Journal: Cell reports

Article Title: The Mef2 transcription network is disrupted in myotonic dystrophy heart tissue dramatically altering miRNA and mRNA expression

doi: 10.1016/j.celrep.2013.12.025

Figure Lengend Snippet: (A) Reduced Mef2a and Mef2c expression in heart tissue from the EpA960; MCM DM1 mouse model (n=3) and individuals with DM1 (n=8) or normal controls (n=4). Gata4 mRNA levels are not affected. Data is normalized to ribosomal protein L30 (Rpl30).(B) Western blot showing reduction in steady state MEF2A protein levels in human DM1 heart samples. CELF1 protein levels are up regulated in these samples, as previously described(Savkur et al., 2001; Timchenko et al., 2001). Quantification of relative band intensities, normalized to GAPDH levels are shown below. (C) Decreased Mef2a and Mef2c expression affects mRNA steady-state levels of Mef2 target genes in mouse DM1 heart tissue. Representative Mef2 target genes show a significant reduction in expression (light red bars) whereas Gata4 target genes are unaffected (light blue bars). *P< 0.05.

Article Snippet: ChIP was performed two times with 2μg of anti-RNA Pol II rabbit polyclonal antibody (Santa Cruz, sc-900) and anti-pan Mef2A and Mef2C goat polyclonal antibody (Santa Cruz, sc-313), anti-Mef2c specific rabbit polyclonal antibody (Cell signaling, 5030) and 2μg of normal rabbit IgG (Santa Cruz, sc-2027) or goat IgG (Santa Cruz, sc-2028) as isotype controls.

Techniques: Expressing, Western Blot

Journal: Cell reports

Article Title: The Mef2 transcription network is disrupted in myotonic dystrophy heart tissue dramatically altering miRNA and mRNA expression

doi: 10.1016/j.celrep.2013.12.025

Figure Lengend Snippet: RNAi-based Mef2 knockdowns coupled with chromatin immunoprecipitation (ChIP) assays identify miRNAs directly regulated by Mef2. (A)Knockdown efficiency of Mef2a and Mef2c siRNAs in HL-1 cardiac cells was determined by qRT-PCR in three independent experiments. Reduced steady state levels of Mef2 mRNA targets Myocardin (Myocd), Myomesin (Myom1) and α-T-catenin (Ctnna3) in response to Mef2A, Mef2C individual or double knockdowns (mean ± SD; n=3). (B) Reduced steady state levels of pri-miRNAs in Mef2 knockdowns. All data is plotted relative to a luciferase control siRNA and expression is normalized to Rpl30. *P< 0.05, N.D, not detected. (C) Reduced interaction of Mef2 with its primary miRNA and mRNA gene targets in DM1 mice. Quantification of genomic DNA in chromatin immunoprecipitates using Mef2 antibody in heart tissue of MCM controls and DM1 mice. The primers used for qRT-PCR assays span the Mef2 binding sites in target primary miRNAs or mRNAs. Each bar represents mean ± SD of the fraction of input detected in the Mef2 precipitates normalized to IgG precipitates (n=3).

Article Snippet: ChIP was performed two times with 2μg of anti-RNA Pol II rabbit polyclonal antibody (Santa Cruz, sc-900) and anti-pan Mef2A and Mef2C goat polyclonal antibody (Santa Cruz, sc-313), anti-Mef2c specific rabbit polyclonal antibody (Cell signaling, 5030) and 2μg of normal rabbit IgG (Santa Cruz, sc-2027) or goat IgG (Santa Cruz, sc-2028) as isotype controls.

Techniques: Chromatin Immunoprecipitation, Knockdown, Quantitative RT-PCR, Luciferase, Control, Expressing, Binding Assay

Journal: Cell reports

Article Title: The Mef2 transcription network is disrupted in myotonic dystrophy heart tissue dramatically altering miRNA and mRNA expression

doi: 10.1016/j.celrep.2013.12.025

Figure Lengend Snippet: (A) Experimental schematic of Mef2c rescue in CUGexp RNA expressing cardiac cells. (B) GFP expression was detected by indirect fluorescence using anti-GFP antibody. RNA foci containing DT960 RNA were detected by FISH using Cy3-labeled probes. Nuclei were counterstained with DAPI. GFP expression or RNA foci formation was not detected in the absence of dox. All images were taken at the same exposure time. Scale bars: 20 m. (C) Induction of DT0 and DT960 containing DMPK mRNA after dox treatment. Each bar represents fold change in expression relative to the DT0 control without dox treatment. Positions of qRT-PCR primers used to quantitate mRNA expression are indicated with red arrows. Data is normalized relative to Rpl30. *P< 0.05. Reduced (D) Mef2A and Mef2C; (E) Myocd and Myom1; and (F) pri-miR-1-1, 1-2, 133a-1 and 133a-2 steady state levels in response to CUGexp RNA expression. Exogenous Mef2c restores the mRNA and miRNA expression in CUGexp RNA expressing cells. Each bar represents fold change in expression relative to DT0 control. a, significantly different from DT0 (infected with control virus). b, significantly different from DT960 (infected with control virus). (G) Model of Mef2-miRNA circuitry in normal and DM1 cardiac cells. In healthy adult cardiomyocytes, Mef2 proteins coordinate expression of cardiac-enriched mRNA and miRNA genes. miRNAs post-transcriptionally down regulate expression of multiple gene targets to maintain adult cardiac gene expression program. This transcriptional circuit becomes defective in DM1 (black panel); Mef2 levels are reduced in response to CUGexp RNA expression, which results in loss of expression of Mef2-driven cardiac genes and miRNAs resulting in developmental reprogramming of gene expression.

Article Snippet: ChIP was performed two times with 2μg of anti-RNA Pol II rabbit polyclonal antibody (Santa Cruz, sc-900) and anti-pan Mef2A and Mef2C goat polyclonal antibody (Santa Cruz, sc-313), anti-Mef2c specific rabbit polyclonal antibody (Cell signaling, 5030) and 2μg of normal rabbit IgG (Santa Cruz, sc-2027) or goat IgG (Santa Cruz, sc-2028) as isotype controls.

Techniques: Expressing, Fluorescence, Labeling, Control, Quantitative RT-PCR, RNA Expression, Infection, Virus, Gene Expression