20 research outputs found

    Disclosing Bias in Bisulfite Assay: MethPrimers Underestimate High DNA Methylation

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    <div><p>Discordant results obtained in bisulfite assays using MethPrimers (PCR primers designed using MethPrimer software or assuming that non-CpGs cytosines are non methylated) versus primers insensitive to cytosine methylation lead us to hypothesize a technical bias. We therefore used the two kinds of primers to study different experimental models and methylation statuses. We demonstrated that MethPrimers negatively select hypermethylated DNA sequences in the PCR step of the bisulfite assay, resulting in CpG methylation underestimation and non-CpG methylation masking, failing to evidence differential methylation statuses. We also describe the characteristics of “Methylation-Insensitive Primers” (MIPs), having degenerated bases (G/A) to cope with the uncertain C/U conversion. As CpG and non-CpG DNA methylation patterns are largely variable depending on the species, developmental stage, tissue and cell type, a variable extent of the bias is expected. The more the methylome is methylated, the greater is the extent of the bias, with a prevalent effect of non-CpG methylation. These findings suggest a revision of several DNA methylation patterns so far documented and also point out the necessity of applying unbiased analyses to the increasing number of epigenomic studies.</p></div

    MethPrimers amplification efficiency is affected by DNA methylation.

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    <p>a) <i>Myogenin</i> and b) <i>PSEN1</i> overall non-CpG methylation. c) and d) Amplification efficiency of primers on samples with differential overall non-CpG methylation was assessed by Real-Time PCR assay using MIPs (light grey columns) or MethPrimers (dark grey columns) for <i>myogenin</i> (c) and <i>PSEN1</i> (d). Bisulphite-treated PCR products and untreated PCR products were used as control of amplification efficiency. Values are expressed as fold n° versus control (C2C12 in 1% 24 hours for <i>myogenin</i>, SK-N-BE in complete medium for <i>PSEN1 used as respective calibrators</i>); <i>myogenin</i> amplification values were normalized using <i>PSEN1</i> PCR product amplification (added to mouse samples and used as exogenous reference) whereas <i>PSEN1</i> amplification values were normalized to <i>myogenin</i> PCR product amplification (in this case added to human samples and used as exogenous reference). *: <i>p</i><0.001 vs. ctrl.</p

    MIPs and MethPrimers result in different CpG methylation patterns.

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    <p>CpG methylation pattern is expressed as percent methylation for each CpG in the investigated region of the mouse <i>myogenin</i> (a-d) and human <i>PSEN1</i> (e-h) promoters. Light grey columns show the values obtained using MIPs, dark grey columns show the values obtained using MethPrimers. Time points for cell cultures are: 48 h for C2C12 10% FCS, SK-N-BE+SAM, SK-N-BE B def.; 24 h for C2C12 1%FCS. Symbols: * <i>p</i><0.05 MIPs vs. MethPrimers; &: <i>p</i><0.05 High Methylated vs. Low Methylated with MIPs; §: <i>p</i><0.05 High Methylated vs. Low Methylated with MethPrimers. Y axes in histograms have a different scale (up to 100% for high methylated samples, up to 50% for low methylated samples) in order to better evidence intra-sequence differences in cytosine methylation analyzed with Methprimers vs. MIPs.</p

    Expanded HTT generates CAG-repeated sRNAs with toxic activity.

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    <p>A. sRNA fraction (<100 nt) were isolated from cells expressing HTT-e1 constructs and equal amounts of each pool were transfected. Both 80*CAG-RNA and 80*CAG-PROT- derived sRNA pools induced death of differentiated SH-SY5Y cells (n = 5; *p<0.05,**p<0.01). 80*CAA-PROT-derived sRNA pools didn't affect SH-SY5Y cell viability. B. The expression of CAG-expanded HTT leads to an increase in CAG-repeated sRNAs of ∌21-nt (sCAG). sCAG levels were quantified using RNU66 as the reference sRNA, and normalized with respect to GFP expression, which indicates the percentage on transfected living cells 24 hours after transfection (n = 4; **p<0.01). C. HTT sRNA toxicity correlates with the length of the CAG expansion, distinguishing pathogenic and non-pathogenic number of CAG repeats (n = 4; * p<0.05, **p<0.01 ***p<0.001. D. HTT sRNA toxicity correlates with the generation of sCAG species (n = 4; **p<0.01). E. Anti-(CAG)<sub>7</sub> sRNA (anti-sCAG) prevents cell damage caused by mutant-<i>HTT</i>-derived sRNA pool. Control sRNA inhibitors did not mitigate sRNA HTT toxicity (n = 4; *p<0.05, **p<0.01, **p<0.001, determinations were performed in quintuplicates). Values represent mean of the ratio expanded-HTT sRNA toxicity vs non-expanded-HTT sRNA toxicity ± SEM. In A. B. C. and D. values represent the mean fold change with respect to the control non-transfected cells ± SEM and are referred to the control cells lacking HTT expression. In all experiments, cells were processed 24 hours after transfection in all the experiments.</p

