Abstract
Developmental and testis-specific expression of the mouse lactate dehydrogenase C (mldhc) gene requires mechanisms for activation in germ cells and repression in somatic cells. Promoter activity restricted to the testis has been demonstrated using in vitro transcription assays with a 60-base pair promoter sequence upstream of the transcription initiation site. This promoter fragment has a TATA box and an overlapping 31-base pair palindromic sequence. Here we have explored the role of the palindrome as a silencer of the ldhc gene in somatic tissues. A gel retardation assay detected two sites within the palindrome that were important for protein binding. A member of the NF-I/CTF family was identified as the protein binding to one of the sites. In transiently transfected mouse L cells, a promoter fragment in which the NF-I site was mutated showed a 4-fold greater activity as compared with the wild-type sequence. Overexpression of the four NF-I proteins, NF-IA, -B, -C, or -X, in mouse L cells transiently transfected with an ldhc promoter-reporter construct resulted in a 20-50% decrease in activity of the wild-type promoter but had no effect when the NF-I binding element in the palindrome was mutated. These results indicate a role for the NF-I proteins in regulation of the mldhc gene.
Highlights
Mechanisms of negative regulation such as chromatin organization, methylation, and DNA-protein interaction increasingly are recognized for a role in differential gene expression [1]
We demonstrate that the NF-I/CTF (CCAAT box transcription factor) protein family binds to the palindrome and functions as a repressor of mouse ldhc gene expression
Identification of Nucleotides within the Palindrome Necessary for Protein Binding in Somatic Tissue Extracts—electrophoretic mobility shift assays (EMSA) was used to demonstrate protein binding to specific domains of the mldhc gene
Summary
Mechanisms of negative regulation such as chromatin organization, methylation, and DNA-protein interaction increasingly are recognized for a role in differential gene expression [1]. In transiently transfected mouse L cells, a promoter fragment in which the NF-I site was mutated showed a 4-fold greater activity as compared with the wild-type sequence.
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