DNase I sensitivity of ribosomal RNA Genes in chromatin and nucleolar dominance in wheat

J. MOE.Biol. (1988) 204, 535-548

DNase I Sensitivity of Ribosomal RNA Genes in Chromatin and Nucleolar Dominance in Wheat W. F. Thompsont Department of Plant Biology Carnegie Institution of Washington 290 Panama Street, Stanford, CA 94305, U.S.A.

and R. B. FlavellS Department of Molecular Genetics AFRC Institute of Plant Science Research (Cambridge Laboratory), Maris Lane Trumpington, Cambridge, CB2 2LQ, U.K. (Received 8 January

1988, and in revised form 2 June 1988)

Ribosomal RNA genes at different nucleolar organizer (NOR) loci in hexaploid wheat are expressed at different levels. The degree of expression of a particular organizer depends on the genetic background, especially on the presence of other NOR loci. For example, when chromosome 1U of Aegilops umbellulata is introduced into the hexaploid wheat cultivar “Chinese Spring” the A. umbellulata NOR accounts for most of the nucleolar activity and seems to suppress the activity of the wheat NOR loci. Even in wild-type “Chinese Spring”, the NOR on chromosome 1B is partially dominant to that on chromosome 6B, since the 1B locus is more active in spite of having fewer genes. We have previously shown that these and other examples of nucleolar dominance in wheat are associated with undermethylation of cytosine residues in certain regions of the dominant rDNA. Here, we show that rRNA genes at dominant loci are organized in a chromatin conformation that renders them more sensitive to DNase I digestion than other rRNA genes. In addition, we have mapped several DNase I-hypersensitive sites in the intergenic spacer region of the rDNA repeating unit. Some of these sites are located near the initiation region for the 45 S rRNA precursor, while others are associated with a series of short direct repeats 5’ to the 45 S rRNA initiation site. The results are discussed in terms of a model in which repeated sequences in the wheat intergenic DNA are presumed to function as upstream promoters and transcriptional enhancers similar to those in Xenopus.

1. Introduction

conclusion that many plants have many more rRNA genes than are required. It follows that rRNA gene activity must be regulated by mechanisms able to maintain an appropriate level of rRNA transcription in the presence of a large excess of potential templates. Cytological evidence indicates that the activities of rRNA genes at different chromosomal loci are regulated differentially, with some loci exhibiting full or partial dominance over others. For example,

Plant cells typically have of the order of ten times more ribosomal RNA genes than animal cells (Ingle et al., 1975; Long & Dawid, 1980), and closely related plant species, races or cultivated varieties often exhibit large variations in rRNA gene number in the absence of obvious phenotypic effects (for example, see Flavell & Smith, 1974; Flavell, 1986). and extensive numbers high gene These intraspecific variation make it difficult to escape the

in the hexamoid

wheat

cultivar

“Chinese

Snring”

the nucleolar organizer (NOR)$ on chromosome I”B t Present address: Department of Botany, North Carolina State University, Raleigh, NC 27695, 1J.S.A. $ Present address: John Innes Institute, Colney Lane, Norwich. Norfolk NR4 7IJH. U.K.

4 Abbreviations used: NOR, nucleolar organizer; kb, 103 bases or base-pairs: CS, cultivar “Chinese Spring”: bp. base-pair(s). 535

002”~%836/88/930.535-14

$03.00/o

0 1988 Aradrmic Press Limited

536

W. F. Thompson

forms a nucleolus that is approximately twice as large as the nucleolus formed on chromosome 6B, even though the 6B NOR contains twice as many rRNA genes as the IB NOR (Flavell & O’Dell, 1976; Martini & Flavell, 1985). Genes at the 6B locus are not permanently inactivated, since in aneuploids lacking chromosome 1B there is a compensatory increase in the volume of the 6B nucleolus. Removal of chromosome 6B likewise leads to an increase in the volume of the IB nucleolus. These and other results (for reviews, see Flavell, 1986; Flavell et aZ., 1986) (1) establish the existence of a dosage compensation mechanism that, keeps total rRNA gene activity roughly constant, over a wide range of rRNA gene numbers, and (2) indicate that rRNA genes at different NOR loci may differ in their potential for expression when combined in the same cell. Nucleolar dominance is best known in interspecific hybrids, where a NOR from one parent may largely or completely suppress the activity of organizer(s) contributed by the other parent. Originally described in Crepis by Navashin (1934), nucleolar dominance has been documented in interspecific hybrids in many plant genera, including Hordeum, Ribes, Solanum, and the (for intergeneric (wheat x rye) hybrid Triticale reviews, see Flavell, 1986; Flavell et al., 1986). In wheat, nucleolar dominance is observed when chromosome 1U from Aegilops umbellulata is crossed into a hexaploid wheat background (Martini et al.: 1982). In the resulting addition-line plants, the 1C NOR forms a large nucleolus, while the wheat NORs form only micronucleoli. Nucleolar dominance also occurs in animals, where a particularly well-studied example is provided by the frog genus Xenopus. In the progeny of interspecific crosses between X. Eaevis and X. borealis the rRNA genes of X. laevis are dominant and, at early stages of development, there is little or no expression of t.he X. borea& rDNA (Honjo & Reeder, 1973; Cassidy & Blackler. 1974). Macleod & Bird (1982) reported that, the X. laevis genes in such interspecies hybrids are present in a chromatin conformation in which they are more sensitive to DNase I digestion than are the rRNA genes contributed by the X. borealis parent. In addition, a localized DNase I hypersensitive site was found in the X. laevis rDNA chromatin in or near a promoter-like sequence in the intergenic region upstream from the main promoter for the 40 S rRNA precursor (La Volpe et al., 1983). This site was also present in chromatin from X. borealis individuals. However, in chromatin from interspecific hybrid individuals only t.he X. laevis rDNA exhibited localized hypersensitivity. Thus, both general DNase sensitivity and at least one specific hypersensitive site correlat,e with nucleolar dominance in Xenopus interspecies hybrids. As part’ of an effort to understand the physical basis of nucleolar dominance in wheat, we have analyzed the DNase I sensitivity of rDNA at different nucleolar organizer loci in nuclei isolated

and R. B. Flavell

from euploid and aneuploid derivatives of hexaploid wheat. In the accompanying paper (Flavell et al., a correlation bet,ween 1988) we demonstrate undermethylation of cytosine residues and the expression of rRNA genes at particular loci in this species, with dominant or partially dominant) loci showing reduced methylation in comparison with recessive, inactive loci. Here we show that some of the rRNA genes at, active, dominant loci art’ organized in a chromatin conformation that renders them more sensitive t,o DNase T digest)ion t,ha,n are other rRNA genes. In addition, we have mapped several DNase I hypersensitive sites in the intergenic space region of the rDNA repeat,ing unit. Some of these sites are located near the initiation region for the 45 S rRNA precursor, while others are associated with a series of short direct) repeats occupying a region of approximately 2 kh 5’ to the 45 S rRNA initiation sit,e. Some of t,hr upstream DNase I sites correspond t,o putative upstream initiation sites mapped by M. Vincentz & K. Is. Flavell (unpublished results) in regions caont,aining sequences homologous with t,he main 45 S rRNA promoter (Barker et al., 1988; Vincentz clr Flavell. unpublished results). The results are discussed in terms of a model, based originally on work with Xenopus, in which repeated sequences in t,he wheat, intergenic DNA are presumed to fun&ion a?; upstream promoters and transcriptional enhancers. 2. Materials

