Epigenetics of developmental regulation of mammalian genes

我们在人类K562细胞中以高分辨率确定了表观遗传修饰特征和染色质结构，包括β-珠蛋白基因座，在该细胞中，胚胎epsilon和胎儿伽马基因表达，而成年β-珠蛋白基因不表达。为了研究这些基因的表观遗传修饰与它们在发育过程中的顺序激活之间的相关性，我们选择了一个体外分化系统，在该系统中，人类CD34+细胞经过两周的分化，最终表达包括成体基因在内的每个珠蛋白基因。因为这些细胞的数量有限，所以我们优化了一种微型染色质免疫沉淀的方案，使用比典型细胞数量更少的细胞。到目前为止，结果与我们在K562细胞中的数据一致，表明在珠蛋白基因表达之前，基因座控制区增强子的早期表观遗传修饰，以及随着基因的激活，跨基因座的活性组蛋白标记的渐进检测。 组蛋白H3和H4乙酰化以及H3K4二甲基化是染色质允许性的标志，通常与后生动物中活跃转录的基因有关。相比之下，H3K9的二甲基化和三甲基化(H3K9me2和ME3)被普遍认为是沉默的异染色质的特征。令人惊讶的是，我们和其他人发现H3K9三甲基化与活跃转录的珠蛋白基因有关。人类细胞中至少有6种酶在不同水平上进行H3K9甲基化。G9a通常与常染赖氨酸9的二甲基化有关，但在体外具有三甲基化活性。利用芯片，我们发现G9a被招募到人β-珠蛋白LCRHSS和活性珠蛋白基因启动子上。G9a和相关的甲基转移酶GLP形成了一个异构体复合体，对它们在体内的活性是必不可少的。为了确定G9a或GLP是否与我们在活跃转录的珠蛋白基因中观察到的H3K9me3有关，我们使用RNAi技术分别和同时下调了它们在红系细胞中的表达，并正在检测对珠蛋白转录和HMTase复杂成分定位的影响。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。

We determined epigenetic modification signatures and chromatin structure at high resolution across 300 Kb of human chromosome 11, including the beta-globin locus, in human K562 cells where the embryonic epsilon and fetal gamma genes are expressed and the adult beta-globin genes are silent. To investigate a correlation between epigenetic modification of these genes and their sequential activation during development, we chose an in vitro differentiation system in which human CD34+ cells are differentiated over two weeks and eventually express each of the globin genes including the adult genes. Because these cells are in limiting number we optimized a protocol for mini-chromatin immunoprecipitation using a lower cell number than is typical. The results thus far are consistent with our data in K562 cells and indicate early epigenetic modification of the locus control region enhancer before the globin genes are expressed and the progressive detection of active histone marks across the locus as the genes are activated. Histone H3 and H4 acetylation and H3 K4 di-methylation are marks of permissive chromatin generally associated with actively transcribed genes in metazoans. In contrast, H3K9 di- and tri-methylation (H3K9me2 and me3) are generally accepted to be features of silent heterochromatin. Surprisingly, we and others found H3 K9 tri-methylation associated with actively transcribed globin genes. There are at least 6 enzymes in human cells that are known to carry out H3 K9 methylation at different levels. G9A is generally associated with euchromatic lysine 9 dimethylation but has trimethylation activity in vitro. Using ChIP, we found that G9A is recruited to the human beta-globin LCR HSs and active globin gene promoters. G9A and the related methyltransferase GLP form a heteromeric complex that is essential for their activity in vivo. To determine whether G9A or GLP are responsible for the H3K9me3 we observed in actively transcribed globin genes, we knocked down their expression singly and concurrently in erythroid cells using RNAi and we are examining the consequences 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.