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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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Promoter analysis of miR-125b-1 and DPF3.(A) Srf ChIP-seq analysis revealed an Srf binding region downstream of mmu-miR-125b-1. Shown are the positions of mmu-miR-125b-1 and the Srf binding motif with its core sequence in red. The Srf ChIP-seq peak region was cloned as mmu-miR-125b-1 promoter into the pGL3basic vector for luciferase reporter gene assay. Srf alone and in combination with its cofactor Myocardin (Myocd) significantly increases the activation of the luciferase beyond activation driven by endogenous Srf. Mutation of the core sequence (GCCA→TAGT) of the Srf binding motif (Mut) abolished activation by Srf and Myocd compared to the wildtype (WT). (B) ChIP-chip showed binding of Nkx2.5 to an evolutionary human-mouse conserved region of the DPF3 core promoter. Depicted is the Nkx2.5 binding element, which were deleted for luciferase reporter gene assays (red). The DPF3 core promoter fused to luciferase alone and in combination with increasing amounts of Nkx2.5 expression vector showed dose-dependent activation by Nkx2.5 beyond the endogenous Nkx2.5 activation. Deletion of the Nkx2.5 binding element (red) abolished activation by Nkx2.5 compared to the wildtype (WT). The empty pcDNA3.1 expression vector and the empty pGL3basic luciferase reporter vector served as controls for transcription factors and reporter constructs, respectively. The resulting p-values are indicated: p<0.001 (***), p<0.01 (**).
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pgen-1001313-g006: Promoter analysis of miR-125b-1 and DPF3.(A) Srf ChIP-seq analysis revealed an Srf binding region downstream of mmu-miR-125b-1. Shown are the positions of mmu-miR-125b-1 and the Srf binding motif with its core sequence in red. The Srf ChIP-seq peak region was cloned as mmu-miR-125b-1 promoter into the pGL3basic vector for luciferase reporter gene assay. Srf alone and in combination with its cofactor Myocardin (Myocd) significantly increases the activation of the luciferase beyond activation driven by endogenous Srf. Mutation of the core sequence (GCCA→TAGT) of the Srf binding motif (Mut) abolished activation by Srf and Myocd compared to the wildtype (WT). (B) ChIP-chip showed binding of Nkx2.5 to an evolutionary human-mouse conserved region of the DPF3 core promoter. Depicted is the Nkx2.5 binding element, which were deleted for luciferase reporter gene assays (red). The DPF3 core promoter fused to luciferase alone and in combination with increasing amounts of Nkx2.5 expression vector showed dose-dependent activation by Nkx2.5 beyond the endogenous Nkx2.5 activation. Deletion of the Nkx2.5 binding element (red) abolished activation by Nkx2.5 compared to the wildtype (WT). The empty pcDNA3.1 expression vector and the empty pGL3basic luciferase reporter vector served as controls for transcription factors and reporter constructs, respectively. The resulting p-values are indicated: p<0.001 (***), p<0.01 (**).

Mentions: We confirmed a panel of observed transcription factor binding sites by qPCR (Figure S5). Using luciferase reporter gene assays, we validated an Srf binding site in the regulatory region of mouse miR-125b-1 as well as an Nkx2.5 binding element in the core promoter region of human DPF3. Mmu-miR-125b-1 is known to be deregulated in heart diseases [44] and was found to be differentially expressed in Srf siRNA knockdown. Figure 6A shows the Srf binding motif and respective Srf ChIP-seq peak within the regulatory region of miR-125b-1. Luciferase reporter gene assays with wildtype and mutated fusion constructs confirm its functionality. Mutation of the potential Srf binding sequence (CAGCCAAC→CATAGTAC) significantly reduced the transcriptional activity of the reporter gene. DPF3 is a novel epigenetic regulator of heart and skeletal muscle development [13]. Within the 1.2kbp promoter region we found three Mef2 matrices and one Nkx2.5 matrix using TRANSFAC MATCH [45]. In case of Mef2a, all three potential binding sites can drive reporter gene expression as reported [13]. Figure 6B shows the binding of Nkx2.5 to the human DPF3 core promoter. Subsequently, co-transfection of reporter construct and increasing amounts of Nkx2.5 expression vector revealed a dose-dependent transcriptional activation by Nkx2.5. In line with this, deletion of the potential Nkx2.5 binding element (TCCACTTTCC) showed that transcriptional activity was indeed mediated through this motif, as activation was lost in the mutated construct.


