Promoter elements and protein factors involved in temporal regulation of sea urchin Strongylocentrotus purpuratus early H1 histone gene

Abstract

In the sea urchin, early and late histone variants are transcribed during distinct time periods. In vivo expression of early H1 histone gene constructs assayed by microinjection showed that the minimal sequences required for proper temporal expression are from {dollar}-{dollar}65 to +39. These sequences include the TATA element, a conserved {dollar}-{dollar}46ACGTACGCA-38 (USE0) element immediately upstream of the TATA box, an initiator (Inr) element at the transcriptional start site and an internal element in the leader region of the mRNA coding sequence. Each of these sequences is recognized by a sequence-specific DNA binding protein detected by DNase I footprinting. The USE0 element was proved to be critical in regulation of the gene. The sequence ACGTACGCA differs by a single base from the late H1 gene USE0 element ACGTCCGCA. This single base pair in the USE0 element plays a role in both the accumulation and repression of early H1 transcripts. An early USE0-specific DNA binding activity exists throughout all developmental stages tested. This activity is composed of non-covalently associated components that can be dissociated in the presence of desoxycholate. The USE0 binding protein was identified as 90Kd polypeptide by southwestern analysis. The Inr and internal element are also characterized as positive sequence elements that can ectopically activate a reporter gene. An Inr binding activity was identified in both early and late nuclear extracts.;The early H1 TATA box sequence, TATATT is conserved in most H1 genes. Mutations in either the TATA box or the Inr have relatively mild effects on transcription of H1{dollar}\alpha{dollar} gene. The double mutant, however, showed almost complete loss of gene activity, suggesting an synergistic interaction between the two elements. We defined distinct TFIID activities in sea urchin nuclear extracts which recognize the two TATA box sequences. The DNA binding component of each class of TATA binding activity was identical. This sea urchin TBP activity was identified as a 40Kd polypeptide.