Transcriptional Control and Lineage Commitment by Pax-5

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DESCRIPTION (provided by the applicant): Pax-5 (BSAP) is an early B cell-specific regulator of transcription and lineage commitment. We have previously shown that Pax-5 is required for transcription of an important B cell-specific gene, mb-l(Ig-alpha). To better understand how Pax-5 binds DNA and regulates transcription in vivo, we collaborated with other investigators to determine the X-ray crystallographic structure of Pax-5 together with its partner protein Ets-1 bound to mb-I promoter DNA. The informative structure provides the basis for testing the functional roles of individual amino acids in Pax-5 for binding different nucleotide sequences, for recruitment of Ets partners to bind target genes in vivo, and for assessing roles of Pax-5 in B cells. To address how Pax-5 controls early B cell development, we will express wild type Pax-5 or mutated proteins that recognize a more restricted subset of DNA sequences in a B cell line, or in B cell progenitors isolated from Pax-5-deficient mice. These "altered specificity" mutant proteins exhibit different patterns of binding to various sites in vitro and are expected to activate or repress different sets of genes in vivo. Other mutations selectively reduce the ability of Pax-5 to recruit Ets partner proteins to bind DNA. We will use these mutants to test the hypothesis that Pax-5:Ets interactions are important for controlling B cell-specific transcription. These experiments will also aid in identifying Ets proteins required for transcriptional activation with Pax-5. Pax-5 has been called a "master regulator" of B lineage commitment because it is essential for normal B lineage progression. Pax-5 blocks "promiscuous" transcription and differentiation, allowing for fixation of the B cell phenotype. Unlike normal B cell progenitors, Pax-5-deficient B cell progenitors attain other cell fates, including natural killer cells and macrophages, in response to cytokines. We will test the abilities of "altered specificity" mutant Pax-5 proteins to activate the B cell program, and block promiscuous differentiation. Our studies will provide important insights concerning the regulation of B cell-specific transcription and B cell development.