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细胞特异性转录和谱系承诺调节剂。我们之前已经证明Pax-5是一个重要的B细胞特异性基因mb- 1 （ig - α）的转录所必需的。为了更好地了解Pax-5如何结合DNA并调节体内转录，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。该信息结构为检测Pax-5中单个氨基酸结合不同核苷酸序列的功能作用、在体内招募Ets伴侣结合靶基因以及评估Pax-5在B细胞中的作用提供了基础。为了解决Pax-5如何控制早期B细胞发育的问题，我们将在B细胞系或从Pax-5缺陷小鼠中分离的B细胞祖细胞中表达野生型Pax-5或突变蛋白，这些蛋白识别更有限的DNA序列亚群。这些“特异性改变”的突变蛋白在体外表现出与不同位点的不同结合模式，并有望在体内激活或抑制不同的基因组。其他突变选择性地降低了Pax-5招募Ets伴侣蛋白结合DNA的能力。我们将使用这些突变体来验证Pax-5:Ets相互作用对控制B细胞特异性转录很重要的假设。这些实验也将有助于鉴定与Pax-5转录激活所需的Ets蛋白。Pax-5被称为B谱系承诺的“主调节剂”，因为它对正常的B谱系进展至关重要。Pax-5阻断“混杂”的转录和分化，允许B细胞表型的固定。与正常的B细胞祖细胞不同，pax -5缺陷的B细胞祖细胞在细胞因子的作用下获得其他细胞命运，包括自然杀伤细胞和巨噬细胞。我们将测试“特异性改变”突变Pax-5蛋白激活B细胞程序和阻断混杂分化的能力。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.