DNase I and nuclease S1 sensitivity of the rabbit Î²1 globin gene in nuclei and in supercoiled...

.J. Mol. Bid. (1985) 184, 195-210

DNase I and Nuclease S1 Sensitivity of the Rabbit fll Globin Gene in Nuclei and in Supercoiled Plasmids

Jean B. Margot and Ross C. Hardison

Department of Molecular and Cell Biology Paul M. Althouae Laboratory

The Pennsylvania State University University Park, PA 16802, U.S.A.

fReceived 30 January 1985, and in revised form 15 March 1985)

We have examined the nuclease sensitivity of the 5’ flanking region of the rabbit 81 globin gene in bone marrow nuclei and in supercoiled plasmids. A DNase I hypersensitive site was found about 100 base-pairs 5’ to the cap site in bone marrow nuclei. S, nuclease can introduce a specific double-strand cut in the DNA in the same region. The presence of the nuclease-hypersensitive region correlates with the active transcription of gene b1 in bone marrow. Treatment with nuclease S, of a supercoiled plasmid containing 1400 base-pairs of 5’ flanking sequences as well as part of the 81 gene reveals a major double-strand cut 400 base-pairs 5’ to the cap site. This cut maps within a stretch of repeating dinucleotides (C-T),, and does not correspond to the in vivo site. Introduction of an RsaI fragment containing the nuclease S,-hypersensitive site into plasmid pBR322 shows that this fragment alone is sufficient to generate the hypersensitive site. Deletion of that RsaI fragment from the fil plasmid reveals another site 1300 base-pairs upstream. Further deletion of this secondary site uncovers numerous other sites, none of which corresponds to the site in nuclei. Chromatin reconstitution wit,h plasmids carrying the 5’ flanking region of bl and histones is capable of suppressing the in vitro nuclease-S,-hypersensitive site at -400 but is incapable of generating the in viva site at - 100. Fine analysis at the nucleotide level of the early events in the digestion with nuclease S, shows that the enzyme attacks preferentially the sequence (G-A),, on the message complementary strand. The region of DNA containing the supercoil-dependent S, site adopts at least three different conformations that can be resolved electrophoretically. These different conformations are detected in linear restriction fragments and may represent non-B DNA or unusual R-form DNA.

1. Introduction

The observation by Weintraub & Groudine (1976) that active chromatin is preferentially digested by DNase I led to numerous studies correlating an “open” chromatin structure with gene activity (Garel & Axel, 1976; Miller et al., 1978; Weintraub et al., 1981; Stalder et al., 1980a,b; Zasloff & Camerini- Otero, 1980). Not only active genes but the regions around them are DNase I-sensitive. These regions or domains of DNase-sensitive chromatin can eit,her be localized to a few kbt as in the case of viral integration (Flint & Weintraub, 1977) or can

t Abbreviations used: kb, 1000 base-pairs; bp, base- pair; nt, nucleotide; HS, hypersensitive site; S,HS, HS sensitive to nucleaee S,: Pu, purine; Py, pyrimidine; N, any nucleotide.

extend as far as 100 kb, as in the case of the ovalbumin gene (Lawson et al., 1982), the a and fi globin genes of chickens (Stalder et al., 1980b) and the heat shock genes of Drosophila (Wu et al., 1979a,b).

In contrast to these broad patterns of DNase I sensitivity, specific sites have been recognized in chromatin that are even more sensitive to DNase I. These hypersensitive sites (HS), which correspond to double-strand cuts of the DNA in the chromat,in fiber, have been mapped at or near the 5’ end of numerous genes. There is a direct correlation between expression and appearance of the HS 5’ to the human y globin gene in fetal erythroid cells. The turning off of y in adult cells is accompanied by the loss of the HS 5’ to this gene (Groudine et al.. 1983). These sites are tissue-specific for t,he most part, (Stalder et al., 198Oa; McGhee et al., 1981; Wu & Gilbert, 1981; Lachman & Mears, 1983: Tuan &

wzz-2836/85/ I ‘to 19,s 16 $03.00/0 195 0 1985 Academic Press Inc. (London) Ltd.

