Cell-type-specific transcription factor interactions with cis-elements present in the mouse LDH/C proximal promoter...

The major aim of this study was to identify cell-type-specific transcription factors present in somatic and germ cells that interact at cis-elements within the lactate dehydrogenase C (LDH/C) proximal promoter region. Electrophoretic mobility shift assays (EMSAs) were performed using wild-type and mutagenized GC-box LDH/C double-stranded (ds) oligonucleotides to probe nuclear extracts from primary germ cell populations, and an LDH/C nonexpressing cell line (Hela cells). Significant differences were observed in the composition of nuclear transcription factors bound to cis-elements within the LDH/C proximal promoter region in somatic versus germ cells. Using the wild-type LDH/C ds oligonucleotide to probe germ cell nuclear extracts, we consistently observed two DNA-protein complexes, one formed by Sp1 proteins and one formed by another uncharacterized germ cell nuclear transcription factor. Methylation interference analysis identified a unique 5'G residue within the GC-box motif as an integral part of the cis-element recognized by this germ cell nuclear transcription factor. In contrast, at least six major DNA-protein complexes were observed in EMSA studies using Hela cell nuclear extracts probed with the LDH/C wild-type oligonucleotide. In these EMSA studies, supershift analyses with antibodies recognizing the somatic Sp1 protein and the GC-box mutated LDH/C ds oligonucleotide demonstrated that only one of the six complexes formed was due to binding of Sp1 proteins. These in vitro EMSA studies have identified several somatic nuclear factors and at least one other germ cell nuclear transcription factor, which in addition to members of the Sp1 protein family, recognized cis-elements within the LDH/C proximal promoter region. Observations presented in this report strongly suggest that these somatic and germ cell nuclear transcription factors play central roles in modulating transactivation or suppression of the mouse LDH/C subunit gene in the respective cell types.