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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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Histone 3 acetylation correlates with target gene expression of Srf and Gata4.(A) For each transcription factor the binding sites were categorized into two groups depending on co-occurrence with histone 3 acetylation (H3ac) in ChIP-chip. Genes marked by Mef2a or Nkx2.5 show significant increased expression levels compared to non-marked genes (Ref) independent of co-occurring H3ac. In contrast, expression levels of genes bound by Gata4 or Srf were only increased when H3ac marks co-occurred. (B) Confirmation of selected target genes of Srf and Gata4 with H3ac dependent expression level. HL-1 Illumina expression levels were confirmed using same amount of cDNA for semi-quantitative PCR (30 cycles) followed by gel electrophoresis and quantitative real-time PCR (40 cycles). Used primer had PCR efficiencies between 1.8–2.0. (C, D) Srf knockdown in HL-1 cells leads to complementary alterations in H3ac marks at Srf binding sites. H3ac-ChIP enrichments after Srf knockdown (siSrf) compared to control siRNA (siNon) were measured with qPCR for two groups of promoter regions (H3ac & Srf binding and no H3ac/Srf). The H3ac enrichment was normalized to Input and IgG controls. Fold changes show significant decrease in H3ac enrichment after Srf knockdown. (E) Confirmation of H3ac dependent expression of Srf target genes by ChIP-seq. Shown are expression levels of transcripts with H3ac and/or Srf binding close to the transcriptional start site (TSS<1.5kb). (F) H3ac reduces downregulation of Srf target genes in its knockdown. Shown are fold changes relative to siNon of downregulated transcripts after Srf knockdown with H3ac and/or Srf binding (TSS<1.5kb). Expression levels (A, E) and fold changes (F) are represented as box plots. Genes showing neither binding of investigated transcription factors nor H3ac are used as reference. The resulting p-values are indicated: p<0.001 (***), p<0.01 (**) and p<0.05 (*).
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pgen-1001313-g003: Histone 3 acetylation correlates with target gene expression of Srf and Gata4.(A) For each transcription factor the binding sites were categorized into two groups depending on co-occurrence with histone 3 acetylation (H3ac) in ChIP-chip. Genes marked by Mef2a or Nkx2.5 show significant increased expression levels compared to non-marked genes (Ref) independent of co-occurring H3ac. In contrast, expression levels of genes bound by Gata4 or Srf were only increased when H3ac marks co-occurred. (B) Confirmation of selected target genes of Srf and Gata4 with H3ac dependent expression level. HL-1 Illumina expression levels were confirmed using same amount of cDNA for semi-quantitative PCR (30 cycles) followed by gel electrophoresis and quantitative real-time PCR (40 cycles). Used primer had PCR efficiencies between 1.8–2.0. (C, D) Srf knockdown in HL-1 cells leads to complementary alterations in H3ac marks at Srf binding sites. H3ac-ChIP enrichments after Srf knockdown (siSrf) compared to control siRNA (siNon) were measured with qPCR for two groups of promoter regions (H3ac & Srf binding and no H3ac/Srf). The H3ac enrichment was normalized to Input and IgG controls. Fold changes show significant decrease in H3ac enrichment after Srf knockdown. (E) Confirmation of H3ac dependent expression of Srf target genes by ChIP-seq. Shown are expression levels of transcripts with H3ac and/or Srf binding close to the transcriptional start site (TSS<1.5kb). (F) H3ac reduces downregulation of Srf target genes in its knockdown. Shown are fold changes relative to siNon of downregulated transcripts after Srf knockdown with H3ac and/or Srf binding (TSS<1.5kb). Expression levels (A, E) and fold changes (F) are represented as box plots. Genes showing neither binding of investigated transcription factors nor H3ac are used as reference. The resulting p-values are indicated: p<0.001 (***), p<0.01 (**) and p<0.05 (*).

Mentions: To explore the influence of histone modifications as an epigenetic mechanism to modulate gene expression, we analyzed our transcription factor binding data in the context of co-occurring histone marks. In a previous study we investigated the localization of four histone modifications, which are known to promote an open chromatin state (H3K9K14ac, H4K5K8K12K16ac, H3K4me2 and H3K4me3) [39]. We found that ∼80% of the respective transcription factor binding events are marked by one or more of these histone modifications, whereas in a randomized simulation only 23% are expected to co-occur (Figure S3). We consequently investigated whether the presence of any of these marks correlates with higher expression levels of direct target genes and found a significant impact for histone 3 acetylation (H3ac) only (Figure 3A and Figure S4). For Nkx2.5 and Mef2a the expression levels of direct targets were significantly higher than the reference group, independent of whether H3ac was present or not. Genes showing neither transcription factor binding nor H3ac were used as a reference. In case of Gata4 and Srf the expression levels of direct targets were only significantly increased when binding sites were additionally marked by H3ac. The enhanced expression levels depending on H3ac co-occurrence is further depicted in Figure 3B, which shows confirmation experiments of nine genes using quantitative PCR. In conclusion, our data provide evidence that acetylation of histone 3 supports the activating function of Gata4 and Srf, which might be mediated via p300. The histone acetyl transferase p300 not only acetylates lysine residues on histone 3 but also on Gata4, thereby enhancing the DNA-binding and activating potential of this transcription factor [40]. The Srf cofactor Myocardin has been reported to recruit p300 to Srf binding sites whereby histone 3 acetylation is induced and gene expression enhanced [41]. Finally, we studied the change of H3ac marks as a consequence of Srf knockdown using ChIP followed by qPCR. Strikingly, we found complementary alterations of H3ac in a panel of relevant promoter regions (Figure 3C and 3D).


