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The cardiac transcription network modulated by Gata4, Mef2a, Nkx2.5, Srf, histone modifications, and microRNAs.

Schlesinger J, Schueler M, Grunert M, Fischer JJ, Zhang Q, Krueger T, Lange M, Tönjes M, Dunkel I, Sperling SR - PLoS Genet. (2011)

Bottom Line: Finally, we confirmed conclusions primarily obtained in cardiomyocyte cell culture in a time-course of cardiac maturation in mouse around birth.In addition to the analysis of the individual transcription factors, we found that histone 3 acetylation correlates with Srf- and Gata4-dependent gene expression and is complementarily reduced in cardiac Srf knockdown.Further, we found that altered microRNA expression in Srf knockdown potentially explains up to 45% of indirect mRNA targets.

View Article: PubMed Central - PubMed

Affiliation: Group Cardiovascular Genetics, Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics, Berlin, Germany.

ABSTRACT
The transcriptome, as the pool of all transcribed elements in a given cell, is regulated by the interaction between different molecular levels, involving epigenetic, transcriptional, and post-transcriptional mechanisms. However, many previous studies investigated each of these levels individually, and little is known about their interdependency. We present a systems biology study integrating mRNA profiles with DNA-binding events of key cardiac transcription factors (Gata4, Mef2a, Nkx2.5, and Srf), activating histone modifications (H3ac, H4ac, H3K4me2, and H3K4me3), and microRNA profiles obtained in wild-type and RNAi-mediated knockdown. Finally, we confirmed conclusions primarily obtained in cardiomyocyte cell culture in a time-course of cardiac maturation in mouse around birth. We provide insights into the combinatorial regulation by cardiac transcription factors and show that they can partially compensate each other's function. Genes regulated by multiple transcription factors are less likely differentially expressed in RNAi knockdown of one respective factor. In addition to the analysis of the individual transcription factors, we found that histone 3 acetylation correlates with Srf- and Gata4-dependent gene expression and is complementarily reduced in cardiac Srf knockdown. Further, we found that altered microRNA expression in Srf knockdown potentially explains up to 45% of indirect mRNA targets. Considering all three levels of regulation, we present an Srf-centered transcription network providing on a single-gene level insights into the regulatory circuits establishing respective mRNA profiles. In summary, we show the combinatorial contribution of four DNA-binding transcription factors in regulating the cardiac transcriptome and provide evidence that histone modifications and microRNAs modulate their functional consequence. This opens a new perspective to understand heart development and the complexity cardiovascular disorders.

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Binding site location and co-occurrence of Gata4, Mef2a, Nkx2.5, and Srf.HL-1 mRNA and miRNA expression profiles are highly comparable to the ones observed in human and mouse hearts. (left) Gene expression levels obtained from HL-1 cells and P0.5 of C57/BL6 mouse heart. (right) Rank-transformed miRNA expression levels in HL-1 cells and human right ventricle. (B) Positional distribution of transcription factor binding sites relative to the transcription start site (TSS). The y-axis shows the number of transcription factor binding sites per transcription factor as bar plots in 2.5kb windows. Most binding sites (∼75%) are located within close proximity to the TSS. (C) Gata4, Mef2a, Nkx2.5 and Srf frequently bind together. Shown is the combinatorial binding of all four transcription factors to 498 common target genes. 91 target genes were bound by all four factors (black), 121 target genes were bound by three (dark gray) and 286 target genes were bound by two transcription factors (gray). The total number of genes solely bound by a single TF are indicated above the respective TF. (D) Odds ratios of pair-wise contingency tables of the occurrence of transcription factor binding sites at one gene. Total numbers of pair-wise occurrences are given. The numbers in white boxes represent the total number of bound genes for the respective transcription factor. Red indicates positive, blue negative correlation. Gata4 and Nkx2.5 had the lowest number of targets (345 for Gata4, 276 for Nkx2.5) but we observed co-binding to 143 genes and their occurrence is therefore highly correlated. Although Mef2a and Srf bind at 320 genes together, they each have a much higher number of target genes.
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pgen-1001313-g001: Binding site location and co-occurrence of Gata4, Mef2a, Nkx2.5, and Srf.HL-1 mRNA and miRNA expression profiles are highly comparable to the ones observed in human and mouse hearts. (left) Gene expression levels obtained from HL-1 cells and P0.5 of C57/BL6 mouse heart. (right) Rank-transformed miRNA expression levels in HL-1 cells and human right ventricle. (B) Positional distribution of transcription factor binding sites relative to the transcription start site (TSS). The y-axis shows the number of transcription factor binding sites per transcription factor as bar plots in 2.5kb windows. Most binding sites (∼75%) are located within close proximity to the TSS. (C) Gata4, Mef2a, Nkx2.5 and Srf frequently bind together. Shown is the combinatorial binding of all four transcription factors to 498 common target genes. 91 target genes were bound by all four factors (black), 121 target genes were bound by three (dark gray) and 286 target genes were bound by two transcription factors (gray). The total number of genes solely bound by a single TF are indicated above the respective TF. (D) Odds ratios of pair-wise contingency tables of the occurrence of transcription factor binding sites at one gene. Total numbers of pair-wise occurrences are given. The numbers in white boxes represent the total number of bound genes for the respective transcription factor. Red indicates positive, blue negative correlation. Gata4 and Nkx2.5 had the lowest number of targets (345 for Gata4, 276 for Nkx2.5) but we observed co-binding to 143 genes and their occurrence is therefore highly correlated. Although Mef2a and Srf bind at 320 genes together, they each have a much higher number of target genes.

