Chromatin structure at the replication origins and transcription-initiation regions of the ribosomal RNA genes of tetrahymena

Cell, Vol. 36,933-942,

April 1994, Copyright

Q 1984 by MIT

co92-@674/84/040933-IO

$02.00/o

Chromatin Structure at the Replication Origins and Transcription-Initiation Regions of the Ribosomal RNA Genes of Tetrahymena Ted E. Palen and Thomas Ft. Cech Department of Chemistry University of Colorado Boulder, Colorado 80309

Summary The chromatin structure of regulatory regions of the extrachromosomal I-RNA genes of Tetrahymena thermophila was probed by nuclease treatment of isolated nuclei. The chromatin near the origins of replication contains hypersensitive sites for micrococcal nuclease, DNAase I, and DNAase II. These sites persist in starved cells, consistent with the origins’ being maintained in an altered chromatin structure independent of DNA replication. The region between the two origins of replication is organized into a phased array of seven nucleosomes, the fourth of which is centered at the axis of symmetry of the palindromic rDNA. The entire transcribed region and 150 bp upstream from the initiation site are generally accessible to nucleases; any histone proteins associated with these regions are clearly not in a highly organized nucleosomal array as seen in the central region. Comparison of the chromatin structures of the central spacer of T. thermophila and T. pyriformis rDNA reveals that deletion or insertion of DNA has occurred in increments of 200 bp. This is taken to imply that there are constraints on the evolution of spacer DNA sequences at the level of the nucleosome. Introductioh In the eucaryotic nucleus the bulk of the DNA is highly compacted in the form of chromatin, yet selected portions of the DNA are made available to the transcriptional and replicational apparati. Ribosomal RNA genes, as well as messenger RNA genes, are associated with histone proteins and, at least in part, are packaged into repeating subunits (reviewed by Mathis et al., 1980; see also Gottschling et al., 1983; Colavito-Shepanski and Gorovsky, 1983; Labhart et al., 1983; Prior et al., 1983). The fundamental problem of how chromatin is transcribed and replicated is therefore common to both mRNA and rRNA genes. Some aspects of the problem may be solved in a similar manner for the two types of genes. A generalized sensitivity to DNAase I is a feature common to transcriptionally active mRNA and rRNA genes (Weintraub and Groudine, 1976; Garel and Axel, 1976; Mathis and Gorovsky, 1978; Stalder et al., 1978). This DNAase I sensitivity appears to reflect an altered nucleosome conformation that may be required for passage of RNA polymerase through chromatin. Another common feature of mRNA genes is the pres-

ence of DNAase l-hypersensitive sites at their 5’ and 3’ ends. In several cases the occurrence of these sites has been correlated with gene activity (reviewed by Elgin, 1981). These sites are nucleosome-free but not necessarily protein-free. The DNA in these regions appears to have an altered secondary structure, perhaps a non-B form of the DNA double helix (Weintraub, 1983). The 5’ sites may be involved in the initiation of transcription on a chromatin template, e.g., by serving as entry sites for transcription factors or RNA polymerase. Most of the information we have about such hypersensitive sites has been derived from studies of genes transcribed by RNA polymerase II. DNAase l-hypersensitive regions and sites of endogenous nuclease activity have been found upstream from the transcription-initiation site in Tetrahymena rDNA chromatin by Bonven and Westergaard (1982). Further study of rRNA genes should be informative, since these genes are transcribed by a different polymerase and have a particularly high rate of transcription. Very little is known about the structure of chromatin in the vicinity of replication origins. Do the proteins involved in replication interact with the origin in a transitory manner, or is some stable DNA-protein interaction set up at the origin to serve as a recognition site for the DNA polymerase and other replication proteins? The SV40 and polyoma virus origins of replication occur in highly accessible, nucleosome-free regions (Varshavsky et al., 1979; Saragosti et al., 1980). However, transcription initiation and T-antigen binding also occur in this region, and analysis of mutant polyoma virus chromatin supports the view that the structure is more directly related to transcription than replication (Herbomel et al., 1981). There is much less information about DNA-protein interactions at nonviral origins of replication The reason is that few such origins have been identified, although the ARSs (autonomously replicating sequences) of yeast are certainly good candidates. The rRNA genes (rDNA) in the macronucleus of Tetrahymena are linear, extrachromosomal molecules containing about 20,000 bp of DNA with a palindromic sequence symmetry (reviewed by Blackburn, 1982). Transcription of the rRNA precursor proceeds bidirectionally from the central region toward the ends (Gall et al., 1977). The site of transcription initiation has been mapped at the nucleotidesequence level in T pyriformis (Niles et al., 1981; Saiga et al., 1982) and T. thermophila (Engberg et al., 1984). In Tetrahymena there is no discernible transcription upstream from the site that encodes the 5’ end of the pre-rRNA (Kister et al., 1983). Replication of the rDNA is under a control separate from that of the bulk macronuclear DNA (Engberg et al., 1972; Nilsson et al., 1977). Yet, like the bulk DNA, each rDNA molecule replicates only once per cell cycle as judged by BUdR-labeling experiments (Engberg et al., 1972; J. Engberg, personal communication). Origins of replication are located in the central nontranscribed spacer, 650 f 300 bp from the center of the rDNA in T. thermophila and closer to the center in T. pyriformis (Truett and Gall, 1977;

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Cech and Brehm, 1981). In both species the origin of replication and initiation site for transcription are separated by several hundred base pairs, making it possible to assign chromatin structure features to one function or the other. Based on the pattern of nuclease cleavage of chromatin, we now propose a model of the chromatin structure of the rDNA central spacer. The center of the r-chromatin is organized in a phased nucleosomal array bordered on each side by nuclease-hypersensitive regions at or near the origins of replication.

