Analysis of the Interaction of Histone H1 with Chromatin In Vivo

高等生物的DNA以一种称为染色质的复杂结构组织起来。染色质的主要功能是促进DNA的压缩，以便它可以被包装到细胞核中。然而，这必须这样做，以使潜在的基因是可访问的调节反应和染色质结构显然在调节真核生物的转录发挥主导作用。接头或H1组蛋白是后生动物染色质核小体亚单位的化学计量组分。这些蛋白质对染色质的整体结构和功能做出了重要贡献，但潜在的分子机制仍不清楚。关于组蛋白H1在核小体上的确切位置，几乎没有直接的结构信息。这些信息对于理解这些蛋白质如何调节基因表达至关重要。 最近，光漂白技术被用来测量动态结合的H1未扰动的染色质在活细胞中。结果表明，大多数H1分子在染色质结合位点之间连续交换，这表明存在“走走停停”机制，其中H1分子在染色质结合位点停留一到两分钟，然后解离并迅速结合到另一个位点。光漂白的研究也可以提供定量测量的结合亲和力H1的DNA在活细胞中。本研究的主要目的是利用这些技术来了解H1与活细胞中基因组DNA的相互作用。将构建含有与绿色荧光蛋白偶联的野生型和突变型H1同种型基因的表达载体。这些构建体将在小鼠细胞中表达，蛋白质的动力学行为将通过光漂白技术进行测定。根据这些研究，将确定这些突变构建体的相对结合亲和力。分子建模，然后将用于开发高分辨率的模型，这些H1同种型的每个核小体的分子相互作用。在这个项目中进行的其他研究包括红白血病分化过程中的H1动力学分析和鉴定体内H1结合蛋白的测定系统的开发。

The DNA of higher organisms is organized in a complex structure called chromatin. A primary function of chromatin is to facilitate the compaction of DNA so that it can be packaged into the nucleus. However, this must be done such that the underlying genes are accessible to regulatory responses and chromatin structure clearly plays a dominant role in regulating much of eukaryotic transcription. The linker or H1 histones are stoichiometric components of the nucleosomal subunits of metazoan chromatin. These proteins make important contributions to the overall structure and function of chromatin but the underlying molecular mechanisms remain unclear. There is little direct structural information regarding the exact location of histone H1 on the nucleosome. This information is crucial to understanding how these proteins might function to regulate gene expression. Recently, photobleaching techniques were used to measure the dynamic binding of H1 to unperturbed chromatin in living cells. The results indicate that most H1 molecules are continuously exchanged between chromatin binding sites suggesting a 'stop-and go' mechanism in which an H1 molecule resides at a chromatin binding site for one or two minutes then dissociates and rapidly binds to another site. Photobleaching studies can also provide quantitative measurements of the binding affinity of H1 to DNA in living cells. The major goal of this research is utilize these techniques to obtain insight into the interaction of H1 with genomic DNA in living cells. Expression vectors containing genes for wild type and mutant H1 isotypes coupled to the green fluorescent protein will be constructed. These constructs will be expressed in mouse cells and the kinetic behavior of the proteins will be assayed by photobleaching techniques. From these studies the relative binding affinities of these mutant constructs will be determined. Molecular modeling will then be used to develop high resolution models of the molecular interaction of each of these H1 isotypes to nucleosomes. Additional studies to be performed in this project include an analysis of H1 dynamics during erythroleukemic differentiation and development of assay systems to identify in vivo H1 binding proteins.