Proc. Nati. Acad. Sci. USA Vol. 82, pp. 4018-4022, June 1985 Biochemistry Torsional stress induces an S1 nuclease-hypersensitive site within the promoter of the Xenopus laevis oocyte-type 5S RNA: gene : (gene expression/transcription factor/DNA-protein interaction) WANDA F. REYNOLDS AND JOEL M. GOTTESFELD Department of Molecular Biology, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, CA 92037 Communicated by James Bonner, February 22, 1985 ABSTRACT The internal promoter of the Xenopus laevis gene promoter adopts an S1 nuclease sensitive conformation oocyte-type 5S RNA gene is preferentially cleaved by S1 and in supercoiled DNA. This altered conformation may be Bal-31 nucleases in plasmid DNA. S1 nuclease sensitivity is similarly induced or stabilized by TFIIIA in linear DNA. largely dependent on supercoiling; however, Bal-31 cleaves These findings provide a clear correlation between an S1 within the 5S RNA gene in linear as well as in supercoiled DNA. nuclease-sensitive conformation and a promoter element. The S1 nuclease-hypersensitive site is centered at position +48-52 of the gene at the 5' boundary of the promoter. A METHODS DNase I-hypersensitive site is induced at this position upon DNAs. For DNase I "footprint" analysis, pXlo 3'A+56 binding of the transcription factor, TFIIIA, specific for the 5S was digested with EcoRI and end-labeled with polynucleotide RNA gene. The somatic-type 5S RNA gene promoter is not kinase (Bethesda Research Laboratories) and ['y-32P]ATP. preferentially cleaved by S1 nuclease or Bal-31 nuclease in The DNA was secondarily digested with HindIII and the supercoiled DNA, nor does TFIIIA induce a DNase I site at 530-bp fragment containing the gene was isolated by poly- position +50. This differential promoter response may be acrylamide gel electrophoresis. The fragment was labeled on related to a 4-fold difference in TFIIIA affinity between the the coding strand, 60 bp 3' to the gene. pXlsll was digested oocyte and somatic 5S RNA genes. with Hpa II and end-labeled with the Klenow fragment of DNA polymerase and [a-32P]dCTP. The DNA was secondar- The 5' flanking sequences oftranscriptionally active genes in ily digested with HindIII and the 380-bp fragment containing chromatin-are often hypersensitive to nucleases. This sensi- the gene was isolated from a polyacrylamide gel. The frag- tivity extends not only to DNase I, micrococcal nuclease, and ment was labeled on the coding strand, 107 bp 5' to the gene. restriction enzymes (1) but also to single-strand-specific Digestion of DNA with S1 and Bal-31 Nucleases. DNA (1-2 reagents such as S1 nuclease and bromoacetaldehyde (2, 3). ,ug) was digested with up to 1 unit of S1 nuclease (Bethesda It has been suggested that hypersensitive regions correspond Research Laboratories or P-L Biochemicals) in a reaction to regulatory sequences and may be sites of interaction with volume of 30 p.1 in a buffer containing 30 mM sodium acetate effector proteins. Upstream elements that are hypersensitive at pH 4.8, 50 mM NaCl, and 1 mM ZnCl2. After a 30-min in active chromatin are in some instances S1 nuclease incubation at 37°C, the reaction was stopped with EDTA (20 hypersensitive in supercoiled DNA. The significance of this mM). The DNA was purified, digested with an appropriate correlation is made apparent by recent evidence that active restriction enzyme, electrophoresed in 1.2% agarose gels in chromatin is subject to torsional stress (4, 5). 1 x TAE buffer (40 mM Tris acetate, pH 7.8/2 mM EDTA), Examples of upstream elements that are hypersensitive as transferred to nitrocellulose filters, and hybridized with a supercoiled DNA include those of the chicken P-globin gene nick-translated probe abutting the restriction site (15). (2, 6), Drosophila heat shock protein 70 (7) and histone genes Plasmids were digested with Bal-31 (Bethesda Research (8), sea urchin histone genes (9), adenovirus major late region Laboratories) (units, as defined by the supplier, indicated in (10), and the simian virus 40 origin/enhancer region (11). figures) for 1 min at 22°C in a 20-,ul reaction volume in a buffer Although these findings are provocative, there is as yet little containing 20 mM Tris HCl at pH 8.1, 100 mM NaCl, 12 mM correlation between hypersensitive sites (HSSs) in CaC12, 12 mM MgCl2, and 1 mM EDTA. After extraction with and known elements. In phenol and precipitation with ethanol, the DNA was digested supercoiled DNA promoter fact, and analyzed as described above. sequences giving rise to a HSS upstream of the Drosophila Mapping of S1 Nuclease Cutting Sites. pXlo8 (5-10 ,ug per heat shock protein 70 gene can be deleted without signifi- lane) was