196 J. B. Margot and R. C. Hardison

London, 1984) and their appearance precedes initiation of transcription (Wu, 1980; Keene et al., 1981; McGinnis et al., 1983; Zaret & Yamamoto, 1984).

The nature of the HS is not known, although studies with simian virus 40 (SV40) mini- chromosomes and the chicken fl globin gene indicate that the DNase I HS corresponds to a region of the chromatin free of nucleosomes (Jakobovits et al., 1980; Saragosti et al., 1980; McGhee et al., 1981). Emerson & Felsenfeld (1984) have isolated a protein fraction that is able to confer DNase I hypersensitivity to a plasmid carrying the chicken PA globin gene, which had been reassembled with histones. One, or more, component of this fraction binds to a locus comparable to the hypersensitive domain of erythrocyte chromatin defined by McGhee et al.

(1981). Nuclease S 1 has been used to probe the

chromatin structure around active genes. In chickens, specific regions of u and /I globin genes are sensitive to S,. These S,-sensitive sites are tissue- specific and their presence reflects the switching pattern of globin gene expression in embryonic and adult’ red cells (Larsen 6 Weintraub, 1982). It is possible that S, is cleaving at single-stranded regions around the genes. This interpretation is supported by the observation that bromo- acetaldehyde, a single-strand-specific reagent, reacts with globin genes in erythrocytes or red blood cell nuclei at positions analogous to the S, HS (Kohwi-Shigematsu et al., 1983). However, experiments in vitro indicate that the prominent S, HS 5’ to the chicken p globin gene does not show the symmetry of strand cleavage expected for single-stranded DNA (Schon et al., 1983). Fine mapping of the S, HS in plasmids containing the 5’ flanking regions of various eukaryotic genes has revealed a preference of S, for relatively long polypurine/poIypyrimidine stretches. An S, HS is located within a (T-C-C-C), stretch for the chicken pro-a2(1) collagen gene (Finer et al., 1984), within a ((X-T), stretch for the human LX globin genes (Shen, 1983), within stretches of (C-T), for some Drosophila heat shock genes (Mace et al., 1983) and for the human Ul RNA genes (Htun et aE., 1984), within a (a),, stretch for the chicken p globin gene (Nick01 & Felsenfeld, 1983; Schon et al., 1983), and wit,hin the sequences (C-A),,, (C-T),, and (G-A)16 for sea urchin histone genes (Hentschel, 1982).

The apparent interdependence between DNase I and S, HS needs to be understood both in nuclei and in isolated plasmids. We have therefore analyzed the organization of the chromatin around the adult fil globin gene of rabbits. We show here that there is a DNase I and an S, HS 5’ to pl in bone marrow nuclei. In supercoiled plasmids, a different S, HS is detected in the 5’ flanking region of 81. This site maps within a stretch of repeating dinucleotides (C-T) 1 2, although the initial nicking by S, occurs preferentially on the opposite strand in the sequence (G-A),*. Chromatin reconstit,ution

experiments indicate that histones mask the supercoil-dependent site. An RsaI fragment containing the (C-T),, S1 HS is sufficient to confer S, hypersensitivity to pBR322. This RsaI fragment can exist in three different structures that can be resolved electrophoretically. These different structures may represent non-B-form DNA.

