AIMS: The transcription factor early growth response-1 (Egr-1) is increased in models of cardiac pathology; however, it is unclear how Egr-1 impacts the heart. We sought to identify how Egr-1 regulates expression of proteins involved in cardiomyocyte calcium homeostasis. METHODS: Protein expression was measured by immunoblotting in control cardiac differentiated H9c2 cells or in H9c2 cells overexpressing wild-type Egr-1 (Egr-1) or an Egr-1 (I293F) mutant. Microspectrofluorimetry of fura-2-loaded cells was used to study calcium dynamics. Chromatin immunoprecipitation with anti-Egr-1 antibody was used to identify Egr-1-associated DNA. RESULTS: Calsequestrin (CSQ) expression was reduced in Egr-1- and profoundly reduced in I293F-expressing cells. Calreticulin, triadin, sarcoendoplasmic reticulum ATPase 2a, phospholamban, and phosphoserine 16-phospholamban expression was unaffected. Calcium release from CSQ-dependent ryanodine-sensitive stores was reduced in Egr-1 and absent in I293F-expressing cells. In contrast, calcium release from calreticulin-dependent inositol 1,4,5-trisphosphate stores was unaffected. In vivo and in vitro chromatin immunoprecipitation demonstrated Egr-1 binding to the CSQ2 promoter. The Egr-1-binding region contains overlapping Egr-1, SP1, and nuclear factor of activated T-cells (NFAT) sites and a CpG island. Reciprocal immunoprecipitation coupled to immunoblots indicated Egr-1:NFAT3 binding was present in all cells lines. Treatment with cyclosporin A, inhibition of DNA methylation using 5-azadeoxycytidine, or inhibition of protein acetylation using sodium butyrate reduced CSQ expression. CONCLUSION: Our data suggest that Egr-1:DNA binding at the promoter, DNA methylation, and protein acetylation are important in CSQ repression. Moreover, we demonstrate that a reduction in CSQ protein is associated with abnormal calcium dynamics. We conclude that Egr-1 acts as a transcriptional repressor at the CSQ promoter, resulting in downregulation of CSQ, the major calcium storage protein that links excitation-contraction coupling in the cardiac sarcoendoplasmic reticulum.
AIMS: The transcription factor early growth response-1 (Egr-1) is increased in models of cardiac pathology; however, it is unclear how Egr-1 impacts the heart. We sought to identify how Egr-1 regulates expression of proteins involved in cardiomyocyte calcium homeostasis. METHODS: Protein expression was measured by immunoblotting in control cardiac differentiated H9c2 cells or in H9c2 cells overexpressing wild-type Egr-1 (Egr-1) or an Egr-1 (I293F) mutant. Microspectrofluorimetry of fura-2-loaded cells was used to study calcium dynamics. Chromatin immunoprecipitation with anti-Egr-1 antibody was used to identify Egr-1-associated DNA. RESULTS: Calsequestrin (CSQ) expression was reduced in Egr-1- and profoundly reduced in I293F-expressing cells. Calreticulin, triadin, sarcoendoplasmic reticulum ATPase 2a, phospholamban, and phosphoserine 16-phospholamban expression was unaffected. Calcium release from CSQ-dependent ryanodine-sensitive stores was reduced in Egr-1 and absent in I293F-expressing cells. In contrast, calcium release from calreticulin-dependent inositol 1,4,5-trisphosphate stores was unaffected. In vivo and in vitro chromatin immunoprecipitation demonstrated Egr-1 binding to the CSQ2 promoter. The Egr-1-binding region contains overlapping Egr-1, SP1, and nuclear factor of activated T-cells (NFAT) sites and a CpG island. Reciprocal immunoprecipitation coupled to immunoblots indicated Egr-1:NFAT3 binding was present in all cells lines. Treatment with cyclosporin A, inhibition of DNA methylation using 5-azadeoxycytidine, or inhibition of protein acetylation using sodium butyrate reduced CSQ expression. CONCLUSION: Our data suggest that Egr-1:DNA binding at the promoter, DNA methylation, and protein acetylation are important in CSQ repression. Moreover, we demonstrate that a reduction in CSQ protein is associated with abnormal calcium dynamics. We conclude that Egr-1 acts as a transcriptional repressor at the CSQ promoter, resulting in downregulation of CSQ, the major calcium storage protein that links excitation-contraction coupling in the cardiac sarcoendoplasmic reticulum.
Authors: Yi Feng; Cody A Desjardins; Olivia Cooper; Akuah Kontor; Sarah E Nocco; Francisco J Naya Journal: PLoS One Date: 2015-05-26 Impact factor: 3.240
Authors: Jayati Mookerjee-Basu; Robert Hooper; Scott Gross; Bryant Schultz; Christina K Go; Elsie Samakai; Jonathan Ladner; Emmanuelle Nicolas; Yuanyuan Tian; Bo Zhou; M Raza Zaidi; Warren Tourtellotte; Shan He; Yi Zhang; Dietmar J Kappes; Jonathan Soboloff Journal: EMBO Rep Date: 2020-03-25 Impact factor: 8.807