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

Overnight T-wave alternans in sleep apnea patients

Sleep apnea (SA) is linked to cardiovascular complications and to an increased risk of sudden cardiac death. Microvolt T-wave alternans (TWA) is a noninvasive electrocardiographic (ECG) index of cardiovascular risk; its rate of occurrence in SA patients remains unknown. Thus, this study investigated the occurrence of TWA in SA patients during night. To this aim, overnight ECG recordings of 16 SA patients were analyzed for TWA identification by means of our heart rate adaptive match filter. Results indicate that overnight TWA was characterized by a low mean amplitude (mean TWA: 6±3 µV). However, higher-amplitude transient TWA episodes (max TWA: 29±21 µV) occurred overnight, sometimes when patients were awake (max TWA: 33±18 µV; 56% of cases) and sometimes when patients were sleeping (max TWA: 24±23 µV; 44% of cases with 13%, 19%, 6% and 6% during sleep stage 1, 2, 3 and 4, respectively). In only 3 subjects (19%) TWA peaks occurred during an SA episode: during obstructive apnea with arousal in two cases (max TWA of 7 µV and 17 µV, during stages 1 and 2, respectively) and during hypoapnea with arousal in one case (max TWA of 6 µV while awake). Thus, SA patients show significant transient overnight TWA episodes, not necessarily occurring during a SA episode

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

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 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 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

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

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

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

<p>The cdk inhibitor p57^kip2, encoded by the <i>Cdkn1c</i> 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 <i>Cdkn1c</i> silencing, causing growth disorders and cancer. We have previously reported that, in skeletal muscle cells, induction of <i>Cdkn1c</i> 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 <i>Cdkn1c</i>. 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 <i>Cdkn1c</i> induction. This, in turn, promotes the release of a repressive chromatin loop between KvDMR1 and <i>Cdkn1c</i> promoter and, thus, the upregulation of the gene. Analysis of the chromatin status of <i>Cdkn1c</i> 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 <i>Cdkn1c</i> 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 <i>Cdkn1c</i> and, possibly, of its aberrant silencing in some pathological conditions.</p