The chromatin of eukaryotic cells is organized in nucleosomes. This organization allows the efficient packaging of chromosomal DNA into the nucleus but limits the access of highmolecular-weight protein complexes of the transcription machinery. At least two different mechanisms enable the eukaryotic cell to relieve nucleosomal repression: the chromatinremodeling complexes (reviewed in references 55 and 57) and reversible histone acetylation. Two recent reports indicate a direct link between these two activities (60, 67). Posttranslational acetylation on conserved lysine residues within the Nterminal regions of nucleosomal histones is assumed to lead to a reduced attraction between chromosomal DNA and histone tails and changed interactions with neighboring nucleosomes or other nonhistone proteins. The resulting local chromatin decondensation increases the accessibility of particular DNA regions for RNA polymerase complexes. Consistent with this idea, transcriptionally active chromatin correlates with histone hyperacetylation (reviewed in references 18, 30, 47, 49, 61, and 62). This model predicts that histone acetyltransferases would promote transcription, while histone deacetylases (HDACs) should act as repressors. In accordance with this model, several transcriptional adapters and coactivators, such as GCN5 (8, 31), p300/CBP (4, 46), TAFII250 (40), SRC-1 (54), and ACTR (10), have been classified as histone acetyltransferases. Five HDACs have been identified in mammalian cells (12,14,56,58,63,64). Three of them, HDAC1, HDAC2, and HDAC3, have significant homology to yeast Rpd3 (44,50,59). HDAC4 and HDAC5 belong to the histone deacetylase A (HDA) family (9, 58). HDAC1 and HDAC2 are found in high-molecularweight complexes associated with adapter proteins like SIN3, SAP18, and SAP30 and nuclear corepressors like N-CoR, SMRT, and 24,32,42,65,66). Recently it was demonstrated that several mammalian transcription factors, such as Mad (21, 24, 32, 52), YY1 (64), hormone-dependent nuclear receptors (24, 42), MeCP2 (26, 43), CBF (27), retinoblastoma protein (Rb) (7, 38, 39), and related pocket proteins (16), can repress transcription by recruiting HDACs to specific promoters. In addition, the aberrant recruitment of HDACs by PLZF, PML, and ETO fusion proteins can interfere with the differentiation of hematopoietic precursor cells in acute promyelocytic leukemia (13,17,19,35).In this study we investigated the potential function of HDACs as transcriptional repressors during the growth arrest of mammalian cells. Using the S-phase-specific mouse thymidine kinase (TK) promoter as a model system, we show that HDAC1 can mediate transcriptional repression via the Sp1 binding site. HDAC1 is associated with Sp1 and binds directly to the C-terminal part of Sp1 that was previously identified as interacting domain for E2F1 (28). Sp1 and E2F1 cooperate in the activation of S-phase-specific promoters (28, 36). Here we show that E2F1 but not E2F4 can compete with HDAC1 binding to Sp1, thereby relieving HDAC1-mediated repression of the TK...

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 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.The tumor suppressor p53 can induce cell cycle arrest or apoptosis in response to a variety of stress signals, such as DNA damage, oncogenic stimuli, or hypoxia (reviewed in reference 49). Activation of p53 occurs by several mechanisms including protein stabilization and modification of the protein by phosphorylation and acetylation. p53 is a transcription factor that recognizes specific binding sites within numerous target genes including mdm2, cyclin G, bax, and p21/WAF1/CIP1 (for reviews see references 5 and 12). While multiple downstream targets are involved in the mediation of apoptotic effects, the main target for p53-induced cell cycle arrest seems to be the p21 gene. p21 has been identified by virtue of its activation by p53 (13), its association with cyclin/cyclin-dependent kinase (CDK) complexes (23, 66), and its up-regulation during senescence (47). Furthermore, the p21 protein was shown previously to interact with the proliferating cell nuclear antigen (PCNA), thereby preventing DNA replication (10). Induction of p21 expression by genotoxic stress and its role during terminal differentiation of various cell types have been investigated intensively. While p21 is activated by p53-dependent mechanisms in response to DNA damage to ensure cell cycle arrest and repair, a variety of agents that promote differentiation, like phorbol ester or okadaic acid, can up-regulate p21 independently of p53 (for a review see reference 16). Similarly, the p21 gene can be activated by transforming growth factor ␤, Ca 2ϩ , lovastatin, or nerve growth factor (16).Recently, a number of reports demonstrated the induction of p21 by inhibitors of histone deacetylases (HDACs), such as sodium butyrate (46), trichostatin A (TSA) (56), suberoylanilide hydroxamic a...

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 a, 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 ax) 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 G,/S border in serum-stimulated cells but not during polyomavirus T antigeninduced transcriptional activation of DNA synthesis enzymes in arrested cells.Enzymes involved in DNA synthesis and precursor production are regulated during growth stimulation and during the cell cycle. Their synthesis is induced at the beginning of the S phase, and in many cases controls at different levels, transcriptional as well as posttranscriptional, are crucial (for reviews, see references 32 and 42). Transcriptional regulation plays the major role when cells are stimulated to grow from an arrested state. Members of a family of transcription factors called E2F were recently found to play a key role in transcriptional regulation of many important genes in the GI phase and at the G,/S boundary of the cell cycle (reviewed in references 4, 15, and 27). E2F was originally identified as a cellular protein binding to the adenovirus E2 promoter, but binding sites for E2F were later also found in cellular promoters and were shown to be important for the transcription of cell cycleregulated genes such as c-myc, dihydrofolate reductase, and thymidine kinase (2,17,27,30,38). E2F is a target for the retinoblastoma tumor suppressor protein (pRB); a related protein, p107 (reviewed in references 4, 15, and 27); and possibly other so-called pocket proteins; complexes formed on binding sites for E2F were also found to c...

No abstract

A mitochondria-free cell extract from Xaccharomyces cerevisiae was used as source of yeast DNA polymerase for the partial purification and characterization of the enzyme. Upon DEAEcellulose chromatography, the total DNA polymerase activity separates into a major fraction, DNA polymerase A, and a minor fraction, DNA polymerase B. The final purification achieved was 150-to 200-fold for DNA polymerase A and 60-to 80-fold for DNA polymerase B. The two polymerases A and B differ from each other in some properties. Both purified enzyme preparations contain only traces of nucleases and very little, if any, terminal transferase. Activity is totally dependent on the presence of DNA, all four deoxyribonucleoside triphosphates and Mg++. With "activated" DNA or with poly d(A-T) as template the DNA polymerases A and B exhibit a n optimal activity. Native DNA preparations promote DNA synthesis to a low degree only, while denatured DNA9 are even less active as templates. The enzymes have a molecular weight around 150 000 ; however, larger aggregates have been observed under certain conditions. DNA polymerases have been purified and characterized from a variety of bacterial and animal sources. These enzymes synthesize DNA from monomeric units, the deoxyribonucleoside triphosphates, the base sequence being determined by a DNA template. The recent extensive studies in Kornberg's laboratory on a homogeneous preparation of Escherichia coli DNA polymerase have clarified much of the mechanism of action of this enzyme [l-61. I n the last few years it has become increasingly clear, however, that the process of DNA replication in the living cell cannot be mediated solely by DNA polymerase but that this enzyme is only part of a more complicated replication machinery. (An extensive discussion of all aspects of DNA synthesis in microorganisms can be found elsewhere [6 a] .)Eukaryotic cells present a further interesting feature of cellular DNA synthesis because these cells contain partially autonomous particles, mitochondria and chloroplasts, which have their own DNA replicating machinery [7].I n the course of our own studies on mitochondria1 DNA polymerase from yeast [S,

No abstract

No abstract