Results Central Spacer of T. thermophila r-Chromatin Nuclease-sensitive sites in the r-chromatin were mapped from the most internal Hind III restriction site of the palindromic rDNA using the indirect end-labeling technique (Wu, 1980; Nedospasov and Georgiev, 1980). Chromatin in isolated nuclei was digested to a small extent (<5% TCA solubility) with either DNAase I, DNAase II, or micrococcal nuclease. The purified DNA was cleaved completely with Hind Ill restriction endonuclease, subjected to gel electrophoresis, and blotted onto nitrocellulose. The blot was then probed with the 190 bp Hind Ill-Alu I DNA fragment shown in Figure 1. This probe was specific for the central spacer of the rDNA, as evidenced by its hybridization to the expected fragments of total T. thermophila DNA that had been cleaved to completion with Hind Ill and to a limited extent with Xba I (Figure 2, lane 5). The locations of micrococcal nuclease cleavage sites were determined from the autoradiograms of Figure 2. All of the cleavage sites appear to be distributed symmetrically :-

(A)

me

_ _-_ __-

lil t I K r K

I. I THI

I

I

I

--_ 1--. H ioot+l

Figure 1. Restriction Indirect End-Labeling

Endonuclease Experiments

Fragments

Used

as Probes

for the

(A) T. thermophila rDNA. (Wavy arrows) pre-rRNA transcription untts. (Vertical dashed line) axis of symmetry of the palindromic molecule. The detailed restriction map, shown for one-half of the central region, and the location of the transcription start site are based on the work of Engberg et al., (1984) and A. Amin. P. Chafloner, R. Peartman, and E. Blackburn (personal communication). (ori) ortgin of replication (see Discussion). (B) T. pyriformis rDNA. Restriction map and location of transcription start site based on data of Niles et al. (1981) and Saiga et al. (1982). A T. thermophila rDNA probe was used for indirect end-labeling (87% homology to corresponding region of T. pyrifonis rDNA). Restriction sites are indicated as (T) Taq I; (Xm) Xmn I; (X) Xba I; (Hh) Hha I; (A) Alu I; (H) Hind Ill; (K) Kpn I; and (HI) Hinf I.

relative to the center of the rDNA. Because of the palindromic symmetry of the rDNA, any single cleavage within the central spacer should produce two bands of hybridization that map to positions equidistant from the center. The occurrence of such a symmetric hybridization pattern therefore does not prove that nuclease-sensitive sites occur in the same position on both halves of each palindromic molecule, although it is consistent with such a model. The chromatin-digestion patterns are dominated by two regions of intense hybridization on each side of the center of the rDNA, each in the vicinity of an Xba I site. These are apparent at very low levels of digestion, when there is an average of less than one cleavage per molecule. They are therefore classified as hypersensitive sites for micrococcal nuclease cleavage. The region around the inner Xba I site consists of two hypersensitive sites separated by a protected area. The region around the outer Xba I site consists of a single hypersensitive site followed by a uniform set of six bands with a 35 bp periodicity. Six additional micrococcal nuclease-sensitive sites form a uniform ladder between the innermost Xba I restriction sites. The distance between adjacent sites averages 200 bp (range = 180-210 bp). Deproteinized T. thermophila nuclear DNA was also subjected to limited micrococcal nuclease treatment, cleaved with Hind Ill, and analyzed by blot hybridization. As shown in lanes 10, 11, 12, and 17 of Figure 2, the central spacer was found to contain some nucleotide sequences preferred by the nuclease, but the cleavage pattern was quite different from that obtained for DNA in nuclei. For example, the exact center of the palindromic rDNA and a region around the more distal Xba I site were hot spots for micrococcal nuclease in the naked DNA but were protected from cleavage in nuclei. Thus while there are sequences in the central spacer that are preferred substrates for micrococcal nuclease, these sequences are not responsible for the pattern of cleavage seen in isolated nuclei. When nuclei were incubated in the absence of added nuclease, the DNA remained intact (lanes 18 and 19). We conclude that endogenous nuclease activity is insignificant under these conditions and that the chromatin cleavage pattern is due to the added micrococcal nuclease. Data used to map DNAase I and DNAase II cleavage sites are shown in Figure 3. The 200 bp repeat in the central region was evident in the DNAase II digests and to some extent in the DNAase I digests, but these nucleases also produced additional cleavages resulting in a background smear. In addition, both nucleases cleaved at the axis of symmetry. These cleavage patterns are consistent with the model of a nucleosomal array, because DNAase I and, to a lesser extent, DNAase II are known to produce intranucleosomal cuts. The regions near the Xba I sites that were hypersensitive to micrococcal nuclease were also sites of DNAase I and DNAase II cleavage in chromatin. The cleavages were not as strong as those produced by micrococcal nuclease,

