Characterization of proximal transcription regulatory elements in the rat phospholamban promoter

Phospholamban is a major regulator of cardiac diastole, with alterations in expression associated with modified cardiac relaxation. To study transcriptional regulation of phospholamban expression, we made reporter constructs that expressed luciferase under control of putative promoter sequences from the rat phospholamban gene. When transfected into neonatal rat cardiomyocytes, constructs containing at least 159 nucleotides preceding the transcription start site were equally active, while truncation to -66/+64 removed all promoter activity. Constructs were more active in cardiomyocytes than in HeLa cells (which do not express phospholamban), but did not show absolute cell-type specificity of expression. Addition of sequences upstream to -4032, all of the intron (7.4 kb), or 3'UTR sequences (0. 8 kb) did not enhance cell-specific expression. To focus on the basal promoter region (-159/-66), a series of deletion constructs were made that identified a novel 35 bp region (-159/-125; Phospholamban Promoter Element 1, PPE1) required for promoter activity in cardiomyocytes. Site-specific mutations identified nucleotides -150/-133 as containing most of the promoter-enhancing activity. While the rat PPE1 is highly conserved (>70%) in four other mammalian phospholamban genes, it does not contain previously characterized regulatory elements. In cardiomyocytes the PPE1 sequence markedly enhanced activity of the SV40 early promoter. A conserved CCAAT element (-83/-79) was also required for promoter activity in both cardiomyocytes and HeLa cells. Exonuclease III footprinting identified protein/DNA interactions in both the extended CCAAT box and PPE1 domains. Gel shift studies identified the CCAAT elements as binding CBF/NF-Y.