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

转录因子Pax-5在B细胞发育的早期阶段表达，并影响几个B细胞特异性基因的表达，其中大多数参与细胞发育和增殖。在过去的资助期间，研究了小鼠Pax-5基因的选择性剪接异构体的存在。除现有的Pax-5a异构体外，还分离到3个新的异构体Pax-5b、5d和5e。在Pax-5b和Pax-5e亚型中，外显子2被剪接掉，导致蛋白质具有不完整的dna结合域。在Pax-5d和Pax-5e中，编码反激活结构域的3′区被删除，取而代之的是一个功能未知的新序列。最近的研究表明，Pax-5d和Pax-5a蛋白水平在正常b细胞中都很高，而在b细胞系中，Pax-5d水平非常低。当正常B细胞被B细胞有丝分裂原脂多糖激活时，Pax-5d蛋白水平迅速下降，而5a蛋白水平没有下降。静止b细胞中Pax-5d蛋白水平高，而增殖b细胞中Pax-5d蛋白水平低，这与Pax-5d作为增殖或生长的主要负调节因子的功能是一致的。另一方面，Pax-5b蛋白仅存在于B细胞发育的后期，包括成熟的B细胞和浆细胞阶段。Pax-5b在发育如此晚的阶段出现是出乎意料的，鉴于其在b细胞发育末期可能发挥的调节作用，这一点很有趣。本项目将研究Pax-5亚型5a、5b和5d的转录活性。这些研究有望导致对b细胞分化过程中调节转录因子Pax-5活性的具体机制的进一步了解。这种机制可能包括其替代亚型的功能活性以及对其蛋白质稳定性的差异调节。Pax-5a蛋白已被证明在产生抗体的细胞的发育过程中发挥重要作用。Pax-5是一种“调节”蛋白，影响这种细胞类型中产生的抗体的数量。Pax-5还在细胞增殖中发挥作用，从而调节细胞中“增殖”决定蛋白的数量，但这一过程尚不清楚。在这个项目中，主要的问题将是：Pax-5a以及另外两种类似Pax-5的蛋白质（命名为Pax-5b和Pax-5d）在抗体产生细胞的发育和激活中的作用是什么？这项研究将深入了解Pax-5a、5b和5d用来确保细胞在正确的时间和正确的位置产生抗体的可能机制。

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.