2. Materials and Methods

(a) DNase I and PstI digestion of isolated nuclei

The bone marrow from adult Sew Zealand white rabbits (Oryctohgus cunicuhs) was disrupted with a Dounce tissue grinder in an excess of cold phosphate- buffered saline (McGhee et al., 1981) and filtered through a nylon cloth. The cells were washed twice before being lysed in reticulocyte standard buffer (0.01 M-Tris. HCl (pH 7.4), 0.01 M-NaCl, 3 mM-Mgcl,) containing 0.5:/, (v/v) NP-40 (Weintraub & Groudine, 1976). The nuclei were washed several times in reticulocyte standard buffer without NP-40 and finally resuspended at A,,, = 20. Pancreatic DNase I (type I; Sigma) digestions of the nuclei suspension were carried out in reticulocvte standard buffer for 10 min at 37°C with increasing amounts of DNase I. The reactions were quenched with EDTA (final concentration 5 mM) and the solution was adjusted to 0.1% (w/v) sodium dodecyl sulfate. 0.5 M- NaCl and 0.1 mg proteinase K/ml (Sigma). After 1 h at 37”C, the DNA was extracted once with phenol, twice with phenol/chloroform (1 : 1, v/v) and precipitated with ethanol. Nuclei to be digested with PstI were resuspended at A 260 = 20 in 10 mM-Tris. HCl (pH 7.5). 100 mM-NaCI. 10 mi%-MgCl,. 1 mM$-mercaptoethanol. 0.1 mM-EDT.4. Portions were digested with 0 to 2.5 units PstT/pl of nuclei suspension for 1 h at 37°C. Quenching of the reaction and purification of the DNAs wm as described above.

(b) DNA restriction digests, blotting and hybridizations

Restriction endonucleases (from Bethesda Research Laboratories or New England Biolabs) were used as recommended by the supplier. Genomic DNA was digested with 0.8 to 1.2 units of enzyme/pg of DNA for 6 to 8 h. The DNA fragments were separated by electrophoresis on agarose gel and transferred to nitrocellulose (Smith & Summers? 1980). The blots were hybridized for 12 to 16 h at 42°C and washed as described by Cheng et al. (1984). The probes (2.5 to 5 rig/ml) were prepared by nick translation to a specific activity of about 2 x lo* cts/ min per pg (Maniatis et al., 1975). 5’ End-labeling of restriction fragments was accomplished according t.o Maxam 8: Gilbert (1980) using polynucleotide kinase and [y-jLP]ATP after dephosphorylation of the ends. 3’ End- labeling was by repair synthesis using reverse transcriptase and the appropriate deoxyribonucleoside [a-32P]triphosphate (Hardison & Margot, 1984). Nucleotide sequencing was according to Maxam & Gilbert, (1977. 1980).

(c) Nuclease S, digestion of nuclei and plasmids

Nuclei prepared as described above were resuspended at A 260 = 20 in low salt buffer containing 3 mM-ZnCl,. 30 mM-sodium acetate (pH 4.5), 30 mM-NaCl, 0.2 mM- EDTA (Larsen & Weintraub, 1982). Portions were incubated for 30 min at 37°C with 0 to 50 units of S,/pl of nuclei suspension. Nuclease R, from Sigma or BRL was

Nuclease Studies of Rabbit Globin Gene 81 1117

used without noticeable differences. Plasmid DNA to be cut with 6, was incubated in low salt buffer for 5 min at 37°C’ with 0.5 unit of S,/pg of DNA. The reaction was immediately extracted twice with phenol. twice with phenol/chloroform (1 : 1, r/v) and once wit,h ether before being precipitated with ethanol. Plasmid DNA to be nicked wit’h S, was handled similarly, except that 0.1 unit of S, was used to digest 1 pg of DXA for 90 s.

Cd 1 Plnxmid su~bclones a,nd plasmid constructions

The isolation and characterization of recombinant 1 bacteriophage clones and plasmid subclones containing the adult rabbit B globin gene (Bl) have been described by Maniatis et aE. (1978) and Lacy et al. (1979). The 0.65 kb BarnHI-EcoRI fragment, containing most of the large intron of fil was subcloned into pBR322. pEB1.95 (EcoRI-BamHI) is a subclone from 1RBG2 and contains about 1.4 kb of 5’ flanking region of PI as well as exon I, intron 1 and most of exon 2. pEBl.95ARsa0.28 is a derirat’ive of pEBl.95 formed by deletion of the 282 bp RsaI fragment between positions -495 and - 212 (negative numbers denote the number of nucleotides 5’ to the cap site). pEB1.95ARsa1.17 was obtained from pEB1.95 by deletion of 2 contiguous RXZI fragments between positions - 1378 and -212.