    Cytotoxic sCAGs are increased in brain regions of HD.

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    <p>A. sCAG levels are increased in affected brain areas from R6/2 HD mouse model compared to control mice. sCAG were quantified by qRT-PCR using RNU6B as the reference sRNA; HC, hippocampus; STR, striatum cortex; CX, cortex; and CB, cerebellum. Values represent mean fold change with respect the control samples ± SEM (n = 3; *p<0.05 ***p<0.001). B. Increased expression of sCAG in HD human brain samples compared to control subjects. CA, caudate; and FC, frontal cortex. RNU66 sRNA was used as reference sRNA. Values represent mean fold change with respect to the control samples ± SEM (n = 3; *p<0.05 ***p<0.001). C. HD-derived sRNA pools induce neuronal toxicity. sRNA pools were isolated from control and HD human brain samples and delivered to differentiated SH-SY5Y cells; cell death was determined 24 hours later. The use of anti-sCAG dramatically reduced the cytotoxic effect. Control sRNA inhibitors (scrambled anti-sRNA) were used as a negative control. Values represent mean of the ratio (HD sRNA toxicity/Control sRNA toxicity) for each condition ± SEM (experiments were performed in quintuplicates, n = 6; *p<0.05). Pools from four control individuals and four patients with HD were used.</p

    CAG-expanded exon 1 of human <i>HTT</i> is toxic at the RNA level.

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    <p>A. CAG-unexpanded (wild-type; 23 CAG repeats) and CAG-expanded (mutant; 80 CAG repeats) constructs of human <i>HTT</i> exon 1 (<i>HTT</i> exon 1) were subcloned into a pIRES-EGFP vector. Each variant was produced as a normal translated form (left) and a form lacking the translation initiation codon (right). The specific role of the expanded protein was analyzed with a construct expressing CAA-expanded HTT-e1. The use of IRES-based bicistronic vectors with a GFP reporter allows monitoring of transfected cells. B. The four different constructs express the mRNA <i>HTT</i>-IRES-GFP (left) and the GFP reporter protein (right). HTT protein is only expressed in the constructs containing the ATG translation initiation codon (right). C. Differentiated SH-SY5Y cells were transfected with the HTT-IRES-GFP vectors and LDH cell toxicity assay was performed 18 h and 24 h after transfection. Expression of CAG-expanded <i>HTT</i> (RNA or protein) resulted in dramatic cell death. CAA-expanded HTT-e1 didn't induce a significant effect on cell viability at the time points analyzed (n = 4; *p<0.05, **p<0.01, ***p<0.001). D. The percentage of dead transfected cells was also determined 36 hours after transfection by counting 200 GFP-negative cells (left) and 200 GFP-positive cells (right), scoring in each case the presence of nuclear fragmentation. Values represent the percentage of cells showing nuclear condensation in each situation ± SD (n = 3; **p<0.01). E. Expression of CAG-expanded <i>HTT</i> RNA induced caspase 9 cleavage. GFP blots highlight the expression of all constructs in transfected cells and polyglutamine (PolyQ) blots show expression of expanded HTT protein. Densitometry determinations of cleaved caspase 9 <i>vs.</i> α-Tubulin were performed on cells lysated 24 hours after transfection. Results are presented as the mean of arbitrary optical density units (O.D. units ± SEM; n = 3; *p<0.05, ***p<0.001). In C. and E., values represent the mean fold change with respect to the control non-transfected cells ± SEM.</p

    sCAG neurotoxic effect is dependent on Dicer and Ago proteins.