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)

Histone 3 acetylation correlates with target gene expression of Srf and Gata4.(A) For each transcription factor the binding sites were categorized into two groups depending on co-occurrence with histone 3 acetylation (H3ac) in ChIP-chip. Genes marked by Mef2a or Nkx2.5 show significant increased expression levels compared to non-marked genes (Ref) independent of co-occurring H3ac. In contrast, expression levels of genes bound by Gata4 or Srf were only increased when H3ac marks co-occurred. (B) Confirmation of selected target genes of Srf and Gata4 with H3ac dependent expression level. HL-1 Illumina expression levels were confirmed using same amount of cDNA for semi-quantitative PCR (30 cycles) followed by gel electrophoresis and quantitative real-time PCR (40 cycles). Used primer had PCR efficiencies between 1.8–2.0. (C, D) Srf knockdown in HL-1 cells leads to complementary alterations in H3ac marks at Srf binding sites. H3ac-ChIP enrichments after Srf knockdown (siSrf) compared to control siRNA (siNon) were measured with qPCR for two groups of promoter regions (H3ac & Srf binding and no H3ac/Srf). The H3ac enrichment was normalized to Input and IgG controls. Fold changes show significant decrease in H3ac enrichment after Srf knockdown. (E) Confirmation of H3ac dependent expression of Srf target genes by ChIP-seq. Shown are expression levels of transcripts with H3ac and/or Srf binding close to the transcriptional start site (TSS<1.5kb). (F) H3ac reduces downregulation of Srf target genes in its knockdown. Shown are fold changes relative to siNon of downregulated transcripts after Srf knockdown with H3ac and/or Srf binding (TSS<1.5kb). Expression levels (A, E) and fold changes (F) are represented as box plots. Genes showing neither binding of investigated transcription factors nor H3ac are used as reference. The resulting p-values are indicated: p<0.001 (***), p<0.01 (**) and p<0.05 (*).
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1001313-g003: Histone 3 acetylation correlates with target gene expression of Srf and Gata4.(A) For each transcription factor the binding sites were categorized into two groups depending on co-occurrence with histone 3 acetylation (H3ac) in ChIP-chip. Genes marked by Mef2a or Nkx2.5 show significant increased expression levels compared to non-marked genes (Ref) independent of co-occurring H3ac. In contrast, expression levels of genes bound by Gata4 or Srf were only increased when H3ac marks co-occurred. (B) Confirmation of selected target genes of Srf and Gata4 with H3ac dependent expression level. HL-1 Illumina expression levels were confirmed using same amount of cDNA for semi-quantitative PCR (30 cycles) followed by gel electrophoresis and quantitative real-time PCR (40 cycles). Used primer had PCR efficiencies between 1.8–2.0. (C, D) Srf knockdown in HL-1 cells leads to complementary alterations in H3ac marks at Srf binding sites. H3ac-ChIP enrichments after Srf knockdown (siSrf) compared to control siRNA (siNon) were measured with qPCR for two groups of promoter regions (H3ac & Srf binding and no H3ac/Srf). The H3ac enrichment was normalized to Input and IgG controls. Fold changes show significant decrease in H3ac enrichment after Srf knockdown. (E) Confirmation of H3ac dependent expression of Srf target genes by ChIP-seq. Shown are expression levels of transcripts with H3ac and/or Srf binding close to the transcriptional start site (TSS<1.5kb). (F) H3ac reduces downregulation of Srf target genes in its knockdown. Shown are fold changes relative to siNon of downregulated transcripts after Srf knockdown with H3ac and/or Srf binding (TSS<1.5kb). Expression levels (A, E) and fold changes (F) are represented as box plots. Genes showing neither binding of investigated transcription factors nor H3ac are used as reference. The resulting p-values are indicated: p<0.001 (***), p<0.01 (**) and p<0.05 (*).
Mentions: To explore the influence of histone modifications as an epigenetic mechanism to modulate gene expression, we analyzed our transcription factor binding data in the context of co-occurring histone marks. In a previous study we investigated the localization of four histone modifications, which are known to promote an open chromatin state (H3K9K14ac, H4K5K8K12K16ac, H3K4me2 and H3K4me3) [39]. We found that ∼80% of the respective transcription factor binding events are marked by one or more of these histone modifications, whereas in a randomized simulation only 23% are expected to co-occur (Figure S3). We consequently investigated whether the presence of any of these marks correlates with higher expression levels of direct target genes and found a significant impact for histone 3 acetylation (H3ac) only (Figure 3A and Figure S4). For Nkx2.5 and Mef2a the expression levels of direct targets were significantly higher than the reference group, independent of whether H3ac was present or not. Genes showing neither transcription factor binding nor H3ac were used as a reference. In case of Gata4 and Srf the expression levels of direct targets were only significantly increased when binding sites were additionally marked by H3ac. The enhanced expression levels depending on H3ac co-occurrence is further depicted in Figure 3B, which shows confirmation experiments of nine genes using quantitative PCR. In conclusion, our data provide evidence that acetylation of histone 3 supports the activating function of Gata4 and Srf, which might be mediated via p300. The histone acetyl transferase p300 not only acetylates lysine residues on histone 3 but also on Gata4, thereby enhancing the DNA-binding and activating potential of this transcription factor [40]. The Srf cofactor Myocardin has been reported to recruit p300 to Srf binding sites whereby histone 3 acetylation is induced and gene expression enhanced [41]. Finally, we studied the change of H3ac marks as a consequence of Srf knockdown using ChIP followed by qPCR. Strikingly, we found complementary alterations of H3ac in a panel of relevant promoter regions (Figure 3C and 3D).

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