Hans Rotheneder

Histone Deacetylase 1 Can Repress Transcription by Binding to Sp1

ABSTRACT The members of the Sp1 transcription factor family can act as both negative and positive regulators of gene expression. Here we show that Sp1 can be a target for histone deacetylase 1 (HDAC1)-mediated transcriptional repression. The histone deacetylase inhibitor trichostatin A activates the chromosomally integrated murine thymidine kinase promoter in an Sp1-dependent manner. Coimmunoprecipitation experiments with Swiss 3T3 fibroblasts and 293 cells demonstrate that Sp1 and HDAC1 can be part of the same complex. The interaction between Sp1 and HDAC1 is direct and requires the carboxy-terminal domain of Sp1. Previously we have shown that the C terminus of Sp1 is necessary for the interaction with the transcription factor E2F1 (J. Karlseder, H. Rotheneder, and E. Wintersberger, Mol. Cell. Biol. 16:1659–1667, 1996). Coexpression of E2F1 interferes with HDAC1 binding to Sp1 and abolishes Sp1-mediated transcriptional repression. Our results indicate that one component of Sp1-dependent gene regulation involves competition between the transcriptional repressor HDAC1 and the transactivating factor E2F1.

Interaction of Sp1 with the growth- and cell cycle-regulated transcription factor E2F.

Within the region around 150 bp upstream of the initiation codon, which was previously shown to suffice for growth-regulated expression, the murine thymidine kinase gene carries a single binding site for transcription factor Sp1; about 10 bp downstream of this site, there is a binding motif for transcription factor E2F. The latter protein appears to be responsible for growth regulation of the promoter. Mutational inactivation of either the Sp1 or the E2F site almost completely abolishes promoter activity, suggesting that the two transcription factors interact directly in delivering an activation signal to the basic transcription machinery. This was verified by demonstrating with the use of glutathione S-transferase fusion proteins that E2F and Sp1 bind to each other in vitro. For this interaction, the C-terminal part of Sp1 and the N terminus of E2F1, a domain also present in E2F2 and E2F3 but absent in E2F4 and E2F5, were essential. Accordingly, E2F1 to E2F3 but not E2F4 and E2F5 were found to bind sp1 in vitro. Coimmunoprecipitation experiments showed that complexes exist in vivo, and it was estabilished that the distance between the binding sites for the two transcription factors was critical for optimal promoter activity. Finally, in vivo footprinting experiments indicated that both the sp1 and E2F binding sites are occupied throughout the cell cycle. Mutation of either binding motif abolished binding of both transcription factors in vivo, which may indicate cooperative binding of the two proteins to chromatin-organized DNA. Our data are in line with the hypothesis that E2F functions as a growth- and cell cycle regulated tethering factor between Sp1 and the basic transcription machinery.

The Tumor Suppressor p53 and Histone Deacetylase 1 Are Antagonistic Regulators of the Cyclin-Dependent Kinase Inhibitor p21/WAF1/CIP1 Gene

ABSTRACT The cyclin-dependent kinase inhibitor p21/WAF1/CIP1 is an important regulator of cell cycle progression, senescence, and differentiation. Genotoxic stress leads to activation of the tumor suppressor p53 and subsequently to induction of p21 expression. Here we show that the tumor suppressor p53 cooperates with the transcription factor Sp1 in the activation of the p21 promoter, whereas histone deacetylase 1 (HDAC1) counteracts p53-induced transcription from the p21 gene. The p53 protein binds directly to the C terminus of Sp1, a domain which was previously shown to be required for the interaction with HDAC1. Induction of p53 in response to DNA-damaging agents resulted in the formation of p53-Sp1 complexes and simultaneous dissociation of HDAC1 from the C terminus of Sp1. Chromatin immunoprecipitation experiments demonstrated the association of HDAC1 with the p21 gene in proliferating cells. Genotoxic stress led to recruitment of p53, reduced binding of HDAC1, and hyperacetylation of core histones at the p21 promoter. Our findings show that the deacetylase HDAC1 acts as an antagonist of the tumor suppressor p53 in the regulation of the cyclin-dependent kinase inhibitor p21 and provide a basis for understanding the function of histone deacetylase inhibitors as antitumor drugs.

Coordinated trans activation of DNA synthesis- and precursor-producing enzymes by polyomavirus large T antigen through interaction with the retinoblastoma protein.

Previously constructed Swiss mouse 3T3 fibroblasts producing polyomavirus large T antigen after addition of dexamethasone were used to study the transcriptional activation by the viral protein of five genes coding for enzymes involved in DNA synthesis and precursor production, namely, dihydrofolate reductase, thymidine kinase, thymidylate synthase, DNA polymerase alpha, and proliferating-cell nuclear antigen. It was found that all these genes, whose expression is stimulated at the G1/S boundary of the cell cycle after growth stimulation by serum addition, are coordinately trans activated when T antigen is induced in cells previously growth arrested by serum withdrawal. Cell lines carrying the information for a mutant form of large T antigen, in which a glutamic acid residue in the binding site for the retinoblastoma protein was changed into aspartic acid, were constructed to test the involvement of an interaction of T antigen with the retinoblastoma protein in this reaction. It was found that the mutated T protein is incapable of stimulating transcription of any one of the genes. The promoter of three of the genes (dihydrofolate reductase, thymidine kinase, and DNA polymerase alpha) unequivocally carries binding sites for transcription factor E2F, suggesting that complexes forming with this growth- and cell cycle-regulating transcription factor are the targets for T antigen. Although there is so far no evidence that thymidylate synthase and proliferating cell nuclear antigen are regulated via E2F, our data indicate that the retinoblastoma protein still is involved in the control of these genes. mRNA for E2F itself increases in amount at the G1/S border in serum-stimulated cells but not during polyomavirus T antigen-induced transcriptional activation of DNA synthesis enzymes in arrested cells.