CHROMATIN HISTONE MODIFICATIONS AND HMG PROTEINS

染色质环或域模型表明环具有 染色体组织中的结构作用和功能 在确定真核基因表达单位中的作用和 他们的功能状态。 主要问题涉及： i) 如何 环的包装决定了不同的基因状态 特定的细胞类型，即永久抑制，潜在活跃 和活跃状态； ii) 染色质变量对 DNA 的影响 拓扑、核小体和染色质结构以及转录 在受限环中和iii)转录的位置 与染色质环和核基质相关的机制。 至 解决问题一和二时，必须使用明确定义的 核小体精确定位在已知 DNA 上的系统 序列并正确间隔。 确定功能 与活性染色质相关的染色质变量应该存在 对转录控制有一定的深度理解 系统。 此时最适合研究这些的基因 通过底物可用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 其 DNA 侧翼序列包含精确的内部 TFIIIA 结合位点和精确的核小体核心定位序列。 辛普森的实验室已经设计出含有串联的质粒 克隆的杂色 Lytechinus variegatus 5S rRNA 基因的重复序列（3 至 50 个） 以及长度为172 bp至207 bp的5'侧翼DNA序列。 一个 50 次重复的闭环将为染色质提供模型 大约 10 kbp 的循环，在循环大小范围内 体内观察到。 我们已经开发出隔离的方法 乙酰化和泛素化组蛋白的不同状态 有方法可以分离富含赖氨酸的组蛋白和 HMG 蛋白质。 在初步研究中我们发现转录 因子 TFIIIA 与乙酰化但不与控制 5S rRNA 基因结合 核小体。 在包含串联重复 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 控制核小体。 这意味着组蛋白乙酰化可以 释放核小体限制的DNA超螺旋以增加自由 环内的负超螺旋，即它可能表现为 DNA 旋转酶。 它也可能是一种定位瞬时 DNA 的机制 34 nm 超螺旋展开和重折叠之间的超螺旋 转录循环中，这可以解释为什么大部分 DNA 染色体处于松弛状态。 两种不同的观点 转录机制占主导地位。 在经典RNA模型中 聚合酶是可移动的并且在基因过程中沿着基因进展 转录。 在另一个模型中，RNA 聚合酶被锚定在 核基质并通过卷绕 DNA 来转录 DNA 附加聚合酶复合物。 我们开发了一种方法 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 在操作上定义的染色质环上或在核中 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.