MiR-20a targets p300 and p300-regulated angiogenic genes.

<p><b>A. Low expression of miR-20a levels in cardiac progenitor cells.</b> Absolute MiR-20a transcript levels in CPCs vs. neonatal cardiomyocytes were measured by qPCR in equivalent 10 ng total RNA samples. <b>B... </b><b>Successful expression of miR20a in CPCs following lentiviral transduction.</b> Ordinate is in log₁₀ scale. U = uninfected. Ct-miR: scrambled sequence. M = miR 20a sequence. ** = p<0.001. *p<0.05. <b>C. miR-20a reduces endogenous p300 transcript levels in CPCs. D... </b><b>MiR-20a reduces Vegf-A protein in CPCs.</b> (Top) representative Western blot. (Bottom) Summary of 3 independent experiments<b>. E. Overexpression of miR-20a slows proliferation of cardiac progenitor cells (CPCs).</b> CPC clones stably expressing GFP and miR-20a or a scrambled sequence (ct-miR) were plated and maintained at 40–60% confluency by serial passage. Cell counts were determined as described in Experimental Procedures. <b>F... </b><b>Mir-20a overexpression represses p300-induced angiogenic genes in CPCs.</b> Angiogenic genes identified as p300-regulated in the myocardial expression profile from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079133#pone-0079133-g002" target="_blank">Figure 2A</a> were assayed by QPCR in CPCs expressing miR-20a or ct-miR. Data represent the average of 3 biological replicates per transcript. Color bar denotes relative expression levels. Grey = expression below detection threshold <b>G. MiR-20a directly targets EP300 3′UTR.</b> See Experimental Procedures for miR-20a binding site sequence and mutant sequence.</p

p300-induced genes repressed by miR-20a transduction <i>in vivo</i>.

<p>A previously defined subset of 86 transcripts specifically upregulated in p300tg vs wt littermate hearts (15) was assessed by qPCR 3 months after transduction with miR-20a or a scrambled sequence. Significantly altered genes constituted more than a third of the total; almost all were repressed. N = 4–6 mice per group. MiR-20a binding sites predicted by TargetScan.</p

A model of the miR20-p300 feedback loop during hypertrophy.

<p>Stress signals acting on the myocardium induce p300 expression, which results in the activation of angiogenic and hypertrophic transcriptional programs leading to cardiac hypertrophy. p300 also induces miR-20a, which provides feedback inhibition of p300, reversing both the angiogenic and hypertrophic programs and providing counter-regulatory control of the hypertrophic stress response.</p

Reciprocal regulation of myocardial VegfA and miR-17∼92 with age.

<p>VegfA and mature miR transcript levels were determined by quantitative PCR in mice at the indicated time points. <b>A. Relative upregulation of miR-17-3p and miR-20a in p300 transgenic mice.</b> The ratios of p300 to wt levels for each transcript are shown. * p<0.05. <b>B. Time dependent inverse regulation of VegfA and miR-20a.</b> For B-D, absolute transcript levels in log 10 are shown. * p<0.05 for comparison between wt and p300. <b>C. Age-dependent downregulation of miR 17-3p and -18a. D. Age-dependent decline in miR-19b and -92. </b><b>E. Increasing myocardial VegfA protein content between 1 and 8 months.</b> Above: representative Western blot showing comparative VegfA levels in wt and p300tg mice at 1 and 8 months of age, with Gapdh as loading control. Below: Quantitation of normalized Vegf protein expression in wt mice at 1 (n = 3) and 8 (n = 2) months.</p

Organ weights in p300tg and wild type littermate mice.

<p>Organ weights and tibia length were measured in 1 month and 5 month old p300 tg and wild type littermates (4 female mice per group. *<i>p</i><0.05. Significant increases in heart weight, heart weight/body weight, and heart weight/tibia length ratios are seen in p300tg mice, as previously reported (Wei <i>et al</i>., 2008).</p

Angiogenic genes differentially expressed in 1-month old p300tg hearts.