and Methods

Seed of the hexaploid wheat variety “C’hinese Spring“ (CS: Sears, 1954) and the “UBD” addition line in which a pair of 117 chromosomes from Aegilops ?LmbeZZulatahave been added to t,he full complement of “Chinese Spring” chromosomes (see Martini et al., 1982) were obt’ained from the Plant Breeding Institute, Cambridge, 1r.K. They were

germinatid

in soil and the seedlings grown under

greenhouse conditions with supplementary lighting to extend the daylength. When the plants were 15 to 25 cm t,all they were harvested for isolation of nuclei. Fresh tissue (20 to 50 g), comprising the younger (lower) portions of actively growing leaves. was used in each extraction and, when possible, harvesting was carried out early in the day in order to obtain mat,erial with a, minimum of starch. Nuclei were isolated as described by Wat>son & Thompson (1986). Briefly, ether-pretreated leaves were homogenized in media containing hexylene glycol in place of sucrose, and purified by centrifugation through step gradients containing Percoll. Extraction buffers contained 1.0 M-hexylene glycol, 10 m&I-Pipes-KOH (pH 7.0). 10 mM-MgCl,, 0.1 mM-EDTA. 5 mw-mercaptoethanol. In most experiments 100 pg sonicated calf thymus DNA/ml was added to reduce the effect of endogenous nuclease activity. In a few of the later experiments reported here. endogenous nuclease activity. was even more effectively inhibit,ed by the inclusion of 10 mnr-o-phenanthroline in homogenization and gradient buffers. This compound chelates zinc. but not magnesium. ions: its use was suggested t,o us by Gordon Lark (personal caommunication). After filtration through a series of nylon meshes. the homogenate was made O.li?; (w/v) in Trit,on X-100 and layered over step gradients of 309: and 604b Perc*oll c:ont,aining 0..5“6 Triton in huffel X-100 and

DNase I Sensitivity

of rRNA Genes in Chromatin

room temperature and then at 65°C prior to several washes in 0.5 x SSC at 65°C (SCC is 0.15 M-NaCl, 0.15 Msodium citrate, pH 7.0). Autoradiography was on Kodak XAR-5 film at -70°C with 1 intensifying screen.

0.5 M-hexylene glycol. Nuclei were pelleted by centrifug&ion at approximately 200 g, resuspended in gradient buffer without Percoll, and pelleted a 2nd time through a similar step gradient. They were then suspended in DNase buffer (250 mM-sucrose, 10 mM-NaCl, 10 mw-Pipes (pH 7-O), 3 mM-MgCl,, 5 mM-mercaptoethanol; Spiker et al., 1983) and treated with bovine pancreatic DNase I Sigma. type III) as described in the Figure legends. Micrococcal nuclease (EC 3.1.31.1; Sigma, grade VI) digestion was carried out similarly, except that the digestion buffer also contained 3 mM-CaCl,. All isolation and incubation buffers contained 5 m&%-sodium butyrate and the proteaxe inhibitors phenylmethylsulfonyl flouride (0.1 mM) and methylmethanesulfonate (0.2% (w/v)). After incubation with DNase, nuclei were pelleted by a brief spin at 2OOg, washed once with ice-cold DNase buffer lacking protease inhibitors, and resuspended in the same buffer for lysis. Lysis, DNA purification, restriction enzyme digests, agarose gel electrophoresis, blotting, and hybridization were carried out essentially as described by Watson & Thompson (1986), except that the concentration of sonicated calf thymus DNA in the hybridization buffer was reduced lo-fold (to 10 fig/ml), and hybridizations done at 65°C in the absence of formamide. After hybridization, filters were washed in 2 x SSC at

“6” repeats

6

“A” repeats

E

537

3. Results (a) Structure of wheat ribosomal RNA genes Figure 1 shows maps illustrating the major rDNA variants in the wheat cultivar “Chinese Spring”, as

determined by restriction mapping experiments with genomic DNA (Appels & Dvorak, 1982, and unpublished data) and by sequence analysis of the rDNA clone pTA71 (Barker et aE., 1988). A single repeating unit of the tandemly arrayed rRNA gene cluster at each locus is illustrated. Each repeating unit is characterized by one EcoRI site and two sites for BamHI. A prominent feature of this and other plant rDNAs is the presence of short, tandemly repeated sequence elements in the intergenic spacer between the 3’ end of the 25 S rRNA gene and the 5’ end of the 18 S gene (for a review, see Flavell, 1986). In wheat, these elements

“D” repeats

_

B

HooIl

8

68-2 tl

7aqI 7bqI

roe I

I kb

6B-I 3 Top1 TuqI

(+I)

raq I

IB-2 t

t

7bqI

TaqI

IB-I fn To9 I

h4)

ToqI

IU n (+5)

Figure 1. Maps illustrating the major rDNA variants in the wheat cultivar “Chinese Spring” and the UBD chromosome addition line. A single repeating unit from a tandemly arrayed rRNA gene cluster is illustrated. Each repeating unit, approximately 9 kb long, is characterized by 1 EcoRI site and 2 sites for BarnHI. Regions encoding mature rRNAs are indicated by filled boxes. The various types of subrepeats in the intergenic region are indicated by the variously stippled boxes, while the principal transcription start site mapped by Vincentz &. Flavell (unpublished results) is indicated by the horizontal arrow above the line. The TaqI and HpaII sites shown are sites of particular interest; for both enzymes many other sites exist that are not shown in the Figure. The HpaII site is that which is most frequently unmethylated in genomic rDNA. The Tap1 sites shown define a fragment containing most of the intergenic region; the absence of the interior TapI site permits this fragment from rDNA variants on chromosome 1B of CS wheat to be distinguished from that of the otherwise similarly sized variants on chromosome 6B. For further discussion, see the text.

W. F. Thompson

*ii38

(variously called spacer repeats, subrepeats, or intergenic repeats) have been divided into several classes based on DNA sequence data (Barker et al., 1988); “A” and “B” repeats are about 135 and 150 bp in length, respectively, while the “C” repeats are approximately 170 bp and the “I)” repeats about 30 bp. All are direct repeats, and to varying degrees all four major classes of intergenic repeats are homologous to sequences near the presumed start of transcription (Vincentz & Flavell, unpublished results). The rDNA repeating units at different loci exhibit length differences attributable to differences in the number of spacer intergenic repeats. Two variants differing by about 130 bp, or the length of one A-type spacer repeat, occur on chromosome 6B in about equal proportions (Appels & Dvorak, 1982). These two variants together account for about 60% of the total rDNA in the cultivar “Chinese Spring” (Flavell & O’Dell, 1976). The major variant on chromosome IB, accounting for about 30% of the total rDNA, has the same overall length as the smaller of the two 6B forms. The larger variant on chromosome IH is present in relatively few copies, accounting for only 2 to 394, of total rDNA, or about, loo/; of the rDKA on chromosome 1B (Flavell et al., 1988). It is longer than the major IB variant by an amount equivalent to the length of four A-typo spacer repeats. Chromosomes IB and 6B account for approximately 90% of the total rDNA in the genome of CS wheat (Flavell & O’Dell, 1976). The remaining 10% on chromosomes 5D and IA is not, discussed in this paper, since these rDNA loci are relatively inactive. Figure 1 also illustrates the predominant form of rDNA on chromosome IU from A. umbellubta. This chromosome is present (together with a complete set of hexaploid wheat chromosomes) in the UBD chromosome addition line used in some of the experiments reported below. 111 rDNA accounts for about 25% of the total rDNA in this addition line. The intergenic spacer in 1 L: rDNA is even longer than that of the longest IB variant), exceeding the basic CS spacer length by an amount corresponding t,o five A-type repeats. These spacer length polymorphisms permit one to distinguish intergenic spacer fragments of different rDNA variants in BamHI or RamHT + EcoRI digests. However, such digests do not permit separation of the smaller form of rDNA on chromosome 6B from the majority of the 1B rDNA. For this purpose Tag1 digests can be used. As shown in Figure I, a TapT restriction site polymorphism increases the length of the largest TaqI fragment, obtained from IB rDNA by about O-385 kb, permitting separation of 1B fragments from 6B fragments containing the same number of spacer intergenic repeats. (b) General

