9506164 Workman While functional transcription studies have implicated both core histones (nucleosome cores) and the linker H1 histones in transcription control, the distinct roles of these proteins and their functional interactions have not been determined. The long term goal of this research is to describe the details of these interactions as well the interactions of transcription factors with these histone complexes. These studies will employ model nucleosome templates to determine basic parameters governing the ability of H1 to regulate transcription factor access to nucleosomal DNA. We will analyze the histone domains and modifications that govern the functions of H1 in transcription repression and activation. Moreover, these studies will utilize purified systems, where activities can be accurately attributed to protein components. These studies will help clarify several issues concerning H1 function and provide mechanistic detail of how transcription factors overcome H1 repression to access nucleosomal DNA at enhancer and promoter elements in cellular chromatin during gene activation. The specific objectives are: 1) Determination of which core histone amino termini and precise acetylated lysines effect H1 binding to nucleosomes and repression of factor binding. Individual core histone amino termini regulating H1 repression will be identified by the formation of hybrid nucleosomes where specific histone tails have been removed. The ability of acetylated forms of these histones to also relieve repression will be determined by the application of novel immunoblotting techniques using antibodies to all or specific acetylated lysines on histones. These methods will determine the specific acetylated histones and in some cases the specific acetylated lysines which alleviated H1 repression. 2) Analysis of H1 domains involved in restricting transcription factor access and the effect of H1 phosphorylation on their activity. The function of histone H1 domains (i.e. the central globular doma in, the C-terminal and N-terminal domains) in repression of transcription factor binding will be determined by assaying the function of these individual domains. The role of H1 phosphorylation will be determined by measuring the repression of factor binding by hyper-phosphorylated H1. In addition, the retention or loss of phosphorylated H1 forms upon transcription factor binding will be determined by western-blots of mobility shift gels using antibodies to total H1 or antibodies specific for hyperphosphorylated or dephosphorylated forms. %%% While functional transcription studies have implicated both core histones (nucleosome cores) and the linker H1 histones in transcription control, the distinct roles of these proteins and their functional interactions have not been determined. The long term goal of this research is to describe the details of these interactions as well the interactions of transcription factors with these histone complexes. These studies will employ model nucleosome templates to determine basic parameters governing the ability of H1 to regulate transcription factor access to nucleosomal DNA. ***