digested with S1 nuclease at low concentrations cantly affecting promoter function in vivo (12). Only in the (0.1-0.2 unit/30 ,ul) for 30 min at 37°C. This nuclease cases of certain (T)ATA homologies (6, 10) and sequences concentration produced less than 5% linear molecules and within the simian virus 40 origin/enhancer region (11) do such ensured a low level of nicking. The DNA was then digested HSSs correspond to identifiable promoter elements. To help with EcoRI and electrophoresed in a 1.2% agarose gel as clarify the biological significance of S1 nuclease hypersensi- above. Sections ofthe agarose gel containing DNA fragments tive structures, a system is needed in which the promoter of interest (see Fig. 3) were excised and the DNA was sequences are well defined and specific transcription factors electroeluted and digested with HindIII. The DNA was have been identified. The Xenopus SS RNA genes provide end-labeled with the Klenow fragment of DNA polymerase such a system. Deletion mutant analysis has delineated a and [a-32P]dATP, followed by electrophoresis in 6% promoter element internal to the 120-base-pair (bp) gene (13). acrylamide nondenaturing gels in TBE buffer (90 mM Tris A transcription factor, TFIIIA, specific for the 5S RNA gene borate, pH 8.3/2 mM EDTA). Labeled fragments were has been shown to bind this promoter region (14). In this excised from the polyacrylamide gel, crush-eluted and elec- report, we present evidence that the oocyte-type 5S RNA trophoresed in sequencing gels (7.6% acrylamide/0.4% bisacrylamide/8.3 M urea/ix TBE). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: bp, base pair(s); HSS, hypersensitive site; TFIIIA, in accordance with 18 U.S.C. §1734 solely to indicate this fact. transcription factor specific for 5S RNA gene. 4018 Downloaded by guest on September 26, 2021 Biochemistry: Reynolds and Gottesfeld Proc. Natl. Acad. Sci. USA 82 (1985) 4019 DNase I Footprinting. TFIIIA was isolated from immature electrophoresed in agarose gels, blotted onto nitrocellulose oocytes as 7S particles consisting of TFIIIA in association and hybridized with a nick-translated probe abutting the with 5S RNA or as purified TFIIIA according to published EcoRI site. This indirect end-labeling procedure (15) revealed procedures (16, 17). Identical footprints were obtained with a repeating pattern ofcleavage sites within the 5S insert (Fig. 7S particles and purified TFIIIA. Singly end-labeled DNA 1B). S1 nuclease preferentially cleaves at the approximate fragments were incubated with saturating amounts of 7S center of each gene and pseudogene. The A+T-rich spacer particles in the presence of RNase A (10 gg/ml) for 10 min sequence, which should be the most likely to "breathe," is followed by DNase I digestion (2 pg/ml for 1 min at 220C). not preferentially cleaved. S1 nuclease cleavage within the 5S The DNA fragments were purified and electrophoresed on gene is largely dependent on negative supercoiling. Only at sequencing gels. the highest nuclease concentration employed was cleavage RESULTS observed within linear 5S DNA (Fig. 1C). Furthermore, the cleavage sites in linear DNA are less defined than within S1 Nuclease Cleavage of 5S Plasmid DNA. We examined the supercoiled DNA. A plasmid containing a single copy of the S1 nuclease sensitivity of a plasmid, pXlo8, containing four 5S gene and the 5' flanking sequences but lacking the tandem repeating units of oocyte-type 5S DNA. As shown in pseudogene gives rise to a similar S1 nuclease sensitivity Fig. lA, each 720-bp repeat contains the 5S gene, a pseudo- profile (Fig. 1D). S1 nuclease cleaves at one site central to the gene, and approximately 500 bp of spacer sequences (18). but does not cleave within The pseudogene is a direct duplication of the first 101-bp of gene the 5' or 3' flanking the gene with 10 base substitutions. Though apparently not sequences. There are a number of S1 nuclease cleavage sites transcribed in vivo, the pseudogene retains transcriptional within the prokaryotic vector sequences. We note, however, activity in cell-free extracts (19). Supercoiled pXlo8 was that within the 720-bp oocyte 5S DNA insert, only one site digested with S1 nuclease at concentrations producing 5-50% linear molecules. The DNA was then digested with A 0 0.1 0.05 0.02 0.01 0.1 0 0.1 0.05 EcoRI, NS.D SCD- A EcoRI H 720 H H H H BamrHI SC- 11073120 -425 Pseudogene 5S gene A+T-rich spacer Si B Supercoiled Linear B Hindlil 0 1 D XIo H 01 1.i Ht _ -Bam - _=, _ H U H w S....fl m so H ml FIG. 1. S1 nuclease cleavage of 5S plasmid DNA. (A) pXlo8 contains four 720-bp repeat units of Xenopus laevis oocyte-type 5S DNA (18) inserted at the HindIII site of pMB9.