A cross-talk between DNA methylation and H3 lysine 9 dimethylation at the KvDMR1 region controls the induction of Cdkn1c in muscle cells

The cdk inhibitor p57kip2, encoded by the Cdkn1c gene, plays a critical role in mammalian development and in the differentiation of several tissues. Cdkn1c protein levels are carefully regulated via imprinting and other epigenetic mechanisms affecting both the promoter and distant regulatory elements, which restrict its expression to particular developmental phases or specific cell types. Inappropriate activation of these regulatory mechanisms leads to Cdkn1c silencing, causing growth disorders and cancer. We have previously reported that, in skeletal muscle cells, induction of Cdkn1c expression requires the binding of the bHLH myogenic factor MyoD to a long-distance regulatory element within the imprinting control region KvDMR1. Interestingly, MyoD binding to KvDMR1 is prevented in myogenic cell types refractory to the induction of Cdkn1c. In the present work, we took advantage of this model system to investigate the epigenetic determinants of the differential interaction of MyoD with KvDMR1. We show that treatment with the DNA demethylating agent 5-azacytidine restores the binding of MyoD to KvDMR1 in cells unresponsive to Cdkn1c induction. This, in turn, promotes the release of a repressive chromatin loop between KvDMR1 and Cdkn1c promoter and, thus, the upregulation of the gene. Analysis of the chromatin status of Cdkn1c promoter and KvDMR1 in unresponsive compared to responsive cell types showed that their differential responsiveness to the MyoD-dependent induction of the gene does not involve just their methylation status but, rather, the differential H3 lysine 9 dimethylation at KvDMR1. Finally, we report that the same histone modification also marks the KvDMR1 region of human cancer cells in which Cdkn1c is silenced. On the basis of these results, we suggest that the epigenetic status of KvDMR1 represents a critical determinant of the cell type-restricted expression of Cdkn1c and, possibly, of its aberrant silencing in some pathological conditions

Chromosome-Protein Interactions in Polyomavirus Virions

In this work, we sought to determine whether the components of the murine polyomavirus capsid establish specific interactions with the minichromosome encapsidated into the mature viral particles by using the cis-diamminedichloroplatinum(II) cross-linking reagent. Our data indicated that VP1, but not minor capsid proteins, interacts with the viral genome in vivo. In addition, semiquantitative PCR assays performed on cross-linked DNA complexes revealed that VP1 binds to all regions of the viral genome but significantly more to the regulatory region. The implications of such an interaction for viral infectivity are discussed

Mitochondrial Localization of PARP-1 Requires Interaction with Mitofilin and Is Involved in the Maintenance of Mitochondrial DNA Integrity*

Poly(ADP-ribose)polymerase-1 (PARP-1) is a predominantly nuclear enzyme that exerts numerous functions in cellular physiology and pathology, from maintenance of DNA stability to transcriptional regulation. Through a proteomic analysis of PARP-1 co-immunoprecipitation complexes, we identified Mitofilin, a mitochondrial protein, as a new PARP-1 interactor. This result prompted us to further investigate the presence and the role of the enzyme in mitochondria. Using laser confocal microscopy and Western blot analysis of purified mitochondria, we demonstrated the mitochondrial localization of a fraction of PARP-1. Further, the effects of overexpressing or down-regulating Mitofilin showed that this protein promotes and is required for PARP-1 mitochondrial localization. We also report several lines of evidence suggesting that intramitochondrial PARP-1 plays a role in mitochondrial DNA (mtDNA) damage signaling and/or repair. First, we show that PARP-1 binds to different regions throughout the mtDNA. Moreover, we demonstrated that the depletion of either PARP-1 or Mitofilin, which abrogates the mitochondrial localization of the enzyme, leads to the accumulation of mtDNA damage. Finally, we show that DNA ligase III, known to be required for mtDNA repair, participates in a PARP-1-containing complex bound to mtDNA. This work highlights a new environment for PARP-1, opening the possibility that at least some of the nuclear functions of the enzyme can be also extended to mtDNA metabolism

PARP-1 interaction with VP1 capsid protein regulates polyomavirus early gene expression.