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RNA Gene Chromatin

Figure 2. Micrococcal

Structure

Nuclease-Sensitive

Sites in T. thermophila

r-Chromatin

Mapped across the Central Spacer

Nuclei isolated from (lanes 1-4) log-phase cells and (lanes 6-9) starved cells were treated with micrococcal nuclease until 0.5%-2.0% of the DNA was rendered acid-soluble. (Lane 5) total T. thermophila DNA digested to completion with Hind Ill and partially with Xba I to serve as a marker for the Xba I restriction sites and to check for probe specificity. (Lanes 10-12) deproteinized nuclear DNA treated with micrococcal nuclease, acid solubilities ranging from 19/o-3% or (lane 17) ~0.5%. (Lanes 14 and 16) purified plasmid pTtd DNA containing the central Hind Ill fragment of the rDNA treated with micrococcal nuclease while supercoiled or (lanes 13 and 15) after Hind Ill cleavage. (Lane 18) DNA purified from isolated nuclei that had been self-digested (incubated at 37°C in the absence of added nuclease) for 60 min or (lane 19) IO min. (M) Molecular weight markers (see Experimental Procedures). Micrococcal nucleasetreated DNA samples (3-10 rg) were digested to completion with Hind Ill, subjected to electrophoresis on a 15% agarose gel, transferred to nitrocellulose, and hybridized with the 190 bp Hind Ill-Alu I fragment of rDNA (Figure 1). The lanes shown here came from two gels, each of which contained chromatin digests and naked DNA controls as well as marker DNA lanes. The schematic to the left of the autoradiogram shows the initiation point of transcription (bent arrow) and the major micrococcal nuclease-cleavage sites in chromatin.

but they were convincing because they occurred in areas that were not preferred in the naked DNA controls (Figure 3A). Although they occurred in the same regions, the DNAase I and micrococcal nuclease hypersensitive sites were not identical. At the inner Xba I site, DNAase I cleaved between the two strong micrococcal nuclease sites. At the outer Xba I site, DNAase I cleavage occurred at a point mainly distal to the restriction site, whereas the strongest micrococcal nuclease cleavage was proximal to the restriction site. We interpret these data to indicate that a -200 bp region at each Xba I site is in a highly altered chromatin structure, with the exact sites of cleavage depending upon the sequence and DNA structure preferences of the nuclease that is used as the probe. The locations of the nuclease cleavage sites were also determined by mapping outward from the center of the rDNA (Figure 4). Because of the dearth of restriction endonuclease recognition sites in this A+T-rich sequence, a 450 bp Taq I-Xmn I fragment was the smallest suitable probe. Most of the regular cleavage pattern in the center of the molecule was within the region covered by the probe

and therefore not clearly seen. However, the two major micrococcal nuclease-hypersensitive regions were observed and their locations confirmed. Each of the two hypersensitive sites in the region around the inner Xba I site covers a -80 bp, while the region near the outer Xba I site covers -200 bp. Additional sites upstream from the initiation point for transcription were again observed, but they could be more accurately mapped from the data of Figure 2.

Log-Phase vs Starved Cells The cleavage patterns obtained with all three nucleases were qualitatively similar for log-phase compared to starved-cell chromatin, the latter representing a state of transcriptional and replicational inactivity. The three micrococcal nuclease-hypersensitive sites occurred in both logphase and starved-cell r-chromatin. There was, however, a difference in their relative intensities. The innermost of the three sites was the least intense in starved cells, whereas in log-phase cells it was as intense as any of the three. This difference was consistently observed with dif-

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Figure 3. DNAase

I and DNAase

II Cleavage

Sites in the Central Spacer

(A) DNAase I. (Lane 1) nuclei self-digested for 60 min at 37°C without added enzyme. (Lanes 2 and 3) plasmid pTtrl DNA treated with DNAase I as supercoiled and linear DNA, respectively. (Lanes 4-6) DNA extracted from nuclei immediately after isolation, purified, and then subjected to digestion; l%, 396, and % acid solubility, respectively. (Lanes 7 and 8) nuclei isolated from log-phase cells or (lanes 9 and 11) starved cells treated wfth DNAase I (0.5%~2% acid solubility). (Lane IO) marker for Xba I sites as in lane 5 of Figure 2. All lanes came from a single gel (two autoradiographic exposures). (B) DNAase II. (Lane 1) marker for Xba I sites. (Lanes 2 and 3) nuclei isolated from starved cells or (lanes 4 and 5) log-phase cells treated with DNAase II (0.52% acid solubility). (Lanes 6 and 10) DNA extracted from nuclei immediately after isolation, purified, and then subjected to digestion (1% and ~05% acid sdubility. respectively). (Lanes 7 and 9) plasmid pTtri DNA treated with DNAase II as supercoiled and linear DNA, respectively. (Lane 8) nuclei selfdigested for 10 min without added enzyme. All lanes came from a single gel (two autoradiograph exposures). DNA samples were treated as described in the legend to Figure 2.

ferent DNA samples and was apparent when either the external hybridization probe (Figure 2) or the central probe (Figure 4A) was used. The other structural features of the central spacer, including the seven phased nucleosomes and the DNAase I- and DNAase II-sensitive sites, were conserved between log-phase and starved-cell chromatin.

