REGULATION OF TRANSCRIPTION OF THE TATA-LESS HUMAN-COMPLEMENT COMPONENT C4 GENE

The 5'-sequences flanking the human complement component C4 genes (C4A and C4B) have been analyzed for their ability to direct expression of a reporter gene in cell lines that constitutively express or do not e xpress C4, No difference in the level of reporter gene expression was detected in cells transfected with C4A- or C4B-specific constructs. A series of reporter constructs containing progressively truncated C4 pr omoter fragments transfected into the hepatocyte Hep G2 cell line, ide ntified the sequence contained within the region -178 to -39 as that a ssociated with maximal reporter gene expression. This region contains consensus binding moths for nuclear factor 1 (-110 to -97), Sp1 (-57 t o -49), and three basic helix-loop-helix (-137 to -132, -98 to -93, an d -78 to -73)-like transcription factors. Electromobility shift assays and DNase I footprinting analysis showed specific DNA-protein interac tions of the C4 promoter at the nuclear factor 1, two E box (-98 to -9 3 and -78 to -73), and Spl binding domains. Site-directed mutagenesis of the Spl binding site resulted in total abrogation of reporter gene expression and mutation of the E box (-78 to -73) resulted in a 8-fold reduction in expression. We conclude that the Spl binding site at pos ition -57 to -49 is critical for accurately initiated, basal transcrip tion of C4.