Poly(ADP-ribose)polymerases are involved in fundamental cellular events as well as they seem to be associated to some viral infection process. In this work, the poly(ADP-ribose)polymerase-1 (PARP-1) role in the polyomavirus life cycle has been investigated. Early viral transcription was reduced by competitive inhibitors of PARPs in Swiss 3T3 cells and almost abolished in PARP-1 knockout fibroblasts and in wild-type fibroblasts when PARP-1 was silenced by RNA interference. In vivo chromatin immunoprecipitation assays showed that poly(ADP-ribosyl)ation (poly(ADP-ribose)) facilitates the release of the capsid protein viral protein 1 (VP1) from the chromatin of infecting virions. In vitro experiments demonstrated that VP1 stimulates the enzymatic activity of PARP-1 and binds non-covalently both protein-free and PARP-1-bound poly(ADP-ribose). Our studies suggest that PARP-1 promotes the complete VP1 displacement from viral DNA favouring the viral early transcription.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

PARP-1 binds <i>in vivo</i> to c-MYC promoter.

<p>A, Schematic representation of the <i>c-MYC</i> promoter region (adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102575#pone.0102575-Wierstra1" target="_blank">[21]</a>). The transcriptional start sites P1 and P2, the Guanine-quadruplex forming element (G–Q), the consensus sequence CCCTCCCC (CT-I₂) are shown. The fragments analyzed in B are indicated below the scheme. B, ChIP analysis of PARP-1 binding to c-MYC promoter. The assays were carried out in quiescent (0) and in 30 minutes serum-stimulated cells, using an antibody specific for PARP-1 or a normal rabbit IgG as a negative control of the immuno-precipitation. Diluted Input and immuno-precipitated samples were subjected to PCR amplification of the indicated fragments of c-MYC (fr.1, fr.2 and fr.3) or GAPDH promoter regions. RHO promoter was used as a negative control of PARP-1 binding. Numbers above the panels indicate the minutes post serum addition (min p.s.a.). Numbers below the panels indicate the cycles of PCR amplification. The results shown are representative of multiple independent experiments.</p

PARP activity promotes an active chromatin status at c-MYC promoter.

<p>A, DNAseI accessibility assays on c-MYC promoter. Quiescent (-FBS) and 30-minutes serum-stimulated (+FBS) fibroblasts were treated or not with the PARP inhibitor PJ-34. The entire nuclei were isolated and treated with 5 or 10 units of DNaseI or left untreated. After purification DNAs were analyzed by semi-quantitative PCR with a primer pair specific for the fragment 2 of c-MYC promoter. Densitometric-values obtained for c-MYC and GADPH promoters were normalized respect to those obtained for RHO promoter. Data are plotted as mean ± SD of four biological replicates. Statistical significances: *: P<0.05; **: P<0.01 (Student's t test). B, ChIP analysis of c-MYC promoter occupancy by SP1 and CTCF. Chromatin samples from cells treated as above were immuno-precipitated with antibodies specific for SP1 or CTCF or normal rabbit IgG. Input and immuno-precipitated samples were subjected to qPCR amplification with a primer pair specific for the region covering the fragment 2 and 3 (fr.2+3) of c-MYC promoter region (c-MYC) and for RHO promoter used as a negative control. The results shown are representative of two independent experiments and plotted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102575#pone-0102575-g003" target="_blank">Figure 3A</a>.</p

PARP-activity is required for histone modifications at c-MYC promoter.

