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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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RNAi–induced knockdown of Gata4, Mef2a, Nkx2.5, and Srf.(A) Knockdown efficiency of Gata4, Mef2a, Nkx2.5 and Srf in HL-1 cells using two different siRNAs was analyzed on protein level by Western Blot 48h after transfection. Histone 3 (H3) served as loading control. In independent experiments similar knockdown efficiencies were obtained. (B) Odds ratios of pair-wise contingency tables of differentially expressed transcripts after RNAi knockdown of the respective transcription factor. Total numbers of pair-wise occurrences are given. The numbers in white boxes represent the total number of deregulated transcripts. Red indicates positive, blue negative correlation. Mef2a shows the lowest number of differentially expressed transcripts (119) probably due to buffering effects of the other Mef2 family members. Despite this fact, Mef2a shares a high number of deregulated transcripts with the other transcription factors. Of note, transcription factors having a high number of common binding targets (see Figure 1D) share only a small number of co-regulated genes in RNAi knockdown. (C) Transcription factor network showing a selection of cardiac relevant genes bound in ChIP-chip and/or ChIP-seq, and significantly differentially expressed in RNAi knockdown experiments of the respective factor. Up- and downregulation of genes is depicted and occurrence of ChIP binding marked by color-coded circles.
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pgen-1001313-g002: RNAi–induced knockdown of Gata4, Mef2a, Nkx2.5, and Srf.(A) Knockdown efficiency of Gata4, Mef2a, Nkx2.5 and Srf in HL-1 cells using two different siRNAs was analyzed on protein level by Western Blot 48h after transfection. Histone 3 (H3) served as loading control. In independent experiments similar knockdown efficiencies were obtained. (B) Odds ratios of pair-wise contingency tables of differentially expressed transcripts after RNAi knockdown of the respective transcription factor. Total numbers of pair-wise occurrences are given. The numbers in white boxes represent the total number of deregulated transcripts. Red indicates positive, blue negative correlation. Mef2a shows the lowest number of differentially expressed transcripts (119) probably due to buffering effects of the other Mef2 family members. Despite this fact, Mef2a shares a high number of deregulated transcripts with the other transcription factors. Of note, transcription factors having a high number of common binding targets (see Figure 1D) share only a small number of co-regulated genes in RNAi knockdown. (C) Transcription factor network showing a selection of cardiac relevant genes bound in ChIP-chip and/or ChIP-seq, and significantly differentially expressed in RNAi knockdown experiments of the respective factor. Up- and downregulation of genes is depicted and occurrence of ChIP binding marked by color-coded circles.

Mentions: Considering the cooperative co-binding of the investigated transcription factors, we were interested in the functional consequence of significantly reducing the quantity of each of the factors. Therefore, we used siRNA technique to reduce the protein levels of investigated transcription factors by more than 70% and studied its consequence for gene transcription. The reduction at mRNA and protein level was monitored by quantitative PCR and Western Blot analysis (Figure S1 and Figure 2A) and the genome-wide effects on transcript levels were measured by expression array analysis (Table S17). All data were based on a total of 4 replicate experiments using duplicates of two different siRNAs per transcription factor. The majority of deregulated transcripts were downregulated in the siRNA treated samples, confirming a primarily activating function of the transcription factors. Performing Annexin assays and Tryptophan Blue staining, we observed an increased apoptosis and cell death in particular when Gata4 or Srf were knocked down (Figure S2), which is in line with previous data [34]–[36].


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)

RNAi–induced knockdown of Gata4, Mef2a, Nkx2.5, and Srf.(A) Knockdown efficiency of Gata4, Mef2a, Nkx2.5 and Srf in HL-1 cells using two different siRNAs was analyzed on protein level by Western Blot 48h after transfection. Histone 3 (H3) served as loading control. In independent experiments similar knockdown efficiencies were obtained. (B) Odds ratios of pair-wise contingency tables of differentially expressed transcripts after RNAi knockdown of the respective transcription factor. Total numbers of pair-wise occurrences are given. The numbers in white boxes represent the total number of deregulated transcripts. Red indicates positive, blue negative correlation. Mef2a shows the lowest number of differentially expressed transcripts (119) probably due to buffering effects of the other Mef2 family members. Despite this fact, Mef2a shares a high number of deregulated transcripts with the other transcription factors. Of note, transcription factors having a high number of common binding targets (see Figure 1D) share only a small number of co-regulated genes in RNAi knockdown. (C) Transcription factor network showing a selection of cardiac relevant genes bound in ChIP-chip and/or ChIP-seq, and significantly differentially expressed in RNAi knockdown experiments of the respective factor. Up- and downregulation of genes is depicted and occurrence of ChIP binding marked by color-coded circles.
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Related In: Results  -  Collection

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pgen-1001313-g002: RNAi–induced knockdown of Gata4, Mef2a, Nkx2.5, and Srf.(A) Knockdown efficiency of Gata4, Mef2a, Nkx2.5 and Srf in HL-1 cells using two different siRNAs was analyzed on protein level by Western Blot 48h after transfection. Histone 3 (H3) served as loading control. In independent experiments similar knockdown efficiencies were obtained. (B) Odds ratios of pair-wise contingency tables of differentially expressed transcripts after RNAi knockdown of the respective transcription factor. Total numbers of pair-wise occurrences are given. The numbers in white boxes represent the total number of deregulated transcripts. Red indicates positive, blue negative correlation. Mef2a shows the lowest number of differentially expressed transcripts (119) probably due to buffering effects of the other Mef2 family members. Despite this fact, Mef2a shares a high number of deregulated transcripts with the other transcription factors. Of note, transcription factors having a high number of common binding targets (see Figure 1D) share only a small number of co-regulated genes in RNAi knockdown. (C) Transcription factor network showing a selection of cardiac relevant genes bound in ChIP-chip and/or ChIP-seq, and significantly differentially expressed in RNAi knockdown experiments of the respective factor. Up- and downregulation of genes is depicted and occurrence of ChIP binding marked by color-coded circles.
Mentions: Considering the cooperative co-binding of the investigated transcription factors, we were interested in the functional consequence of significantly reducing the quantity of each of the factors. Therefore, we used siRNA technique to reduce the protein levels of investigated transcription factors by more than 70% and studied its consequence for gene transcription. The reduction at mRNA and protein level was monitored by quantitative PCR and Western Blot analysis (Figure S1 and Figure 2A) and the genome-wide effects on transcript levels were measured by expression array analysis (Table S17). All data were based on a total of 4 replicate experiments using duplicates of two different siRNAs per transcription factor. The majority of deregulated transcripts were downregulated in the siRNA treated samples, confirming a primarily activating function of the transcription factors. Performing Annexin assays and Tryptophan Blue staining, we observed an increased apoptosis and cell death in particular when Gata4 or Srf were knocked down (Figure S2), which is in line with previous data [34]–[36].

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