UHF-1, a factor required for maximal transcription of early and late sea urchin histone H4 genes: analysis of promoter-binding sites.

Abstract

A protein, denoted UHF-1, was found to bind upstream of the transcriptional start site of both the early and late H4 (EH4 and LH4) histone genes of the sea urchin Strongylocentrotus purpuratus. A nuclear extract from hatching blastulae contained proteins that bind to EH4 and LH4 promoter fragments in a band shift assay and produced sharp DNase I footprints upstream of the EH4 gene (from -133 to -106) and the LH4 gene (from -94 to -66). DNase I footprinting performed in the presence of EH4 and LH4 promoter competitor DNAs indicated that UHF-1 binds more strongly to the EH4 site. A sequence match of 11 of 13 nucleotides was found within the two footprinted regions: AGGGGGCGCACTC. Methylation interference and footprinting experiments showed that UHF-1 bound to the two sites somewhat differently. DNA-protein UV cross-linking studies indicated that UHF-1 has an electrophoretic mobility on sodium dodecyl sulfate-acrylamide gels of approximately 85 kDa and suggested that additional proteins, specific to each promoter, bind to each site. In vitro and in vivo assays were used to demonstrate that the UHF-1-binding site is essential for maximal transcription of the H4 genes. Deletion of the EH4 footprinted region resulted in a 3-fold decrease in transcription in a nuclear extract and a 2.6-fold decrease in expression in morulae from templates that had been injected into eggs. In the latter case, deletion of the binding site did not grossly disrupt the temporal program of expression from the injected EH4 genes. LH4 templates containing a 10-bp deletion in the consensus region or base substitutions in the footprinted region were transcribed at 14 to 58% of the level of the wild-type LH4 template. UHF-1 is therefore essential for maximal expression of the early and late H4 genes.The sea urchin genome contains several histone gene sets which are differentially regulated during development (reviewed in references 23 and 43). The early embryonic genes, organized as a unit containing a gene for each of the five histones, are reiterated several hundred-fold per haploid genome in a tandem array. The late gene set of each haploid genome is composed of 5 to 12 genes for each nucleosomal histone (4,29,30,42) and at least two Hi genes (32,33,37), organized in small irregular clusters or found as single genes. The amount of early RNA increases approximately 10-fold from the 16-cell stage to early blastula and then decreases rapidly so that little early histone mRNA remains by the gastrula stage (41,46,67). Late gene transcripts are found at low levels in the egg and, depending on the particular gene, increase to maximum levels at the mid-blastula and later stages (2,3,18,24,29,31,32,37,42,49). Nuclear run-on assays indicate that the basis of these changes in mRNA levels is predominantly transcriptional; during blastulation, early gene template activity decreases and late gene transcription increases (31,62,70). Measurements of histone RNA synthesis rate and turnover in intact embryos are consistent with changes in the level of transcrip...

Modulator factor-binding sequence of the sea urchin early histone H2A promoter acts as an enhancer element.

“…The octamer binding factor involved in the faithful expression of the early H2B gene activates, in fact, the early H2B promoter in early and late cleavage embryos (24). By contrast, the sequence elements for temporal regulation of the early H3, H1, and H4 histone genes are placed in the 5' flanking regions (19)(20)(21)(22). However, only in the case of the early histone H1 gene has a cis-acting element that regulates the temporal expression been identified (20).…”

“…The transcriptional elements required for temporal and maximal expression for each of the five genes have been identified by gene transfer methodology (18) and by promoter binding studies. From all of these studies it appears that each gene has evolved its own specific mechanism for proper temporal expression during development (19)(20)(21)(22)(23)(24).…”

Histone gene transcription: A model for responsiveness to an integrated series of regulatory signals mediating cell cycle control and proliferation/differentiation interrelationships

“…Biological requirements for histone proteins are reflected by two functional classes of genes in the multi-gene family of histone coding sequences. The cell cycle regulated histone genes are expressed during S-phase to support DNA replication [Sierra et al, 1983;Kroeger et al, 1987;Pauli et al, 1987;van Wijnen et al, 1989;Dailey et al, 1988;Lee et al, 1991;Ramsey-Ewing et al, in press]. In contrast the cell cycle independent histone genes, also referred to as "variant" or "differentiation specific," are expressed throughout the cell cycle or are tran-during the early part of S-phase.…”

Sea urchin early histone H2A modulator binding factor 1 is a positive transcription factor also for the early histone H3 gene.

“…§1734 solely to indicate this fact. (13)(14)(15) set up an in vitro transcription system to identify transcriptional sequence elements in the promoter of both early and late histone H4 genes of S. purpuratus.…”