Cf. Mctiernan et al., Characterization of proximal transcription regulatory elements in the rat phospholamban promoter, J MOL CEL C, 31(12), 1999, pp. 2137-2153
Phospholamban is a major regulator of cardiac diastole, with alterations in
expression associated with modified cardiac relaxation. To study transcrip
tional 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 cardiomyocy
tes, constructs containing at least 159 nucleotides preceding the transcrip
tion 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 absolu
te 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 enhan
ce cell-specific expression, To focus on the basal promoter region (-159/-6
6), a series of deletion constructs were made that identified a novel 35 bp
region (-159/-125: Phospholamban Promoter Element 1, PPE1) required for pr
omoter activity in cardiomyocytes, Site-specific mutations identified nucle
otides -150/-133 as containing most of the promoter-enhancing activity. Whi
le the rat PPE1 is highly conserved (>70%) in four other mammalian pbosphol
amban genes, it does not contain previously characterized regulatory elemen
ts. In cardiomyocytes the PPE1 sequence markedly enhanced activity of the S
V40 early promoter, A conserved CCAAT element (- 83/-79) was also required
for promoter activity in both cardiomyocytes and HeLa cells, Exonuclease II
I footprinting identified protein/DNA interactions in both the extended CCA
AT box and PPE1 domains. Gel shift studies identified the CCAAT elements as
binding CBF/NF-Y (C) 1999 Academic Press.