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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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The impact of Srf and H3ac on gene expression in mouse hearts during cardiac maturation.(A) Cardiomyocyte maturation over three developmental stages around birth (E18.5, P0.5 and P4.5) with respect to alignment of myofibrils and cell-cell contacts. Paraffin sections of mouse hearts double labeled with antibodies against α-Actinin and Connexin 43 (Cx43) and examined under the confocal microscope. The α-Actinin stained myofibrils (in green, second panel) elongate and assemble throughout the maturation process while Cx43 (in red, third panel) forms distinct punctuations, which become larger and brighter. Nuclei are visualized in blue by DAPI counterstaining and merged pictures are shown in the lower panel. Scale bar 10µm. (B) Confirmation of the dependency of expression levels on H3ac and Srf binding in mouse hearts using the time-series E18.5, P0.5 and P4.5. Expression levels as well as ChIP enrichment were analyzed by quantitative real-time PCR and normalized to Hprt and Input, respectively. Shown changes of expression levels as well as Srf and H3ac binding over time are in general significant. Values are given in percentages relative to E18.5. For Pitx2 and Nrp2 the additive effect of several measured genomic regions is shown.
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pgen-1001313-g004: The impact of Srf and H3ac on gene expression in mouse hearts during cardiac maturation.(A) Cardiomyocyte maturation over three developmental stages around birth (E18.5, P0.5 and P4.5) with respect to alignment of myofibrils and cell-cell contacts. Paraffin sections of mouse hearts double labeled with antibodies against α-Actinin and Connexin 43 (Cx43) and examined under the confocal microscope. The α-Actinin stained myofibrils (in green, second panel) elongate and assemble throughout the maturation process while Cx43 (in red, third panel) forms distinct punctuations, which become larger and brighter. Nuclei are visualized in blue by DAPI counterstaining and merged pictures are shown in the lower panel. Scale bar 10µm. (B) Confirmation of the dependency of expression levels on H3ac and Srf binding in mouse hearts using the time-series E18.5, P0.5 and P4.5. Expression levels as well as ChIP enrichment were analyzed by quantitative real-time PCR and normalized to Hprt and Input, respectively. Shown changes of expression levels as well as Srf and H3ac binding over time are in general significant. Values are given in percentages relative to E18.5. For Pitx2 and Nrp2 the additive effect of several measured genomic regions is shown.

Mentions: In a further attempt to confirm our results gathered in cell culture, we studied Srf and H3ac binding and their influence on gene expression in mouse hearts in a time-series during cardiac maturation at three developmental stages E18.5, P0.5 and P4.5 around birth. From the fetal to the postnatal stage, the heart adapts to the body circulation and cardiomyocytes mature. During this process the heart increases in size (Figure 4A), the cells elongate, myofibrils align and cell-cell contacts become bipolar. Immunostaining of α-Actinin-1 and Connexin 43 illustrates hypertrophy of cardiomyocytes, assembly of the sarcomeric z-discs and development of gap junction [42]. Based on ChIP-chip/seq results for Srf and H3ac in HL-1 cells, we analyzed promoter binding regions of genes and miRNAs relevant for this process using ChIP followed by quantitative real-time PCR. The selection comprises (Figure 4B): Dmpk (kinase of myogenin), Slmap (sarcomeric protein), Picalm (clathrin assembly protein), miR-133a (cardiac and muscle-specific miRNA), the growth factor Igf1 and its receptor Igf1r, Pitx2c (cardiac transcription factor), and Nrp2 (interactor of Vegf). We found a high correlation between the changes of Srf and H3ac binding and the gene expression levels over time. In case of Pitx2c and Nrp2 we identified multiple binding events in HL-1 cells by ChIP-chip/seq of which their functionality could be confirmed by common changes over time in the mouse model (Figure 4B). Taken together, these data support an important role for the co-occurrence of Srf and H3ac in the regulation of the cardiac maturation process and underline the influence of histone modifications.


