Molecular and functional characterization of the promoter region of the mouse LDH/C gene: enhancer-assisted, Sp1-mediated transcriptional activation.
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.
Yang
J
J
Department of Anatomy, Morehouse School of Medicine, Atlanta, GA 30310-1495, USA.
Thomas
K
K
D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D018983:DNA Footprinting; D004742:Enhancer Elements, Genetic; D005809:Genes, Regulator; D006801:Humans; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase / Q000235:genetics*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D016385:TATA Box; D015533:Transcriptional Activation
D007527:Isoenzymes; D016329:Sp1 Transcription Factor; D004247:DNA; D007770:L-Lactate Dehydrogenase; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase