B cell development in the bone marrow proceeds in a stepwise process from multipotent hematopoietic stem cells (HSCs) to committed immature B lymphocytes. Research has elucidated a B cell-specific regulatory network of transcription factors necessary for the development of early B cells. These proteins include Early B cell Factor 1 (EBF1), which drives B lineage specification and commitment. Although much is known about EBF1, there are many unanswered questions concerning its regulation in B cells and its potential for protein- protein interactions. Several lines of evidence point to Zfp521 (Evi3/ZNF521/EHZF) as a co-regulator of gene transcription with EBF1. Zfp521 interacts physically with EBF1 and can repress its activity in vitro. Zfp521 is co- expressed with EBF1 in developing B cells. However, little is known concerning functions of Zfp521 in B cell development. Specifically, the mechanisms by which Zfp521 modulates activities of EBF1 and the basis and consequences of physical interactions between Zfp521 and EBF1 are unknown. We have demonstrated that early B cell development is perturbed greatly in the absence of Zfp521. This is evidenced by a lack of expression of markers of late pro-B cells and pre-B cells and decreased immature B cells in bone marrow of Zfp521-deficient mice. As a mechanism that integrates the activities of both Zfp521 and EBF1, recent studies in our laboratory using newly developed antibody reagents detected Zfp521 binding to promoters that also bind EBF1. Co-occupancy of Zfp521 and EBF1 on these genes suggests a new model for co-regulation of B lymphopoiesis. In this manner, EBF1, the B lineage determination factor, is regulated by Zfp521 in developing B cells. Hypotheses: Zfp521 is critically important for B cell lymphopoiesis and function. We propose that it regulates the B cell program via EBF1-dependent and -independent mechanisms. Zfp521 interacts with EBF1 to limit its DNA binding, but Zfp521 also assembles complexes with EBF1 on target gene DNA. Determining the basis of interactions between EBF1 and Zfp521 at the molecular level is critical for understanding how the B cell regulatory network generates mature B cells capable of secreting antibodies in humoral immune responses. Therefore, we will: Determine how Zfp521 regulates gene transcription in developing B cells. Determine how Zfp521 functions in the B cell regulatory network by identifying its binding sites in pro- B cells. Define structural requirements and functional consequences of Zfp521 interactions with EBF1.
This project focuses on the functions of a novel nuclear protein, Zfp521, in B cell development and function. ZFp521 is required for the efficient production of antibody-producing B cells. Mice lacking Zfp521 have fewer B cells relative to normal mice. In part, Zfp521 functions by regulating EBF1, the B cell lineage determination/commitment factor. Zfp521 inhibits DNA binding by EBF1, but also binds DNA together with EBF1 in the context of chromatin in B cells. Zfp521 is a proto-oncogene that, when over-expressed, generates B cell leukemias and lymphomas, but little is known concerning these mechanisms.
