Loredana Verdone

Chromatin remodeling during Saccharomyces cerevisiae ADH2 gene activation.

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.

Hyperacetylation of chromatin at the ADH2 promoter allows Adr1 to bind in repressed conditions

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.

Snf1/AMPK regulates Gcn5 occupancy, H3 acetylation and chromatin remodelling at S. cerevisiae ADY2 promoter

The ability of cells to respond to changes in their environment is mediated by transcription factors that remodel chromatin and reprogram expression of specific subsets of genes. In Saccharomyces cerevisiae, changes in carbon source lead to gene induction by Adr1 and Cat8 that are known to require the upstream function of the Snf1 protein kinase, the central regulator of carbon metabolism, to exert their activating effect. How Snf1 facilitates transcription activation by Adr1 and Cat8 is not known. Here we show that under derepressing conditions, deletion of SNF1 abolishes the increase of histone H3 acetylation at the promoter of the glucose-repressed ADY2 gene, and as a consequence profoundly affects the chromatin structural alterations accompanying transcriptional activation. Adr1 and Cat8 are not required to regulate the acetylation switch and show only a partial influence on chromatin remodelling at this promoter, though their double deletion completely abolishes mRNA accumulation. Finally, we show that under derepressing conditions the recruitment of the histone acetyltransferase Gcn5 is abolished by SNF1 deletion, possibly explaining the lack of increased histone H3 acetylation and nucleosome remodelling. The results highlight a mechanism by which signalling to chromatin provides an essential permissive signal that is required for activation by glucose-responsive transcription factors.

A translational signature for nucleosome positioning in vivo.

In vivo nucleosomes often occupy well-defined preferred positions on genomic DNA. An important question is to what extent these preferred positions are directly encoded by the DNA sequence itself. We derive here from in vivo positions, accurately mapped by partial micrococcal nuclease digestion, a translational positioning signal that identifies the approximate midpoint of DNA bound by a histone octamer. This midpoint is, on average, highly A/T rich (∼73%) and, in particular, the dinucleotide TpA occurs preferentially at this and other outward-facing minor grooves. We conclude that in this set of sequences the sequence code for DNA bending and nucleosome positioning differs from the other described sets and we suggest that the enrichment of AT-containing dinucleotides at the centre is required for local untwisting. We show that this signature is preferentially associated with nucleosomes flanking promoter regions and suggest that it contributes to the establishment of gene-specific nucleosome arrays.

H4 acetylation does not replace H3 acetylation in chromatin remodelling and transcription activation of Adr1‐dependent genes

Histone acetylation regulates gene expression. Whether this is caused by a general increase in nucleosome fluidity due to charge neutralization or by a more specific code is still matter of debate. By using a set of glucose‐repressed Adr1‐dependent genes of Saccharomyces cerevisiae, whose transcription was previously shown to require both Gcn5 and Esa1, we asked how changes of histone acetylation patterns at the promoter nucleosomes regulate chromatin remodelling and activation. When the signal of glucose reduction reaches the cells, H4 acetylation is kept constant while an increase of H3 acetylation occurs, in an Adr1‐ and Gcn5‐dependent manner. In cells lacking Gcn5 activity, the H3 acetylation increase does not occur and an unexpected increase of histone H4 acetylation is observed. Nevertheless, chromatin remodelling and transcription activation are impaired, suggesting that acetylation of H3 and H4 histones plays different roles.

DNA topoisomerase I controls the kinetics of promoter activation and DNA topology in Saccharomyces cerevisiae.

Inactivation of the nonessential TOP1 gene, which codes for Saccharomyces cerevisiae DNA topoisomerase I, affects the rate of transcription starting at the ADH2 promoter. For both the chromosomal gene and the plasmid-borne promoter, mRNA accumulation is kinetically favored in the mutant relative to a wild-type isogenic strain. The addition of ethanol causes in wild-type yeast strains a substantial increase in linking number both on the ADH2-containing plasmid and on the resident 2 microns DNA. Evidence has been obtained that such an in vivo increase in linking number depends on (i) the activity of DNA topoisomerase I and of no other enzyme and (ii) ethanol addition, not on the release from glucose repression. A direct cause-effect relationship between the change in supercoiling and alteration of transcription cannot be defined. However, the hypothesis that a metabolism-induced modification of DNA topology in a eukaryotic cell plays a role in regulating gene expression is discussed.

Increased cerebellar volume and BDNF level following quadrato motor training.

Using whole-brain structural measures coupled to analysis of salivary brain-derived neurotrophic factor (BDNF), we demonstrate sensory motor training-induced plasticity, including cerebellar gray matter volume increment and increased BDNF level. The increase of cerebellar volume was positively correlated with the increase of BDNF level.

Poly(ADP-Ribosyl)ation Affects Histone Acetylation and Transcription.

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.

Aspects of nucleosomal positional flexibility and fluidity

Nucleosomes have been considered until recently to be stable and uniquely localized particles. We focus here on two properties of nucleosomes that are emerging as central attributes of their functions: mobility and multiplicity of localization. The biological relevance of these phenomena is based on the fact that chromatin functions depend on the relative stability of nucleosomes, on their covalent or conformational modifications, their dynamics, their localization, and the density of their distribution. In order to understand these complex behaviors both the structure of the nucleosome core particles and the informational rules governing their interaction with defined DNA sequences are here taken into consideration. The fact that nucleosomes solve the problem of how to locate a specific interaction site on a potentially infinite combination of sequences, with interactions recurring to a controlled level of informational ambiguity and stochasticity, is discussed. Nucleosomes have been shown to slide along DNA. This novel facet of their behavior and its implications in chromatin remodeling are reviewed.