Nucleic Acids Research, Vol 25, Issue 11 2213-2220, Copyright © 1997 by Oxford University Press
J Yang and K Thomas
Molecular and functional studies of the LDH/C 5' upstream promoter elements
were undertaken to elucidate the molecular mechanisms involved in temporal
activation of LDH/C gene expression in differentiating germ cells. Ligation
mediated-PCR (LM-PCR) gene walking techniques were exploited to isolate a
2.1 kb fragment of the mouse LDH/C 5' promoter region. DNA sequence
analysis of this isolated genomic fragment indicated that the mouse LDH/C
promoter contained TATA and CCAT boxes as well as a GC-box (Sp1-binding
site) situated upstream from the transcription start site. PCR-based in
vivo DNase I footprinting analysis of a 600 bp fragment of the proximal
LDH/C promoter region (- 524/+38) in isolated mouse pachytene spermatocytes
identified a single footprint over the GC-box motif. Three DNase I
hypersensitive sites were also detectable in vivo, in a region containing
(CT)n(GA)n repeats upstream from the CCAT box domain. Functional
characterization of the promoter region was carried out in a rat C6 glioma
cell line and an SV40 transformed germ cell line (GC-1 spg) using wild-type
and mutated LDH/C promoter CAT reporter constructs. These studies provide
experimental evidence suggesting that transcriptional activation of the
LDH/C promoter is regulated by enhancer-mediated coactivation of the Sp1
proteins bound to the GC-box motif footprinted in vivo in pachytene
spermatocytes.
ARTICLES
Molecular and functional characterization of the promoter region of the mouse LDH/C gene: enhancer-assisted, Sp1-mediated transcriptional activation
Department of Anatomy, Morehouse School of Medicine, Atlanta, GA 30310- 1495, USA.
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