Chromatin Structure In Regulation Of Mammalian Gene Expr

哺乳动物基因表达调控中的染色质结构

• 批准号: 7334688
• 负责人: Ann Dean
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7334688
• 关键词: Chromatin; Structure; Regulation; Mammalian; Gene

In all organisms, regulated gene expression is used to specify complex patterns of development. The vertebrate beta-globin family of genes provides a rich opportunity to study fundamental properties of developmentally regulated gene expression. The human a-globin genes are organized within a 70 kb region in which the 5? to 3? order of the genes, epsilon, G-gamma, A-gamma, delta and beta, mirrors their temporal induction during development. The beta-globin locus control region (LCR) functions as a strong enhancer for all the genes. The LCR includes four erythroid specific DNase I hypersensitive sites, HS1-HS4, 10 to 60 Kb upstream of the globin genes that are the sites of interaction of a cohort of erythroid specific and ubiquitous transcription activators. We use biochemistry and cell and molecular biology approaches to understand the regulation of this human gene locus. Our experimental systems include (1) a manipulable in vivo model episomal system, (2) endogenous chromosomes in human and mouse erythroid cell lines and mouse embryonic stem cells (ES cells), and (3) human a-globin YAC transgenic mice and mice carrying endogenous globin locus alterations introduced by homologous recombination in ES cells. Over the last few years we have contributed to an understanding that the co-activator complexes recruited to the beta-globin LCR access the distant promoters both by spreading across the intervening DNA to create a domain of histone modifications and by physically interacting with the target genes, referred to as looping. Recently, we have investigated insulator-enhancer antagonism. Insulators are DNA elements that are proposed to limit LCR activity by blocking any effect on non-target genes. One of our studies using replicating episomes in vivo showed that a chromatin insulator, 5?HS4 from the chicken beta globin locus, blocked formation of a domain of histone hyperacetylation when placed between the LCR HS2 globin enhancer and a globin gene. We discovered that enhancer blocking correlated with a significant depletion of nucleosomes in the core region of the insulator. Thus, one means by which insulators function is to creation an altered nucleosome structure incompatible with spreading of histone modifications across chromatin. We tested insulator function of a site analogous to chicken 5?HS4, human 5?HS5. An ectopic HS5 inserted between the LCR and the globin genes on a transgene in mice had no affect on histone acetylation in the LCR, but blocked histone acetylation, and transcription, of the downstream genes consistent with our studies in episomes. These studies support the idea that HS5 in the human globin locus has a portable enhancer blocking activity. Flanking the human globin LCR and genes are more distant DNase I hypersensitive sites that loop together and are proposed to be the boundaries of the globin transcriptional domain. We studied epigenetic modification and chromatin structure at high resolution across 400 Kb of chromosome 11 including the globin locus and upstream and downstream flanking regions. We observed distinctive signatures of histone H3K4, K36 and K9 methylation leading us to conclude that mono-methylation is a widely distributed mark on permissive chromatin and that hyper-methylated forms appear at highly transcribed genes at the expense of the mono-methyl mark, consistent with a transcription coupled mechanism of enzymatic conversion. However, H3K36me3 was strongly detected in transcribed coding as well as non-coding regions of the locus leading us to propose that in mammals H3K36me3 is a stable mark on sequences transcribed at any level. The human globin locus flanking hypersensitive sites did not correspond to transitions in histone methylation (or nuclease sensitivity) calling into question the role of the potential boundaries. At this juncture, it seems that new models of enhancer-insulator antagonism are needed in this evolving field. We continue to use biochemical and genetic approaches to gain insight into how genes are activated by remote enhancers and how enhancer activity is restricted to the appropriate gene. It is increasingly clear that mutations affecting long range gene activation, chromatin remodeling and histone modifying proteins play a role in mis-regulation of genes that contribute to genetic diseases and cancers. Thus, understanding how chromatin is regulated during development and differentiation may offer insights into the treatment of numerous diseases. Although we focus on understanding regulation of the human globin locus at the molecular level, the underlying mechanisms of long range gene regulation have widespread relevance to mammalian development in health and disease.