Promoter Region of T. thermophila r-Chrometin Distal to the hypersensitive region around the outer Xba I site there was a 400 bp region that contained only a single micrococcal nuclease-cleavage site in chromatin (-386 + 10 bp with respect to the initiation point for transcription). In contrast, this region contained multiple cleavage sites for the nuclease in naked DNA (lanes lo-16 of Figure 2). The data are most easily explained by the site at -386 occurring in the linker between two nucleosomes. Continuing outward from the axis of symmetry, the r-chromatin had additional cleavage sites at -206, -153, -93, and -3 bp (all f 10 bp). The pattern of nuclease-accessible sites in chromatin bears a strong resemblance to that in naked DNA, although cleavage at -153 and -206 was enhanced in chromatin relative to the deproteinized nuclear DNA controls. The four unprotected sites are too closely spaced to delineate nucleosomes. Therefore, any proteins bound to the DNA in this region are either located at variable positions from molecule to molecule or else do

not afford much protection from the nuclease. Major DNAase I- and II-hypersensitive regions were centered at about -200 bp.

Accessibility in the Transcribed Region Nuclease-sensitive sites in the transcribed region were mapped from the Xba I site 713 bp upstream from the initiation site for transcription. Indirect end-labeling was done with the 200 bp Xba I-Hha I probe shown in Figure 1. All three nucleases produced a general background smear of hybridization that extended several thousand base pairs across the transcribed region to the end of the rDNA. A sample of the data is shown in Figure 5. Except for the 427 bp band due to cross-hybridization of the probe with the Xba I-Xba I fragment, the digestion patterns produced by all three nucleases on either log-phase or starved-cell chromatin can best be characterized as featureless. The lack of discrete protected regions in the chromatin digests is taken to indicate that all or most of the sequences in the transcribed region are accessible to the nucleases at one time or another.

T. pyriformis r-Chromatin Nuclease-sensitive sites were mapped across the central spacer of T. pyriformis r-chromatin using a cloned T. thermophila rDNA probe. As shown in lanes 7-l 1 of Figure

Ribosomal 937

A

RNA Gene Chromatin

lM2345

Structure

B

12345M678

Figure 5. Nuclease matin

Figure 4. Nuclease-Sensitive Sites in T. themrophila outward from the Center of the Molecule

r-Chromatin

Mapped

(A) Micrococcal nuclease digestion of (lane 1) plasmid pTtrl DNA linearized by treatment with Hind Ill endonuclease, (lane 2) deproteinized nuclear DNA, (lane 3) nuclei from starved cells, and (lane 4) nuclei from log-phase cells. (Lane 5) deproteinized nuclear DNA digested to completion with Taq I. (B) DNAase II digestion of (lane 1) deproteinized nuclear DNA, (lane 2) nuclei from starved cells, (lane 3) nuclei from log-phase cells, and (lane 4) supercoiled plasmid pTtrl DNA. DNAase I digestion of (lane 5) nuclei from log-phase cells, (lane 6) nuclei from starved cells, (lane 7) linearized plasmid pTtrl DNA, and (lane 8) deproteinized nuclear DNA. All DNA samples were in the range of
6a, the central 1000 bp is accessible to micrococcal nuclease at 200 bp intervals in chromatin. A different pattern of nuclease-sensitive sites is evident in the naked DNA (lanes 5-6) and these sites are highly protected in the chromatin. The central region is therefore interpreted to be organized as an array of five phased nucleosomes. This array is bordered by a region that is hypersensitive to micrococcal nuclease both in chromatin and in the naked DNA. This is in contrast to the situation in T. thermophila, where the hypersensitive sites that border the phased nucleosomes are not preferred targets for the nuclease in naked DNA. The conclusion that the T. pyriformis sites reflect an altered chromatin structure is strengthened by the observation that they are hot spots for DNAases I and II in chromatin but not in naked DNA (Figure 6B).

Discussion A model for the chromatin structure of the central nontranscribed soacer of the rDNA is aiven in Fiaure 7. In T.

