Analysis of Pax2 target genes involved in metanephric kidney morphogenesis

描述(申请人提供)：人类基因组包含了在胚胎发育过程中形成数千个特化细胞和组织的所有信息。尽管所有细胞都含有相同的基因，但细胞分化需要在发育过程中选择性地激活和抑制基因。如果不能正确地调控这种基因选择性的过程，就会导致癌症、发育异常、细胞退化和许多其他疾病状态。细胞分化过程中的基因激活和抑制模式是由表观遗传机制指定的，这些机制是可遗传的，并受到修改。真核细胞染色质由包裹在组蛋白八聚体上的DNA组成。通过乙酰化、磷酸化、甲基化或泛素化来修饰核心组蛋白尾部可以显著改变局部染色质结构和基因表达的可能性。积累的生化和遗传证据表明，组蛋白H3和H4的特定赖氨酸残基上的甲基化可以决定一个基因是否仍然可以被转录机制访问，或者它是否被沉默成紧密包装的异染色质。这种组蛋白的表观遗传修饰可以解释胚胎发育过程中可遗传的细胞记忆，并可能极大地影响疾病和衰老细胞的基因表达模式。Pax2基因编码一种DNA结合蛋白，对于早期发育的肾脏和泌尿生殖道是必不可少的。Pax2功能丧失会导致完全性肾发育不全。然而，Pax2的过度表达与多种肾脏疾病有关，包括癌症和多囊肾病。最近，我们发现了一种连接Pax2和哺乳动物组蛋白H3赖氨酸4甲基转移酶复合体的蛋白质。为了了解这种复合体如何调节肾脏模式和疾病，我们必须确定Pax2直接调控的基因，并确定Pax2/DNA相互作用的位置。这个提议将采取系统的方法来定义Pax2靶基因和小鼠基因组中的Pax2结合位点。我们将展示这些结合位点的表观遗传印迹模式随Pax2活性的变化。利用胚胎组织和细胞培养模型，我们将测试Pax2/DNA相互作用的意义，并表征这些位置上组蛋白甲基化的修饰。这些实验将定义决定细胞命运的发育调控基因如何以可遗传的方式改变染色质结构。

DESCRIPTION (provided by applicant): The human genome contains all of the information to make thousands of specialized cells and tissues during embryonic development. Even though all cells contain the same genes, cellular differentiation requires the selective activation and suppression of genes during development. Failure to correctly regulate this process of gene selectivity results in cancer, developmental abnormalities, cellular degeneration, and many other disease states. Gene activation and suppression patterns during cellular differentiation are specified by epigenetic mechanisms that are heritable and subject to modifications. Eukaryotic chromatin consists of DMA wrapped around a histone octamer. Modification of the core histone tails by acetylation, phosphorylation, methylation or ubiquitination can dramatically alter the local chromatin structure and the potential for gene expression. Accumulated biochemical and genetic evidence indicates that methylation at specific lysine residues of histones H3 and H4 can determine whether a gene remains accessible to the transcription machinery or whether it is silenced into tightly packaged heterochromatin. Such epigenetic modifications of histones could account for a heritable cellular memory during embryonic development and could greatly affect gene expression patterns in diseased and aging cells. The Pax2 gene encodes a DNA binding protein and is essential for specifying the kidney and urogenital tract early in development. Loss of Pax2 function results in complete renal agenesis. Yet, overexpression of Pax2 is associated with a variety or renal diseases, including cancer and polycystic kidney disease. Recently, we identified a protein that links Pax2 to a mammalian histone H3 lysine 4 methyltransferase complex. In order to understand how this complex regulates renal patterning and disease, we must identify genes that are directly regulated by Pax2 and determine the sites of Pax2/DNA interactions. This propsal will take a systematic approach to define Pax2 target genes and Pax2 binding sites within the mouse genome. We will demonstrate changes in epigenetic imprinting patterns at these binding sites in response to Pax2 activity. Using both embryonic tissues and cell culture models, we will test the significance of Pax2/DNA interactions and characterize the modifcations of histone methylation at these sites. These experiments will define how a developmental regulatory gene that specifies cell fate can alter chromatin structure in a heritable way.