Transcriptional Control and Lineage Commitment by Pax-5

描述(由申请人提供)：Pax-5(BSAP)是一种早期B细胞特异的转录和谱系承诺调节因子。我们以前已经证明，Pax-5是重要的B细胞特异性基因mb-L(Ig-α)转录所必需的。为了更好地了解Pax-5如何在体内结合DNA和调节转录，我们与其他研究人员合作，确定了Pax-5及其伴侣蛋白Ets-1与mb-I启动子DNA结合的X射线晶体结构。该信息结构为测试Pax-5中单个氨基酸结合不同核苷酸序列的功能、招募ETS伙伴在体内结合靶基因以及评估Pax-5在B细胞中的作用提供了基础。为了说明Pax-5如何控制早期B细胞的发育，我们将在B细胞系或从Pax-5缺陷小鼠分离的B细胞祖细胞中表达野生型Pax-5或识别更受限的DNA序列子集的突变蛋白。这些“改变的特异性”突变蛋白在体外表现出不同的结合模式，并有望在体内激活或抑制不同的基因集。其他突变选择性地降低了Pax-5招募ETS伙伴蛋白与DNA结合的能力。我们将使用这些突变体来检验Pax-5：ETS相互作用对于控制B细胞特异性转录的重要性的假设。这些实验还将有助于确定Pax-5转录激活所需的ETS蛋白。Pax-5被称为B系承诺的“主要调节器”，因为它对正常的B系进程是必不可少的。Pax-5阻断了“杂乱”的转录和分化，允许固定B细胞的表型。与正常的B细胞前体细胞不同，Pax-5缺陷的B细胞前体细胞在细胞因子的反应下获得其他细胞的命运，包括自然杀伤细胞和巨噬细胞。我们将测试“改变的特异性”突变的Pax-5蛋白激活B细胞程序的能力，并阻止混杂分化。我们的研究将为B细胞特异性转录和B细胞发育的调控提供重要的见解。

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.