Accessibility

in the Transcribed

Region of the rGhro-

Micrococcal nuclease digestion was performed on (lane 1) deproteinized nuclear DNA, (lanes 2 and 3) nuclei from starved cells, and (lane 5) Hind Ill-cleaved plasmid pTtrl DNA. M&coccal nuclease digestion of nuclei from log-phase cells (data not shown) produced a pattern indistinguishable from that of starved-cell nuclei. (Lane 4) endogenous nuclease control. All DNA samples were digested to completion with Xba I restriction endonuclease prior to electrophoresis. The 200 bp Xba I-Hha I fragment (shown in black in the schematic) was used as the hybridization probe. The probe cross-hybridized with the Xba I-Xba I fragment, producing the band at 0.4 kb. In the schematic, the axis of symmetry is at the bottom, the transcription unit is lightly shaded, and the end of the rDNA molecule is at the top.

thermophila the central 1400 bp is organized into seven nucleosomes that occupy specific sites. These are typical nucleosomes in the sense that the 200 bp repeat of micrococcal nuclease-accessible sites is exactly that found in bulk chromatin (Gottschling et al., 1983). In T. pyriformis, the array contains five nucleosomes. In both cases the exact center of the rDNA is in the middle of a nucleosome. The limits of the phased nucleosome array are defined by regions that are hypersensitive for micrococcal nuclease and DNAases I and II. These hypersensitive regions reflect some special DNALprotein interaction at or near the origins of replication.

Nucleosome Phasing The nucleosomes in the central region appear to be phased, i.e., they occupy specific sites on the DNA. The bands that comprise the nucleosomal ladder are as sharp as bands produced by restriction endonucleases (Figures 2 and 6) so the micrococcal nuclease sites are precise to within IO bp. Reports of nucleosome phasing in some other systems have been complicated by the occurrence of micrococcal nuclease cleavages that were due to the sequence preference of the nuclease (Nedospasov and Georgiev, 1980; Keene and Elgin, 1981; reviewed by McGhee and Felsenfeld, 1983). In the present case, the

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910M

M234M567891011

e-

kbp

21.2

2.91

:s: 1.58 1.38 1.18 ,947 ,831 ,564 .404 ,327 ,231 ,141

Figure 6. Nuclease-Sensitive

Sites in the Central Spacer of the r-Chromatin

of T. pyriformis

(A) Micrococcal nuclease digestion. (Lane 1) deproteinized DNA incubated with the nuclease in absence of Ca+*. (Lanes 2 and 3) nuclei self-digested for 15 min or 60 min, respectively, at 37°C without added nuclease. (Lane 4) DNA digested to completion with Hind III and partially with Kpn I, which cleaves -165 bp to either side of the center of the rDNA; bands were visible on longer exposure. (Lanes 5 and 6) deproteinized T. pyriformis DNA digested with the nuclease. (lanes 7-l 1) nuclei from log-phase cells digested with decreasing amounts of the nuclease. (B) (Lane 1) same as lane 4 in A. DNAase II digestion was performed on (lanes 2-4) nuclei from log-phase cells and (lanes 5-6) deproteinized T. pyriformis DNA. DNAase I digestion was performed on (lanes 7-6) nuclei from log-phase cells and (lanes 9-10) deproteinized DNA. All DNA samples were treated extensively with Hind Ill restriction endonuclease prior to electrophoresis. The 240 bp Hind Ill-Hind Ill fragment of T. thermophila rDNA (Figure 1) was used as the hybridization probe. The wavy lines indicate the omission of -110 bp.

evidence for phasing is unambiguous because of the anticorrelation between the cleavage patterns of naked DNA and chromatin. (It remains possible, however, that the exact site of cleavage within each nucleosome linker is directed by the DNA sequence.) Another potential artifact that might give a false appearance of phasing has been described by McGhee and Felsenfeld (1983); this problem can occur at high levels of micrococcal nuclease digestion, but is not a consideration at the very low levels used in the present study. Other clear examples of nucleosome phasing have been reported (e.g., Worcel et al., 1983). While the nucleosomes do occupy specific sites in the central spacer DNA, this does not necessarily mean that the histones have an affinity for specific DNA sequences. The hypersensitive sites near the origin of replication presumably reflect an altered DNA structure and/or binding of nonhistone proteins to specific sequences. These sites define the boundaries of a 1400 bp region which just fits seven nucleosomes. In T. pyriformis the sites define a 1000 bp region that is packaged into five nucleosomes. Such an explanation for phasing is a version of that mentioned by Nedospasov and Georgiev (1980) and developed in more detail by Kornberg (1981). The sequence of T. thermophila rDNA in this central region (Kiss and Pearlman, 1981) is not a simple repeated sequence like that of the satellite DNAs that comprise centromeric heterochromatin in eucaryotes. It is possible

to find some short, imperfect repeats that are located within &lo bp of the same spot in each phased nucleosome. However, these sequences are not conserved in T. pyriformis rDNA (Engberg, 1983) so it is doubtful that they have any relationship to nucleosome phasing. Even if the nucleosome phasing were a consequence of boundary conditions imposed by the origins of replication, the phasing might still serve a function. For example, the highly ordered nucleosome array might be organized in a higherorder structure that brought the two origins of replication together in a very precise orientation.

