CHROMATIN STRUCTURE & FUNCTION HISTONE MODIFICATION & HM

染色质环或结构域模型表明，环具有 结构在染色体组织中的作用和功能 在确定真核基因表达单位和 它们的功能状态。主要问题涉及：i)如何 环路的包装决定了一种不同的基因状态 特定细胞类型，即永久抑制、潜在活动 和活性状态；II)染色质变量对DNA的影响 拓扑、核小体和染色质结构及其转录 在受限制的环中和iii)转录的位置 与染色质环和核物质有关的机械。至 回答问题一和问题二必须使用定义明确的 核小体精确定位在已知DNA上的系统 序列，并且间隔正确。确定…的功能 与活性染色质相关的染色质变量应该 对转录调控有一定的深入了解 系统。在这个时候，最适合研究这些的基因 通过底物可获得性进行基因控制的广泛问题 转录因子是5S rRNA基因。5S rRNA基因与 其DNA侧翼序列包含一个精确的内部TFIIIA 结合部位和精确的核小体核心定位序列。 辛普森的实验室已经改造出含有串联序列的质粒 大斑狼毒5S rRNA基因的重复序列(3~50+) 和长度为172bp至207bp的5‘侧翼DNA序列。一个 50个重复的闭合环将为染色质提供一个模型 大约10KBP的环路，在环路大小的范围内 活体观察。我们已经开发出方法来分离出 乙酰化组蛋白和泛素化组蛋白的不同状态 分离富含赖氨酸的组蛋白和HMG的方法是可用的 蛋白质。在初步研究中，我们发现转录 因子TFIIIA与乙酰化的5S rRNA基因结合，但不控制5S rRNA基因 核小体。在包含简单重复的5S的闭合圆圈中 RRNA基因我们现在有初步证据表明 乙酰化核小体的连接数小于乙酰化核小体 控制核小体。这意味着组蛋白乙酰化可以 释放核小体约束的DNA超螺旋以增加自由 环内的负超卷曲，即它可能表现为DNA 回旋酶。这也可能是一种定位瞬时DNA的机制 34 nm超线圈的展开和再折叠之间的超线圈 在转录循环中，这可能解释了为什么大部分DNA在 染色体处于一种放松的状态。两种不同的观点 转录机制占主导地位。在经典的RNA模型中 聚合酶是可移动的，并沿着基因在 抄写。在另一种模型中，RNA聚合酶锚定在 核基质，并通过卷绕DNA来转录DNA 附着的聚合酶复合体。我们已经开发了一种方法来处理 选择性标记参与转录和转录的RNA聚合酶 将确定标记的活性RNA聚合酶是否定位 在操作上确定的染色质环或在核中 矩阵。

The chromatin loop or domain model suggests that loops have structural roles in the organization of chromosomes and functional roles in determining the units of eukaryotic gene expression and their functional states. Major questions relate to: i) how the packaging of loops determine the different gene states of a particular cell type i.e. permanently repressed, potentially active and active states; ii) the effects of chromatin variables on DNA topology, nucleosome and chromatin structures and on transcription in constrained loops and iii) the location of the transcriptional machinery relative to chromatin loops and nuclear matices. To address questions i and ii it is essential to use a well-defined system in which nucleosomes are precisely located on known DNA sequences and are correctly spaced. To determine the functions of chromatin variables associated with active chromatin there should be some depth of understanding of transcriptional control of the system. At this time the gene best suited for studies of these broad questions of gene control through substrate availability to transcription factors is the 5S rRNA gene. The 5S rRNA gene with its DNA flanking sequences contains a precise internal TFIIIA binding site and a precise nucleosome core positioning sequence. Simpson's laboratory has engineered plasmids that contain tandem repeats (3 to 50+) of the cloned Lytechinus variegatus 5S rRNA gene and 5' flanking DNA sequences of lengths 172 bp to 207 bp. A closed loop of 50 repeats would provide a model for a chromatin loop of about 10 kbp which is within the rang of loop sizes observed in vivo. We have developed methods to isolate the different states of acetylated and ubiquitinated histones and methods are available to isolate very lysine rich histones and HMG proteins. In preliminary studies we find that the transcription factor TFIIIA binds to acetylated but not to control 5S rRNA gene nucleosomes. In closed circles containing tandemly repeated 5S rRNA genes we now have initial evidence to indicate that the linking number of acetylated nucleosomes is smaller than that of control nucleosomes. This implies that histone acetylation can release nucleosome constrained DNA supercoils to increase the free negative supercoiling within the loop i.e. it may behave as a DNA gyrase. It may also be a mechanism for localizing transient DNA supercoiling between unfolding and refolding of 34 nm supercoils in a transcribing loop, which may explain why the bulk of DNA in chromosomes is in a relaxed state. Two differing views of the mechanism of transcription prevail. In the classical model RNA polymerases are mobile and progress along genes during transcription. In the other model RNA polymerases are anchored to the nuclear matrix and transcribe DNA by reeling it through the attached polymerase complex. We have developed a method for the selective labelling of RNA polymerases engaged in transcription and will determine whether labeled active RNA polymerases are localized on the operationally defined chromatin loops or in the nuclear matrix.