Epigenetic and Developmental Regulation of Mammalian Genes

重新激活成人红系细胞中胎儿珠蛋白的表达是改善诸如镰刀细胞病和β-地中海贫血等血红蛋白疾病的宝贵目标。我们使用人CD34+红系祖细胞来探索与红细胞生成过程中珠蛋白基因调控相关的染色质修饰和转录因子的表达模式。使用成人原代CD34+细胞进行组蛋白修饰和转录组谱分析，这些细胞与细胞因子组合培养，产生低水平和高水平的γ-珠蛋白mRNA和胎儿血红蛋白(HBF)。为了进行比较，我们确定了胚胎、胎儿和成人的珠蛋白转录本和蛋白质表达模式。染色质免疫沉淀分析显示，只有在高HBF培养条件下，RNA聚合酶II占位和组蛋白尾部修饰与转录激活一致。转录组图谱研究表明，与高HBF相关的核转录因子的表达发生了可重复的变化。因此，在成人红系细胞中，细胞因子介导的信号转导导致珠蛋白基因和蛋白表达模式的显著变化，这些变化与不同的组蛋白修饰以及红系转录因子的核重新编程有关。 单个珠蛋白基因如何建立阶段特异性增强子通信尚不清楚，大多数研究都是在非染色体环境中进行的。我们正在使用小鼠ES细胞中的同源重组来解决这个问题。我们在ES细胞中靶向小鼠胚胎epsilon y基因，然后用重组酶介导盒交换将其替换为epsilon y基因，其中所选择的启动子转录因子基序已被破坏或改变。大多数突变都略微干扰了epsilon y的转录，表明它们对LCR/epsilon的通讯是必不可少的。β-珠蛋白CACCC位点的替换是epsilon y转录的强烈上调，并导致成体特异性因子EKLF被招募到启动子，这与CACCC元件在阶段特异性珠蛋白基因表达中的重要作用是一致的。此外，我们在LCR和epsilon y之间插入了cHS4绝缘体，cHS4的插入严重降低了胚胎y和β-H1基因的转录，并导致RNA polII和非基因转录产物在绝缘体中积累。这一观察结果对我们继续探索的LCR/增强剂和染色质绝缘体的工作机制具有重要的意义。这些实验是新颖的，因为它们改变了正常染色体环境中的转录因子招募和染色质组织，并将提供关于LCR和珠蛋白基因在体内如何沟通的信息。

Reactivation of fetal globin expression in adult erythroid cells is a prized goal to ameliorate hemoglobinopathies such as Sickle Cell Disease and beta-thalassemia. We used human CD34+ erythroid progenitor cells to explore chromatin modifications and transcription factor expression patterns that are associated with globin gene regulation during erythropoiesis. Histone modification and transcriptome profiling were performed using adult primary CD34+ cells cultured with cytokine combinations that produced low versus high levels of gamma-globin mRNA and fetal hemoglobin (HbF). Embryonic, fetal, and adult globin transcript and protein expression patterns were determined for comparison. Chromatin immunoprecipitation assays revealed RNA polymerase II occupancy and histone tail modifications consistent with transcriptional activation only in the high-HbF culture condition. Transcriptome profiling studies demonstrated reproducible changes in expression of nuclear transcription factors associated with high HbF. Thus, cytokine-mediated signal transduction in adult erythroid cells causes significant changes in the pattern of globin gene and protein expression that are associated with distinct histone modifications as well as nuclear reprogramming of erythroid transcription factors. How individual globin genes establish stage specific enhancer communication is unknown and most studies have been performed in a non-chromosomal environment. We are using homologous recombination in mouse ES cells to address this question. We targeted the mouse embryonic epsilon y gene in ES cells and then used recombinase mediated cassette exchange to replace it with epsilon y genes in which selected promoter transcription factor motifs had been destroyed or altered. Most mutations modestly perturbed epsilon y transcription, indicating that, individually, they are dispensable for LCR/epsilon communication. Substitution of the beta-globin CACCC site was a strong up-regulator of epsilon y transcription and resulted in recruitment of the adult specific factor EKLF to the promoter, consistent with an important role for the CACCC element in stage specific globin gene expression. In addition we inserted the cHS4 insulator between the LCR and epsilon y. The cHS4 insertion severely reduced transcription of embryonic y and beta-h1 genes and resulted in RNA pol II and non-genic transcript accumulation in the insulator. This observation has important implications for the mechanism by which LCR/enhancers and chromatin insulators work which we continue to explore. These experiments are novel since they alter transcription factor recruitment and chromatin organization in a normal chromosomal setting and will provide information on how the LCR and globin genes communicate in vivo.