Origins of Replication Flanking the seven phased nucleosomes in the center.of the T. thermophila r-chromatin are regions 230 bp in size that contain hypersensitive sites for all three nucleases. The general locations of the nuclease-hypersensitive sites are in good agreement to those mapped on T. thermophila rDNA by Bonven and Westergaard (1982) using a different technique. By analyzing the chromatin digests in parallel with partial restriction endonuclease digests of naked DNA, we have been able to map the sites at the nucleotide sequence level (fl0 bp). We interpret these hypersensitive sites to reflect an altered chromatin structure at the origins of replication that are maintained in the nonreplicating rDNA of starved cells. The designation of these regions as origins of replication is based on three considerations, First, the regions extend

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RNA Gene Chromatin

Structure

-6 Figure 7. Model for the Chromatin

Structure

ioo

-around the Center of the rDNA of T. thermophila

(Top) and T. pyriformis

(Bottom)

( j ) Axis of symmetry of the palindromic rDNA. (P) Initiation point for transcription. (H) Hind III; (HI) Hinf I; (X) Xba I sites. The stippled regions within the restriction map represent regions of sequence homology between the two rDNAs. The extent of homology is (a) 87%, (b) 72%, (c) 61%, (d) 78%, (e) 80%, and (f) 74%. The mottled boxes around the Xba I sites in T. thermophila correspond to the direct repeated sequences; d’ and f’ are 82% and 99% homologous to d and f, respectively. In the schematics of chromatin structure, the regions most inaccessible to nucleases are shaded the darkest. Round symbols represent -200 bp protected regions, presumably nucleosomes. Open areas are hypersensitive to micrococcal nuclease. The thick black arrows just upstream from the transcriptional initiation site represent major micrococcal nuclease cleavage sites in both chromatin and naked DNA. The large open arrows above and below the schematics designate DNAase I- and II-hypersensitive regions. The “nucleosomes” with an asterisk correspond to “protected regions of the chromatin that are not digested in the naked DNA controls, so designation of these regions as nucleosomes is by analogy to T. thermophila.

from 640 to 870 bp from the center of the molecule, a good match to the location of the origins as determined by electron microscopy (650 +- 300 bp from the center; Cech and Brehm, 1981). Second, the restriction fragment extending from the Taq I site near the center to the inner Xba I site (Figure 1) and the adjacent Xba I-Xba I fragment act as ARSs on yeast plasmids, but the more distal Xba I-Taq I fragment does not (Kiss et al., 1981; R. Pearlman, personal communication). Third, this is a region of high sequence conservation between T. thermophila and T. pyriformis, whereas the sequences organized in the phased nucleosomes have little homology (Engberg, 1983; see Figure 7). None of these observations allows us to exclude the possibility that the similar sequence around the outer Xba I site in T. thermophila rDNA represents another pair of origins. (In fact, the detailed micrococcal nuclease cleavage pattern of the hypersensitive region in T. pyriformis rDNA more closely resembles that of the outer hypersensitive region in T. thermophila.) In Xenopus laevis, spacer sequences considerably upstream from the site encoding the 5’ end of the pre-rRNA are important for transcription (Moss, 1983; Busby and Reeder, 1983). In Tetrahymena there is no discernible transcription of any sequences upstream of the rRNA coding region (Kister et al., 1983) unlike the situation in Xenopus (Moss, 1983). It remains possible that the hypersensitive sites near the origins of replication in the Tetrahymena rDNA could be serving a role in transcription as well as (or instead of) a role in replication. For example, they could serve as storage or entry sites for factors that affect the transcription of the ribosomal genes (Busby and Reeder, 1983). Another possibility is that the hypersensitive sequences could be sites of topoisomerase attachment (Bonven and Westergaard, 1982). By altering the superhelical density in some constrained domain of the linear

rDNA, such topoisomerases could affect the transcriptional and/or replicational state of the gene. Based on measurements of replicating rDNA in the electron microscope, it seemed that the T. pyriformis rDNA origin(s) of replication were more centrally located than those in T. thermophila (Truett and Gall, 1977; Cech and Brehm, 1981). Now, based on homology at the level of chromatin structure as well as nucleotide sequence, it appears that the T. pyriformis origins are more centrally located, but only by 200 bp. As judged by nuclease probes, the chromatin structure around the rDNA origin of replication bears a marked similarity to that at yeast centromeres (Bloom and Carbon, 1982). The similarity includes the 230 bp nonnucleosomal region flanked by micrococcal nuclease-hypersensitive sites, and the adjacent phased nucleosomes. This is an unexpected correspondence. It is unlikely that rDNA minichromosomes have the equivalent of a centromere, since they undergo neither mitosis nor meiosis. The yeast centromeres have very weak or, in some cases, undetectable ARS activity, although it has been suggested that they have an independently regulated origin of replication that is activated only during mitosis and meiosis (Tschumper and Carbon, 1983). The chromatin structure of the rDNA origin also resembles that of ARSI of yeast (C. M. Long, C. M. Brajkovich, and J. F. Scott, personal communication) as it occurs on a multiple-copy plasmid (Zakian and Scott, 1982). It remains to be seen to what extent such structural features are general for eucaryotic origins of replication. Promoter and Transcribed Region In starved-cell nuclei, the transcribed region of the r-chromatin has the periodic accessibility to micrococcal nuclease that is indicative of a nucleosome structure (Gottschling et al., 1983). In nuclei of transcriptionally active