features

of nuclei and chromatin

Nuclei were isolated by the hexylene glycol/ Percoll gradient procedure described in Materials

and R. B. Flavell and Methods. Wheat leaf nuclei examined in the electron microscope were similar in appearance to pea nuclei isolated by similar techniques (Watson & Thompson, 1986). The structure of the nucleoplasm and the nucleolus appears to be normal, although the Triton X-100 t’reatfment severely disrupted both inner and outer nuclear membranes, as observed for other plant nuclei (Guilfoyle et aZ.. 1986; Watson & Thompson, 1986). Partial digestion with micrococcal nuclease produced sharply defined oligonucleosome ladders similar to those of Spiker et al. (1983). A periodicity of 180 bp per nucleosome was obtained both from stained gels and from blots hybridized wit,h an rDNA probe (data not shown). Thus, the average nucleosome spacing in wheat leaf chromatin is similar to that, in a wide variety of organisms (Eissenberg et al., 1985; Reeves, 1984: Spiker, 1984). and the average spacing for the majority of the rDNA is not detectably different from that) of total chromatin. In experiments designed t,o assay DNase 1 sensitivity, port’ions of isolated nuclei were digested with increasing amounts of DNase I and/or with a const,ant amount of enzyme for various t.imes. DNA4 was then purified from each portion. restrict,etl, fractionated by electrophoresis on agarose gels and blotted on to nitrocellulose. To check for thra expected differential sensitivity of DNA emboding active genes. such blots were hybridized with a cDNA probe (~2437; Baulcombe &, Buffard. 1983) that encodes an mRNA known to br present a.1 moderat,el,v high levels in wheat leaf cells. Haulcombe et nl. (1987) have shown that this cDiSA has sequence homology t)o yeast carhoxypeptidase 1’. and that it represents a gene present at, low copy number (possibly a single copy) in each of thr t,hree genomes of hexaploid wheat. Figure 2 shows t,hat# most of the DNA complementary to ~2437 in each of the three genomes disappears at low uoncaen t,ra,tions of DNase T. Probing the same blot-s with an rDNA probe showed that, bulk rDNA in these nuclear preparat~ions was much more stable> tcr DNase digestion. Thus. the majority of the DNA sequences complementary to 1~2437 are contained in chromatin that is highly accessible to I)Nase il while the hulk of the ribosomal RSA genes ar(h contained in chromatin t)hat is relatively resist,ant t,o digestion wit,h this enzyme, In this and all our other experiment’s, the degrrp of digestion of bulk rDNA closely approximates that for total nuclear DNA as monitortld 1)~. ethidium staining of the gels (daba not shown). Ribosomal RNA genes in Tetrahymevw Dictyosteliwa and several animal systems have been reported t’o be differentially sensitive t,o DNase 1 (for example, see Mathis & Gorovsky. 1978; Parish et aE., 1986; Bird et al., 1981; Macleod & Bird, 1982). However, the lack of differential sensitivity for bulk rDNA in wheat, is consistent with previous observations on plant) rDNA (Leber & Hemleben. 1979; Murray & Thompson, 1981). Differential DNase I sensitivity in a small subset of’ the 5000 to 10,000 rRNA genes present in higher plant, genomrhs

DNase Z Sensitivity

of rRNA Genes in Chromatin

forms a very large nucleolus, and the nucleoli formed at the wheat NORs are greatly reduced in size compared to those in euploid wheat (Martini & Flavell, 1985; Martini et al., 1982). Since the A. umbellulata 1U rDNA repeats contain longer intergenic spacers than any of the predominant forms of wheat rDNA, it is possible to distinguish the 1U rDNA from the wheat rDNA in this addition line. Thus, we were able to ask whether the 1U chromosome, which appears to account for the vast majority of nucleolar activity in the addition line, contains rDNA chromatin more sensitive to DNase I than that of the wheat chromosomes in the same cells. Results from two sets of experiments with CS+ 1U addition-line DNA are shown in Figure 3. In both cases nuclei from leaves were isolated and portions digested with increasing concentrations of DNase I prior to purification of the DNA. In Figure 3(a) the resulting DNAs were digested with

Units DNase I /ml: (a)

(b) 9.0-

BamHI

Figure 2. Comparison of DNase I sensitivity for a protein-encoding gene that is transcribed in wheat leaves with that of bulk rDNA. Portions of CS wheat leaf nuclei were incubated with the indicated concentrations of DNase I for 36 min at 15°C prior to purification of DNA. Equal amounts of each DNA were then digested to completion with BarnHI and fractionated by electrophoresis on a neutral agarose gel. (a) Nitrocellulose blot of this gel hybridized with nick-translated insert from ~2437, a cDNA clone isolated by D. Baulcombe that represents a polyadenylated RNA known to be present in wheat leaves. (b) The same blot after rehybridization with the rDNA probe, pTA71. Exposure time for ~2437 was 8 days. while that for pTA71 was 6 h. The 3 principal bands seen with ~2437 are attributable to sequences homologous to ~2437 in each of the 3 genomes of hexaploid wheat, and each band probably represents sequences present only once or twice in a given genome (see the text.).

would

be difficult

to detect in any of the plant

rDNA

experiments

reported

wished to subsets of advantage repeat’ing distinguish

539

to date.

Therefore

we

investigate the DNase I sensitivity of rRNA genes at different NOR loci, taking of the length differences between the units at different loci that allow us to them by gel blot analysis.

