Degradation of cyclin A is regulated by acetylation.

Cyclin A accumulates at the onset of S phase, remains high during G(2) and early mitosis and is degraded at prometaphase. Here, we report that the acetyltransferase P/CAF directly interacts with cyclin A that as a consequence becomes acetylated at lysines 54, 68, 95 and 112. Maximal acetylation occurs simultaneously to ubiquitylation at mitosis, indicating importance of acetylation on cyclin A stability. This was further confirmed by the observation that the pseudoacetylated cyclin A mutant can be ubiquitylated whereas the nonacetylatable mutant cannot. The nonacetylatable mutant is more stable than cyclin A WT (cycA WT) and arrests cell cycle at mitosis. Moreover, in cells treated with histone deacetylase inhibitors cyclin A acetylation increases and its stability decreases, thus supporting the function of acetylation on cyclin A degradation. Although the nonacetylatable mutant cannot be ubiquitylated, it interacts with the proteins needed for its degradation (cdks, Cks, Cdc20, Cdh1 and APC/C). In fact, its association with cdks is increased and its complexes with these kinases display higher activity than control cycA WT-cdk complexes. All these results indicate that cyclin A acetylation at specific lysines is crucial for cyclin A stability and also has a function in the regulation of cycA-cdk activity.

Regulation of E2F1 activity by acetylation.

During the G(1) phase of the cell cycle, an E2F-RB complex represses transcription, via the recruitment of histone deacetylase activity. Phosphorylation of RB at the G(1)/S boundary generates a pool of 'free' E2F, which then stimulates transcription of S-phase genes. Given that E2F1 activity is stimulated by p300/CBP acetylase and repressed by an RB-associated deacetylase, we asked if E2F1 was subject to modification by acetylation. We show that the p300/CBP-associated factor P/CAF, and to a lesser extent p300/CBP itself, can acetylate E2F1 in vitro and that intracellular E2F1 is acetylated. The acetylation sites lie adjacent to the E2F1 DNA-binding domain and involve lysine residues highly conserved in E2F1, 2 and 3. Acetylation by P/CAF has three functional consequences on E2F1 activity: increased DNA-binding ability, activation potential and protein half-life. These results suggest that acetylation stimulates the functions of the non-RB bound 'free' form of E2F1. Consistent with this, we find that the RB-associated histone deacetylase can deacetylate E2F1. These results identify acetylation as a novel regulatory modification that stimulates E2F1's activation functions.

E1A directly binds and regulates the P/CAF acetyltransferase.

The P/CAF protein has intrinsic histone acetyltransferase (HAT) activity and is capable of binding the transcriptional co-activator CBP. Here we show that P/CAF can regulate transcription and that this function is independent of its binding to CBP. The HAT domain of P/CAF has transcriptional activation potential in yeast. In mammalian cells P/CAF can stimulate transcription of the RSV promoter, using the activity of its HAT domain. We show that the adenovirus protein E1A targets P/CAF and sequesters its transcriptional activity. Binding of E1A to P/CAF is direct, independent of CBP and requires residues within E1A conserved region 1. We find that the P/CAF binding residues in E1A are within a motif shown to be essential for efficient disruption of myogenesis by E1A. The fact that E1A can directly bind and regulate the activity of P/CAF, independently of its regulation of CBP, highlights an important role for P/CAF in the process of cell differentiation.

The acetyltransferase activity of CBP stimulates transcription.

The CBP co-activator protein possesses an intrinsic acetyltransferase (AT) activity capable of acetylating nucleosomal histones, as well as other proteins such as the transcription factors TFIIE and TFIIF. In addition, CBP associates with two other TSs, P/CAF and SRC1. We set out to establish whether the intrinsic AT activity of CBP contributes to transcriptional activation. We show that a region of CBP, encompassing the previously defined histone AT (HAT) domain, can stimulate transcription when tethered to a promoter. The stimulatory effect of this activation domain shows some promoter preference and is dependent on AT activity. Analysis of 14 point mutations reveals a direct correlation between CBP's ability to acetylate histones in vitro and to activate transcription in vivo. We also find that the HAT domains of CBP and P/CAF share sequence similarity. Four conserved motifs are identified, three of which are analogous to motifs A, B and D, found in other N-acetyltransferases. The fourth motif, termed E, is unique to CBP and P/CAF. Mutagenesis shows that all four motifs in CBP contribute to its HAT activity in vitro and its ability to activate transcription in vivo. These results demonstrate that the AT activity of CBP is directly involved in stimulating gene transcription. The identification of specific HAT domain motifs, conserved between CBP and P/CAF, should facilitate the identification of other members of this AT family.

Drosophila NURF-55, a WD repeat protein involved in histone metabolism.