pR.28-1 was constructed by introduction of the 282 bp R.saI fragment (positions -495 to -212) into pBR322. The insert,ion is at the ScnI site. so t.ransformants were selected for Tc’ and Ap”. The orientation of this fragment (shown in Fig. 5) is such t,hat nucleotide -494 (it.s left border) is closer to PVUI in pBR322 than its right border (nt -213). pR.28-3 has 3 such RsaI fragments tandemly repeated in the same orientation, whereas pR.28-2 has 2 tandemly repeatrd RsaI fragments in the opposite orientat,ion (HW the diagram in Fig. 5).

(e) C’hsomatin assrmbly in vitro

(‘ovalently closed. relaxed plasmid was prepared by t,reatment of the supercoiled form with type I DNA topoisomerase (BRL) for 30 min at 37°C at a ratio of I unit//pg J)N’A. The DNA was extracted with phenol, phenol/chloroform (I : I. v,/r) and ether before precipitation with et,hanot. Chromatin assembly was carried out. as described by Nelson ef al. (1981) using 0.3 pg of relaxed, covalentl,v calosed DNA (form Tr). 0.3 pg of calf thymus core histones (type 11-S; Sigma), 0.3 pg of polyglutamic acid (Sigma) and 2 units of topoisomerase I in 20 mM-Tris. HCl (pH 7.6). 150 mM-NaCl, O.Ol’-& NP-40. 1 mw-EDTA. lW,, (v/v) glycerol. After 45 min at 30°C. the reactions were stopped by addition of 3 pt of a mixture cont,aining V;, sodium dodecyl sulfat,e. 0.25 M-

EDTA. 509,, glycerol. and the superhelical state of the DNA was analyzed by electrophoresis. Alternatively. 100 units of S, were added to the reconstitution mixture per fig of DSA. Samples were taken at various times and extract’ed with phenol. The DNA was digested with an appropriate restriction rndonurlease, run on a 1% (w/v) agarose gel and blotted to nit,rocellulose. The blots were hybridized t.o a 485 bp P&I-BamHI fragment corresponding to axon 1. intron 1 and most, of exon 2 of \]I.

3. Results

(a) Il~Vas~ I hypwaensitiw site 5’ to /I1 in nuclei

In these studies, we have focused our attention on the rabbit globin gene 81. which is expressed in

fetal as well as adult life. Bone marrow nuclei from adult rabbits were isolated and digested with increasing amounts of pancreatic Dpljase I. The DNA was purified, digested with EcoR1 and the DNA fragments separated by agarose gel electrophoresis. The DIiA was blotted onto nitrocellulose and hybridized to the pBamHT-EcoRI 0.65 probe (Fig. 1). This experiment enables us to scan a, region starting at the end of intron 2 and extending l-1 kb into the 5’ flanking region for any posnible HH. Figure 1 shows strong hybridization of the probe with a 2.6 kb band, which corresponds to the intact EcoRT genomic fragment. At higher concrnt~rations of DNase I. the intensity of the 2.6 kb band decreases and a smaller, weaker band appears. Based on the size of this sub-band (I.2 kb). we can localize this cut to a region 100 bp 5’ to the cap site. This location was confirmed by digestion of the DXA with Hind111 instead of EcoRI, which revealed a sub-band of about 7.5 kb: this matches the location of the DNase I cut det*ermined above (results not shown). The l-2 kb sub-band is not very strong but it has been observed reproducibly. Since

