和活体动物中描述了调节大鼠乳糖酶基因肠道特异性表达的分子机制。我们现在将直接关注的表征分子机制介导人类乳糖酶基因的持久性和非持久性和乳糖酶在啮齿类动物的时间下降。初步研究表明，乳糖酶基因多态性区域的DNA序列可以结合核蛋白，增强乳糖酶启动子活性。我们假设与乳糖酶持久性相关的DNA多态性通过与特定核蛋白转录因子的差异相互作用来调节成人肠细胞中乳糖酶基因的转录。因此，研究目标旨在确定人类乳糖酶持久性的遗传决定因素的机制作用。在目标1中，我们将描述与人类乳糖酶基因转录持久性相关的DNA多态性的功能作用。乳糖酶基因多态性的功能表征将在细胞培养和转基因小鼠体内进行研究。在目标2中，我们将定义调节人类乳糖酶持久性基因转录的分子机制。本研究的目的是鉴定和表征能够与与乳糖酶持久性相关的乳糖酶基因多态性区域的DNA序列相互作用的核蛋白，并表征核蛋白是否具有调节乳糖酶转录的功能。在Aim 3中，为了补充我们对人类乳糖酶持久性机制的研究，我们将进一步阐明啮齿动物乳糖酶时间下降的调节机制。了解调节人类乳糖酶基因表达的机制可能为导致上皮细胞消化功能丧失或不适当增加的胃肠道疾病提供未来新的诊断、治疗或预后策略。我们的体外和体内实验方法的结合将阐明与乳糖酶持久性和非持久性相关的遗传决定因素的作用。公共卫生相关性：成人发病乳酸缺乏症，乳糖酶不持续性，使世界上大部分成年人不能耐受过量食用牛奶和其他乳制品。然而，在一些成年人中，高水平的乳糖酶活性在成年期持续存在，这可能是由于遗传了一种基因突变，这种突变阻止了乳糖酶表达的正常成熟下降。对消化性乳糖酶基因表达调控机制的了解可能为肠道疾病中消化酶表达的增强提供新的策略，并将对理解其他基因与衰老的调控具有广泛的意义。

DESCRIPTION (provided by applicant): Lactase non-persistence (adult-type hypolactasia) results in digestive system malabsorption of lactose, the carbohydrate macronutrient present in milk. Human DNA sequence variants associated with intestinal lactase persistence and non-persistence have recently been identified. This research project focuses on characterization of the genetic determinants of intestinal lactase persistence in humans. We have previously characterized molecular mechanisms regulating intestine-specific expression of the rat lactase gene in cell culture and in living animals. We will now direct attention to characterization of the molecular mechanisms mediating human lactase gene persistence and non- persistence and the temporal decline of lactase in rodents. Preliminary studies have shown that DNA sequences in the region of lactase gene polymorphisms can bind nuclear proteins and enhance lactase promoter activity. We hypothesize that DNA polymorphisms associated with lactase persistence function to regulate lactase gene transcription in adult human enterocytes via differential interaction with specific nuclear protein transcription factors. Research objectives are therefore aimed at defining the mechanistic roles of genetic determinants of human lactase persistence. In Aim 1, we will characterize a functional role for DNA polymorphisms associated with persistence of lactase gene transcription in humans. Functional characterization of lactase gene polymorphisms will be investigated in cell culture and in vivo in transgenic mice. In Aim 2, we will define the molecular mechanisms regulating human lactase persistence gene transcription. The goals of this aim will be to identify and characterize nuclear proteins capable of interacting with DNA sequences in the region of lactase gene polymorphisms associated with lactase persistence and to characterize whether the nuclear proteins function to regulate lactase transcription. In Aim 3, to complement our investigation of the mechanism of lactase persistence in humans, we will further elucidate mechanisms regulating the temporal decline of lactase in rodents. Knowledge of mechanisms regulating human lactase gene expression may provide future novel diagnostic, therapeutic or prognostic strategies for gastrointestinal disorders that result in loss or inappropriate gain of epithelial cell digestive function. Our combination of in vitro and in vivo experimental approaches will elucidate roles for the genetic determinants associated with lactase persistence and non-persistence. PUBLIC HEALTH RELEVANCE: Adult-onset hypolactasia, lactase non-persistence, renders much of the world's adult human population intolerant of excessive consumption of milk and other dairy products. In some adults, however, high levels of lactase enzyme activity persist in adulthood presumably due to inheritance of a genetic mutation that prevents the normal maturational decline in lactase expression. Knowledge of mechanisms regulating expression of the digestive lactase gene may provide novel strategies for enhancing expression of digestive enzymes in intestinal diseases and will have broad implications for understanding regulation of other genes with aging.