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Mef2c is an essential regulatory element required for unique expression of the cardiac-specific CARK gene.

Wang H, Chen C, Song X, Chen J, Zhen Y, Sun K, Hui R - J. Cell. Mol. Med. (2007)

Bottom Line: Mutational analysis revealed five conserved cis-acting elements in this 151-bp long minimal promoter.Mutational and loss-of-functional analysis and co-transfection studies indicated that MEF2 binding region is the most critical cis-acting element in the CARK promoter, and CARK transcription level can be down-regulated by MEF2C antisense.Binding to the MEF2 sites by Mef2c protein was confirmed by electrophoretic mobility shift assay and competition and supershift electrophoretic mobility shift assays.

View Article: PubMed Central - PubMed

Affiliation: Sino-German Laboratory for Molecular Medicine, Ministry of Education, FuWai Cardiovascular Hospital and Cardiovascular Institute, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.

ABSTRACT
The cardiac ankyrin repeat kinase (CARK) gene, also named TNNI3K for its interaction with cardiac troponin I, is both a unique expression and heart-enriched gene. To understand the mechanisms of CARK gene expression and regulation, we first cloned the full-length mRNA sequence and mapped the transcription start site of the mouse CARK gene and characterized its promoter regions. Two transcriptional isoforms of the CARK gene were identified in mouse heart tissue. Truncation analysis of the CARK promoter identified a minimal 151 bp region that has strong basal transcription activity. Mutational analysis revealed five conserved cis-acting elements in this 151-bp long minimal promoter. Mutational and loss-of-functional analysis and co-transfection studies indicated that MEF2 binding region is the most critical cis-acting element in the CARK promoter, and CARK transcription level can be down-regulated by MEF2C antisense. Binding to the MEF2 sites by Mef2c protein was confirmed by electrophoretic mobility shift assay and competition and supershift electrophoretic mobility shift assays.

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Mutational analysis of putative transcription factor binding sites of mouse CARK promoter.(A) Homology comparison of minimal promoter region of mouse CARK gene with human, chimpanzee, dog and rat revealed the presence of several conserved putative transcription factor binding sites, including Tbx5, SRE, M-CAT box, MEF2 and GATA4.(B) The putative transcription factor binding sites are shown in grey background and the modified nucleotides are underlined, the substitutes are illustrated below.(C) Five site-directed mutants were prepared in the –151/+79 promoter fragment (pF4-151-luc), as described under ‘Materials and methods’.(D) These mutated constructs were introduced into primary rat cardiomyocytes for transient expression assays. Firefly luciferase activities expressed had been normalized on the basis of Renilla luciferase activity. Data shown are the means ± S. D., n= 3.
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fig04: Mutational analysis of putative transcription factor binding sites of mouse CARK promoter.(A) Homology comparison of minimal promoter region of mouse CARK gene with human, chimpanzee, dog and rat revealed the presence of several conserved putative transcription factor binding sites, including Tbx5, SRE, M-CAT box, MEF2 and GATA4.(B) The putative transcription factor binding sites are shown in grey background and the modified nucleotides are underlined, the substitutes are illustrated below.(C) Five site-directed mutants were prepared in the –151/+79 promoter fragment (pF4-151-luc), as described under ‘Materials and methods’.(D) These mutated constructs were introduced into primary rat cardiomyocytes for transient expression assays. Firefly luciferase activities expressed had been normalized on the basis of Renilla luciferase activity. Data shown are the means ± S. D., n= 3.

Mentions: Cis-acting element response sites in CARK minimal promoter region were identified by sequence analysis using the CardioSignalScan program (http://www.cardiosignal.org/tools/scan.html) [14]. Putative sequence-conserved TFBSs were Tbx5 (−95/−80), SRE (−86/−73), M-CAT (−47/−33), MEF2 (−22/−12) and GATA4 (−9/+1; Fig. 4A). Site-directed mutagenesis was performed to modify each binding site based on the pF4–151-luc reporter using the QuickChange mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. Wild-type and modified sequences of each binding site in CARK promoter region were indicated in Fig. 4B. Five mutant luciferase reporter constructs (mut-Tbx5-luc, mut-SRE-luc, mut-MCAT-luc, mut-MEF2-luc and mut-GATA4-luc) were verified by DNA sequencing to confirm the integrity and the presence of the desired mutations in the constructs (Fig. 4C).


