Proc. Natl. Acad. Sci. USA Vol. 84, pp. 1550-1554, March 1987 Botany Light-regulated changes in DNase I hypersensitive sites in the rRNA genes of Pisum sativum (peas/chromatin/photoregulation/DNA/nucleolar organizer) LON S. KAUFMAN*, JOHN C. WATSONt, AND WILLIAM F. THOMPSON: Carnegie Institution of Washington, 290 Panama Street, Stanford, CA 94305 Communicated by Winslow R. Briggs, November 10, 1986 (receivedfor review May 23, 1986) ABSTRACT We have examined the rDNA chromatin of transcribed genes (8), including the ribosomal RNA genes of Pisum sativum plants grown with or without exposure to light Tetrahymena (9-11), Xenopus (12), and Drosophila (13). for the presence of DNase I hypersensitive sites and possible However, DNase I hypersensitive sites have not yet been developmental changes in their distribution. Isolated nuclei reported in plant chromatin, and developmental changes in from pea seedlings were incubated with various concentrations the pattern of DNase I hypersensitivity have not been of DNase I. To visualize the hypersensitive sites, DNA purified reported for rDNA chromatin in any system. from these nuclei was restricted and analyzed by gel blot In the work reported here, we have examined the rDNA hybridization. We find that several sites exist in both the coding chromatin from pea buds for the presence of DNase I and noncoding regions of rDNA repeating units. Several of the hypersensitive sites. Using conditions that minimize the sites in the nontranscribed spacer region are present in the light activity of endogenous nucleases and proteases, we find that but are absent in the dark. Conversely, the hypersensitive sites DNase I hypersensitive sites exist in rDNA chromatin of within the mature rRNA coding regions are present in the dark isolated pea nuclei. When similar experiments are performed but absent in the light. There are two maojor'length variants of with nuclei isolated from the buds of dark-grown or light- the rRNA genes in P. sativum var. Alaska. The sites in the grown seedlings, we find several sites in the rRNA coding nontranscribed spacer region that appear during the light regions that are present in nuclei from dark-grown plants but treatment occur only in the shorter ofthese two length variants not in nuclei isolated from light-grown material. Conversely, in this cultivar. several DNase I hypersensitive sites in the nontranscribed spacers are present in nuclei from light-treated plants but are The cellular content of cytoplasmic rRNA in several plant absent from those of dark-grown seedlings. species is regulated by light (1), and light-regulated changes in the rate of nuclear rRNA gene transcription have been MATERIALS AND METHODS demonstrated in pea (2), Lemna (3), and barley (4). The overall transcriptional organization of rRNA genes is similar Seeds of Pisum sativum L. cv. Alaska (W. Altee Burpee, in plants and animals, but the plant genes tend to be more Werminster, PA) were imbibed for 5 hr and grown on two highly repeated than their animal counterparts (5). Ribosomal layers ofwater-soaked Kimpac (Kimberly-Clarke, Rosewell, RNA gene copy numbers can also vary substantially even GA) at 270C, 85% relative humidity, in absolute darkness among closely related genotypes within a species, indicating (14). Dark-grown plants were kept in these conditions for 7 that the number of nuclear rRNA genes in plants often days. Light-grown or light-treated plants were kept in the exceeds that needed to supply rRNA to the cytoplasmic dark for 4 days and then transferred to white light (102 ribosome pool (6). The presence of a large excess of gene ILmolmM2 sec-1; cool white fluorescent) for an additional 3 copies suggests that a mechanism must exist for determining days. The buds and hooks of the 7-day-old plants were which copies are transcriptionally active and which are silent. harvested into ice-cold containers under dim green light Regulation of rRNA gene expression by light may therefore (dark-grown plants) or in fluorescent room light (light-grown involve a recruitment ofmore genes into the transcriptionally plants). active pool. Nuclei were isolated as described by Watson and Thomp- The activity of individual rDNA loci (nucleolar organizers) son (15) except that the extraction and gradient buffers has been shown to be genetically determined (6, 7). For contained 5 mM sodium butyrate, 10 mM o-phenanthroline, example, the nucleolar organizer of the rDNA Aegilops 0.2% methylmethanethiosulfonate, and 0.1 mM phenylmeth- umbellulata chromosome 1 is dominant over all four Triticum ylsulfonyl fluoride. The final nuclear pellet was resuspended loci in chromosome addition lines containing this chromo- in DNase I buffer (250 mM sucrose/10 mM NaCl/10 mM some in a Triticum aestivum background (6). Since the rDNA Pipes, pH 7.0/3 mM MgCl2/3 mM CaCl2/5 mM sodium loci in these hybrids have rDNA repeats of different lengths butyrate/0.2% methylmethanethiosulfonate/0.1 mM phenyl- and distinguishable restriction patterns, molecular