Epigenetic and Developmental Regulation of Mammalian Genes

我们正在使用人类红系K562细胞作为模型，试图了解修饰组蛋白的酶活性的功能。虽然H3K9me2和ME3是沉默的异染色质的典型特征，但我们和其他人发现H3K9三甲基化与活跃转录的珠蛋白基因相关。此外，负责常染赖氨酸9二甲基化的甲基转移酶G9a被招募到β-珠蛋白LCR和活性珠蛋白基因启动子中。为了确定G9a和/或其合作伙伴甲基转移酶GLP是否与活跃转录的珠蛋白基因中的H3K9me3有关，我们利用RNAi技术单独和同时降低了这些蛋白在红系细胞中的表达。我们正在研究这些蛋白质敲除对珠蛋白转录和HMTase复合体成分定位的影响。此外，我们将敲除K4 HMT复合体成分Ash2L，并检测其对转录以及K4和K9甲基化的影响，以确定K4和K9甲基化标记是否在功能上相关。 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。这些实验是新颖的，因为它们改变了正常染色体设置中的转录因子招募，并将提供关于LCR和珠蛋白基因在体内如何沟通的信息。

We are using human erythroid K562 cells as a model to try to understand the function of enzymatic activities that modify histones. Although H3K9me2 and me3 are typically features of silent heterochromatin, we and others found H3 K9 tri-methylation associated with actively transcribed globin genes. Further, G9A, the methyl-transferase responsible for euchromatic lysine 9 dimethylation, is recruited to the beta-globin LCR and active globin gene promoters. To determine whether G9A and/or its partner methyltransferase GLP are responsible for the H3K9me3 in actively transcribed globin genes, we reduced expression of these proteins singly and concurrently in erythroid cells using RNAi. We are examining the consequences of these protein knock downs for globin transcription and localization of HMTase complex components. In addition, we will knockdown the K4 HMT complex component Ash2L and examine the effect on transcription and K4 and K9 methylation to determine whether K4 and K9 methylation marks are functionally related. 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. These experiments are novel since they alter transcription factor recruitment in a normal chromosomal setting and will provide information on how the LCR and globin genes communicate in vivo.