Spacer DNA sequences upstream of the T-A-T-A-A-A-T-A sequence are essential for promotion of H2A histone gene transcription in vivo.

Abstract

The control region of a sea urchin H2A histone gene may be functionally dissected into at least three DNA segments, which we have termed modulator, selector, and initiator elements. While the initiator and in particular the selector containing the T-A-T-A-A-A-T-A sequence are specificity elements that dictate the generation of faithful 5' ends to H2A mRNA, the modulators control the rate at which these specificity elements operate [Grosschedl, R. & Birnstiel, M. L. (1980) Proc. Nat]. Acad. Sci. USA 77, 1432USA 77, -1436. By functional tests of in vitro mutated histone DNA in the Xenopus oocyte we have now discovered that the segment E of the A+T-rich spacer DNA lying at a considerable distance upstream of the conservative In prokaryotes, the role of regulatory proteins and their interaction with specific DNA sequences in transcription is being elucidated by the process of classifying the various phenotypes produced by mutation of the regulatory sequences and the genes coding for regulator proteins. This classical genetical approach is complemented by well-defined in vitro transcriptional systems and protein-binding studies. In principle, this method can also be used for animal viruses, which readily express their genetic information in the host cell (cf. ref. 1). Study of the control of transcription of eukaryotic chromosomal genes has been facilitated by the development of surrogate genetics techniques (2, 3) in which in vitro sequence manipulation of the cloned genes is followed by functional tests of the mutants within the living cell (4-6).We previously used this approach to study the transcriptional control of a H2A histone gene of the sea urchin Psammechinus miliaris (6). Short sections [54-59 base pairs (bp)] were deleted from the H2A 5' mRNA leader coding sequence and sequences preceding it. Transcriptional changes in the expression of the modified genes injected into the nucleus of centrifuged Xenopus oocytes (cf. ref. 7) were then examined. From the various mRNA "phenotypes" produced we concluded that RNA polymerase II promoters contain both specificity and ratedetermining elements that were termed; in order of their occurrence on the DNA: modulator, selector, and initiator elements (6). One such modulator, or rate-determining element, was delineated as being coincident with the previously de- Construction of Spacer Mutant Clones. The strategy for the construction of spacer mutants was similar to that described (6). The containment conditions were P2 and EK1 according to the National Institutes of Health guidelines.Spacer deletion mutants h22AD and h22AE. Three DNA components were prepared. Component a consisted of the left-hand portion of the h22 DNA up to a Taq I site, in the case of deletion D near amino acid codon 105 of the H3 gene (9), in the case of deletion E near nucleotide position -524 in the pre-H2A spacer (see Fig. 1). These components were prepared by a partial cleavage of the 4-kilobase (kb) HindIII (9), delimited on the right by the Hpa II site at position -130 in the pre-H2A ...

Simian virus 40 early- and late-region promoter functions are enhanced by the 72-base-pair repeat inserted at distant locations and inverted orientations.

“…at this location and orientation to induce a distinctive DNase I-hypersensitive site was the cause of its inability to enhance the inactivated SV40 early and late promoters in mutant XSLR23 needs further study. DISCUSSION All eucaryotic promoters recognized by RNA polymerase II contain an essential region located between nucleotides +1 and -110; that region is analogous to the +1 to -35 region of procaryotic promoters (25,31) in its involvement in RNA polymerase binding and transcription initiation (10,15,16,19,20,23). However, some eucaryotic promoters also require a sequence upstream of the -110 region for efficient transcription (4,10,15).…”

“…DISCUSSION All eucaryotic promoters recognized by RNA polymerase II contain an essential region located between nucleotides +1 and -110; that region is analogous to the +1 to -35 region of procaryotic promoters (25,31) in its involvement in RNA polymerase binding and transcription initiation (10,15,16,19,20,23). However, some eucaryotic promoters also require a sequence upstream of the -110 region for efficient transcription (4,10,15). The sea urchin histone H2A gene promoter, for example, requires a sequence that occurs 184 to 524 nucleotides upstream of the site of transcription initiation, and significantly this region functions normally even if it is inverted (15).…”

“…However, some eucaryotic promoters also require a sequence upstream of the -110 region for efficient transcription (4,10,15). The sea urchin histone H2A gene promoter, for example, requires a sequence that occurs 184 to 524 nucleotides upstream of the site of transcription initiation, and significantly this region functions normally even if it is inverted (15). The SV40 early-region promoter also requires a sequence that occurs between 112 and 255 nucleotides upstream ofthe initiation sites of the early mRNAs (4,10).…”

“…RNA polymerase II promoters also require nucleotides in the -30 to -110 region; their removal or substantial modification abolishes or greatly reduces transcription, e.g., the herpes simplex virus thymidine kinase promoter (18,19), the human a-globin (20) and rabbit P-globin promoters (16), and the simian virus 40 (SV40) early-region promoter (10). Two others, the sea urchin histone H2A and SV40 early-region promoters, have absolute requirements for a sequence located between nucleotides -184 and -524 and nucleotides -112 and -255, respectively (4,10,15). By contrast, the human aglobin (20) and the herpes simplex virus thymidine kinase (18) promoters show no dependence on the nucleotide sequence in this region.…”

“…By contrast, the human aglobin (20) and the herpes simplex virus thymidine kinase (18) promoters show no dependence on the nucleotide sequence in this region. Of particular interest is the finding that the essential sequence in the -184 to -524 region of the sea urchin histone H2A promoter and the -112 to -255 region of the SV40 early promoter can function even when they are inverted relative to the other promoter components (10,15,22). The nucleotide sequence between nucleotides -112 and -255 that is needed for SV40 early-region promoter function contains a tandemly repeated 72-base-pair (bp) sequence; this sequence can also augment correct transcription initiation from the rabbit (2) and human (J. Sklar and P. Berg, unpublished data) 3-globin promoters, the herpes simplex virus thymidine kinase promoter (7; M. Fromm, Ph.D. thesis, Stanford University, Stanford, Calif., 1983), the conalbumin and ovalbumin promoters (22), and the adenovirus 2 late promoter (22).…”