Analysis of Pax2 target genes involved in metanephric kidney morphogenesis

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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 epigentic 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.