(c) DNase Z sensitivity is correlated with rRNA gene activity in an addition line containing chromosome 1rJ from A. umbellulata Cytological observations on wheat plants in which chromosome 1U from A. umbellulata has been added to the CS wheat background by plantbreeding techniques have shown that the nucleolar organizer on the 1U chromosome is dominant to those on wheat chromosomes in the same nucleus. In such addition lines, the A. umbellulata NOR

prior

to analysis,

while

Figure

3(b) shows a

different set of DNAs analyzed after double digestion with BamHI and EcoRI. In BamHI digests, the 5.7 kb band is observed only in the addition-line DNAs and is thus specific to the 1U rDNA. The corresponding BamHI fragments of wheat rDNA form a doublet at about 5.3 kb, while both the Aegilops and the wheat rDNA contain a 3.7 kb fragment. The band at 9 kb in these experiments probably arises from incomplete digestion at the BamHI site in the 25 S rRNA gene. The failure of some copies of the rDNA repeat to cut with BamHI at this site may reflect cytosine methylation in the CG dinucleotide formed when the 3’ end of the BamHI site (GGATCC) occurs adjacent to a guanosine residue (Siegel & Kolacz, 1983). Incomplete digestion at this site, but not at the BamHI site in the 18 S gene, has also been observed in pea (Jorgensen et al., 1987) and pumpkin (Sigel & Kolacz, 1983) rDNAs. The band pattern is more complex in the double digests, but again one fragment, at 4.8 kb, is specific to the 1U rDNA, and the same 3.7 kb to both wheat and fragment is common A. umbellulata genes. However, in this case the wheat rDNA bands from the intergenic region are resolved into fragments of about 4.4 and 4.5 kb. The 4.4 kb variants are found on chromosome 1R as well as in some of the genes on chromosome 6B, while the 4.5 kb fragments come from a longer spacer length

variant

on chromosome

6B. The band

at 4.6 kb is presumed to arise from incomplete digestion attributable to methylation at the BamHT site in the wheat 25 S rRNA gene, as discussed above in connection with BamHI single digests. In contrast, the A. umbeklata 1U rDNA in these addition-line plants is completely digested by BamHI.

Figure 3 shows that DNase I digestion of fragments specific to the A. umbellulata 1U rDNA proceeds more rapidly than that of the other fragments. Scans of the autoradiograms (Fig. 3(c)) suggest that most of this differential digestion

540

W. F. Thompson and R. B. FlavelE

(a)

Cc) Minutes

of incubatmn I-

CS

I+

2+

4+

S+

16+

32+

32-

CS

(b) Untts

DNase 0

I/ml

0.001

0.003

0.01

0.03

0.10

0.30

cs

Mlgratton

--+

Figure 3. Sensitivity of Aegilops and I’riticum rDNA in the UBD chromosome addition line containing A wnbellulata chromosome 1U in a normal CS wheat background. (a) Isolated nuclei were incubated with ( +) or without (- ) 3 unit,s DNase I/ml at 37°C for the indicated number of minutes prior to lysis and DNA purification. Equal amounts of each DNA preparation were then digested with BumHI, electrophoresed, and blotted t,o nitrocellulose. The blot was then hybridized with 32P-labeled pTA71. Portions of CS DNA from untreated nuclei were included for comparison. (b) A similar experiment was carried out with a different set of UBD leaf nuclei. In this case the nuclease treatments were carried out at 15°C for 60 min with the indicated concentrations of DNase I and the purified DNA samples were digested with both BamHI and EcoRI prior to electrophoresis. (c) Densitometric scans of an autoradiogram from a shorter exposure of the same blot used for Fig. 3(b). Scans are shown for DBase concentrations of (from top to bottom) 0, 0903. 0.01, and 0.1 units/ml.

occurs at low DNase I inputs. Additional digestion does not markedly change the relative proportions of the 1Uspecific fragments to the other fragments.

Thus, we conclude that a significant portion, but not all, of the 1U rDNA is in chromatin that is differentially sensitive to DNase I digestion. We cannot determine whether or not the II; rDNA in the 3.6 kb BamHI-EcoRI fragment is also subject. to differential DNase 1 digestion. Figure 3 shows no evidence of differential sensitivity in the 3.6 kb band. However, since the 1U chromosome contains only about 20% of the total rDNA in these plants (Martini et al., 1982), and not all 111 rRNA genes are in sensitive chromatin, any differentially sensitive 1U chromatin would probably contribute less than 10% of the DNA in the 3.6 kb band. Preferential digestion of such a small fraction would not be detected in Figure 3. To ensure that the differential sensitivity of the lU-specific

chromatin

bands

reflects

a

property

of

the

complex rather than special sensitivity

of the 1U DNA

sequences

per se, we repeated

the

DNase I digestion series using purified DNA in place of isolated nuclei. Figure 4 shows that, when

purified DNA was digested, the IL-specific band remained in approximately the same ratio t,o the wheat bands as in undigested caontrols, whih differential digestion of this band was again observed in isolat)ed nuclei. Thus. difl’erential sensitivity of the 1U rDNA is related t,o structures present in isolated nuclei t’hat are removed or destroyed during DNA purification (d) Gene variants with longer intergenic spacers have, greater IlNase I sensitivity Having

established

t,hat

at

least

some

of thrb

A. umbellulata 1C genes were present in a nucleasesensitive

chromatin

conformation,

we wished

t,o

look for possible differences in sensitivity within hhe population of genes on the 1IJ chromosome. When DNA from the IJRD addition line is cut with HpaTl in addition to BamHI and EcoR,I, three rDNA fragments are observed in high stoichiomet,ry among the ffpaI1 products. These fragments arise from cutting by @pa11 at preferentially unmethj;lated sites in the intergenic spacer, and t~helr different

lengths

reflect

t,he

presence

of

three

DNase I Sensitivity NUCLEI c

I

2

of rRNA Genes in Chromatin

the intensity of this band relat,ive to that of the other two. Although the data from such HpaII digests concern only that subset of genes with an unmethylated CG at this particular HpaII site, these observations nevertheless strongly support our previous conclusion from Figure 3 that only a subset of the 1U rRNA genes are differentially sensitive to DNase I, and further that the longer variant form contains the highest proportion of DNase-sensitive genes. The difference in sensitivity between long and short rDNA variants on the 1U chromosome is paralleled by differences in sensitivity of length variants within the rDNA complement of CS wheat itself. In Figure 6 we present data obtained from TaqI digests. As originally shown by Appels & Dvorak (1982), TaqI sites are frequent throughout wheat rDNA, except in the central portion of the intergenic spacer. Thus, most of the spacer can be cut out as a single fragment, larger than any of the TaqI fragments (see Fig. 1). As illustrated in Figure 6, TuqI digests of CS wheat reveal four size classes of large fragments. Studies with aneuploid lines have shown that the 3.55 and 3.1 kb fragments are derived from chromosome lB, while the 2.8 and 2.6 kb bands are carried on chromosome 6B. The 1B genes lack a TuqI site that is present in the spacer of the 6B genes (see Fig. 1); therefore the 3.1 kb fragments are derived from genes with the same spacer length as the 2.6 kb 6B gene. Only the

DNA 3

c

I

1

NUCLEI

Migration

541

minor

-

1B variant,

with

its four

extra

intergenic

Figure 4. Differential DNase I sensitivity of AegiZops rDNA is seen in nuclei but not in isolated DNA. Nuclei from leaves of UBD seedlings were isolated and treated with increasing concentrations of DNase as described for Fig. 2(b). DNA was purified from all samples. Several portions of the control sample (not previously exposed to DNase I) were digested with different concentrations of DNase I and the DNase I was inactivated by heating at 60°C. All samples were then restricted with BarnHI, electrophoresed, blotted, and hybridized with pTA71, as described for Fig. 3. An autoradiogram of the blot is present’ed in the top panel and scans of the lanes “1” (representing about 50% digestion of the 5.3 kb Triticum band in each case) are presented in the bottom panel. DNase treatments for lanes 1 were 0403 units/ml for 60 min at 15°C for nuclei and 0.001 units/ml for 12 min at 37°C in the case of purified DNA.