tests can methylsulfonyl fluoride/10 mM o-phenanthroline/5 mM 2- be designed to examine features of chromatin structure (e.g., mercaptoethanol) at a concentration of =250 jxg per ml of sensitivity to exogenous DNase I) and cytosine methylation DNA. ofthe DNA of each locus. Nucleolar dominance is correlated DNase I (Sigma) was added to aliquots of the nuclear with a more DNase I sensitive chromatin conformation and suspension to give final concentrations of 8.0, 2.4, 0.8, and a hypomethylation ofthe Aegilops rDNA (ref. 6; W.F.T. and R. B. Flavell, unpublished data). Abbreviations: NTS, nontranscribed spacer; L and S variants, longer It is well established in animal systems that DNase I and shorter variants of rDNA. hypersensitive sites are frequently associated with actively *Present address: Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60680. tPresent address: Department of Botany, University of Maryland, The publication costs of this article were defrayed in part by page charge College Park, MD 20742. payment. This article must therefore be hereby marked "advertisement" *Present address: Departments of Botany and Genetics, North in accordance with 18 U.S.C. §1734 solely to indicate this fact. Carolina State University, Raleigh, NC 27695. 1550 Botany: Kaufman et al. Proc. Natl. Acad. Sci. USA 84 (1987) 1551 0.2 units/ml. Incubation was for 10 min at 370C. Additional RESULTS aliquots ofnuclei were also incubated at 37TC with no enzyme (sham digest) and at 0C with no enzyme (control digest). In the garden pea, there are 3000-4000 copies of the rRNA After 10 min, the reaction was stopped by the addition of a genes (18). Most of these genes, together with spacer se- large volume of ice-cold DNase I buffer lacking both phen- quences between them, are present in large tandem arrays at ylmethylsulfonyl fluoride and methylmethanethiosulfonate. one of the two nucleolar organizer loci (19). The basic The nuclei were pelleted immediately and resuspended in a monomer repeating unit of -9 kilobases (kb) contains the small volume of the buffer. (Phenylmethylsulfonyl fluoride 18S, 5.8S, and 25S rRNA cistrons and a "nontranscribed" and methylmethanethiosulfonate were omitted at this stage to spacer (NTS) =3 kb long (Fig. 1). About one-third of each avoid interference with subsequent proteinase K treatment.) NTS is occupied by a tandem array of short (180 bp) repeated The resuspended nuclei were lysed by the addition of 1 vol of elements, called the subrepeat array, which occurs just 2x lysis buffer (200 mM Tris HCl, pH 8.0/50 mM EDTA/1 downstream from the 3' end of the 25S rRNA gene. M NaCl/2% Sarkosyl) and the lysate was treated with In many pea varieties, including cv. Alaska, two major proteinase K (0.5 mg/ml) at 550C for 1.5 hr. DNA was length classes of rDNA are observed (17, 20), and the purified by banding in cesium chloride/ethidium bromide different forms are thought to be located at different nucleolar gradients as described (15). Purified DNA was exhaustively organizers (20, 21). The number of elements in this subrepeat digested with either BamHI (Bethesda Research Laborato- array is variable both between cultivars and between loci ries) or EcoRI (Bethesda Research Laboratories) in the ionic within a cultivar. In P. sativum cv. Alaska, the longer (L) conditions recommended by the supplier. The DNA restric- variant contains nine elements per subrepeat array, while the tion fragments were size-fractionated on agarose gels, trans- shorter (S) variant subrepeat arrays contain seven elements ferred to either GeneScreen (New England Nuclear) or Nylon each. Although other variants exist (16), they are present in 66 (Schleicher & Schuell), and hybridized with the appropri- much lower copy numbers and do not contribute significantly ate DNA probe as described (15). to the data presented here. Restriction digests of genomic Bands in the hybridization profiles that occurred in either DNA indicate several differences in sequence between the L control or sham digests were discounted when mapping and S variants (17). For example, the L variant, but not the DNase I hypersensitive sites. These bands probably repre- S variant, contains two closely spaced EcoRI sites within the sent restriction fragments from minor length variants known subrepeat array. This difference in sequence and the differ- to be present at low copy number in the pea genome (16). ences in length between the two variants allow us to distin- Control experiments were conducted to be certain that the guish between them in gel blot hybridization studies. DNase I hypersensitive sites mapped reflect properties ofthe DNase I hypersensitive sites were mapped by using the chromatin complex rather than properties of naked DNA. method of indirect end-labeling (22, 23). Small hybridization Both dark-grown and light-grown seedling buds and hooks probes are used that hybridize only to one end ofthe genomic were used to prepare total cell DNA or DNA from isolated restriction fragment being mapped.