Tissue-specific evolutionarily conserved regulation of gene expression

实际上，多细胞生物体中的每个细胞都含有相同的DNA基因组，但每种细胞类型的外观和功能都不同，以履行其在有机体中所需的角色。正是基因组中每个基因在每种细胞类型中表达方式的不同，解释了细胞功能的巨大差异。尽管经过数百万年的进化，脊椎动物的外表千差万别，但它们基本上是由相似的细胞类型和器官组成的。来自不同脊椎动物物种的大脑、肝脏和心脏在同一器官内的共同基因表达多于同一物种内不同器官之间的基因表达。这种共性表明，在每个组织中调节基因表达的机制在进化过程中是高度保守的。然而，我们缺乏对这些机制的了解，也无法准确定位基因组中大多数不在基因中但负责这种非凡保守的区域。*这项建议将通过识别活性的基因表达增强子来研究指定组织特异性基因表达的机制，这些基因表达增强子在4.5亿年的进化中功能上是保守的。识别这些增强子具有挑战性，因为它们很短，通常位于基因组中没有基因的区域，序列保守性较低。尽管根据从鱼类到人类等脊椎动物中发现的非常相似的DNA序列，预测了少量具有发育重要性的增强子，但这一少量并不能解释这些物种的基因表达在全基因组范围内的显著保守。这项拟议的研究不仅可以更好地理解如何识别复杂基因组中的表达增强子，而且还可以更好地了解哪些DNA结合蛋白调节不同细胞环境中的基因表达。*这项建议的目标是：*1)通过使用计算方法和基因组范围的增强子活性的实验验证来确定胚胎干细胞、滋养层细胞和成纤维细胞中的活性增强子，确定基因表达是如何在不同类型的细胞中调节的。*2)通过研究基因表达水平和鉴定10种脊椎动物成纤维细胞中的活性增强子，确定增强子活性和基因表达是如何通过进化调节的。*这将是第一次在进化过程中研究增强子活性，也是第一次在脊椎动物基因组中鉴定活性增强子。这种创新的方法将重点放在可以在细胞培养中保持良好特征的细胞类型上，以发展研究方法，但从长远来看，我们将把我们的专业知识应用于活生物中更复杂的情况，以了解基因表达是如何通过发育控制的，以及这种情况在进化过程中是如何变化的。

Virtually every cell in a multicellular organism contains the same DNA genome yet each celltype looks and functions differently to perform its required role in the organism. It is the differences in the way each gene in the genome is expressed in each cell type that accounts for these dramatic differences in cellular functions. Even though there is a wide variety in appearance between vertebrates separated by millions of years of evolution, they are fundamentally made up of similar cell-types and organs. Brain, liver and heart from different vertebrate species share more common gene expression within the same organ than between different organs within the same species. This commonality indicates that the mechanisms that regulate gene expression in each tissue are highly conserved through evolution. We lack, however, an understanding of these mechanisms and are not able to pinpoint most of the regions of the genome that are not in genes, but that are responsible for this remarkable conservation.****This proposal will investigate the mechanisms that specify tissue-specific gene expression through identifying active enhancers of gene expression that are functionally conserved through 450 million years of evolution. Identifying these enhancers is challenging as they are short and often located in regions of the genome without genes where sequence conservation is low. Although a small number of developmentally important enhancers have been predicted based on very similar DNA sequences found in vertebrates ranging from fish to humans, this small number does not account for the remarkable genome-wide conservation in gene expression across these species. The proposed research will allow for a better understanding not only of how to identify enhancers of expression in complex genomes but also which DNA-binding proteins regulate gene expression in different cellular contexts. ****The objectives of this proposal are; ***1) To determine how gene expression is modulated in different cell types by identifying active enhancers in embryonic stem cells, trophoblasts and fibroblasts using computational approaches and genome-wide experimental validation of enhancer activity. ***2) To determine how enhancer activity and gene expression is modulated through evolution by investigating gene expression levels and identifying enhancers active in fibroblasts from 10 vertebrate species. ***This will be the first investigation of enhancer activity across evolution and the first genome-wide identification of active enhancers in vertebrate genomes. This innovative approach will focus on well characterized cell types which can be maintained in cell culture to develop the research methods, but over the long term we will apply our expertise to more complex situations in living organisms to develop an understanding of how gene expression is controlled through development and how this has changed during evolution.*** **