DNase I HS site

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Figure 1. DNase I hypersensitive site 5’ t,o PI in nuclei. The lower part of the Figure shows the autoradiogram resulting from hybridization of the RamHT-EcoRI 0.65 probe to rabbit genomic DNA. This DNA was obtained from isolated bone marrow nuclei that were digested with 0 pg, 0.5 pg, 1 pg, 5 pg, 7.5 pg and 10 pg of DNase I/ml (lanes 1 through 6, respectively). After digestion with EcoRI, the DNA samples were run on a I?, agarose gel and blotted to nitrocellulose. ARBG5 DNA (Lacy Pt al., 1979) was cut with EcoRI and used as a molecular weight marker; the sizes (in kb) and positions of thr markers i*re indicated at the left of the autoradiogram. .A map of the EcoRI (E) and BamHI (B) sites around /?l is presented at the top of the Figure.


not all of the cells in the bone marrow are erythroid, this will have the effect of reducing the signal in the sub-band (smaller fraction of active /?l globin genes) while increasing the intensity of the 2.6 kb band (which represents the sum of the active and inactive genes).

We have not been able to identify any HS that would map 5’ to the embryonic 83 or /I4 globin genes in adult bone marrow. These genes are expressed exclusively in embryonic erythrocytes and are not transcribed during the adult life of the rabbit (Rohrbaugh & Hardison, 1983; Rohrbaugh et al., 1985). When blots like that in Figure 1 were hybridized with fi3-specific and P4-specific probes, no sub-bands were observed; only a progressive decline in the intensity of the main bands was seen (data not shown). Therefore, within the limits of detection of our assay, we can conclude that in bone marrow there is a correlation between the active t,ranscription of the j?l globin gene and the presence of a HS in its 5’ flanking region.

(b) Nuclease 8, and PstI hypersensitive sites in nuclei

We have probed the 5’ flank of /?l with two other nucleases. S, was chosen because it has been shown to introduce specific double-strand cuts in the chicken /?” globin gene (Larsen & Weintraub, 1982) and PstI because it maps within the DNase I HS of fll. Bone marrow nuclei were digested with increasing amounts of S, or PstI. The DNA was cut with EcoRI, separated on an agarose gel, blotted and hybridized to the BamHI-EcoRI 0.65 probe. The resulting autoradiogram (Fig. 2) shows a strong 2.6 kb band corresponding to the intact EcoRI fragment. At higher concentrations of S,, a weak sub-band appears with a molecular weight of 1.2 kb, which locates the HS to 100 bp 5’ to the cap site. ?r’o S, HS was found 5’ to the embryonic gene p4 (results not shown). Given the relatively weak signal of the sub-band, we cannot rule out’ the possibility of the presence of secondary sites near fll such as those observed for the 8” globin gene of chickens (Larsen & Weintraub, 1982). However, the general increase in hybridization observed below the 1.2 kb sub-band in the S,-treated lanes could reflect S,-sensitive sites within the gene.

Digestion of nuclei with PstI reveals a sub-band of 1.2 kb, which corresponds to the PstI site 95 bp 5’ to the cap site. In a control experiment, we tested whether the PstI site 560 bp upstream from 04 was equally sensitive in bone marrow nuclei. No sub-band could be detected, however, indicating that the PstI site 5’ to pl is in a more sensitive region of chromatin than is the PstI site 5’ to 84. Therefore, as for DNase I, there is a correlation bet’ween the active transcription of /?l and the sensitivity of its 5’ flanking region to S, and PstI.

(c) Mapping of S, cuts in supercoiled plasmids

The presence of an S, I-IS 5’ to pl in nuclei prompted us to look for a similar site in cloned,

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Figure 2. S, and PstI hypersensitive sites 5’ to /?I nuclei. Bone marrow nuclei were digested with S, (0. 0. 0.1, 1, 15. 10, 20 and 50 units/pi) or with PatI (0, 0. 0.05, 0.25. 0.5 and 2.5 units/@). The purified DNAs were cut with EcoRI and blot-hybridized as in Fig. 1. A map of the EcoRI (E), BumHI (B) and PstI (P) sites around /?l is shown at the top of the Figure and the autoradiogram obtained after hybridization to the BumHI-EcoRI 0.65 probe is displayed below. AR/?65 DNA cut with EcoRI was used as molecular weight marker. The P&I-EcoRI sub-band also provides an internal marker of 1200 bp.