repeats, is actually longer than either of the 6B forms. From the data in Figure 6 it is clear that the Tag1 spacer fragment of the minor 1B variant is much more sensitive to DNase I than is the same region of the predominant, but shorter, variant at the same locus. Additional experiments indicate that the longer of the two 6B fragments is also somewhat more sensitive than its shorter counterpart. An indication of this differential sensitivity can be seen in Figure 7 (compare panels (1) and (3)) and a similar difference has been seen in a nullisomic stock lacking any 1B rRNA genes (data not shown). The DNase sensitivity difference

different 1U rDNA spacer length variants (3.2, 2.95, and 2.75 kb; Flavell et al., 1988). None of these three EcoRI-HpaII fragments is derived from the wheat rDNA present in the UBD nuclei, since they do not comigrate with fragments from CS DNA (2.9

sensitivity in rDNA variants with longer intergenic spacers. Given an equal per-nucleotide probability of cutting by DNase I, longer fragments would be cut more rapidly even in the absence of any difference in chromatin conformation. Such an effect should be strictly proportional to length, however, and the fragments under consideration here are very close in size. In Figures 3 and 4 for example, the sensitive fragment from the IU chromosome of

and 2.7 kb). In addition, we show in the accompanying paper (Flavell et al., 1988) that the presence of the 1U chromosome in UBD plants produces such a high degree of CCGG methylation in the wheat rDNA loci that HpaII cleavage becomes undetectable. Figure 5 shows that the longest of these 1U variants is preferentially sensitive to DNase I in isolated UBD nuclei, since as DNase

digestion

proceeds

there

is a decrease

in

between the 6B variants is not as large as that between the two 1B variants, or as that between the A. umbellulata 1U and wheat rDNA. However, it is consistent with the general pattern of greater

A. umbelzulata is less than 10% larger than the wheat rDNA fragments against which it is being compared. In Figure 5 the HpaI1 fragments differ by about 15%, while in Figure 6 t’he Tag1 fragment

W. F. Thompson and R. B. Flavell

542 Umts DNcse I/mi

IO

Hpon

B

t

0.3 -

+

+

f

E

cs

0 -

-

t

t

f

HpolI

El

B

Figure 5. Differential DNase I sensitivity of longer variants of Aegilopa rDNA in the UBD addition line. DKA from nuclei keated with the indicated concentrations of DNase I was purified and incubated with ( + ) or without ( - ) HpaII buffer at 37°C for 6.5 h prior to overnight digestion with BamHI and EcoRI. The bands below the major rDNA bands result from HpaII cutting at the most frequently unmethylated HpnII site, as indicated in the map below the Figure. The 3 different sizes are derived from variants of Aegilopa rDNA that differ in the number of elements in the subrepeat array located between the HpaII and EcoRI sites (see Fig. 1). This and all other HpaII sites are methylated in essentially all copies of the Triticum rDNA in this addition line (Flavell et aE.. 1988), so in UBD DNA these bands are derived only from Aegdops rDNA. (Note that in CS DNA, illustrated on the right, there are only 2 HpuII bands and both migrate slightly faster than the corresponding bands in the URD lanes.) For further discussion, see t,he text. B. RumHI; E, EcoRI.

Units

DNase I/ml

0.02

0.06

02

06

2

0

0

HPOII

‘“1.

,-

,h,

ruq I

(+I)

Taq 1 (0)

3.7

i

I 2 Jnits DNase ; IT, ih!

Figure 6. Differential DNase I sensitivity of a minor rDNA variant at the chromosome 1B NOR in CS wheat. DSA samples prepared from leaf nuclei incubated for 30 min at 15°C with the indicated concentrations of DNase T were digested with TuqI and fractionated on a 1.6% neutral agarose gel. Blots were hybridized with 32P-labeled pTA71. (a) An intermediate autoradiographic exposure is shown, together with maps illustrating the differences between the largest Tug1 fragments of the major rDNA variants at NOR loci on chromosomes 1B and 6B. (b) A plot of data obtained b? densitometric scanning of longer (for lB-1) or shorter (for lB-2, 6B-1 and 6B-2) autoradiographic exposures of the blot shown in (a).

DNase

I Sensitivity

of rRNA

1 (4)

w

Migration

Ir )

Figure 7. Effect of prior DNase I digestion in nuclei on sensitivity of DNase-resistant rDNA sequences. DNA from control (( 1) and (2)) and DNase-treated ((3) and (4)) nuclei was incubated in HpaII buffer with ((2) and (4)) or without ((1) and (3)) HpaII and was then digested to completion at 65°C with TapI. For the samples illustrat,ed, DNase treatment was for 16 min at 37°C and 2 units/ml. The 2 lower molecular weight bands correspond to the 2 length variants on chromosome 6B, while the largest band is the major variant (lB-2) on chromosome 1B. The minor variant on chromosome 1B (lB-1; cf. Fig. 6) is too faint to detect at exposures suitable for scanning the major bands. HpuII

from t,he minor 1B variant is about 15% longer than its counterpart from the major 1B variant. Since these length differences are very small in relation to the large differences we see in DNase I sensitivity (and since, as already noted, we do not observe any differential sensitivity of different variants when digesting purified DNA), we conclude that our DNase I sensitivity measurements reflect differences in the chromatin conformation rather than simply a “target size” effect. (e) DNase I sensitivity is associated with undermethylation

at CCGG sites

We have shown that there is a correlation between rRNA gene activity and the methylation status of the rDNA as measured by digestion of purified DNA with methylation-sensitive restriction enzymes (Flavell et al., 1983, 1988). Although digestion of CS wheat DNA with MspI reduces all the rDNA to small fragments (~2 kb, and mostly much smaller), a very large fraction is fully resistant to cleavage by HpaII. Since HpaII is known to be inhibited by methylation of the internal cytosine residue in the sequence CCGG while MspT recognizes the same sequence but is