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)

Promoter analysis of miR-125b-1 and DPF3.(A) Srf ChIP-seq analysis revealed an Srf binding region downstream of mmu-miR-125b-1. Shown are the positions of mmu-miR-125b-1 and the Srf binding motif with its core sequence in red. The Srf ChIP-seq peak region was cloned as mmu-miR-125b-1 promoter into the pGL3basic vector for luciferase reporter gene assay. Srf alone and in combination with its cofactor Myocardin (Myocd) significantly increases the activation of the luciferase beyond activation driven by endogenous Srf. Mutation of the core sequence (GCCA→TAGT) of the Srf binding motif (Mut) abolished activation by Srf and Myocd compared to the wildtype (WT). (B) ChIP-chip showed binding of Nkx2.5 to an evolutionary human-mouse conserved region of the DPF3 core promoter. Depicted is the Nkx2.5 binding element, which were deleted for luciferase reporter gene assays (red). The DPF3 core promoter fused to luciferase alone and in combination with increasing amounts of Nkx2.5 expression vector showed dose-dependent activation by Nkx2.5 beyond the endogenous Nkx2.5 activation. Deletion of the Nkx2.5 binding element (red) abolished activation by Nkx2.5 compared to the wildtype (WT). The empty pcDNA3.1 expression vector and the empty pGL3basic luciferase reporter vector served as controls for transcription factors and reporter constructs, respectively. The resulting p-values are indicated: p<0.001 (***), p<0.01 (**).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040678&req=5

pgen-1001313-g006: Promoter analysis of miR-125b-1 and DPF3.(A) Srf ChIP-seq analysis revealed an Srf binding region downstream of mmu-miR-125b-1. Shown are the positions of mmu-miR-125b-1 and the Srf binding motif with its core sequence in red. The Srf ChIP-seq peak region was cloned as mmu-miR-125b-1 promoter into the pGL3basic vector for luciferase reporter gene assay. Srf alone and in combination with its cofactor Myocardin (Myocd) significantly increases the activation of the luciferase beyond activation driven by endogenous Srf. Mutation of the core sequence (GCCA→TAGT) of the Srf binding motif (Mut) abolished activation by Srf and Myocd compared to the wildtype (WT). (B) ChIP-chip showed binding of Nkx2.5 to an evolutionary human-mouse conserved region of the DPF3 core promoter. Depicted is the Nkx2.5 binding element, which were deleted for luciferase reporter gene assays (red). The DPF3 core promoter fused to luciferase alone and in combination with increasing amounts of Nkx2.5 expression vector showed dose-dependent activation by Nkx2.5 beyond the endogenous Nkx2.5 activation. Deletion of the Nkx2.5 binding element (red) abolished activation by Nkx2.5 compared to the wildtype (WT). The empty pcDNA3.1 expression vector and the empty pGL3basic luciferase reporter vector served as controls for transcription factors and reporter constructs, respectively. The resulting p-values are indicated: p<0.001 (***), p<0.01 (**).
Mentions: We confirmed a panel of observed transcription factor binding sites by qPCR (Figure S5). Using luciferase reporter gene assays, we validated an Srf binding site in the regulatory region of mouse miR-125b-1 as well as an Nkx2.5 binding element in the core promoter region of human DPF3. Mmu-miR-125b-1 is known to be deregulated in heart diseases [44] and was found to be differentially expressed in Srf siRNA knockdown. Figure 6A shows the Srf binding motif and respective Srf ChIP-seq peak within the regulatory region of miR-125b-1. Luciferase reporter gene assays with wildtype and mutated fusion constructs confirm its functionality. Mutation of the potential Srf binding sequence (CAGCCAAC→CATAGTAC) significantly reduced the transcriptional activity of the reporter gene. DPF3 is a novel epigenetic regulator of heart and skeletal muscle development [13]. Within the 1.2kbp promoter region we found three Mef2 matrices and one Nkx2.5 matrix using TRANSFAC MATCH [45]. In case of Mef2a, all three potential binding sites can drive reporter gene expression as reported [13]. Figure 6B shows the binding of Nkx2.5 to the human DPF3 core promoter. Subsequently, co-transfection of reporter construct and increasing amounts of Nkx2.5 expression vector revealed a dose-dependent transcriptional activation by Nkx2.5. In line with this, deletion of the potential Nkx2.5 binding element (TCCACTTTCC) showed that transcriptional activity was indeed mediated through this motif, as activation was lost in the mutated construct.

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