Abstract
The sarco(endo)plasmic reticulum Ca-ATPases (SERCAs) belong to a family of active calcium transport enzymes encoded by the SERCA1, 2, and 3 genes. In this study, we describe the complete structure of the human SERCA2 gene and its 5’ -regulatory region. The hSERCA2 gene is located in chromosome 12 position q24.1 in Contig NT_009770.8, spans 70 kb, and is organized in 21 exons intervened by 20 introns. The last two exons of the pre-mRNA produce by alternatively splicing the cardiac/slow-twitch muscle-specific SERCA2a isoform and the ubiquitous SERCA2b isoform. The sequence of the proximal 225-bp regulatory region of the SERCA2 genes is 80% G+C-rich and is conserved among human, rabbit, rat, and mouse species. It contains a TATA-like-box, an E-box/USF sequence, a CAAT-box, four Sp1 binding sites, and a thyroid hormone responsive element (TRE). There are two other conserved regulatory regions located between positions -410 to -661 bp and from -919 to -1410 bp. Among the DNA cis-elements present in these two regulatory regions there are potential binding sites for: GATA-4, -5, -6, Nkx-2.5/Csx, OTF-1, USF, MEF-2, SRF, PPAR/RXR, AP-2, and TREs. Upstream from position -1.5 kb, there is no significant homology among the SERCA2 genes cloned. In addition, the human gene has several repeated sequences mainly of the Alu and L2 type located upstream from position -1.7 kb, spanning in a continuous fashion for more than 40 kb. In this study, we report the cloning of 2.4 kb of 5’-regulatory region and demonstrate that the proximal promoter region is sufficient for expression in cardiac myocytes, and the region from -225 to -1232 bp contains regulatory DNA elements which down-regulate the expression of the SERCA2 gene in neonatal cardiomyocytes.
Highlights
The sarcoplasmic reticulum (SR) Ca2+ transport ATPase activity determines the rate of contraction and relaxation of cardiac, skeletal, and smooth muscle
The SERCA2 gene encodes two isoforms produced by alternative splicing (SERCA2a and SERCA2b) which are expressed in a tissuespecific fashion[3,4]
Search for the SERCA2 gene in the human genome using BLAST analysis software from NCBI of the human SERCA2a and 2b mRNA sequences revealed that the entire gene is located within a fragment of 122,605 bp-long in the PAC clone RPC 13-305I20
Summary
The sarcoplasmic reticulum (SR) Ca2+ transport ATPase activity determines the rate of contraction and relaxation of cardiac, skeletal, and smooth muscle. Constructs containing at least 1.2 kb of regulatory sequences showed three- to fivefold lower transcriptional activity, suggesting the presence of DNA cis-elements down-regulating the transcription of the gene in neonatal rat cardiomyocytes in culture. The genomic clone contains 41,909 bp of 5’-flanking sequence upstream of the transcription initiation site, including the promoter region.
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