Genes in Chromatin

543

unaffected by methylation of the internal cytosine (McClelland & Nelson, 1985), we infer that much of the rDNA is methylated at the internal cytosine residue in most of its CCGG sequences. However, a fraction of the rDNA can be cut at one or more sites by HpaII, indicating that not all CCGG sites are methylated in all copies of the rDNA. For any given rDNA variant class, the fraction sensitive to digestion (i.e. cut at least once) by HpaII is related to the activity of the corresponding nucleolar organizer as estimated from nucleolar volume measurements (Flavell et al., 1988). Against this background, it was of interest to determine whether or not the same sets of genes were simultaneously DNase I-sensitive and undermethylated. The hypothesis that DNase-sensitive genes are also less methylated would predict that the proportion of rDNA restriction fragments that are fully methylated should be greater in DNA derived from DNase-treated nuclei, since the most nuclease-sensitive rDNA sequences should have been at least partially degraded by DNase I. We attempted to test this hypothesis by comparing the intensities of rDNA bands in BamHI or EcoRI digests with and without additional HpaII digestion. In all cases we observed some cutting by HpaII even after extensive DNase I treatment, indicating that some unmethylated HpaII sites are present even in the most DNase-resistant rDNA. This approach does not permit us to distinguish between HpaII cuts in different regions of the rDNA repeat, however, and the effects of a specific undermethylation in a particular region would be easily obscured by a relatively small number of unmethylated CCGG sites distributed randomly among a large number of rDNA repeats. Indeed, by focussing on the intergenic spacer region rather than on the entire rDNA repeat unit, we do see the reductions in HpaII sensitivity in DNase-treated samples that would be predicted if the same set of and undergenes is both DNase sensitive methylated. Such an experiment is shown in Figure 7, in which we have again taken advantage of the fact that the largest Tag1 fragments from wheat rDNA are derived entirely from the major intergenic spacer (see Fig. 1 and Appels & Dvorak, 1982). If the genes that are most sensitive to DNase I are also the least methylated, we would predict that HpaII digestion would have a much greater effect on the ratios of band intensities in DNA from control nuclei than it would have in DNA from DNase-treated nuclei. Figure 7 shows this is the case. Both the 1B and larger 6B TaqI fragments are more sensitive than tJhe smaller 6B fragment to DNase I alone (compare panels ( 1) and (3)) or to HpaII alone (compare panels (1) and (2)); when HpaII is used on DNA from nuclei treated with DNase I it has relatively little additional effect (compare panels (3) and (4)). We interpret these data as indicating that, although unmethylated HpaII sites exist in many genes besides those which are sensitive to DNase I, DNase sensitivity defines

W. 4’. Thompson and R. B. Flavell

544

a subset of genes whose intergenic spacer sequences are significantly less methylated than bulk rDNA. (f) Discrete sites in the intergenic region are hypersensitive to DNase I In most of the DNase I cleavage experiments reported above it was possible, at least on longer autoradiographic exposures, to see a set of discrete bands migrating faster than the major rDNA restriction fragments. We suspected that these small fragments might reflect cutting by DNase I at discrete hypersensitive sites. This hypothesis has been confirmed by experiments with small indirect end-labeling probes that permit us to detect these sites with greater sensitivity and to map their positions in relation to known restriction sites in the rDNA repeats. We present below an analysis of such sites in the intergenic spacer. The probes used were BE900, a 0.9 kb BarnHI-EcoRI subfragment located near the 3’ end of the wheat 25 S rRNA gene, and XB380, a 380 bp XbaIBamHI fragment derived from the 5’ end of the pea 18 S rRNA gene. BE900 was obtained from pTA71.25 (a subclone in pUC8 of the BE900 fragment from pTA71), and XB380 was isolated BE900 Units DNase I /ml 0,02 0.06 0.2

0.6

from pHBl.3 (a cloned 1.3 kb fragment from Pisum satiwum rDNA described by Watson et al. (1987)). The sequence of the 18 S rRNA gene is known to be highly conserved (Appels & Dvorak. 1982: Eckenrode et al., 1985; Messing et al., 1984), and the two restriction sites defining XB380 are conserved in wheat and pea (Jorgensen et al., 1987). In BamHI-digested wheat, nuclear DKA BE900 hybridizes to the left-hand end of the BamHT fragment’ containing the intergenic spacer (see map in Fig. 8). DNase cleavages within this fragment. but, outside of the region corresponding to t!he probe, will produce hybridizing fragments between 0.9 and 4.8 kb in length; such fragments must have one end at t,he left-hand BamHI site and the other end at, a site of 1)Nase cleavage. Similarly. fragment’s between 0.38 and 53 kb in length and hybridizing to XB380 must extend from the righthand BamHT site leftward to a DNase cleavage s&e. Figure 8 shows examples of hybridization profiles for both probes. Tt is clear that a substantial number of rather discrete hypersensitive sites exist in the intergenic spacer. Although not’ all sites are visualized equally well by each probe, the two maps agree quite well m the region in which they overlap and we feel confident that the data provide a good Xi3380

probe

2.4

0

linits 002

HpoIl

DNase I/ml, 0,06 0.2

probe ‘3.6

2.4

b/pan

5.3 -

5.3

3.7

I.6

XB380

6

E

Hpan

B

13

Figure 8. Mapping DNase I hypersensitive sites in C6 rDNA by indirect end-labeling. BamHI digests

of DNA preparations from nuclei treated with the indicated concentrations of DNase I (30 min at 15°C) were fractionated by electrophoresis and blotted to nitrocellulose. Blots were hybridized with nick-translated fragments isolated from wheat or pea rDNA subclones as described in Materials and Methods and indicated in the maps below the Figure. The positions of hypersensitive sites are indicated by the unlabeled lines on the right-hand sides of the autoradiograms and by the inverted triangles in the maps corresponding to each probe. For comparison the map of the entire rDNA repeat at the bottom of the Figure shows the locations of intergenic repeats, the most frequently unmethylated H&I site, and the presumed transcription start (based on S, mapping) as determined by Barker et al. (1988), Flavell it al. (1988). and Vincentz & Flavell (unpublished results). For further discussion, see the text. B. BarnHI: E. EcoRI.

DNase I Sensitivity

of rRNA Genes in Chromatin

representation of site distribution in this region of CS wheat rDNA. In other experiments (not shown) we have established that similar but not identical’ patterns are observed for rDNA from N6BT6A plants, and that discrete bands are not produced when purified DNA is digested with DNase I under conditions identical with those in which bands are produced by digesting isolated nuclei. Hypersensitive sites are found to be associated with intergenic repeats in the intergenic spacer, 5’ to the site at which data of Vincentz t Flavell (unpublished results) have suggested transcription is initiated. Eleven DNase I sites occur regularly at intervals of 130 to 140 bp in this region. This periodicity is in excellent agreement with the 135 bp periodicity of the A-type subrepeats as determined from sequence analysis (Barker et al., 1988). Further upstream, there are three additional sites that appear to be spaced at slightly longer intervals; these probably correspond to the 150 bp B-type repeats described by Barker et al. (1988). We believe that each intergenic repeat element has the potential to form a DNase I hypersensitive site under appropriate conditions. In addition, we find two other prominent sites between the subrepeat array and the beginning of the 18 S rRNA coding region. One is located just upstream, and the other just downstream, from the presumed transcription start (Fig. 8). Our data average together the profiles of hundreds of rDNA repeating units, and the cleavage sites we have mapped are not necessarily all exposed in each unit. However, the fraction of rDNA cut at discrete sites (as opposed to being randomly degraded) seems relatively high, and the principal effect of increasing DNase I digestion in experiments such as that in Figure 8 seems to be to shift DNA from higher molecular weight bands to discrete bands of lower molecular weight. We postulate, therefore, that most of the DNase I cuts in all our experiments occur at a limited number of specific hypersensitive sites rather than randomly throughout the rDNA.