Mef2c is an essential regulatory element required for unique expression of the cardiac-specific CARK gene.

Wang H, Chen C, Song X, Chen J, Zhen Y, Sun K, Hui R - J. Cell. Mol. Med. (2007)

Mutational analysis of putative transcription factor binding sites of mouse CARK promoter.(A) Homology comparison of minimal promoter region of mouse CARK gene with human, chimpanzee, dog and rat revealed the presence of several conserved putative transcription factor binding sites, including Tbx5, SRE, M-CAT box, MEF2 and GATA4.(B) The putative transcription factor binding sites are shown in grey background and the modified nucleotides are underlined, the substitutes are illustrated below.(C) Five site-directed mutants were prepared in the –151/+79 promoter fragment (pF4-151-luc), as described under ‘Materials and methods’.(D) These mutated constructs were introduced into primary rat cardiomyocytes for transient expression assays. Firefly luciferase activities expressed had been normalized on the basis of Renilla luciferase activity. Data shown are the means ± S. D., n= 3.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3823491&req=5

fig04: Mutational analysis of putative transcription factor binding sites of mouse CARK promoter.(A) Homology comparison of minimal promoter region of mouse CARK gene with human, chimpanzee, dog and rat revealed the presence of several conserved putative transcription factor binding sites, including Tbx5, SRE, M-CAT box, MEF2 and GATA4.(B) The putative transcription factor binding sites are shown in grey background and the modified nucleotides are underlined, the substitutes are illustrated below.(C) Five site-directed mutants were prepared in the –151/+79 promoter fragment (pF4-151-luc), as described under ‘Materials and methods’.(D) These mutated constructs were introduced into primary rat cardiomyocytes for transient expression assays. Firefly luciferase activities expressed had been normalized on the basis of Renilla luciferase activity. Data shown are the means ± S. D., n= 3.
Mentions: Cis-acting element response sites in CARK minimal promoter region were identified by sequence analysis using the CardioSignalScan program (http://www.cardiosignal.org/tools/scan.html) [14]. Putative sequence-conserved TFBSs were Tbx5 (−95/−80), SRE (−86/−73), M-CAT (−47/−33), MEF2 (−22/−12) and GATA4 (−9/+1; Fig. 4A). Site-directed mutagenesis was performed to modify each binding site based on the pF4–151-luc reporter using the QuickChange mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. Wild-type and modified sequences of each binding site in CARK promoter region were indicated in Fig. 4B. Five mutant luciferase reporter constructs (mut-Tbx5-luc, mut-SRE-luc, mut-MCAT-luc, mut-MEF2-luc and mut-GATA4-luc) were verified by DNA sequencing to confirm the integrity and the presence of the desired mutations in the constructs (Fig. 4C).

Bottom Line: Mutational analysis revealed five conserved cis-acting elements in this 151-bp long minimal promoter.Mutational and loss-of-functional analysis and co-transfection studies indicated that MEF2 binding region is the most critical cis-acting element in the CARK promoter, and CARK transcription level can be down-regulated by MEF2C antisense.Binding to the MEF2 sites by Mef2c protein was confirmed by electrophoretic mobility shift assay and competition and supershift electrophoretic mobility shift assays.

View Article: PubMed Central - PubMed

Affiliation: Sino-German Laboratory for Molecular Medicine, Ministry of Education, FuWai Cardiovascular Hospital and Cardiovascular Institute, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.

ABSTRACT
The cardiac ankyrin repeat kinase (CARK) gene, also named TNNI3K for its interaction with cardiac troponin I, is both a unique expression and heart-enriched gene. To understand the mechanisms of CARK gene expression and regulation, we first cloned the full-length mRNA sequence and mapped the transcription start site of the mouse CARK gene and characterized its promoter regions. Two transcriptional isoforms of the CARK gene were identified in mouse heart tissue. Truncation analysis of the CARK promoter identified a minimal 151 bp region that has strong basal transcription activity. Mutational analysis revealed five conserved cis-acting elements in this 151-bp long minimal promoter. Mutational and loss-of-functional analysis and co-transfection studies indicated that MEF2 binding region is the most critical cis-acting element in the CARK promoter, and CARK transcription level can be down-regulated by MEF2C antisense. Binding to the MEF2 sites by Mef2c protein was confirmed by electrophoretic mobility shift assay and competition and supershift electrophoretic mobility shift assays.

Show MeSH