Mentions: We used the cardiomyocyte cell line HL-1 to study the global transcription network driven by the DNA-binding transcription factors Gata4, Mef2a, Nkx2.5 and Srf. The mRNA as well as miRNA expression profiles of beating HL-1 cells are highly comparable to the one observed in mouse hearts at P0.5 (Pearson correlation coefficient of 0.95, Figure 1A left) and human right ventricle (Pearson correlation coefficient of 0.90, Figure 1A right; data unpublished). The use of a cell line enabled us to cope with the technical limitations of our different approaches (ChIP-chip/seq, miRNA-seq, siRNA knockdown, microarrays) and supported a single cell-type specific study. We validated the key findings in a time-course of mouse hearts during the cardiac adaptation and maturation period around birth at E18.5, P0.5 and P4.5.


The cardiac transcription network modulated by Gata4, Mef2a, Nkx2.5, Srf, histone modifications, and microRNAs.

Schlesinger J, Schueler M, Grunert M, Fischer JJ, Zhang Q, Krueger T, Lange M, Tönjes M, Dunkel I, Sperling SR - PLoS Genet. (2011)

Binding site location and co-occurrence of Gata4, Mef2a, Nkx2.5, and Srf.HL-1 mRNA and miRNA expression profiles are highly comparable to the ones observed in human and mouse hearts. (left) Gene expression levels obtained from HL-1 cells and P0.5 of C57/BL6 mouse heart. (right) Rank-transformed miRNA expression levels in HL-1 cells and human right ventricle. (B) Positional distribution of transcription factor binding sites relative to the transcription start site (TSS). The y-axis shows the number of transcription factor binding sites per transcription factor as bar plots in 2.5kb windows. Most binding sites (∼75%) are located within close proximity to the TSS. (C) Gata4, Mef2a, Nkx2.5 and Srf frequently bind together. Shown is the combinatorial binding of all four transcription factors to 498 common target genes. 91 target genes were bound by all four factors (black), 121 target genes were bound by three (dark gray) and 286 target genes were bound by two transcription factors (gray). The total number of genes solely bound by a single TF are indicated above the respective TF. (D) Odds ratios of pair-wise contingency tables of the occurrence of transcription factor binding sites at one gene. Total numbers of pair-wise occurrences are given. The numbers in white boxes represent the total number of bound genes for the respective transcription factor. Red indicates positive, blue negative correlation. Gata4 and Nkx2.5 had the lowest number of targets (345 for Gata4, 276 for Nkx2.5) but we observed co-binding to 143 genes and their occurrence is therefore highly correlated. Although Mef2a and Srf bind at 320 genes together, they each have a much higher number of target genes.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040678&req=5

