POSITIVE AND NEGATIVE TRANSCRIPTIONAL REGULATORY ELEMENTS IN THE EARLY H4 HISTONE GENE OF THE SEA-URCHIN, STRONGYLOCENTROTUS-PURPURATUS

The early H4 (EH4) histone gene of the sea urchin, Strongylocentrotus purpuratus, is shown to contain at least five positive-responding sequence elements and one negative-responding site which control the level of in vitro transcription in an embryonic nuclear extract. The positive acting elements are: 1) the UHF-1 region, located between - 133 and - 102 (the site of a strong footprint, due at least in part to the binding of an 85 kD protein factor termed UHF-1); 2) the H4 specific element (H4SE), situated between - 62 and - 39; 3) a sequence corresponding to a TATA box between - 33 and - 26 (TAACAATA); 4) the transcriptional initiation site; and 5) an internal sequence element found between + 19 and + 50. Deletion of, or base changes in, the UHF-1, H4SE, initiation, or internal sequence sites resulted in significant decreases in transcription. Base subtitutions in the TATA-like sequence had much less effect, resulting in no more than a 2-fold decrease in transcription, and there was no evidence that alternative initiation sites are utilized in the mutant templates. The negative element (termed the UHF-3 nucleotides - 75 and - 56. Base subtitutions in this area result in templates that were transcribed at a level 1.2 - 2.0-fold higher than the wild-type gene. Transcription levels of double UHF-1/H4SE and UHF-1/INR mutants were those expected from additive effects of the individual mutations and there was no suggestion of synergism.