The dual mechanism of BSAP mediated transcriptional repression on PU.1 activity

Hematopoiesis is a process by which a multipotent cell differentiates into distinct lineages including B lymphocytes. Two transcription factors important for proper hematopoietic development are PU.1 and BSAP. Loss of either factor impairs B cell development by altering gene expression. A locus which serves as a paradigm for gene regulation is the Ig kappa gene. This locus is regulated in a temporal and tissue specific fashion during B cell differentiation. The goal for this project was to further elucidate the transcriptional mechanisms regulating the Ig kappa 3′ enhancer. Here we show that BSAP can repress PU.1 activity by two distinct mechanisms. In the first mechanism, BSAP can repress PU.1 activity independent of other factors. This repression does not influence PU.1 DNA binding or the PU.1 ETS or PEST domains. Instead, BSAP targets a portion of the PU.1 transactivation domain and requires the B SAP inhibitory domain. The mechanism appears to involve a competition for the coactivator p300. In the second mechanism, BSAP can repress the activity of the Igk3′ enhancer by targeting PU.1 activity. This BSAP-mediated repression mechanism does not involve p300, the PU.1 transactivation or PEST domains. Instead, the PU.1 Ets DNA binding domain is the target. BSAP requires multiple BSAP domains for maximal repression, suggesting that multiple protein interactions are required. One interaction involves the PU.1 Ets domain with the B SAP paired box domain. This may be important in nucleating B SAP to the enhancer as BSAP can form a higher order enhancesome complex with other proteins. This complex does not occur in the absence of B SAP and suggests that BSAP can form an inactive enhancesome complex. We also show that BSAP can slightly repress expression of the endogenous Ig kappa locus, suggesting that BSAP can function in vivo to regulate kappa transcription. Lastly, transgenic constructs were prepared in mice to study the activity of the entire enhancer as compared to the enhancer core in vivo