pgen-1001313-g001: Binding site location and co-occurrence of Gata4, Mef2a, Nkx2.5, and Srf.HL-1 mRNA and miRNA expression profiles are highly comparable to the ones observed in human and mouse hearts. (left) Gene expression levels obtained from HL-1 cells and P0.5 of C57/BL6 mouse heart. (right) Rank-transformed miRNA expression levels in HL-1 cells and human right ventricle. (B) Positional distribution of transcription factor binding sites relative to the transcription start site (TSS). The y-axis shows the number of transcription factor binding sites per transcription factor as bar plots in 2.5kb windows. Most binding sites (∼75%) are located within close proximity to the TSS. (C) Gata4, Mef2a, Nkx2.5 and Srf frequently bind together. Shown is the combinatorial binding of all four transcription factors to 498 common target genes. 91 target genes were bound by all four factors (black), 121 target genes were bound by three (dark gray) and 286 target genes were bound by two transcription factors (gray). The total number of genes solely bound by a single TF are indicated above the respective TF. (D) Odds ratios of pair-wise contingency tables of the occurrence of transcription factor binding sites at one gene. Total numbers of pair-wise occurrences are given. The numbers in white boxes represent the total number of bound genes for the respective transcription factor. Red indicates positive, blue negative correlation. Gata4 and Nkx2.5 had the lowest number of targets (345 for Gata4, 276 for Nkx2.5) but we observed co-binding to 143 genes and their occurrence is therefore highly correlated. Although Mef2a and Srf bind at 320 genes together, they each have a much higher number of target genes.
Mentions: We used the cardiomyocyte cell line HL-1 to study the global transcription network driven by the DNA-binding transcription factors Gata4, Mef2a, Nkx2.5 and Srf. The mRNA as well as miRNA expression profiles of beating HL-1 cells are highly comparable to the one observed in mouse hearts at P0.5 (Pearson correlation coefficient of 0.95, Figure 1A left) and human right ventricle (Pearson correlation coefficient of 0.90, Figure 1A right; data unpublished). The use of a cell line enabled us to cope with the technical limitations of our different approaches (ChIP-chip/seq, miRNA-seq, siRNA knockdown, microarrays) and supported a single cell-type specific study. We validated the key findings in a time-course of mouse hearts during the cardiac adaptation and maturation period around birth at E18.5, P0.5 and P4.5.

Bottom Line: Finally, we confirmed conclusions primarily obtained in cardiomyocyte cell culture in a time-course of cardiac maturation in mouse around birth.In addition to the analysis of the individual transcription factors, we found that histone 3 acetylation correlates with Srf- and Gata4-dependent gene expression and is complementarily reduced in cardiac Srf knockdown.Further, we found that altered microRNA expression in Srf knockdown potentially explains up to 45% of indirect mRNA targets.

View Article: PubMed Central - PubMed

Affiliation: Group Cardiovascular Genetics, Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics, Berlin, Germany.

ABSTRACT
The transcriptome, as the pool of all transcribed elements in a given cell, is regulated by the interaction between different molecular levels, involving epigenetic, transcriptional, and post-transcriptional mechanisms. However, many previous studies investigated each of these levels individually, and little is known about their interdependency. We present a systems biology study integrating mRNA profiles with DNA-binding events of key cardiac transcription factors (Gata4, Mef2a, Nkx2.5, and Srf), activating histone modifications (H3ac, H4ac, H3K4me2, and H3K4me3), and microRNA profiles obtained in wild-type and RNAi-mediated knockdown. Finally, we confirmed conclusions primarily obtained in cardiomyocyte cell culture in a time-course of cardiac maturation in mouse around birth. We provide insights into the combinatorial regulation by cardiac transcription factors and show that they can partially compensate each other's function. Genes regulated by multiple transcription factors are less likely differentially expressed in RNAi knockdown of one respective factor. In addition to the analysis of the individual transcription factors, we found that histone 3 acetylation correlates with Srf- and Gata4-dependent gene expression and is complementarily reduced in cardiac Srf knockdown. Further, we found that altered microRNA expression in Srf knockdown potentially explains up to 45% of indirect mRNA targets. Considering all three levels of regulation, we present an Srf-centered transcription network providing on a single-gene level insights into the regulatory circuits establishing respective mRNA profiles. In summary, we show the combinatorial contribution of four DNA-binding transcription factors in regulating the cardiac transcriptome and provide evidence that histone modifications and microRNAs modulate their functional consequence. This opens a new perspective to understand heart development and the complexity cardiovascular disorders.

Show MeSH
Related in: MedlinePlus