Sp1 and Sp3 transcription factors are required for trans-activation of the human SERCA2 promoter in cardiomyocytes.
OBJECTIVE
The sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) is essential to the removal of cytosolic calcium following cardiac contraction, and its abundance and activity are significantly altered during perinatal development and in failing myocardium. The objective of the current study was to identify cis regulatory elements and nuclear transcription factors responsible for transactivating SERCA2 gene expression in cardiomyocytes.


METHODS
Primary cultures of neonatal rat ventricular myocytes were transiently transfected with luciferase (LUX) reporter gene constructs containing deletions of the SERCA2 promoter or which harbored mutations in consensus Sp1 transcription factor binding sites. Cotransfection assays, electrophoretic mobility shift, and supershift assays were also performed to delineate the regulatory role of specific transcription factors.


RESULTS
We identified a putative AP-1-like element and a consensus Egr-1 binding site, but neither Egr-1 nor 12-O-tetradecanoylphorbol 13-acetate (TPA) significantly modified human SERCA2 promoter activity in vitro. Maximal activity of the SERCA2 promoter required the proximal 177 bp, and strong activation was observed with a 125-bp construct, within which an Sp1 site and a CAAT box were important. Mutation analysis also revealed the importance of two Sp1 sites between -125 and -200. Sp1 and Sp3 transcription factors were subsequently identified to bind to oligonucleotide probes corresponding to only the two most proximal Sp1 sites.


CONCLUSIONS
These studies provide direct evidence that regulation of human SERCA2 gene expression in cardiomyocytes depends on transactivation events elicited by Sp1 and Sp3 transcription factors.
Brady
Marc
M
National Heart and Lung Institute Division, Faculty of Medicine, Imperial College London, Guy Scadding Building, Dovehouse Street, London SW3 6LY, UK.
Koban
Maren U
MU
Dellow
Kimberley A
KA
Yacoub
Magdi
M
Boheler
Kenneth R
KR
Fuller
Stephen J
SJ
D000818:Animals; D001483:Base Sequence; D000252:Calcium-Transporting ATPases / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051381:Rats; D017208:Rats, Wistar; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D016329:Sp1 Transcription Factor / Q000235:genetics*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000235:genetics*; D015533:Transcriptional Activation; D014162:Transfection / Q000379:methods
C506948:Atp2a2 protein, rat; D004268:DNA-Binding Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; C497123:Sp3 protein, rat; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506946:ATP2A2 protein, human; D000252:Calcium-Transporting ATPases