Mechanisms for Stat 3-Mediated Gamma-Globin Gene Silenc*

描述（由申请人提供）： 本申请旨在验证γ珠蛋白基因的5 '非翻译区中的顺式作用元件阵列和包括Stat 3 β在内的同源转录因子在γ珠蛋白沉默中的调节功能。顺式元件的集合包括位于-14、+9和+26的3个Stat 3 DNA元件，位于+26的1个先前被分配负调节功能的加塔-1元件，以及位于+26的1个推定的HoxB 2结合AT富集元件。我们最近通过定点诱变和共转染分析证明了+9顺式元件是Stat 3 β介导的γ珠蛋白启动子抑制所必需的。此外，在本申请中作为初步数据呈现的后续实验显示重组Stat 3在体外结合+9和+26顺式元件。我们接下来探索了Stat 3 β通过与其他转录因子如HoxB 2（一种已知的γ启动子活性抑制剂）的蛋白质-蛋白质相互作用抑制γ珠蛋白启动子的可能性。我们发现Stat 3与HoxB 2、加塔-1和CBP之间存在潜在的相互作用。这表明Stat 3 β对γ珠蛋白的抑制可能涉及与HoxB 2的协同作用和与加塔-1的功能性拮抗作用。基于这些初步的观察，我们假设“一个Stat 3含有多蛋白复合物通过一个假定的γ-珠蛋白沉默域相互作用，以抑制γ基因”。该假设将在四个具体目标中进行测试，1）通过染色体整合的g启动子中的突变分析来确定推定的γ-珠蛋白沉默结构域内的顺式元件的功能意义。2)证明体内Stat 3结合到假定的γ-珠蛋白沉默结构域，并通过染色质免疫沉淀和质谱鉴定Stat 3阻遏物复合物的组分。3)开发抑制Stat 3介导的γ珠蛋白抑制的方法，作为通过两种方法诱导HbF产生的治疗策略。第一种方法将测试Stat 3cz功能性抑制Stat 3 γ的能力。其次，我们将测试新的抗Stat 3结合肽去抑制y基因启动子的能力。4)最后，我们将测试用活化的Stat 3阻断肽和新型短链脂肪酸联合治疗以增加g基因表达的功效。因此，该项目提供了一个系统的分析γ珠蛋白沉默，提供了一个独特的机会，开发一种新的基于基因的治疗，以增加镰状细胞病和库利氏贫血的HbF表达。

DESCRIPTION (provided by applicant): This application is aimed at validating a regulatory function for an array of cis-acting elements in the 5'untranslated region of the gamma globin gene and cognate transcription factors including Stat3beta in gamma globin silencing. The collection of cis elements includes three Stat3 DNA elements at -14, +9 and +26, one GATA-1 element at +26 previously assigned a negative regulatory function, and a putative HoxB2 binding AT-rich element at +26. We have recently demonstrated by site directed mutagenesis and co-transfecfion analysis that the +9 cis-element is required for Stat3beta-mediated repression of the gamma globin promoter. Furthermore, subsequent experiments presented as preliminary data in this application show recombinant Stat3 binding to the +9 and +26 cis elements in vitro. We next explored the possibility that Stat3beta inhibits gamma globin promoter via protein-protein interactions with other transcription factors such as HoxB2, a known inhibitor of gamma promoter activity. We found potential interactions between Stat3 with HoxB2, GATA-1 and CBP. This suggests that inhibition of the gamma globin by Stat3beta may involve synergy with HoxB2 and functional antagonism with GATA-1. Based on these preliminary observations we hypothesize that "a Stat3-containing multi-protein complex interacts through a putative gamma-globin silencing domain to repress the gamma gene". This hypothesis will be tested in four specific aims, 1) Determine the functional significance of cis elements within a putative gamma-globin silencing domain by mutation analysis in chromosomally integrated g promoters. 2) Demonstrate in vivo Stat3 binding to the putative gamma-globin silencing domain and identify components of the Stat3 repressor complex by chromatin immunoprecipitation and mass spectrometry. 3) Develop methods to inhibit Stat3-mediated gamma globin repression as a therapeutic strategy for inducing HbF production by two approaches. The first approach will test the ability of Stat3cz to functionally inhibit Stat3 gamma. Secondly we will test the ability of novel anti-Stat3 binding peptides to de-repress the y gene promoter. 4) Finally we will test the efficacy of combined treatment with activated Stat3 blocking peptides and novel short chain fatty acids to augment g gene expression. This project thus offers a systematic analysis of gamma globin silencing that offers a unique opportunity to develop a novel gene-based therapy to increase HbF expression in Sickle cell disease and Cooley's anemia.