POTENTIAL ROLE OF TFII-I IN IMMUNODEFICIENCY

TFII-II是一种重要的多功能转录因子，在多个基因中将事件与转录联系起来。TFII-I在构成上与Bruton酪氨酸激酶(BTK)有关，BTK是一种对正常B细胞功能至关重要的非受体酪氨酸激酶，因为它的突变会导致人类的X-连锁无丙球蛋白血症(XLA)和小鼠的X-连锁免疫缺陷(XID)。我们认为，TFII-I是连接BTK介导的信号与B细胞转录的一个重要的新成分。此外，威廉综合征(WS)中TFII-I基因缺失，这是一种多系统表现的神经发育障碍，包括瓣膜上动脉狭窄、婴儿期高钙血症、智力低下和认知缺陷。因此，TFII-I似乎与两种遗传性疾病有关：威廉综合征和X连锁无丙种球蛋白血症(XLA)。从这些研究中获得的知识可能有助于我们更好地理解BTK依赖的关键途径，该途径将B细胞受体介导的信号转导与B细胞特异性转录联系起来。这些研究也可能最终帮助确定受BTK突变影响的潜在靶基因(S)。重要的是，这些研究可能会在神经发育障碍(如WS)和免疫发育障碍(如XLA)之间建立可能的联系。为了更好地了解TFII-I在BTK介导的免疫应答中的作用，我们首先定位了TFII-I与BTK的物理和功能相互作用的重要区域(S)。我们将通过缺失和点突变来确定TFII-I中与BTK相互作用的重要区域(S)，然后通过定点突变、磷酸肽、指纹图谱和质谱分析相结合的方法，在体外和体内定位TFII-I中被BTK磷酸化的酪氨酸位点。我们还将在功能瞬时转染实验中分析这些突变体。为了确定TFII-I在B细胞中的功能及其与BTK的生化相互作用，我们将采用体内转录分析。为了确定TFII-I在B细胞中的功能及其与BTK的生化相互作用，我们将采用体内转录分析，然后通过免疫共沉淀和异位表达突变形式的TFII-I在B细胞中进行相互作用研究。我们还将在B细胞系中稳定表达野生型和突变型TFII-I和BTK，并从鸡B细胞中基因删除TFII-I。最后，为了确定TFII-I在无BTK和激活BTK存在的情况下的定位，首先，我们将TFII-I的各种突变体与BTK在COS细胞中共表达。随后，我们将使用来自野生型、XID和BTK-/-小鼠的新鲜分离的原代脾B细胞，研究在没有B细胞受体信号和存在B细胞受体信号的情况下TFII-I的定位和酪氨酸磷酸化。

TFII-II is an important multi-functional transcription factor that links events to transcription in several genes. TFII-I is constitutively associated with Bruton's tyrosine kinase (Btk), a non-receptor tyrosine kinase that is essential for normal B cell function, as its mutation causes X-linked agammaglobulinemia (XLA) in humans and X-linked immune deficiency (xid) in mice. We propose that TFII-I is an important and novel component in linking Btk-mediated signaling to transcription in B cells. Furthermore, the TFII-I gene gets deleted in William's syndrome (WS) which is a neuro-developmental disorder with multi-system manifestations, including supravalvar aortic stenosis, hypercalcemia in infancy, mental retardation and cognitive defects. Thus, TFII-I appears to be involved in two genetic disorders: William's Syndrome and X-linked agammaglobulinemia (XLA). Knowledge gained from these studies may help us better understand a critical Btk dependent pathway that links B cell receptor mediated signal transduction to B cell specific transcription. These studies may also ultimately help identify potential target gene(s) that are affected by mutations in Btk. Importantly, these studies may establish possible connections between the neuro-developmental disorders (as in WS) and immuno-developmental disorders (as in XLA). Toward a better understanding of TFII-I function in Btk mediated immune response, we will first map the region(s) in TFII-I important for its physical and functional interactions with BTK. We will determine by deletion and point mutation the region(s) in TFII-I that is important for its interaction with Btk, followed by mapping the sites in TFII-I that are tyrosine phosphorylated by Btk in vitro and in vivo by a combination of site directed mutagenesis, phosphopeptide, finger printing, and mass spectrometric analysis. We will also analyze these mutants in functional transient transfection assays. To determine the functions of TFII-I and its biochemical interactions with Btk in B cells, we will employ in vivo transcriptional analysis. To determine the functions of TFII-I and its biochemical interactions with Btk in B cells, we will employ in vivo transcriptional analysis followed by the interaction studies by co- immunoprecipitation and ectopic expression of mutant forms of TFII-I in B cells. We will also stably express wild type and mutant forms of TFII-I, and Btk in B cell lines, and genetically delete TFII-I from chicken B cells. Finally, to ascertain the localization of TFII-I in the absence and in the presence of non-activated versus activated Btk, first, we will co-express various mutants of TFII-I with Btk in COS cells. Subsequently, we will employ freshly isolated primary splenic B cells derived from wild type, xid and Btk-/- mice and study the localization and tyrosine phosphorylation of TFII-I in the absence and in presence of B cell receptor signaling.