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

描述(由申请人提供)： 该应用的目的是验证伽马珠蛋白基因5‘非翻译区中的一系列顺式作用元件和包括Stat3β在内的同源转录因子在伽马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的三个STAT3 DNA元件，一个位于+26的GATA-1元件先前被指定为负调控功能，以及一个假定的HoxB2结合的富含AT的元件(+26)。我们最近通过定点突变和共转染分析证明，+9顺式元件是Stat3β介导的抑制伽马珠蛋白启动子所必需的。此外，在本申请中作为初步数据提出的后续实验表明，重组STAT3在体外与+9和+26顺式元件结合。接下来，我们探索了Stat3β通过与其他转录因子(如已知的伽马启动子活性抑制剂HoxB2)之间的蛋白质-蛋白质相互作用来抑制伽马珠蛋白启动子的可能性。我们发现STAT3与HoxB2、GATA-1和CBP之间存在潜在的相互作用。这表明，Stat3β对γ珠蛋白的抑制可能与HoxB2协同作用和GATA-1的功能拮抗作用有关。基于这些初步观察，我们假设“一个含有STAT3的多蛋白复合体通过一个假定的伽马珠蛋白沉默结构域相互作用，从而抑制伽马基因”。这一假设将在四个具体目标上得到验证：1)通过对染色体整合的g启动子进行突变分析，确定顺式元件在假定的伽马珠蛋白沉默结构域中的功能意义。2)通过染色质免疫沉淀和质谱鉴定STAT3抑制物复合体的成分，证实STAT3在体内与可能的伽马珠蛋白沉默结构域结合。3)开发抑制STAT3介导的伽玛珠蛋白抑制的方法，作为通过两种途径诱导HBF产生的治疗策略。第一种方法将测试Stat3cz对STAT3伽马的功能抑制能力。其次，我们将测试新的抗STAT3结合多肽对y基因启动子的抑制能力。4)检测活化的STAT3封闭肽与新型短链脂肪酸联合作用对g基因表达的影响。因此，该项目提供了对伽马珠蛋白沉默的系统分析，这为开发一种新的基于基因的治疗方法以增加镰刀细胞病和库利氏贫血患者HBF的表达提供了一个独特的机会。

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.