cell 940

log-phase cells, this periodic chromatin structure is largely lost and the transcribed region becomes much more accessible to micrococcal nuclease (Borchsenius et al., 1981; Gottschling et al., 1983; Palen and Cech, 1983) although the histone:DNA ratio is the same for r-chromatin and bulk chromatin (Colavito-Shepanski and Gorovsky, 1983). In the present study using the indirect end-labeling method, no discrete pattern of nuclease cleavage or protection was observed for the transcribed region even in the starved-cell r-chromatin. This indicates that the nucleosomes in the gene region are not located in specific positions like those in the central portion of the molecule. The general accessibility of the DNA to micrococcal nuclease continued -150 bp upstream from the initiation point for transcription in both species. In addition, there are some nuclease-hypersensitive sites in this region. Classification of this region as the RNA polymerase I promoter is based on analogy to other rRNA genes (e.g., Kohorn and Rae, 1983; Learned et al., 1983; Busby and Reeder, 1983). The altered chromatin structure in this region is maintained even in starved cells, where transcription is 95% reduced (Sutton et al., 1979).

Can Chromatin Structure Influence the Evolution of Spacer DNA? The central spacer of T. thermophila rDNA is considerably larger than that of T. pyriformis rDNA. Examination of the nudleotlde sequences (Niles et al., 1981; Niles, pers. comm.; Engberg et al., 1984; A. Amin, P. Challoner, R. Pearlman,‘” and E. Blackburn, personal communication) reveals that the extra nucleotides in T. thermophila are accommodated in three separate regions. (We refer to these as insertions in T. thermophila rDNA, but they could be deletions in T. pyriformis rDNA.) In each case the number of extra nucleotides in the block is an approximate multiple of 200 bp. Upstream from the transcription initiation site between conserved sequences a and c in Figure 7 there is an insertion that expands a 51 bp region in T. pyriformis to 187 bp; this appears to allow formation of another nucleosome’ in T. thermophila. Further upstream there is a 401 bp insertion that represents an approximate repeat of the sequence around the origin of replication. This region is organized as a nonnucleosomal hypersensitive region, similar to that found at the origin, plus one presumptive nucleosome. Finally, in the center of the molecule between the innermost micrococcal nucleasehypersensitive sites, T. thermophila has an additional 370 bp that adds two nucleosomes to the phased array of five found in T. pyriformis. One possible explanation for these data is that the molecular events that produce DNA insertion or deletion, such as unequal cross-over, occur preferentially between nucleosomes. (This would require the micronuclear halfpalindrome copy of the rDNA to have the same chromatin structure as the macronuclear palindromic copies, a situation about which we have no information.) A similar argument for evolution at the level of chromatin structure

has been proposed by Maio et al. (1977) for highly repeated satellite DNA sequences. Another possibility is that there could be selective pressure for stretches of DNA to occur in lengths that can be stably packaged into chromatin. Deletions or insertions that cannot be readily accommodated into an integral number of chromatin subunits would result in abnormally long linkers between nucleosomes, increasing susceptibility to damage by endogenous nucleases and chemicals. Such selective pressure would apply even to nonfunctional DNA sequences. Experimental Growth

Procedures

of Cells and Isolation

of Nuclei

Tetrahymena them-rophila strain B VII were grown at 30°C as described by Gottschling et at. (1983). T. pyriformis strain GL (amicronucleate) were obtained from E. Niles and grown by the same protocol except at 28°C. Cells were labeled with ?--thymidine (150 &i/l) during the entire growth period. Cells were starved in 50 mM Tris-HCI (pH 7.5) for 18 hr as described by Gottschling et al. (1983). Nuclei were isolated by the NP-40 detergent method described by Zaug and Cech (1980) except that aurintricarboxyfic acid was omitted from the TMS (0.01 M Tris-HCI, pH 7.5, 0.01 M MgCI,. 0.003 M CaCl*, 0.25 M sucrose). The nuclei from two 1 I cultures were combined and resuspended in TMS to give a final concentration of 3-5 x 10’ macronuclei per ml.

Treatment

of Nuclei with Nucleases

Nuclei were digested with micrococcal nuclease at 37OC in TMS buffer as described by Pafen and Cech (1983) except that nuclei in 0.2-0.3 ml reaction volumes (200-300 rg DNA per reaction) were treated with 0.04 U for IO set or with 0.1 U for 15. 20, or 30 sec. The resulting DNA samples had TCA solubilities ranging from <0.5% to 5%. Nuclei in TMS were treated with DNAase I (Sigma #D-4783, 2000 Kunitz U/mg) for 1 .O min at 25OC after being prewarmed for 1 .O min at 25’C. Treatment with 0. 0.005.0.01, 0.1, 0.2, and 1 .O Kunitz U resulted in DNA samples with TCA solubilities of approximately 0, ~0.5. 1, 4, 8. and 25%, respectively. Prior to treatment with DNAase II, 0.2-0.3 ml aliquots of nuclei in TMS were diluted with 1 .O ml of 0.01 M Tris-HCI (pH 7.5). 0.25 M sucrose. Nuclei were pelleted by centrifugation (Eppendorf 5414 centrifuge, 2 min), the supernatant was removed, and the pellet was resuspended in 0.3 ml of 0.01 M Tris-HCI (pH 7.5) 0.25 M sucrose. Each sample was treated with DNAase II (Sigma #DN-II-HP, 24,910 Kunitz U/mg) for 1 hr at 25’C. Treatment with 0. 100, 200, 400, 800, and 1800 Kunitz U resulted in DNA samples with TCA solubilities of approximately 0, 0.1, 0.25. 0.5, 4.0, and 15%, respectively. All reactions were stopped by the addition of EDTA to a final concentration of 50 mM and SDS or sarcosine to a final concentration of 0.2%. Determination of TCA solubility and purification of DNA was as described previously (Gottschling et al.. 1983).

