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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031138-10
Application #
2176030
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1982-09-30
Project End
1997-05-31
Budget Start
1994-06-01
Budget End
1995-05-31
Support Year
10
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Southern California
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041544081
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Wu, Frank; Lee, Amy S (2002) CDP and AP-2 mediated repression mechanism of the replication-dependent hamster histone H3.2 promoter. J Cell Biochem 84:699-707
Wu, F; Lee, A S (2001) YY1 as a regulator of replication-dependent hamster histone H3.2 promoter and an interactive partner of AP-2. J Biol Chem 276:28-34
Lau, J S; Baumeister, P; Kim, E et al. (2000) Heterogeneous nuclear ribonucleoproteins as regulators of gene expression through interactions with the human thymidine kinase promoter. J Cell Biochem 79:395-406
Naeve, G S; Zhou, Y; Lee, A S (1995) Identification of a 68 kDa protein species as a specific DNA-binding component of the H3abp complex interacting with the histone H3.2 G1/S regulatory domain. Nucleic Acids Res 23:475-84
Li, L J; Naeve, G S; Lee, A S (1993) Temporal regulation of cyclin A-p107 and p33cdk2 complexes binding to a human thymidine kinase promoter element important for G1-S phase transcriptional regulation. Proc Natl Acad Sci U S A 90:3554-8
Naeve, G S; Sharma, A; Lee, A S (1992) Identification of a 10-base pair protein binding site in the promoter of the hamster H3.2 gene required for the S phase dependent increase in transcription and its interaction with a Jun-like nuclear factor. Cell Growth Differ 3:919-28
Kim, Y K; Lee, A S (1992) Identification of a protein-binding site in the promoter of the human thymidine kinase gene required for the G1-S-regulated transcription. J Biol Chem 267:2723-7
Kim, Y K; Lee, A S (1991) Identification of a 70-base-pair cell cycle regulatory unit within the promoter of the human thymidine kinase gene and its interaction with cellular factors. Mol Cell Biol 11:2296-302
Kim, Y K; Wells, S; Lau, Y F et al. (1988) Sequences contained within the promoter of the human thymidine kinase gene can direct cell-cycle regulation of heterologous fusion genes. Proc Natl Acad Sci U S A 85:5894-8
Lee, A S; Wells, S; Delegeane, A M (1986) Methylation analysis of a plasmid containing a mammalian cell cycle regulatory sequence after transient transfection into the host cell. Biochem Biophys Res Commun 135:942-9

Showing the most recent 10 out of 11 publications