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    <p>A. Dicer knockdown inhibits the generation of sCAGs produced by the expression of 80*CAG HTT-e1. sCAG levels were normalized to RNU66 levels. GFP blots indicate the expression of the HTT-constructs (n = 3; interaction p-value = 0.000138; F = 46.220). B. In the same experiments, cell viability and caspase 9 cleavage analysis show that Dicer depletion mitigates cell death induced by expanded <i>HTT</i> (n = 5; interaction p-value = 0.000135; F = 18.263). C. Ago2 depletion mitigates the toxicity of sRNA obtained from mutant <i>HTT</i> expressing cells (n = 3; interaction p-value = 0.011; F = 10.821). D. sCAG efficiently associate to Ago2 <i>in vivo</i>. <i>HTT</i>-expressing constructs were transfected on cells stably expressing Flag-Ago2. Flag IP demonstrate that sCAG binds to Ago2 complex. No significant binding was detected in control IP experiments (α-V5). The plot shows the mean ratio of sCAG levels in FLAG IP <i>vs.</i> control V5 IP (n = 3; *p<0.05). E. The expression of Flag-Ago2 in cells depleted for endogenous Ago2 partially, but significantly, rescued CAG-expanded HTT toxic effect (n = 3; *p<0,05). Values represent the mean of the ratio expanded-HTT sRNA toxicity vs non-expanded-HTT sRNA toxicity ± SEM in each experimental condition. Toxicity levels are referred to the control cells lacking HTT expression. In A. B. and C., values represent the mean fold change with respect to the control, non-transfected cells ± SEM. Cells were processed 24 hours after double transfection in all the experiments.</p

    sCAGs induce post-transcriptional gene silencing in genes with CTG regions.

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    <p>A. Hela cells were cotransfected with firefly luciferase expressing vectors containing the indicated nucleotide sequences in its 3â€Č-UTR, the specific HTT-e1 expressing vectors or the (CAG)7 siRNA and Renilla luciferase plasmid to normalize data. Assays were performed 24 hours after transfection. Data were first normalized to the 100% of luminescence obtained with the control luciferase vector, lacking 3â€ČUTR inserts (n = 3; *p<0.05, p**p<0,01). B,C. Levels of ADORA2A and MEIS2 transcripts in SH-SY5Y cells transfected with normal and expanded HTT vectors. MRIP was used as endogenous control. qRT-PCR was performed in cells fixed 24 hours after transfection. (n = 3; *p<0.05). Values represent the mean fold change with respect to the control, non-transfected cells ± SEM. D. Western blot showing reduced MEIS2 protein levels in differentiated SH-SY5Y expressing expanded <i>HTT</i> RNA 24 hours after transfection. The graph shows the densitometry determination of MEIS2 levels <i>vs</i> ÎČ-Actin. Results represent the mean arbitrary optical density change normalized to the mean value obtained in non-transfected cells (n = 4; *p<0.05).</p

    sCAG toxicity is variable in different human cells, preferentially affecting neuronal viability.

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    <p>A. Exogenous administration of (CAG)<sub>7</sub> siRNAs interfere with cell viability depending on the cell type (n = 3, p<0.0083). HMEC, HPDE and UROTSA cell lines were used as a source for breast, pancreatic and bladder primary human cells. Differentiated SH-SY5Y cells were used as a post-mitotic neuronal cell model (n = 3; one-way ANOVA *p<0.05 ***p<0.001; F = 15.203). B. The toxic effect of (CAG)7 siRNA is dependent on the type of differentiation. SHSY5Y cells were subjected to several neuronal differentiation protocols, and CAG)<sub>7</sub> or scrambled sequences (control siRNA) were administered in each situation. SH-SY5Y sensitivity to (CAG)7 significantly differs in each differentiation condition (*), excepting for TPA standard differentiation condition (#), whose effect wasn't significantly different from the effect observed under TPA long exposure conditions (One-way ANOVA; F = 63.926). (n = 3, p<0.0083). MTT assays were performed 48 hours after transfection. Graphs show relative cell survival indicated as the ratio between cell viability in cells transfected with controls siRNA <i>vs</i> cell viability in cells transfected with (CAG)7. Values indicate the mean ratio ± SEM of three independent experiments.</p
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