4. Discussion (a) Functional

significance of DNase hypersensitive sites

Since the DNase I sensitivity of rDNA at different chromosomal loci closely parallels previously described differences in nucleolar activity, we believe that a DNase-sensitive chromatin activity or conformation is associated with potential for activity of particular groups of rRNA genes in vivo. From the results in Figure 8 we believe that much of the DNase digestion occurring in experiments such as that in Figure 3 is occurring at many discrete sites of hypersensitivity in the intergenic DNA. This conclusion is consistent with findings for many animal genes that localized sites of DNase hypersensitivity are associated with potential or actual gene expression (e.g. Eissenberg

545

et al., 1985), but is one of only a few such observations reported to date for plant systems (Steinmiiller et al., 1986; Kaufman et al., 1987; Paul et al., 1987; Wurtzel et al., 1987). There are striking parallels between the locations of DNase hypersensitive sites and the sequence organization of the intergenic DNA. Barker et al. (1988) report the complete nucleotide sequence of this region and discuss its organization into several regions containing repeated motifs. In particular, there is a large array of 135 bp direct repeats (A repeats) near the center of the intergenic region, and a set of three similar sequences (B repeats) upstream from the A repeat array. Elsewhere (Barker et al., 1988; Vincentz & Flavell, unpublished results) we have discussed evidence that the A and B repeats include sequences related to those around the principal transcription initiation site for the 45 S rRNA precursor. Figure 8 shows DNase I hypersensitive sites mapping in each of the B repeats and an array of sites associated with the A repeat array. We have mapped 11 sites associated with A repeats. There are 12 A repeats in the intergenic region of the plasmid clone pTA71 sequenced by Barker et al. (1988), which probably contains a gene belonging to the 6B-1 variant class. However the 6B-2 and lB-2 variants (see Fig. 1) have only 11 A repeats, and these two variants account for a majority of the genes in the genome of CS wheat. Thus it seems likely that each of the A repeats has the potential to form a DNase I hypersensitive site. Presumably most genes contain only one or a few A repeats in the hypersensitive conformation at any one time, although as discussed further below we believe that active genes or genes in the nucleolus with the potential to become active probably have a higher percentage of their intergenic repeats in a hypersensitive conformation. It is noteworthy that DNase cutting in the B repeats occurs at lower DNase I concentrations and perhaps somewhat more frequently than cutting at individual A repeats. The B repeats contain more extensive homology to the initiation region, and nuclease S, mapping has revealed the presence of several transcripts whose 5’ ends map to the B repeat region (Vincentz & Flavell, unpublished results). Thus, these repeats are comparable to the spacer promoters in Xenopus (Reeder, 1984; Moss et al., 1985; DeWinter & Moss, 1986), and the prominent DNase I hypersensitive sites in the wheat B repeats may be compared to the major DNase I site reported by La Volpe et al. (1983) in the upstream spacer promoter of Xenopus rDNA. Further, the hypersensitive sites in the A repeat array may be compared to the region of less discrete DNase I sensitivity observed by La Volpe et al. in the array of 60/81 bp enhancer-like elements downstream from the Xenopus spacer promoter. The array of discrete sites in wheat rDNA can also be compared to similar arrays of discrete sites, also associated with intergenic repeated sequence arrays, in the rDNA chromatin of Drosophila melanogaster

546

u’. F. Thompson

(Udvardy et al., 1984) and Pisum sativum (Kaufman et al., 1987). The Drosophila rrpcats cont,ain sequences homologous t,o those around the start site for t’he major transcription rRXA precursor and can initiat,e transcription both in vitro (Kohorn & Rae. 1982) and irr GPO (Murtif & Rae, 1985). Two other DIXasr I sites were mapped downstream from the A repeat array. One is locxat>ed near t’he start of the 45 S rR)NA transcaript and another is located further downstrea,m within t*he 46 S precursor sequence. The site within the 4.5 S precursor is within the more 5’ of t’hc two 172/l 74 bp repeats designated by Barker it nl. (1988) as the CI repeats. These repeats also have homology to sequences in the initiat,ion region, alt,hough it) is less ext,ensive t’han that in the B or A repeats. There is a short region of sequence within the (’ repeats that is very different between the two copies. and it is possible t’hat the DNase sensit,ive site in the 5’ copy may be with dyad associated with a 23 bp sequence symmetry that is present in this copy but not t.he other. The I)Nase 1 site near the start of the 45 S transcript is also c~lose to a pair of 30 bp repeat> sequences designated 1) repeats by t3arkc.r et (~1. (1988). We do not believe this sitfl actually lies within a I> repeat’, but the limit.rd resolution of agarose gel c,lrc,trol,horesis does not permit) a definitive conc~lusion on t’his point. However, the 1)Nasc I cut is c~lt~arly within a 170 bp stret,ch of Dr\‘A upstream from the t’ranscription initiat)ion sit,e t)hat differs from the rest of the int.ergenic I)NA in that it contains (‘p(: dinuc+ot~ides much less frequently t,han C:p(‘ dinuclrot~ides (Barker et nl.. 19X8). Kaufman et nl. (1987) observed changes in the pattern of I)Nase hypersensitivity in pea rI)NA chromatin that suggest that the presr>nce of certain sites is closely asso&tcd with rRNA gene ac$ivit,J in developing pea buds. An array of discrete sites comparable to the sites associat)ed with A repeats in wheat r1)SX was seen in both of the two major rDXVA variants in hoi h light and dark-grown pea buds. However. three sites were seen in one variant, only. in the actively growing buds of illuminat,cd plants. buds in which rRSA synthesis is proceeding at a high rate. These three dcvt~lopmer~tally variable sites were in posit)ions c*ornparable t)o t,he positions of thr wheat sit.es discussed in the preceding paragraph, which arc’ loc~atcd just 5’ and 3’ to the start of t’ranscriptiori. I)N* ase I hyprrsrnsit’ive To summarize. sit,es in \vheat, rl)NX arc’ associated \vit,h sprcifca sequence motifs that are homologous to sequences in the t ransrription init iat ion region: t’here are (alose parallels between the wheat. l)ro.sophil*. and Xcnopzrs systems in terms of srquence organization and I)Nasr sensitivity patterns: a remarkable overall similarit,! also rxist,s brtwreri the srqu~ri~~e organization and I)Nase I cautt ing-patt’erns iii wheat ittld p(‘a rl)NA: and drvrloprnent~al changes are obsrrvrd at I)Naw I sites in pea rDS.4

and R. B. Flavell corresponding to the most promoter-proximal sites in wheat. We believe these different lines of evidence, together with the observation by Vincent2 and Flavell (unpublished results) that the 5’ ends of several intergenic transcripts map near the DNase T sites in the B repeats, provide st,rong rircumstantial evidence that DNase I hypersensitive sites in the intergenic region of wheat rDNA reflect DNA-protein interactions important to rRSA gene expression and involve DXA sequences with promoter or enhancer-like activities. DNase hypersensitive sites may be creat’ed by changes in DNA conformation or alterations in nuclrosomr structure resulting from the binding of factors related t,o transcriptional activity (Elgin, 1984; Weintraub, 1985: Eissenberg et al., 1985). Since. as will be discussed further below. the DNA sequences at hypersensitive sites contain homologies to the main rR,NA promoter, one viable possibility is that they are created by the binding of RSA polyrnerase I transcription complexes. or of some factor necessary to the format’ion of such complexes. Among the possible accessorv fact,ors topoisomerase I deserves serious coniideration (Sollner-Webb & Tower, 1986). Topoisomerase T has been shown to be concentrated in t’he nucleolus (Muller et al., 1985) and some type of topoisomerase activity is likely t20 be required for transcription of rI)SA in r,i~o (Brill et al., 1987: Garg et al., 1987). l>Nase I hypersensitive sites have been described at topoisomerasr 1 binding sites in t’he ribosomal RXA genes of several organisms (Bonven et al.. 1985: Anderson et nl., 1985; Ness et nl.. 1986). Topoisomerase act,ivity may create localized changes in the degree of supercoiling and torsional strain that may facailitate transcription either directly or by influencing the binding of factors required for transcriptional initiat’ion. (b) DBase

