A complex network of transcription factor interactions controls the process by which B lymphocytes develop from hematopoietic stem cells (HSCs). Pax5 has been identified as the critical transcription factor controlling B cell commitment throughout B lymphopoiesis. These analyses revealed that Pax5 commits early hematopoietic progenitors to the B cell pathway by repressing B-lineage-inappropriate genes and by simultaneously activating B-cell-specific genes. By comprehensive cDNA microarray analysis, 110 Pax5-repressed and 170 Pax5-activated genes have been identified, demonstrating that Pax5 controls a complex regulatory network involved in B cell signaling, adhesion, migration and immune function. The goal of the project proposed here will be to systematically analyze all direct Pax5 target genes in the entire genome during B cell development. Recently, a mouse strain carrying a biotin tag at the C-terminus of Pax5 has been generated. We will use this mouse strain for genome-wide identification of Pax5-binding sites by chromatin precipitation and Solexa 1G ultrahigh-throughput DNA sequencing. Islands of chromatin modifications have been shown to provide an efficient way of identifying active and repressed promoters and cis-regulatory elements. Here we propose to use chromatin immunoprecipitation (ChIP)-sequencing to map the entire mouse genome for Pax5-controlled promoters and cis-regulatory elements. Integration of this information with the identification of Pax5-binding sites will provide us with a genome-wide map of functional Pax5-binding sites, from which a list of all direct target genes of Pax5 during B cell development will be obtained. The project proposed here will constitute an essential piece of a larger group effort, which aims at elucidating the gene regulatory network interactions controlling B cell development.
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