<p>A, Histone H3K4me3 levels at c-MYC promoter assessed by ChIP-qPCR. Chromatin samples were obtained from quiescent (-FBS) or 30 minutes serum-stimulated (+FBS) fibroblasts treated or not with PJ-34, and immuno-precipitated with an antibody specific for H3K4me3. Values relative to the occupancy of c-MYC promoter (fragment fr.2+3) were normalized respect to those of GAPDH promoter and expressed as percentages of Input chromatin. The results shown are representative of two independent experiments and the error bars represent the SD of three technical replicates. B, Acetyl histone H3 (AcH3) and phosphoacetyl histone H3 (pAcH3) levels at c-MYC promoter assessed by ChIP-qPCR. Quiescent fibroblasts were pre-treated with TSA or Anisomycin (Anis) then serum-stimulated for 30 minutes in the presence or absence of PJ-34. Chromatin samples were immuno-precipitated with antibodies specific for AcH3 or pAcH3. The reported values are expressed as in A.</p

PARP-1 is required for the normal accumulation of c-MYC mRNA upon serum stimulation.

<p>A, PARP-1 levels assessed by western blot in quiescent FB1329 fibroblasts transfected with the control (siGFP) or the specific (siPARP-1) siRNAs. TUBULIN was used as a loading control. Left panel shows the results of a representative experiment; right panel shows the averages and the Standard Deviations (SD) of densitometric values of PARP-1 signals normalized respect to TUBULIN, derived from three independent experiments. B, c-MYC expression assayed by RT-qPCR in quiescent (0) and serum-stimulated (for 30 or 60 minutes) siRNA-trasfected fibroblasts. c-MYC expression levels were normalized relatively to TBP expression and reported as fold increase respect to the control quiescent sample. The error bars represent the Standard Deviation of three biological replicates. Statistical significance: *: P<0.05 (Student's t test).</p

Anisomycin-stimulated phosphoacetylation restores c-MYC expression in the absence of PARP activity.

<p>c-MYC and GAPDH expression levels were assessed by RT-qPCR. Quiescent fibroblasts pre-treated with TSA or Anisomycin (Anis) or not-treated (NT), were serum-stimulated for 30 or 60 minutes in the presence or absence of PJ-34. Total RNAs were purified and the expression values obtained for c-MYC and GAPDH were normalized respect to TBP and plotted as relative to the control sample corresponding to quiescent cells. The error bars represent the SD of three biological replicates. Statistical significance: *: P<0.05.</p

Poly(ADP-ribosyl)ation of c-MYC promoter is mediated by PARP-1.

<p>A, Chromatin poly(ADP-ribosyl)ation at c-MYC promoter investigated by ChIP assays. Chromatin samples from quiescent cells (-FBS) or from cells stimulated for 30 minutes (+FBS) in the presence or absence of the PARP inhibitor PJ-34, were immuno-precipitated with an antibody specific for poly(ADP-ribose) (PAR) or a normal mouse IgG. Input and immuno-precipitated samples were subjected to qPCR with primers specific for the fragment including both fr.2 and fr.3 (fr.2+3) of c-MYC promoter region (c-MYC). The RHO promoter was used as negative control of the binding. The results were plotted as % of Input and are representative of two independent experiments. The error bars represent the SD of three technical replicates. B, PARP-1 binding to c-MYC promoter assessed by ChIP. Chromatin samples were obtained as described in A and immuno-precipitated with an antibody specific for PARP-1 or a normal rabbit IgG. Quantitative PCR reactions were performed using the same primers as in A. Results were reported as mean ± SD of two independent experiments. C, Poly(ADP-ribosyl)ation of mouse c-MYC promoter investigated by ChIP assays in mouse fibroblasts stably knocked-down for PARP-1. Chromatin samples from quiescent cells (0) or from cells stimulated for 30 minutes, were immuno-precipitated with an antibody specific for poly(ADP-ribose) (PAR) or a normal mouse IgG. Input and immuno-precipitated samples were subjected to qPCR. The results were plotted as % of Input. Results shown are representative of two independent experiments. The error bars represent the SD of three technical replicates. min p.s.a means minutes post serum addition.</p