MED12 regulates a transcriptional network of calcium-handling genes in the heart
Kedryn K. Baskin, Catherine A. Makarewich, Susan M. DeLeon, Wenduo Ye, Beibei Chen, Nadine Beetz, Heinrich Schrewe, Rhonda Bassel-Duby, Eric N. Olson
Kedryn K. Baskin, Catherine A. Makarewich, Susan M. DeLeon, Wenduo Ye, Beibei Chen, Nadine Beetz, Heinrich Schrewe, Rhonda Bassel-Duby, Eric N. Olson
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Research Article Cardiology Cell biology

MED12 regulates a transcriptional network of calcium-handling genes in the heart

Abstract

The Mediator complex regulates gene transcription by linking basal transcriptional machinery with DNA-bound transcription factors. The activity of the Mediator complex is mainly controlled by a kinase submodule that is composed of 4 proteins, including MED12. Although ubiquitously expressed, Mediator subunits can differentially regulate gene expression in a tissue-specific manner. Here, we report that MED12 is required for normal cardiac function, such that mice with conditional cardiac-specific deletion of MED12 display progressive dilated cardiomyopathy. Loss of MED12 perturbs expression of calcium-handling genes in the heart, consequently altering calcium cycling in cardiomyocytes and disrupting cardiac electrical activity. We identified transcription factors that regulate expression of calcium-handling genes that are downregulated in the heart in the absence of MED12, and we found that MED12 localizes to transcription factor consensus sequences within calcium-handling genes. We showed that MED12 interacts with one such transcription factor, MEF2, in cardiomyocytes and that MED12 and MEF2 co-occupy promoters of calcium-handling genes. Furthermore, we demonstrated that MED12 enhances MEF2 transcriptional activity and that overexpression of both increases expression of calcium-handling genes in cardiomyocytes. Our data support a role for MED12 as a coordinator of transcription through MEF2 and other transcription factors. We conclude that MED12 is a regulator of a network of calcium-handling genes, consequently mediating contractility in the mammalian heart.

Authors

Kedryn K. Baskin, Catherine A. Makarewich, Susan M. DeLeon, Wenduo Ye, Beibei Chen, Nadine Beetz, Heinrich Schrewe, Rhonda Bassel-Duby, Eric N. Olson

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Figure 5

MED12 facilitates cooperative transcription of calcium-handling genes in the heart.

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MED12 facilitates cooperative transcription of calcium-handling genes in...
(A) Upstream regulator analysis (URA) of differentially expressed genes in CTL and cKO ventricles. Bars in green are transcription factors known to regulate calcium-handling genes. (B) Circos plot demonstrating transcription factor binding sites (TFBS) within MED12 target genes. Genes containing TFBS for CEBP, MEF2, CREB, EGR1, STAT3, and STAT1 are categorized by function, and the increased width of connecting ribbons indicates more TFBS (Supplemental Table 2 contains the data used to generate the plot). (C) Chromosomal map of Atp2a2 with TFBS and ChIP-PCR assay primers. (D) MED12 ChIP-PCR results for select calcium-handling genes. PCR assay number corresponds to sequences on chromosomal maps for the indicated genes (see Supplemental Figure 4). Regions devoid of transcription factor binding sites were used as negative controls (nc). n = 4. (E) Coimmunoprecipitation of MED12 with MEF2 in nuclei isolated from CTL and cKO cardiomyocytes. (F) MED12-MEF2 ChIP-reChIP on Atp2a2 and Ryr2 promoters, PCR assay number corresponds to sequences on chromosomal maps (see C and Supplemental Figure 4). n = 4. (G) RNA Polymerase II and MEF2 ChIP-seq signals at the Atp2a2 locus in CTL and cKO ventricles. MEF2 motif locations are shown in green. (H) MEF2 luciferase reporter assays performed in COS-7 cells. n = 3 independent experiments. Luc, luciferase. (I) Coexpression studies performed in HL-1 cardiomyocytes. n = 3 independent experiments. Data are average ± SEM. *P < 0.01 by 2-tailed Student’s t test (D, F, I) or one-way ANOVA with post-hoc Tukey test (H).