Tissue-specific evolutionarily conserved regulation of gene expression

事实上，多细胞生物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在生物体中发挥其所需的作用。正是基因组中每个基因在每种细胞类型中表达方式的差异，导致了细胞功能的巨大差异。尽管经过数百万年的进化，脊椎动物之间在外观上存在很大差异，但它们基本上是由相似的细胞类型和器官组成的。不同脊椎动物的大脑、肝脏和心脏在同一器官内比在同一物种的不同器官之间具有更多的共同基因表达。这一共性表明，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理解，也无法确定基因组中大部分不在基因中的区域，但它们负责这种显著的保护。****该提案将通过鉴定在4.5亿年的进化中功能保守的基因表达活性增强子来研究指定组织特异性基因表达的机制。鉴定这些增强子具有挑战性，因为它们很短，并且通常位于基因组中没有序列保守性低的基因的区域。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，已经预测了一小部分对发育重要的增强子，但这一小部分并不能解释这些物种之间基因表达的显着全基因组保护。提出的研究将允许更好地理解，不仅如何识别复杂基因组的表达增强子，而且dna结合蛋白调节基因表达在不同的细胞环境。****本建议的目标是：***1)通过计算方法和全基因组增强子活性的实验验证，鉴定胚胎干细胞、滋养层细胞和成纤维细胞中的活性增强子，确定基因表达在不同细胞类型中的调节机制。***2)通过研究10种脊椎动物成纤维细胞的基因表达水平和鉴定活性增强子，确定增强子活性和基因表达是如何通过进化调节的。***这将是首次在进化过程中对增强子活性进行研究，也是首次在脊椎动物基因组中对活性增强子进行全基因组鉴定。这种创新的方法将专注于可以在细胞培养中保持的特征良好的细胞类型，以开发研究方法，但从长远来看，我们将把我们的专业知识应用于更复杂的生物体中，以了解基因表达是如何通过发育控制的，以及这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.*** **