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purified DNA from that region. pEB1.95 is a subclone from JRBG2 containing 1.4 kb of 5’ flanking region, exon 1, intron 1 and most of exon 2 of the rabbit /?l globin gene. When supercoiled pEB1.95 is digested with S,, it is very rapidly nicked. Upon further digestion, most of the DNA is converted to its linear form (Fig. 3(a)). The very rapid conversion of the supercoiled form to the open circular form indicates the presence of a site or sites highly sensitive to S,. To determine the number of cuts and whether the cut(s) is localized, pEB1.95 was digested with Si followed either by BamHI or EcoRI (Fig. 3(b)). The ethidium bromide-stained patterns in Figure 3(b) (lanes 2 and 3) indicate that S, cuts pEB1.95 in only one region located about 900 bp from the BamHI site and about 1000 bp from the EcoRI site. This means that the Si HS in the plasmid is between 400 to 500 bp from the cap site. The same Si HS was seen over a range of 30 to 300 m&r-Na+ and 37°C to 50°C at S, to DNA ratios of O-5 to 5 units/pg. At higher ratios (20 units/pg), other bands begin to appear at 37°C and in 30 mM- Na’. When the temperature of the digest with the higher S, to DNA ratio is raised to 42°C or 50°C.

Nuclease Studies of Rabbit Globin Gene Bl

1 2 3 4 5 6 7 8 9 IO 11 12

oc

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Figure 3. S, digesbion of pEB1.95. (a) Plasmid DNA (I pg) was digested with 0, 0.01. 0.025. 0.05, 0.1, 0.25, 0.5, 1 .O. 5, 10 or 20 units of R, (lanes 1 to 11. respectively). 1 cut with Hind111 and pBR322 cut with HphI were used as molecular weight markers (lane 12). OC. open circular; L, linear: SC, supercoiled DNA. (b) Plasmid DNA (2 pg) was digested with rither 1 unit of S, followed by BumHI (lane 2) or 1 unit of S, followed by EcoRI (lane 3). Lane : is a marker lane caontaining a digest of 1 and pBR322 DNA; sizes are given in bp. The arrowheads indicate the fragments generaM 1)~ S, and the respective restriction endonuclease.

the specific S, HS disappears, while the plasmid is progressively degraded (data not shown).

We determined the location of the S, cleavage site more precisely by labeling the 5’ ends generated by an S, cut (Fig. 4). The labeled DNA was then cut with PvuII. PstI or M&I and the fragments separated by polyacrylamide gel electrophoresis. Autoradiography of the non-denaturing gel (Fig. 4) reveals a 440 bp band in the S,-PvuII digest and a 340 bp band in the S,-PstI digest. These fragment sizes would place the S, cut about 400 bp before the cap site of fil. A precise location of the cut was obtained by running t’he S,-Mnll fragment (labeled at, Sl) on a denaturing polyacrylamide gel along with a pRR322 sequencing ladder. In these conditions. only the message synonymous (top or sense strand) IS detected. The autoradiogram in Figure 4 shows heterogeneity in the S, cutting patt,ern, as at least eight bands appear. The distribution of the int’ensity of the bands is a,symmetric. extending from 84 to 92 nt with a maximum around 90 nt. The data from the S,-MnZI fragment map the S, cut to a stretch of 14 repeating dinucleotides, (C-T),,, based on the sequence of allele I of j31 (Dierks et aZ., 1981). The sequence of allele 2 of Bl (the gene used in this study) differs from the sequence of allele 1 in a few positions in this region including the deletion of two C-T dinucleotides. (The relevant sequence data are in Fig. 10). Therefore, the double-strand S, cut

mapped in Figure 4 is within t’he sequence (C-T),,, and the maximum cutting occurs in the cent’er of the (C-T),, stretch. When the S,-cut. end-labeled plasmid is digested with RsaT (a restriction endonuclease cutting on both sides of the S, HS; see the map in Fig. 8), two bands of about 90 bp and 190 bp are produced. Analysis of the I>XA of t)he 190 bp band on a sequencing gel alongside a sequencing ladder reveals the presence of a clustjer of bands between 190 nt and 194 nt, which confirms the location of the S, HS (results not shown).