Treatment

of Deproteinized

DNA with Nucleases

DNA was isolated from nuclei that had not been treated with nucleases. For micrococcal nuclease treatment, the DNA was resuspended in 10 mM Tris-HCI (pH 7.5) 10 mM MgC12, 3 mM Car& 0.7 mM EDTA. Each reaction mixture contained 0.2-0.3 ml of DNA (200-300 rg DNA) and was incubated for 29 set at 37OC either without added micrococcal nuclease or with 0.01, 0.02,0.03,0.04, or 0.05 U. For DNAase I treatment, DNA was resuspended as described and incubated for 20 set at 25°C with 0. 0.01, 0.1, 0.2, or 1 .O U. For the DNAase II treatment, DNA in 10 mM Tris-HCI (pH 7.5) was incubated with no added nuclease or with 150 Kunitz U DNAase II for 1.5, 2.5, 5. 10. 20, 30, or 80 min at 25°C. All three nucleasa treatments resulted in TCA solubilities of
Gel-Blot

Hybridization

The following is a summary of our methods; details are given by Gottschling et at (1983). DNA was subjected to electrophoresis in 1.5% agarose gels,

Ribosomal 941

RNA Gene Chromatin

Structure

23 cm long and 3 mm thick. In all cases, DNA from chromatin digests and deproteinrzed DNA controls were present on the same gel. The DNA was blotted onto nitrocellulose (Schleicher and Schuell BA83. 0.20 pm pore size). Hybridization was carried out in a 50% formamide-containing buffer at 40”-42°C for the T. thermophila DNA blots, with washes at 37°C. For T. pyriformrs DNA blots, hybridization with T. thermophila probe was at 32’C with washes at 28°C. Blots were exposed to Kodak XAR-5 X-ray film for 2 days to 2 weeks at -70°C with an intensifying screen. Probes were derived from pTtrl, which contains the central Hind III fragment of T. thermophila rDNA cloned in pBR322, or from pRP58. which contarns the Xba I-Hind Ill fragment of T. thermophila rDNA cloned in pACYC184 The pTtrl plasmid was obtained from P. Challoner and E. Blackburn and pRP58 from R. Pearlman. Restriction fragments were gelpurified prior to being labeled by nick translation. Molecular weight markers were Hind Ill-Eco RI fragments of A DNA and Taq I fragments of 6x174 replrcative form II DNA. In both cases DNA fragments were end-labeled wrth y-?ATP and polynucleotide kinase. Frequent interspersion of lanes containing marker DNA with the experimental samples made it possible to align precisely the lanes within each autoradiogram and between autoradiograms.

We are most grateful to Ron Pearlman. Peter Challoner, Elizabeth Blackburn, and Jan Engberg for provrdrng DNA sequences prior to therr publicatron and for plasmid DNAs. This work was supported by grants from the National Institutes of Health and the Biomedical Research Support Grant Program, Division of Research Resources, NIH. T. R. C. is recrprent of a Research Career Development Award from the National Cancer Institute, Department of Health and Human Services. The costs of publication of this arkcle were defrayed in part by the payment of page charges. Thus article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. December

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Jetrahymena

Acknowledgments

Recerved

Engberg, J., Din, N., Saiga, H., and Higashrnakagawa, T. (1984). Nucleotrde sequence of the 5’terminal coding region for pre-rRNA and mature 17s rRNA in Jetrahymena thermophila rDNA. Nucl. Acids Res., in press.

9, 1983; revised

February

1, 1984

Kiss, G. B., Amin, A. A., and Pearlman, R. E. (1981). Two separate regions of the extrachromosomal ribosomal DNA of Jetrahymena thermophila enable autonomous replication of plasmrds In Saccharomyces cerevisiae. Mol. Cell. Brol. 7, 535-543. Krster, K.-P., Mtiller, B., and Eckert, W. A. (1983). Complex endonucleolytrc cleavage pattern during early events in the processing of pre-rRNA in the lower eukaryote. Jetrahymena thermophila. Nucl. Acids Res. 17, 34873502. Kohorn, B. D.. and Rae, P M. M. (1983). Localization of DNA sequences promoting RNA polymerase I activity in Drosophila. Proc. Nat. Acad. SCI. USA 80, 3265-3268. Kornberg, R. (1981). The location of nucleosomes statrstrcal? Nature 292, 579-580.

in chromatrn:

specific

or

Labhart, P., Ness, P., Banz. E., Parrsh, R.. and Keller, T. (1983). Model for the structure of the active nucleolar chromatin. Cold Sprrng Harbor Symp. Quant. Brol. 47, 557-564.

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RNA