在所有生物体中，受调控的基因表达被用来指定复杂的发育模式。脊椎动物β-珠蛋白基因家族为研究发育调控基因表达的基本特性提供了丰富的机会。人类α-珠蛋白基因的组织内的70 kb的区域，其中5？到3？基因的顺序，β，G-γ，A-γ，δ和β，反映了它们在发育过程中的时间诱导。β-珠蛋白基因座控制区（LCR）作为所有基因的强增强子发挥作用。LCR包括四个红细胞特异性DNA酶I超敏位点，HS 1-HS 4，在珠蛋白基因上游10至60 Kb处，其是红细胞特异性和普遍存在的转录激活因子的群组的相互作用位点。我们使用生物化学和细胞和分子生物学方法来了解这个人类基因位点的调控。我们的实验系统包括（1）可操作的体内模型附加体系统，（2）人和小鼠红系细胞系和小鼠胚胎干细胞（ES细胞）中的内源性染色体，和（3）人α-珠蛋白YAC转基因小鼠和携带通过ES细胞中的同源重组引入的内源性珠蛋白基因座改变的小鼠。在过去的几年里，我们已经促成了这样一种理解，即被招募到β-珠蛋白LCR的辅激活因子复合物通过在间插DNA中扩散以产生组蛋白修饰的结构域以及通过与靶基因的物理相互作用（称为成环）来访问远端启动子。 最近，我们研究了绝缘体增强剂拮抗作用。绝缘子是DNA元件，其被提议通过阻断对非靶基因的任何影响来限制LCR活性。我们的一项研究使用复制游离体在体内显示，染色质绝缘体，5？来自鸡β球蛋白基因座的HS 4在置于LCR HS 2球蛋白增强子和球蛋白基因之间时阻断组蛋白超乙酰化结构域的形成。我们发现增强子阻断与绝缘子核心区域的核小体显著耗尽相关。因此，绝缘子发挥作用的一种方式是产生一种改变的核小体结构，这种结构与组蛋白修饰在染色质中的扩散不相容。我们测试绝缘子功能的网站类似鸡5？HS 4，人类5？HS5。在小鼠中，在LCR和转基因上的珠蛋白基因之间插入的异位HS 5对LCR中的组蛋白乙酰化没有影响，但阻断了下游基因的组蛋白乙酰化和转录，这与我们在附加体中的研究一致。这些研究支持了人珠蛋白基因座中的HS 5具有便携式增强子阻断活性的观点。 人球蛋白LCR和基因的侧翼是更远的DNA酶I超敏位点，它们环在一起，被认为是球蛋白转录结构域的边界。我们研究了表观遗传修饰和染色质结构在高分辨率的400 Kb的11号染色体，包括珠蛋白基因座和上游和下游侧翼区。我们观察到组蛋白H3 K4、K36和K9甲基化的独特特征，这使我们得出结论，单甲基化是容许染色质上广泛分布的标记，并且高甲基化形式以单甲基标记为代价出现在高度转录的基因中，这与酶促转化的转录偶联机制一致。然而，H3 K36 me 3在转录的编码区以及非编码区的基因座中被强烈检测到，这使我们提出在哺乳动物中H3 K36 me 3是在任何水平转录的序列上的稳定标记。人类珠蛋白基因座侧翼超敏位点不对应于组蛋白甲基化（或核酸酶敏感性）的转变，这对潜在边界的作用提出了质疑。在这个节骨眼上，似乎在这个不断发展的领域需要新的增强子-绝缘子拮抗模型。 我们继续使用生物化学和遗传学的方法来深入了解基因是如何被远程增强子激活的，以及增强子的活性如何被限制在适当的基因上。越来越清楚的是，影响长距离基因激活、染色质重塑和组蛋白修饰蛋白的突变在导致遗传疾病和癌症的基因的错误调节中起作用。因此，了解染色质在发育和分化过程中是如何调节的，可以为许多疾病的治疗提供见解。虽然我们专注于在分子水平上了解人类珠蛋白基因座的调控，但长期基因调控的潜在机制与哺乳动物的健康和疾病发育具有广泛的相关性。