The Drosophila nucleosome remodeling factor (NURF) is a protein complex of four distinct subunits that assists transcription factor-mediated chromatin remodeling. One NURF subunit, ISWI, is related to the transcriptional regulators Drosophila brahma and yeast SWI2/SNF2. We have determined peptide sequences and isolated cDNA clones for a second NURF component (the 55-kDa subunit). Immunological studies show that p55 is an integral subunit of NURF and is generally associated with polytene chromosomes. The predicted sequence of p55 reveals a WD repeat protein that is identical with the 55-kDa subunit of the Drosophila chromatin assembly factor (CAF-1). Given that WD repeat proteins related to p55 are associated with histone deacetylase and histone acetyltransferase, our findings suggest that p55 and its homologs may function as a common platform for the assembly of protein complexes involved in chromatin metabolism.

Displacement of sequence-specific transcription factors from mitotic chromatin.

The general inhibition in transcriptional activity during mitosis abolishes the stress-inducible expression of the human hsp70 gene. Among the four transcription factors that bind to the human hsp70 promoter, the DNA-binding activities of three (C/EBP, GBP, and HSF1) were normal, while Sp1 showed reduced binding activity in mitotic cell extracts. In vivo footprinting and immunocytochemical analyses revealed that all of the sequence-specific transcription factors were displaced from promoter sequences as well as from bulk chromatin during mitosis. The correlation of transcription factor displacement with chromatin condensation suggests an involvement of chromatin structure in mitotic repression. However, retention of DNase I hypersensitivity suggests that the hsp70 promoter was not organized in a canonical nucleosome structure in mitotic chromatin. Displacement of transcription factors from mitotic chromosomes could present another window in the cell cycle for resetting transcriptional programs.

Zinc(II) ions selectively interact with DNA sequences present at the TFIIIA binding site of the Xenopus 5S-RNA gene.

It has been known for some time that zinc, as well as most transition metal ions, is capable of binding to the DNA bases. However, little is known about the presence and distribution of metal binding sites along naturally occurring genomic DNA molecules. In this paper, the interaction of zinc with the Xenopus 5S-RNA gene has been studied and several metal binding sites have been identified on the basis of the changes in chemical reactivity observed in the presence of the metal. The strongest zinc-binding sites of the Xenopus 5S-RNA gene correspond to GGG trinucleotide repeats. Some GG dinucleotides also show a significant affinity for zinc. Interestingly, the binding site for TFIIIA, a zinc-finger transcription factor, contains several sites with strong zinc affinity. In particular, a TGGGA sequence which is essential for the binding of TFIIIA shows the strongest affinity for zinc. The conformational properties of this DNA sequence, together with the high electronegative potential of GGG runs, is likely to determine its strong affinity for zinc. The possible biological relevance of these results is discussed.

Reversion of a simian virus 40 enhancer mutant depends on the growth conditions.

The simian virus 40 (SV40) enhancer contains three 8-bp purine-pyrimidine (R:Y) alternating sequences (Z-motifs) which are known to adopt the left-handed Z-DNA conformation in vitro. Mutations at these three Z-motifs seriously impair enhancer function. Reversion of one of these mutants (dpm12) is studied in this paper. The results indicate that, depending on growth conditions, recovery of the enhancer function is achieved through different mechanisms. Mutant viruses growing in solid-agar medium do not revert. On the other hand, revertants obtained in liquid medium contain a duplication of the enhancer sequences, showing no recovery of the original Z-motifs.

Identification of sequence elements contributing to the intrinsic curvature of the mouse satellite DNA repeat.

In this paper, the contribution of different sequence elements to the intrisic curvature of the mouse satellite DNA repeat was investigated. This DNA fragment contains nineteen groups of three or more consecutive adenines which are only poorly phased with respect to the helical repeat. The mouse satellite DNA repeat shows a sinusoidal pattern of cleavage by the hydroxyl radical; the waves of reactivity are phased with respect to the A-tracts. Some interesting observations arise from a detailed analysis of these cleavage patterns: a) the maxima of hydroxyl radical cleavage are more periodically spaced along the DNA sequence than the A-tracts themselves. As a consequence, the position of each maximum with respect to the A-tract is variable; b) the sequence 5' TGGAATATG/AA 3' shows a sinusoidal pattern of hydroxyl radical cleavage. This sequence shows a retarded migration in polyacrylamide gels indicating that it is actually intrinsically curved. These results are discussed in view of the current models for DNA curvature.

A new ionizable chromophore of 1,4-bis(alkylamino)benzo[g]phthalazine which interacts with DNA by intercalation.

The tricyclic heteroaromatic nucleus of 1,4-bis(alkylamino)benzo[g]phthalazine can be protonated at physiological pH, depending on the nature of the side chains. The interaction of the 3-methoxypropyl derivative with calf thymus and closed, circular DNA has been studied with UV-vis spectroscopy and NMR. The effect of drug binding on the topology of closed, circular DNA was determined by topoisomerase-I catalyzed relaxation of the complex followed by gel electrophoresis. The results strongly support intercalative binding and suggest that this series of compounds are promising targets for anticancer activity evaluation.