The sizes of the S,-PstI, S,-PvuII and S,-MuZI fragments shown in Figure 4 were determined by electrophoresis of the double-st,randed molecbules on non-denaturing gels and by electrophoresis of the single-stranded molecules on denaturing sequencing gels. In all three cases, the apparent size of each double-stranded fragment was 19 to 30 nt larger than the size determined for the corresponding single-stranded fragment. This could be explained by the presence of a single-stranded tail on t’he double-stranded molecule at the SC;1 site. which would retard the mobility of the fragment, through the non-denaturing gel. However. this is unlikely. since S1 would be expected to remove the single- stranded tail (Rushizky, 1981). An alternate explanation is that the double-stranded D?r’A at, the S, site is in an unusual conformation that has a slower electrophoretic mobility. Experiments described below (section (f)) confirm that rrstrict.ion

J. B. Margot and R. C. Ha&son

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Figure 4. Mapping of the double-strand cut by S1 in supercoiled plasmid pEB1.95. The top part of the Figure shows the strategy used to determine the location of the SI cut. The bottom left panel corresponds to the autoradiogram of the &-P&I and S,-PvuII digests electrophoresed in non-denaturing conditions (5% polyacrylamide gels). pBR322 DNA cut with Hinff was used as the molecular weight markers. The bottom right panel shows an autoradiogram of the S,-MnZI fragment run in denaturing conditions (8 M-urea/B% polyacrylamide gel) along with a pBR322 sequencing ladder.

fragments containing the S, hypersensitive region do migrate slower than expected on polyacrylamide gels.

(d) Effects of insertion and removal of a short DNA fragment containing the S, hypersensitive site

A DNA segment containing the (C-T),, stretch with surrounding sequences is sufficient to generate an St HS in a supercoiled plasmid. This was shown by isolating the 282 bp RsaI fragment containing the pEB1.95 S, HS and introducing it into pBR322 at the ScaI site. Three plasmids (pR.28-1, pR.28-2 and pR.28.3) corresponding to the three different arrangements shown in Figure 5(a) were obtained (note that the ligation of this RsaI fragment into a ScaI site generates Sea1 sites at both ends). When pR.28-1 is cut with S1 and EcoRI, the ethidium bromide-stained pattern in Figure 5(b), lane 3, shows

three bands of 4650 bp, 3950 bp and 700 bp. These bands correspond, respectively, to the linear form of the plasmid, the large EcoRI-S, fragment (which contains most of pBR322) and the small S,-EcoRI fragment. Digestion of pR.28-2 with S1 and EcoRT shows that S, cuts twice in the plasmid (Fig. 5(b), lane 4). The first’ cut (labeled as a) generates two fragments of about 880 bp and 4100 bp, and the second cut (labeled as b) generates two fragments of about 600 bp and 4300 bp. Similarly, digestion of pR.28-3 with S, (Fig. 5(b), lane 5) generates one cut in each of the RsaI inserts. Based on the molecular weight of these fragments, the S, sites are mapped precisely to where the (C-T), 2 stretches are in all three plasmids. When more than one Rsal insert is present, the ethidium bromide-stained pattern shows that all S1 HSs are hit with equal frequency, indicating codominance between identical (C-T),, stretches. Also, the intensity of the S,-generated bands is inversely proportional to the number of RsaI inserts in a particular plasmid. This is due to the fact that when Si nicks a plasmid at a particular (C-T),, stretch, this plasmid is relaxed and the other (C-T),, stretches are no longer preferred targets. Since only one of the S, HSs is cleaved, the presence of multiple S, sites per molecule merely spreads the signal over a larger number of bands. These data show that the RsaI fragment contains sufficient sequences to generat)e an S, HS comparable to that in pEB1.95 (Fig. 5(b), compare lanes 2 and 3).

