Giorgio Camilloni

Substantial Histone Reduction Modulates Genomewide Nucleosomal Occupancy and Global Transcriptional Output

The basic unit of genome packaging is the nucleosome, and nucleosomes have long been proposed to restrict DNA accessibility both to damage and to transcription. Nucleosome number in cells was considered fixed, but recently aging yeast and mammalian cells were shown to contain fewer nucleosomes. We show here that mammalian cells lacking High Mobility Group Box 1 protein (HMGB1) contain a reduced amount of core, linker, and variant histones, and a correspondingly reduced number of nucleosomes, possibly because HMGB1 facilitates nucleosome assembly. Yeast nhp6 mutants lacking Nhp6a and -b proteins, which are related to HMGB1, also have a reduced amount of histones and fewer nucleosomes. Nucleosome limitation in both mammalian and yeast cells increases the sensitivity of DNA to damage, increases transcription globally, and affects the relative expression of about 10% of genes. In yeast nhp6 cells the loss of more than one nucleosome in four does not affect the location of nucleosomes and their spacing, but nucleosomal occupancy. The decrease in nucleosomal occupancy is non-uniform and can be modelled assuming that different nucleosomal sites compete for available histones. Sites with a high propensity to occupation are almost always packaged into nucleosomes both in wild type and nucleosome-depleted cells; nucleosomes on sites with low propensity to occupation are disproportionately lost in nucleosome-depleted cells. We suggest that variation in nucleosome number, by affecting nucleosomal occupancy both genomewide and gene-specifically, constitutes a novel layer of epigenetic regulation.

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.

ABFI contributes to the chromatin organization of Saccharomyces cerevisiae ARS1 B-domain

The involvement of the ABFI transcription factor in organizing the chromatin structure of the Saccharomyces cerevisiae ARS1 region has been previously postulated. We studied the ARS1 chromatin structure both on the chromosome and on plasmids carrying wild type or mutated ABFI binding sites, using a recently developed no-background technique for nucleosome mapping, coupled with high resolution micrococcal nuclease in vivo footprinting. We show that ABFI protein acts as a boundary element of chromatin structure, by limiting the invasion by nucleosomes toward the essential A-domain.

Sirtinol Treatment Reduces Inflammation in Human Dermal Microvascular Endothelial Cells

Histone deacetylases (HDAC) are key enzymes in the epigenetic control of gene expression. Recently, inhibitors of class I and class II HDAC have been successfully employed for the treatment of different inflammatory diseases such as rheumatoid arthritis, colitis, airway inflammation and asthma. So far, little is known so far about a similar therapeutic effect of inhibitors specifically directed against sirtuins, the class III HDAC. In this study, we investigated the expression and localization of endogenous sirtuins in primary human dermal microvascular endothelial cells (HDMEC), a cell type playing a key role in the development and maintenance of skin inflammation. We then examined the biological activity of sirtinol, a specific sirtuin inhibitor, in HDMEC response to pro-inflammatory cytokines. We found that, even though sirtinol treatment alone affected only long-term cell proliferation, it diminishes HDMEC inflammatory responses to tumor necrosis factor (TNF)α and interleukin (IL)-1β. In fact, sirtinol significantly reduced membrane expression of adhesion molecules in TNFã- or IL-1β-stimulated cells, as well as the amount of CXCL10 and CCL2 released by HDMEC following TNFα treatment. Notably, sirtinol drastically decreased monocyte adhesion on activated HDMEC. Using selective inhibitors for Sirt1 and Sirt2, we showed a predominant involvement of Sirt1 inhibition in the modulation of adhesion molecule expression and monocyte adhesion on activated HDMEC. Finally, we demonstrated the in vivo expression of Sirt1 in the dermal vessels of normal and psoriatic skin. Altogether, these findings indicated that sirtuins may represent a promising therapeutic target for the treatment of inflammatory skin diseases characterized by a prominent microvessel involvement.

In vivo analysis of chromatin following nystatin-mediated import of active enzymes into Saccharomyces cerevisiae

In vivo DNA-protein interactions are usually studied at the molecular level using DNA-degrading agents of low molecular weight. In order to be useful, macromolecular probes of chromatin structure, such as enzymes must first cross the cell membrane. In this paper we describe the introduction and evaluation of macromolecules with enzymatic activity into yeast spheroplasts treated with the polyene antibiotic nystatin. We report the low resolution analysis of chromatin structure in the promoter region of the Saccharomyces cerevisiae gene encoding DNA topoisomerase I by this technique using micrococcal nuclease and restriction enzymes.

Structure of RNA polymerase II promoters. Conformational alterations and template properties of circularized Saccharomyces cerevisiae GAL1-GAL10 divergent promoters.

