Factors modulating gamma globin gene expression

持续胎儿血红蛋白表达的镰状细胞疾病的改善表明，能够重新激活γ-珠蛋白缺失表达的治疗方法应该被探索。基因转移能够逆转成年红系细胞从伽马到β-珠蛋白的转换的转录因子是治疗这种毁灭性疾病的一种方法。我们 已经确定了一种蛋白质复合体，即阶段选择蛋白(SSP)，它是这种方法的有力候选者。SSP由普遍存在的转录因子CP2/LBP-1a、红系限制性成分p22NF-E4和稳定两者之间相互作用的伴侣蛋白Aly组成。我们已经证明，p22 NF-E4作为伽马基因表达的激活剂发挥作用，在胎儿/成人基因竞争的背景下，促进伴随而来的成人β基因表达的下调。在BetaYAC转基因小鼠中强制表达p22NF-E4推迟了胎儿/成人的转换，但无法推翻控制伽马基因沉默的内在机制。我们已经证明，SSP的功能是通过与辅助激活因子和一般转录因子的直接蛋白质/蛋白质相互作用来调节的。基于这些发现，我们推测SSP通过改变与这些元件的结合而将基因座控制区(LCR)招募到伽马启动子中。在特定的目标1中，我们将在胎儿红细胞生成的细胞和动物模型中建立转录因子与LCR和伽马启动子的关联模式，并检测在这些背景下p22 NF-E4表达增加的影响。我们将确定LCR和伽马启动子在胎儿红系细胞中是否在物理上接近，以及这是否受到SSP复合体的影响。在具体目标2中，我们将剖析SSP的各个组件对复合体整体功能的相对贡献，包括它们在因子结合和LCR/启动子相互作用中的作用。具体目标3将集中在LCR/伽马-启动子的相互作用和因子在破坏内在伽马基因沉默的突变的背景下的占据，以及p22NF-E4在这方面的表达的影响。综上所述，这些研究将确定SSP复合体在修饰与LCR和γ启动子相关的因子方面的作用，并确定SSP的主要功能是否是直接将LCR招募到启动子。最终，它们可能会为镰状细胞疾病提供新的治疗策略。

Amelioration in sickle cell disease seen with persistent fetal hemoglobin expression suggests that therapies capable of reactivating gamma-globin gone expression should be explored. Gene transfer of a transcription factor capable of reversing the gamma- to beta-globin switch in adult erythroid cells represents one approach to treatment of this devastating disorder. We have identified a protein complex, the stage selector protein (SSP) that is a strong candidate for this approach. The SSP consists of the ubiquitous transcription factor CP2/LBP-1a, p22NF-E4, an erythroid-restricted component, and ALY, a chaperone protein that stabilizes the interaction between the other two. We have demonstrated that p22 NF-E4 functions as an activator of gamma-gene expression, and in the setting of fetal/adult gene competition promotes a concomitant down-regulation of adult beta-gene expression. Enforced expression of p22NF-E4 in betaYAC transgenic mice delayed the fetal/adult switch but was unable to override the intrinsic mechanisms that govern gamma-gene silencing. We have shown that the function of the SSP is mediated by direct protein/protein interactions with co-activators and general transcription factors. Based on these findings, we postulate a role for SSP in recruiting the locus control region (LCR) to the gamma-promoter through changes in factor binding to these elements. In Specific Aim 1, we will estabh'sh the pattern of transcription factor association with the LCR and gamma-promoter in cellular and animal models of fetal erythropoiesis and examine the effects of increased expression of p22 NF-E4 in these contexts. We will determine whether the LCR and gamma-promoter are in close physical proximity in fetal erythroid cells and if this is influencedby the SSP complex. In Specific Aim 2, we will dissect the relative contribution of the individual components of the SSP to the overall function of the complex, including their role in factor binding and LCR/promoter interaction. Specific Aim 3 will focus on LCR/gamma-promoter interactions and factor occupancy in the setting of a mutation that disrupts intrinsic gamma-gene silencing, and the effects of p22NF-E4 expression in this context. In summary, these studies will define the role of the SSP complex in modifying factor association with the LCR and gamma-promoter, and determine whether the predominant function of the SSP is to directly recruit the LCR to the promoter. Ultimately they may provide new therapeutic strategies for sickle cell disease.