The objective of this program is to understand the regulation of the cell cycle dependent human histone genes, a multi-gene family of moderately repeated sequences with differences in the structure, organization and regulation of the various copies. The tight coupling of histone gene expression with DNA replication and the stringent requirement of histones to package newly replicated DNA into nucleosomes account for the involvement of histone gene expression in control of cell growth and cell division. Studies carried out during the current funding period have identified sequences that influence specificity, level and cell cycle regulation of histone gene transcription and have confirmed the modular organization of histone gene promoters. A series of proximal and distal H4 histone promoter elements have been examined by deletion analysis, site directed mutagenesis and construction of chimeric genes assayed in vitro, by transfection into mammalian cells and in transgenic mice. Protein-DNA interactions in the proximal promoter have been established in intact cells at single nucleotide resolution and several nuclear protein factors have been characterized in vitro. In vitro protein-DNA interactions with the most proximal promoter element have been shown to be stringently cell cycle controlled in diploid cells and deregulated (constitutive) in transformed and tumor cells where loss of binding occurs only with the onset of differentiation. These results serve as the basis for ongoing and proposed studies which are: 1) To further define and characterize sequences in histone gene promoters that modulate levels of transcription during the cell cycle and are responsible for the downregulation of transcription during differentiation; 2) To continue the identification and characterization of protein-DNA interactions in intact cells in the 5' promoter of a histone gene; 3) To isolate and characterize promoter binding factors that interact with proximal and distal 5' regulatory elements; 4) To assess the contributions of factor regulation to the control of histone gene transcription by a) determining the role of phosphorylation in factor- regulatory sequence interactions, and b) cloning cDNAs for histone gene promoter binding factors to determine the extent to which their regulation influences histone gene transcription; and 5) To examine the potential involvement of nuclear structure with the regulation of histone gene expression by determining the relationship of histone gene transcription to nuclear matrix association of the genes and by exploring the possibility that the nuclear matrix may concentrate and/or localize histone promoter binding factors.
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