GROWTH REGULATION OF REPLICATION-DEPENDENT GENES

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This is a proposal to study the mechanisms that govern the G1-S regulated transcription of two replication-dependent genes, using the well characterized histone and thymidine kinase (tk) genes as model systems. The major goal of this proposal is to identify the regulatory factors and to examine the protein interactions at the G1-S control elements mediating this regulation. It has been documented that these genes are transcriptionally activated at the G1-S phase border by a mechanism that depends on specific sequence elements located in their promoters. We have recently succeeded in localizing the cis-regulatory control elements that confer the G1-S transcription regulation in vivo. For the H3.2 gene, a hamster nuclear factor, designated H3 abp1, binds a G1-S regulatory site GGCGAGTCAG which resembles a Jun protein binding site. Recently, the Jun proteins are shown to be specifically required for entrance into S-phase in both serum stimulated and asynchronously growing fibroblasts. We now determined that the H3 abp1 complex is related to but is immunologically and functionally distinct from the previously described Jun/CREB/ATF1 proteins. Further, we discovered that H3 abp1 specifically binds to the H3 promoter and its binding activity is biphasic and rises sharply at the G1-S border. A major thrust of this proposal is the purification of the H3 abp1. The H3 apb1 synthesis profile, binding properties, cell-cycle dependent posttranslational modifications and interactions with other co-activators or any known cell cycle regulated proteins will be examined. To provide direct evidence for a functional link between H3 abp1 and H3.2 regulation, the ability of the purified H3 abp1 to stimulate H3 transcription in vitro and in vivo and the effect of reduced- or over-expression of H3 abp1 will be investigated. For the tk system, a 14 bp protein binding site has been identified as a G1-S regulatory unit and its activity is enhanced by an adjacent CCAAT site. The binding activities of one of the protein complexes interacting with the G1-S regulatory site changes sharply at the G1-S border. Our proposed studies are aimed at defining the components of these protein complexes. In addition, we test the hypothesis that the human tk and histone H1 CCAAT site may share common regulatory factor mediating their simultaneous increase in S-phase transcription. These studies will provide the fundamental information on the complex, interdependent molecular events which mediate stringent regulation of cell cycle progression. Our new direction includes expansion of our studies into continuously cycling cells separated by centrifugal elutriation and compared that to serum stimulated cells. This will provide important information on how cells adjust G1-S transcriptional control under different physiological conditions.