CAREER: Mechanisms Regulating the Activity of the Pax-5 Transcription Factor During B-cell Development

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Zwollo, P The transcription factor Pax-5 is expressed during the early stages of B-cell development and influences the expression of several B cell-specific genes, most of which are involved in cell development and proliferation. Over the past grant period, the existence of alternatively spliced isoforms of the murine Pax-5 gene was investigated. In addition to the existing isoform (Pax-5a), three new isoforms, Pax-5b, 5d, and 5e, were isolated. In isoforms Pax-5b and Pax-5e, exon 2 has been spliced out, resulting in proteins with an incomplete DNA-binding domain. In Pax-5d and Pax-5e, the 3' region which encodes the transactivating domain, is deleted and replaced by a novel sequence with unknown function. Recent studies revealed that both Pax-5d and Pax-5a protein levels are high in normal B-cells, in contrast to cell lines of the B-cell lineage, where Pax-5d levels are very low. When normal B cells were activated by the B-cell mitogen lipopolysaccharide, the protein level of Pax-5d, but not 5a, decreased rapidly. The high protein levels of Pax-5d in resting B-cells, as well as its low levels in proliferating B-cells, would be in agreement with it functioning as a dominant negative regulator of proliferation or growth. Pax-5b protein, on the other hand, is only present during the later stages of B-cell development, including the mature B and plasma cell stages. The presence of Pax-5b so late in development was unexpected and is interesting in light of its possible regulatory roles during the terminal stages of B-cell development. In this project, the transcriptional activities of Pax-5 isoforms 5a, 5b, and 5d will be investigated. These studies are expected to lead to an increased understanding of specific mechanisms that regulate activity of the transcription factor Pax-5 during B-cell differentiation. Such mechanisms may include both the functional activities of its alternative isoforms as well as the differential regulation of their protein stability. The protein Pax-5a has been shown to play important roles during the development of antibody-producing cells. Pax-5 is a "regulator" protein that affects the amount of antibodies that are made in this cell type. Pax-5 also plays a role in cell proliferation, thus regulating the amount of "proliferation" determining proteins in the cells, but this process is not well understood. In this project, the main question will be: what is the role of Pax-5a, as well as two other Pax-5 like proteins (named Pax-5b and Pax-5d) in the development and activation of antibody producing cells. This research will provide insight into possible mechanisms that Pax-5a, 5b, and 5d use to make sure that cells produce antibodies at the right time and in the right place.