wnsiticCty

an,d nuclrolar

dominnnw

DNase sensitive sites are not dist,ributed at randotn bet,ween rRXA genes at different loci, since not all loci are equally sensitive to I)Nasr treatment. In the 1 I’ addition line t)hc 1 V rDR’A is much more sensitive than either of the major wheat) rDXA variant’s, while in (X wheat the 1 R locus contains more DNasr-sensitive genes than does the 6B locus. Sensitivity thus decreases in the order II’ > 1 I3 > 6K. The freyuency of unmrthylatcd CCGC: sites in the rT>NA of t,hese loci follows t,he same pattern. with the 1 LT genes of addition-line plantas containing t’hr highest’ proportion of unmrthylated sites and a similar relationship between the 1B and 6B loci in euploid CK plants (Flavell et nl.. 1988). Sincae activity of t,hr different loci varies in the same manner, we caoncludr t,hat the loci that are the most) active in a particular genetic background also have more genes with unmethylatcd (XXX: sit*rs and a DSase T-sensitive chromatin conformation. The differences we have observed between loci may reflecat differences in the number of hypersensitive sit’rs per gene. (iiven an equal probability of

DNase I Sensitivity

of P-RNA Genes in Chromatin

cleavage at each site, genes with more sites should be cleaved more rapidly. Figure 8 shows that discrete bands are observed over a wide range of DNase concentrations. Assuming the probability of DNase cleavage to be the same for all sites regardless of their location, DNase cuts should appear first in a population of genes with more sites accessible to the enzyme. Thus, we believe the pattern of bands we see at low levels of digestion is attributable primarily to cutting within a population of genes containing many hypersensitive sites per gene, while that at higher levels of digestion reflects cutting at rare hypersensitive sites in the remainder of the population. The differential sensitivity of genes at active loci in experiments such as those of Figure 3 would then be explained by assuming the hypersensitive sites we observe in Figure 8 reflect the binding of factors that enhance transcription, so that genes with more factors have more sites and greater potential for transcriptional activity. These findings are consistent with a model, developed originally from findings in Xerwpus (Reeder, 1984; Moss et al., 1985), suggesting that genes with more intergenic repeats compete more effectively for factors present in limiting concentration and required for transcriptional competence or activity. This competition would be the basis for nucleolar dominance, since rDNA loci able to sequester a large proportion of the available factor would effectively suppress the activity of other loci in the same cell. Among the wheat rDNA variants under discussion here, that on the 1U chromosome has the largest number of intergenic repeats. The rRNA genes at this locus show much greater nucleolusforming activity, as well as a much higher sensitivity to DNase I and lower degree of methylation than other genes in the addition-line plants. The 111 NOR also appears to suppress the activity of the wheat NOR loci in these plants, since they form only micronucleoli and exhibit higher levels of methylation than their counterparts in euploid CS plants. In normal hexaploid CS plants the NOR on chromosome IB locus displays partial dominance over the 6B NOR, since the nucleolus formed on chromosome 1B is roughly twice as big as that on chromosome 6B, and again this activity difference is paralleled by differences in DNase sensitivity and methylation. This is true in spite of the fact that the 6B NOR contains roughly twice as many genes as the 1B NOR (Martini et al., 1982), indicat,ing that nucleolar activity is relatively little influenced by the number of genes at a locus. At first it may seem strange that the 1B locus should exhibit dominance over the 6B locus, since the majority of genes on chromosome 1B do not have any more intergenic repeats than those on chromosome 6B (see Fig. 1). However, about 10% of the genes at the 1B locus contain several additional intergenic repeats (the lB-1 genes; see Fig. 1). These genes are considerably more sensitive to DNase I and less extensively methylated than

547

other genes at either locus (see Fig. 6 and Flavell et al., 1988). We believe the dominance of the 1B NOR is attributable to the presence of the lB-1 genes. However, it is not only the lB-1 genes that show enhanced DNase sensitivity. Although the lB-1 genes exhibit larger effects, some of the lB-2 genes also show increased DNase I sensitivity relative to the rDNA on chromosome 6B (Figs 6 and 7). This behavior is an apparent exception to the correlation of intergenic-repeat number with DNase sensitivity, since the lB-2 genes do not have more repeats than 6B genes. We can offer two speculations as possible explanations of this apparent discrepancy. One hypothesis is that the nucleotide sequences of the intergenic repeats in the 1B genes might differ from those of the 6B genes so as to give all the 1B genes, including the lB-2 type, a higher inherent affinity for limiting transcription factors. In this case differences in sequence composition rather than repeat number would be the determining factor for gene activity. Alternatively, the greater DNase sensitivity of some lB-2 variant genes might be attributable to their association with lB-1 genes. We suppose that limiting transcription factors, which initially produce hypersensitive sites by binding to intergenie repeats in rDNA, are most concentrated around the active genes in the nucleolus. Because of the high concentration of factors in their vicinity, other nearby genes might occasionally bind a factor and thus contain DNase I hypersensitive sites. This idea is consistent with our observation that DNase-sensitive genes seem to comprise only a fraction of the total at any given locus. As noted in the discussion of Figure 3, the differential sensitivity of the 1U rDNA in A. umbellulata is probably attributable to a fraction of the 1U genes being digested by quite small amounts of DNase, with the remaining 1U genes being relatively resistant to digestion. The notion of a discrete subpopulation of sensitive genes is also supported by the reduction in HpaII sensitivity that occurs upon partial DNase I digestion (see Fig. 7), where it appears that the same subpopulation of genes is both DNase sensitive and undermethylated. If one assumes that all the genes in a particular length class at a particular locus have identical spacer and promoter sequences, the idea that only a few of them participate in nucleolus formation requires some mechanism independent of DNA sequence per se for determining which genes are to be included. Indeed, there seems to be a virtually a priori requirement for some such mechanism, since the large and variable numbers of rRNA genes in plants argue strongly that these genes are usually well in excess of actual requirements. One possibility is that nucleolar assembly is a co-operative process that proceeds along the chromosomes until a limiting factor of some sort is exhausted. The amount of factor concentrated in the vicinity of the NOR would determine the size and eventual activity of the nucleolus; this amount. in turn,

W. F. Thompson and R. B. Flavell

548

would be a function of the number and sequence of promoter and enhancer-like elements in the rRNA genes in a particular portion of the rDNA tandem array. Regions such as that containing the lB-1 variant genes would then act as “nucleolation sites” initiating nucleolus formation, with some adjoining lB-2 variants included in the resulting nucleolus because of their proximity to the lB-1 genes. Studies with isolated nucleoli may help to resolve this issue. We thank Mike O’Dell and Linda Roberts for technical assistance on the east and west sides of the Atlantic, respectively, Dave Baulcombe for pDB2437, and John Watson for pHB1.3 and the fragment XB380. W.F.T. acknowledges also many helpful discussions with John Watson and Lon Kaufman. Support for this work was provided by the Carnegie Institution of Washington, the AFRC, and USDA Competitive Research grants 85CRCRl-1559 and 86-CRCR-1-1910 to W.F.T.

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by R. Laskey