Mechanisms of Transcriptional Regulation in Stem Cells

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DESCRIPTION (provided by applicant): Stem cells must deal with two critical, yet opposing, forces; the need for continual self-renewal and the ability to respond to differentiation signals with large scale changes in gene expression. Many studies have established that regulation of proliferation and differentiation is heavily influenced by chromatin structure. The overall goals of this proposal are to determine the molecular mechanisms responsible for establishing and maintaining patterns of active and inactive chromatin, and thus patterns of gene expression, in stem cells. A highly innovative aspect of this proposal is the combination of chromatin immunoprecipitation with the use of several different types of mouse genomic microarrays. The first set of arrays will contain spotted CpG islands and other regulatory regions that are conserved between the mouse and human genomes. The second type of array will consist of oligonucleotides that encompass an entire mouse chromosome. We will begin our studies by employing the arrays to identify active vs. inactive promoters, using antibodies to RNA polymerase II and modified histones (Aim 1). We will test the hypothesis that different forms of gene regulation predominate in embryonic stem cells vs. in embryoid bodies. These experiments will provide a global snapshot of the amount and location of active vs. inactive chromatin in undifferentiated vs. differentiating embryonic stem cells and will also provide information as to which genes turn on and off in a differentiation-specific manner. In Aim 2A, we will identify targets genes of Oct4, a transcription factor that is known to be critical for maintaining pluripotency of ES cells. After identification of Oct4 target genes, we will characterize the mechanisms by which Oct4 regulates these genes, specifically testing the hypothesis that Oct4 functions as a transcriptional repressor in ES cells. The knowledge gained in Aim 1 concerning the characteristics of active vs. inactive chromatin will be critical for these studies. In Aim 2B, we will characterize the molecular mechanisms responsible for the stem cell-specific regulation of the Oct4 gene.