Histone proteins play structural and functional roles in all nuclear processes. They undergo different types of covalent modifications, defined in their ensemble as epigenetic because changes in DNA sequences are not involved. Histone acetylation emerges as a central switch that allows interconversion between permissive and repressive chromatin domains in terms of transcriptional competence. The mechanisms underlying the histone acetylation-dependent control of gene expression include a direct effect on the stability of nucleosomal arrays and the creation of docking sites for the binding of regulatory proteins. Histone acetyltransferases and deacetylases are, respectively, the enzymes devoted to the addition and removal of acetyl groups from lysine residues on the histone N-terminal tails. The enzymes exert fundamental roles in developmental processes and their deregulation has been linked to the progression of diverse human disorders, including cancer.Key words: gene expression, transcription, HATs, HDACs, nucleosome.

We have analyzed at both low and high resolution the distribution of nucleosomes over the Saccharomyces cerevisiae ADH2 promoter region in its chromosomal location, both under repressing (high-glucose) conditions and during derepression. Enzymatic treatments (micrococcal nuclease and restriction endonucleases) were used to probe the in vivo chromatin structure during ADH2 gene activation. Under glucose-repressed conditions, the ADH2 promoter was bound by a precise array of nucleosomes, the principal ones positioned at the RNA initiation sites (nucleosome +1), at the TATA box (nucleosome -1), and upstream of the ADR1-binding site (UAS1) (nucleosome -2). The UAS1 sequence and the adjacent UAS2 sequence constituted a nucleosome-free region. Nucleosomes -1 and +1 were destabilized soon after depletion of glucose and had become so before the appearance of ADH2 mRNA. When the transcription rate was high, nucleosomes -2 and +2 also underwent rearrangement. When spheroplasts were prepared from cells grown in minimal medium, detection of this chromatin remodeling required the addition of a small amount of glucose. Cells lacking the ADR1 protein did not display any of these chromatin modifications upon glucose depletion. Since the UAS1 sequence to which Adr1p binds is located immediately upstream of nucleosome -1, Adr1p is presumably required for destabilization of this nucleosome and for aiding the TATA-box accessibility to the transcription machinery.

We report that in vivo increased acetylation of the repressed Saccharomyces cerevisiae ADH2 promoter chromatin, as obtained by disrupting the genes for the two deacetylases HDA1 and RPD3 , destabilizes the structure of the TATA box‐containing nucleosome. This acetylation‐dependent chromatin remodeling is not sufficient to allow the binding of the TATA box‐binding protein, but facilitates the recruitment of the transcriptional activator Adr1 and induces faster kinetics of mRNA accumulation when the cells are shifted to derepressing conditions.

The effects of supercoiling on the topoisomerization reaction by eukaryotic DNA topoisomerases I have been analyzed. The systems used were: DNA topoisomerase I from wheat germ, chicken erythrocyte and calf thymus on a 2.3 kb DNA fragment which encompasses the immunoglobulin .kappa.-light chain (L .times. promoter of the mouse plasmacytoma MPC11; S. cerevisiae DNA topoisomerase I on a 2.2 kb DNA fragment from the same organism with encompasses the regulatory and the coding region of the ADH II gene; wheat germ DNA topoisomerase I on the plasmid pUC18. It was found in every system that lack of torsional stress prevents topoisomerization of the substrate. A simple regulatory model of DNA topoisomerase I function, based on topological considerations, is presented.

Poly(ADP-ribosyl)ation (PARylation) is a posttranslational protein modification catalyzed by members of the poly(ADP-ribose) polymerase (PARP) enzyme family. PARylation regulates a wide variety of biological processes in most eukaryotic cells including energy metabolism and cell death, maintenance of genomic stability, chromatin structure and transcription. Inside the nucleus, cross-talk between PARylation and other epigenetic modifications, such as DNA and histone methylation, was already described. In the present work, using PJ34 or ABT888 to inhibit PARP activity or over-expressing poly(ADP-ribose) glycohydrolase (PARG), we show decrease of global histone H3 and H4 acetylation. This effect is accompanied by a reduction of the steady state mRNA level of p300, Pcaf, and Tnfα, but not of Dnmt1. Chromatin immunoprecipitation (ChIP) analyses, performed at the level of the Transcription Start Site (TSS) of these four genes, reveal that changes in histone acetylation are specific for each promoter. Finally, we demonstrate an increase of global deacetylase activity in nuclear extracts from cells treated with PJ34, whereas global acetyltransferase activity is not affected, suggesting a role for PARP in the inhibition of histone deacetylases. Taken together, these results show an important link between PARylation and histone acetylation regulated transcription.

The effects of supercoiling on the cleavage reaction by eukaryotic DNA topoisomerases I (wheat germ, chicken erythrocyte, and calf thymus) have been analyzed on DNA fragments (0.96 and 2.3 kilobases) encompassing an immunoglobulin .kappa. light-chain promoter. In one topological condition of the substrate, the absolutely relaxed state, cleavage was found to be impeded. This finding defines the topology-dependent step of the eukaryotic DNA topoisomerase I reaction and shows that for the cleavage reaction topology is more critical than sequence effects. These findings suggest a simple model for the regulation of the DNA topoisomerase I reaction based on topological factors, which may explain the regulatory function of the enzyme in in vivo eukaryotic transcription.