Limits...
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.

Show MeSH

Related in: MedlinePlus

miRNAs and their impact on the Srf-driven transcription network.(A) RNAi knockdown of Srf in HL-1 cardiomyocytes results in 42 differentially expressed miRNAs (49 loci) (Table S10). Target prediction of these mostly downregulated miRNAs revealed 192 differentially expressed genes, with a higher fraction of upregulated genes (57% of all upregulated genes) compared to downregulated genes (44% of all downregulated genes). (B) Direct Srf targets represent only a small fraction of all differentially expressed genes in Srf knockdown (orange and blue). Targets of differentially expressed miRNAs impact 45% (dark grey) with a partial overlap of direct Srf targets (orange). Approximately 50% of differential expression is driven by other secondary effects (light grey). (C) Exemplary network of indirect gene regulation by miRNAs. The genes Igfbp5, Nfic and Ctnnal1, which are not directly bound by Srf, are predicted targets for a set of downregulated miRNAs and are found to be upregulated in the Srf knockdown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3040678&req=5

pgen-1001313-g005: miRNAs and their impact on the Srf-driven transcription network.(A) RNAi knockdown of Srf in HL-1 cardiomyocytes results in 42 differentially expressed miRNAs (49 loci) (Table S10). Target prediction of these mostly downregulated miRNAs revealed 192 differentially expressed genes, with a higher fraction of upregulated genes (57% of all upregulated genes) compared to downregulated genes (44% of all downregulated genes). (B) Direct Srf targets represent only a small fraction of all differentially expressed genes in Srf knockdown (orange and blue). Targets of differentially expressed miRNAs impact 45% (dark grey) with a partial overlap of direct Srf targets (orange). Approximately 50% of differential expression is driven by other secondary effects (light grey). (C) Exemplary network of indirect gene regulation by miRNAs. The genes Igfbp5, Nfic and Ctnnal1, which are not directly bound by Srf, are predicted targets for a set of downregulated miRNAs and are found to be upregulated in the Srf knockdown.

Mentions: Considering that only a small proportion of differentially expressed genes in loss-of-function experiments are direct targets of the respective transcription factors, we were interested in studying the impact of miRNAs as secondary effectors (Figure 5A, 5B). Again we focused on the transcription factor Srf, which is known to regulate cardiac relevant miRNAs like miR-1 and miR-133 [26], [43]. We investigated Srf binding using ChIP-seq technology to map Srf binding sites potentially regulating miRNAs. We found 22 miRNAs from the miRNA database miRBase with Srf binding within a region of 10kb. This includes the previously described miR-208 Srf binding site, as well as other well-known muscle relevant miRNAs like miR-1, miR-125b, miR-133, miR-143 and miR145 (Table S9). Second, we performed Srf knockdown using two different siRNAs and quantified the miRNA expression levels by miRNA-seq (Table S10 and Table S18). We observed 42 miRNAs (49 loci) to be differentially expressed in both siRNA experiments, including miR-208, miR-125b and miR-21. The analysis revealed that most of the miRNAs were downregulated (78%) supporting the role of Srf as an miRNA activator (Figure 5A).


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)

miRNAs and their impact on the Srf-driven transcription network.(A) RNAi knockdown of Srf in HL-1 cardiomyocytes results in 42 differentially expressed miRNAs (49 loci) (Table S10). Target prediction of these mostly downregulated miRNAs revealed 192 differentially expressed genes, with a higher fraction of upregulated genes (57% of all upregulated genes) compared to downregulated genes (44% of all downregulated genes). (B) Direct Srf targets represent only a small fraction of all differentially expressed genes in Srf knockdown (orange and blue). Targets of differentially expressed miRNAs impact 45% (dark grey) with a partial overlap of direct Srf targets (orange). Approximately 50% of differential expression is driven by other secondary effects (light grey). (C) Exemplary network of indirect gene regulation by miRNAs. The genes Igfbp5, Nfic and Ctnnal1, which are not directly bound by Srf, are predicted targets for a set of downregulated miRNAs and are found to be upregulated in the Srf knockdown.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1001313-g005: miRNAs and their impact on the Srf-driven transcription network.(A) RNAi knockdown of Srf in HL-1 cardiomyocytes results in 42 differentially expressed miRNAs (49 loci) (Table S10). Target prediction of these mostly downregulated miRNAs revealed 192 differentially expressed genes, with a higher fraction of upregulated genes (57% of all upregulated genes) compared to downregulated genes (44% of all downregulated genes). (B) Direct Srf targets represent only a small fraction of all differentially expressed genes in Srf knockdown (orange and blue). Targets of differentially expressed miRNAs impact 45% (dark grey) with a partial overlap of direct Srf targets (orange). Approximately 50% of differential expression is driven by other secondary effects (light grey). (C) Exemplary network of indirect gene regulation by miRNAs. The genes Igfbp5, Nfic and Ctnnal1, which are not directly bound by Srf, are predicted targets for a set of downregulated miRNAs and are found to be upregulated in the Srf knockdown.
Mentions: Considering that only a small proportion of differentially expressed genes in loss-of-function experiments are direct targets of the respective transcription factors, we were interested in studying the impact of miRNAs as secondary effectors (Figure 5A, 5B). Again we focused on the transcription factor Srf, which is known to regulate cardiac relevant miRNAs like miR-1 and miR-133 [26], [43]. We investigated Srf binding using ChIP-seq technology to map Srf binding sites potentially regulating miRNAs. We found 22 miRNAs from the miRNA database miRBase with Srf binding within a region of 10kb. This includes the previously described miR-208 Srf binding site, as well as other well-known muscle relevant miRNAs like miR-1, miR-125b, miR-133, miR-143 and miR145 (Table S9). Second, we performed Srf knockdown using two different siRNAs and quantified the miRNA expression levels by miRNA-seq (Table S10 and Table S18). We observed 42 miRNAs (49 loci) to be differentially expressed in both siRNA experiments, including miR-208, miR-125b and miR-21. The analysis revealed that most of the miRNAs were downregulated (78%) supporting the role of Srf as an miRNA activator (Figure 5A).

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