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)

The impact of Srf and H3ac on gene expression in mouse hearts during cardiac maturation.(A) Cardiomyocyte maturation over three developmental stages around birth (E18.5, P0.5 and P4.5) with respect to alignment of myofibrils and cell-cell contacts. Paraffin sections of mouse hearts double labeled with antibodies against α-Actinin and Connexin 43 (Cx43) and examined under the confocal microscope. The α-Actinin stained myofibrils (in green, second panel) elongate and assemble throughout the maturation process while Cx43 (in red, third panel) forms distinct punctuations, which become larger and brighter. Nuclei are visualized in blue by DAPI counterstaining and merged pictures are shown in the lower panel. Scale bar 10µm. (B) Confirmation of the dependency of expression levels on H3ac and Srf binding in mouse hearts using the time-series E18.5, P0.5 and P4.5. Expression levels as well as ChIP enrichment were analyzed by quantitative real-time PCR and normalized to Hprt and Input, respectively. Shown changes of expression levels as well as Srf and H3ac binding over time are in general significant. Values are given in percentages relative to E18.5. For Pitx2 and Nrp2 the additive effect of several measured genomic regions is shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040678&req=5

pgen-1001313-g004: The impact of Srf and H3ac on gene expression in mouse hearts during cardiac maturation.(A) Cardiomyocyte maturation over three developmental stages around birth (E18.5, P0.5 and P4.5) with respect to alignment of myofibrils and cell-cell contacts. Paraffin sections of mouse hearts double labeled with antibodies against α-Actinin and Connexin 43 (Cx43) and examined under the confocal microscope. The α-Actinin stained myofibrils (in green, second panel) elongate and assemble throughout the maturation process while Cx43 (in red, third panel) forms distinct punctuations, which become larger and brighter. Nuclei are visualized in blue by DAPI counterstaining and merged pictures are shown in the lower panel. Scale bar 10µm. (B) Confirmation of the dependency of expression levels on H3ac and Srf binding in mouse hearts using the time-series E18.5, P0.5 and P4.5. Expression levels as well as ChIP enrichment were analyzed by quantitative real-time PCR and normalized to Hprt and Input, respectively. Shown changes of expression levels as well as Srf and H3ac binding over time are in general significant. Values are given in percentages relative to E18.5. For Pitx2 and Nrp2 the additive effect of several measured genomic regions is shown.
Mentions: In a further attempt to confirm our results gathered in cell culture, we studied Srf and H3ac binding and their influence on gene expression in mouse hearts in a time-series during cardiac maturation at three developmental stages E18.5, P0.5 and P4.5 around birth. From the fetal to the postnatal stage, the heart adapts to the body circulation and cardiomyocytes mature. During this process the heart increases in size (Figure 4A), the cells elongate, myofibrils align and cell-cell contacts become bipolar. Immunostaining of α-Actinin-1 and Connexin 43 illustrates hypertrophy of cardiomyocytes, assembly of the sarcomeric z-discs and development of gap junction [42]. Based on ChIP-chip/seq results for Srf and H3ac in HL-1 cells, we analyzed promoter binding regions of genes and miRNAs relevant for this process using ChIP followed by quantitative real-time PCR. The selection comprises (Figure 4B): Dmpk (kinase of myogenin), Slmap (sarcomeric protein), Picalm (clathrin assembly protein), miR-133a (cardiac and muscle-specific miRNA), the growth factor Igf1 and its receptor Igf1r, Pitx2c (cardiac transcription factor), and Nrp2 (interactor of Vegf). We found a high correlation between the changes of Srf and H3ac binding and the gene expression levels over time. In case of Pitx2c and Nrp2 we identified multiple binding events in HL-1 cells by ChIP-chip/seq of which their functionality could be confirmed by common changes over time in the mouse model (Figure 4B). Taken together, these data support an important role for the co-occurrence of Srf and H3ac in the regulation of the cardiac maturation process and underline the influence of histone modifications.

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