A DNA fragment encompassing the Saccharomyces cerevisiae GAL1‐‐GAL10 divergent promoters (914 bp) has been circularized in vitro with T4 DNA ligase. We have defined a set of conditions that allows the production of a series of nine topoisomers covering a range from relaxed to highly negatively supercoiled DNA. Topoisomers were recovered in pure form from agarose gels and were analysed singly for the presence of sites sensitive to the single strand‐specific endonuclease Pl. In this way, the occurrence of conformational alterations as a function of the linking deficiency of the closed DNA domain has been determined. Interestingly, sites of Pl hypersensitivity localize on the three sequences identified as relevant for the in vitro transcription of the GAL1 moiety of the divergent promoter: the upstream activator sequence (UAS), the TATA sequence, and the RNA initiation site (RIS). In vitro transcription with purified S. cerevisiae RNA polymerase II shows that activation of transcription parallels the appearance of conformational alterations on the UAS, the TATA and the RIS sequences.

The conformation of constitutive DNA interaction sites for eukaryotic DNA topoisomerase I on intrinsically curved DNAs

The analysis of the sites which are cleaved constitutively and preferentially by eukaryotic DNA topoisomerase I on two intrinsically curved DNAs reveals the conformational features that provoke the cleavage reaction on the curve-inducing sequence elements in the absence of supercoiling. This analysis is based on the observation (Caserta et al. (1989) Nucleic Acids Res. 17, 8521-8532 and (1990) Biochemistry 29, 8152-8157) that the reaction of eukaryotic DNA topoisomerase I occurs on two types of DNA sites: sites S (Supercoiled induced) and sites C (Constitutive, whose presence is topology-independent). We report that sites C are abundant on the intrinsically curved DNAs analyzed. The DNAs studied were two intrinsically curved segments of different origin: the Crithidia fasciculata kinetoplast DNA and the bent-containing domain B of the Saccharomyces cerevisiae ARS1. On these DNA segments DNA topoisomerase I cleaves at the junctions between the poly(A) tracts and mixed-sequence DNA. Analysis of the conformation of the double helix around the cleavage sites has revealed that the reaction occurs in correspondence of a defined DNA conformational motif. This motif is described by the set of Eulerian angular values that define the axial path of DNA (helical twist, deflection angle, direction) and of the orthogonal components of wedge (roll and tilt).

RNA polymerase I transcription silences noncoding RNAs at the ribosomal DNA locus in Saccharomyces cerevisiae.

ABSTRACT In Saccharomyces cerevisiae the repeated units of the ribosomal locus, transcribed by RNA polymerase I (Pol I), are interrupted by nontranscribed spacers (NTSs). These NTS regions are transcribed by RNA polymerase III to synthesize 5S RNA and by RNA polymerase II (Pol II) to synthesize, at low levels, noncoding RNAs (ncRNAs). While transcription of both RNA polymerase I and III is highly characterized, at the ribosomal DNA (rDNA) locus only a few studies have been performed on Pol II, whose repression correlates with the SIR2-dependent silencing. The involvement of both chromatin organization and Pol I transcription has been proposed, and peculiar chromatin structures might justify “ribosomal” Pol II silencing. Reporter genes inserted within the rDNA units have been employed for these studies. We studied, in the natural context, yeast mutants differing in Pol I transcription in order to find whether correlations exist between Pol I transcription and Pol II ncRNA production. Here, we demonstrate that silencing at the rDNA locus represses ncRNAs with a strength inversely proportional to Pol I transcription. Moreover, localized regions of histone hyperacetylation appear in cryptic promoter elements when Pol II is active and in the coding region when Pol I is functional; in addition, DNA topoisomerase I site-specific activity follows RNA polymerase I transcription. The repression of ncRNAs at the rDNA locus, in response to RNA polymerase I transcription, could represent a physiological circuit control whose mechanism involves modification of histone acetylation.

FOB1 affects DNA topoisomerase I in vivo cleavages in the enhancer region of the Saccharomyces cerevisiae ribosomal DNA locus

In Saccharomyces cerevisiae the FOB1 gene affects replication fork blocking activity at the replication fork block (RFB) sequences and promotes recombination events within the rDNA cluster. Using in vivo footprinting assays we mapped two in vivo Fob1p-binding sites, RFB1 and RFB3, located in the rDNA enhancer region and coincident with those previously reported to be in vitro binding sites. We previously provided evidences that DNA topoisomerase I is able to cleave two sites within this region. The results reported in this paper, indicate that the DNA topoisomerase I cleavage specific activity at the enhancer region is affected by the presence of Fob1p and independent of replication and transcription activities. We thus hypothesize that the binding to DNA of Fob1p itself may be the cause of the DNA topoisomerase I activity in the rDNA enhancer.

Acetylation and Accessibility of rDNA Chromatin in Saccharomyces cerevisiae in Δtop1 and Δsir2 Mutants

The insertion of reporter genes in the ribosomal DNA (rDNA) locus of Saccharomyces cerevisiae causes their transcriptional repression. This kind of transcriptional silencing depends on proteins such as Sir2p and Top1p, and has been shown to be mediated by chromatin. While Sir2p modifies nucleosomes directly through its histone deacetylase activity, little is known about changes in the chromatin structure that occur at the rDNA locus when TOP1 is deleted. Here, we show that the absence of Top1p causes increased histone acetylation at the rDNA locus. Moreover, rDNA chromatin becomes more accessible in a similar manner in both top1 and sir2 mutant strains.