<p>Microarray was performed on LV myocardial tissue from 3 mice per genotype, one array per mouse. Data were analyzed using GeneSpring software and filtered by p value (≤0.05) and extent of differential expression (>1.5x up or <0.66x reduced). Gene list corresponds to heatmap in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079133#pone-0079133-g002" target="_blank">Figure 2A</a>.</p

MiR-20a reduces actinin content in cardiomyocytes.

<p>Lentiviral-transduced cardiomyocytes expressing a control sequence (<b>A</b>, ct-miR) or miR-20a (<b>B</b> and <b>C</b>) together with EGFP (green) were visualized with anti-GATA-4 (magenta) and pan-alpha-actinin (red) antibodies and nuclear DNA was stained with DAPI (blue)<b>.</b> Note that cells expressing the miR-20a-EGFP lentivirus (<b>B and C,</b> arrows) have greatly reduced staining for actinin, compared with adjacent non-transduced cells (<b>B and C,</b> asterixes), or cells taking up the scrambled sequence (compare EGFP+ cells in <b>A</b>). Original magnification = 32x.</p

Impact of tissue type, p300 content and genomic context on miR-17∼92 cluster expression.

<p><b>A. Comparative expression of 7 members of the miR-17∼92 cluster in normal tissues.</b> Tissues were harvested from mice at 3 months of age and RNA was quantitated for the indicated microRNAs. <b>B. Comparative miR-17∼92 cluster expression in normal murine heart.</b> LA: left atrium. RA: right atrium. LV: left ventricle. RV: right ventricle. Transcript levels expressed per ng total RNA. Note relatively higher expression of 3′ members of the cluster, miR-20a, miR-19b, and miR-92. <b>C. Gain of p300 induces miR-20a.</b> Above: p300 immunoblot of cardiac myocyte lysates 48 h after transduction with Adp300 or a control virus (AdGFP). (below) p300 and miR-20a transcript levels are both increased following transduction of Adp300 but not AdGFP. <b>D. Gain of p300 induces multiple members of miR-17∼92 cluster.</b> Relative expression of miR-17∼92 cluster members and an unrelated microRNA, miR-199, in Adp300- vs AdGFP-expressing cardiac myocytes. For A through C, N = 4 per group. * = p<0.05. <b>E. Genomic structure of miR17∼92 cluster.</b> Red arrow = cluster position on murine chromosome 14. Shown are locations and sequences of canonical MEF2 and GATA binding sites upstream of miR-20a.</p

MiR-20a represses p300 and p300-driven angiogenic transcription <i>in vivo</i>.

<p><b>A. Sustained <i>in vivo</i> transduction of miR20a.</b> 5-day old wt pups were transduced with the indicated lentiviruses and miR20a expression was detected by qPCR in left ventricular myocardial lysates at2 weeks. <b>B. Reduced p300 in miR-20a-transduced hearts.</b> p300 protein was quantitated in the same lysates in (A) by Western blot 3 months after transduction with miR-20a. n.d.u = normalized densitometry levels; * = p<0.05, n = 5. <b>C. Representative Western blot of data summarized in (B).</b> p300 and GAPDH bands were separately detected on 6% and 10% gels, respectively, with all 12 bands per protein quantified from a single autoradiogram. <b>D. miR-20a reverses myocardial p300-induced gene expression profile.</b> Absolute expression levels (log₁₀) of p300-induced angiogenic genes in wt and p300tg mice with indicated treatment (n = 4–8/condition).</p

Statistics pertaining to HPRD growth, experimental types for protein–protein interactions and a breakdown of PTMs

<p><b>Copyright information:</b></p><p>Taken from "Human protein reference database—2006 update"</p><p>Nucleic Acids Research 2005;34(Database issue):D411-D414.</p><p>Published online 28 Dec 2005</p><p>PMCID:PMC1347503.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () Growth of HPRD over the last 3 years with respect to protein entries, protein–protein interactions and PTMs. () Distribution of protein–protein interactions in HPRD based on the type of the experimental method. () Distribution of various types of PTMs in HPRD. The percentage of the respective PTM is indicated only when it is greater than or equal to 2