Sea urchin early and late H4 histone genes bind a specific transcription factor in a stable preinitiation complex.

Abstract

Early embryonic H4 (EH4) and H2B (EH2B) and late embryonic H4 (LH4) histone genes were transcribed in vitro in a nuclear extract from hatching blastula embryos of the sea urchin Strongylocentrotus purpuratus. The extract was prepared by slight modifications of the methods of Morris et al. (G. F. Morris, D. H. Price, and W. F. Marzluff, Proc. Natl. Acad. Sci. USA 83:3674-3678, 1986) that have been used to obtain a cell-free transcription system from embryos of the sea urchin Lytechinus variegatus. Achievement of maximum levels of transcription of the EH4 and LH4 genes required a 5-to 10-min preincubation of template with extract in the absence of ribonucleoside triphosphates. This preincubation allowed the formation of a stable complex which was preferentially transcribed compared with a second EH4 or LH4 template that was added 10 min later. Although the EH4 gene inhibited both EH4 and LH4 gene transcription in this assay and although the LH4 gene inhibited both EH4 and LH4 genes, neither of these genes inhibited transcription of the EH2B gene. Preincubation with the EH2B gene had no effect on the transcription of subsequently added EH4 or LH4 genes. Using this template commitment assay, we showed that the site of binding of at least one essential factor required for transcription of both EH4 and LH4 genes was located between positions -102 and -436 relative to the 5' terminus of the EH4 mRNA. Moreover, deletion of this region resulted in a reduction in EH4 gene transcription in vitro. The sea urchin embryonic nuclear extract, therefore, should prove very helpful in the assay and purification of sea urchin gene-specific trans-acting factors, in the analysis of the cis-acting sequences with which they interact, and in biochemical studies on the formation of stable transcription complexes.The histone genes of the sea urchin are among the best known examples of genes which are differentially regulated during early embryogenesis (for a review, see reference 41). During the cleavage and blastula stages of the sea urchin embryo, dramatic changes occur in the levels of mRNAs derived from the early embryonic (or a) genes and the late embryonic (or 1, y, and 8) genes. The early embryonic genes, which are reiterated several hundred-fold in a tandem array of a unit containing a gene for each of the five histones, are transcribed during oogenesis and between the 16-cell and midblastula stages (for reviews, see references 26 and 41). The level of early histone mRNAs per embryo increases about 10-fold during the cleavage and early blastula period and then rapidly decreases so that by the gastrula stage there is little remaining early mRNA (39,43,58). The late embryonic histone genes of Strongylocentrotus purpuratus and Lytechinus pictus are present in far fewer copies (2 to 12 members per genome, depending on the histone species) and are distributed in irregular clusters (9,30,31,33,34,40). The late mRNAs are found in low levels in the egg, but in contrast to the early mRNA species, they do not increase to their maximum level...

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

“…This element, corresponding to the site upstream of -102 in the EH4 gene previously identified (60,61), is shown to be essential for maximal in vitro transcription of both genes in the nuclear extract and for maximal embryonic expression of the EH4 gene after injection into Lytechinus pictus eggs. Deletion of the sequence element, however, did not grossly change the in vivo temporal expression pattern.…”

“…In addition, the in vivo assay has been used to identify which sequence elements have a role in setting the level of expression of S. purpuratus EH3 (13) and H1-p (39) genes and the Psammechinus miliaris late H2B-2.1 gene (1). A complementary approach to identify transcriptional sequence elements is possible with the use of an in vitro transcription assay using embryonic nuclear extracts (1,51,60,61 …”

“…In addition, the in vivo assay has been used to identify which sequence elements have a role in setting the level of expression of S. purpuratus EH3 (13) and H1-p (39) genes and the Psammechinus miliaris late H2B-2.1 gene (1). A complementary approach to identify transcriptional sequence elements is possible with the use of an in vitro transcription assay using embryonic nuclear extracts (1,51,60,61). Although the histone gene temporal expression programs seen in vivo were not reproduced when genes were transcribed in nuclear extracts from different embryonic stages (61), the in vitro transcription system does provide a useful assay for positive and negative cis-acting transcriptional elements.…”

“…A complementary approach to identify transcriptional sequence elements is possible with the use of an in vitro transcription assay using embryonic nuclear extracts (1,51,60,61). Although the histone gene temporal expression programs seen in vivo were not reproduced when genes were transcribed in nuclear extracts from different embryonic stages (61), the in vitro transcription system does provide a useful assay for positive and negative cis-acting transcriptional elements. We have demonstrated with this assay that there are multiple positively acting elements upstream of the S. purpuratus early H4 (EH4) gene and that EH4 and late H4 (LH4) templates form a stable transcription complex when incubated with the nuclear extract (61).…”

“…Although the histone gene temporal expression programs seen in vivo were not reproduced when genes were transcribed in nuclear extracts from different embryonic stages (61), the in vitro transcription system does provide a useful assay for positive and negative cis-acting transcriptional elements. We have demonstrated with this assay that there are multiple positively acting elements upstream of the S. purpuratus early H4 (EH4) gene and that EH4 and late H4 (LH4) templates form a stable transcription complex when incubated with the nuclear extract (61). Since the EH4 and LH4 genes could compete against each other in formation of the complex, we concluded that the two genes shared a requirement for at least one transcription factor.…”