Analysis of the Interaction of Histone H1 with Chromatin In Vivo

点击翻译按钮获取中文摘要

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.