Our data indicate that the position of the S, HS is different in nuclei and in supercoiled plasmids. Tn order t’o determine whether a secondary S, site in the plasmid might, correspond to the site seen in nuclei: the 282 bp RsaI fragment containing t’he (C-T),, stretch was deleted on pEB1.95. The new plasmid, pEB1.95ARsa0.28, was digested with S, and BumHI and run on an agarose gel (Fig. 5(b), lane 7). Staining of the gel revealed bands of 1500 bp and 4200 bp, as well as the linear plasmid of 5700 hp. The first two bands define a new site. still in the rabbit DNA insert, about 1.3 kb 5’ to the cap site (in pEB1.95). Finer mapping (not shown) localized this secondary site to a region about. 120 bp downstream from the EcoRT site, but a search of the sequence did not reveal any special feature that could account for this secondary HK. Deletion of this secondary site (pEB1.95ARsal. 17) revealed multiple distinct bands as well as apparently random cutting (Fig. 5(b), lane 8). As none of the stronger minor sites appeared to map close t,o the in viuo S, site, the EcoRI-RamHT fragment from pEH I .95ARsal. 17 was isolated after a light S, treatment of the plasmid and either 5’ or 3’ end-labeled at’ the RamHI site. This would allow detection of bands too weak to be seen by staining with ethidium bromide. Autoradiography of this preparat’ion electrophoresed on a sequencing gel did not reveal any localized nicking of the upper or lower strand in this fragment (data not shown). Therefore, even though there is a hierarchy of sites susceptible to cleavage by S, (site at - 400 > site

Nudeuse Studies of Rabbit Gtobin Gene fll 201

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pR.28-

~R.28-

Figure 5. S, sensitivity of different plasmid constructs. (a) The line drawings on the left show the orientation of 1, 2 and 3 copies of the 282 bp RsaI fragment inserted at the (S) ScaI site in pBR322 (broken lines). The circular drawing on the right shows the orientation of the 282 bp RsuI fragment in pR.28-1 in relation to the (E) EeoRI and (B) BumHI sites in pBR322. The direction of the arrow indicates the right border of the RsaI fragment (see Materials and Methods). When more than 1 copy of the RsaI fragment is inserted into pBR322, a, b, c, d and e indicate the position of the respective S, HSs. (b) Ethidium bromide-stained patterns of plasmids cut with S, and BumHI (lanes 2.6, 7 and 8) or S, and EcoRI (lanes 3, 4 and 5). The plasmids are pEBl.95 (lanes 2 and 6) pR.28-1 (lane 3), pR.28-2 (lane 4), pR.28-3 (lane 5), pEB1.95ARsu0.28 (lane 7), pEB1.95ARsu1.17 (lane 8). Lane 1 is a molecular weight marker line containing 1. cut with HindIII, pBR322 cut with Ace1 and pBR322 cut with HphI. The small arrowheads indicate the linear forms of the plasmids and the large arrowheads indicate the fragments generated by S1 and the restriction endonuclease. When more than 1 S, site is present, the 2 fragments generated by every S, and EcoRI cut are indicated by the same letter (a to e) used to identify each of the S, HSs in (a). The broad, unlabeled band observed in most lanes, but particularly in lanes 3 and 7. is not an S,-generated linear fragment (if it were, a second fragment corresponding to the rest of t,he plasmid would be produced; this is not seen). These broad bands are not’ seen reproducibly in all experiments.

at -1300), none of the S, sites seen in the supercoiled plasmid corresponds to the site seen in nuclei.

(e) Nuclease S, sensitivity of plasmids reconstituted into chromatin in vitro

The experiments reported by Weintraub (1983) have suggested that pure DNA sequences with an altered secondary structure (i.e. S, HS) do not lose this altered structure once integrated into the chromosomal DNA of L cells. Also, when a supercoiled plasmid carrying the chicken PA globin gene was reconstituted irt. vitro with histones, the reconstituted chromatin retained its ability to form S,-sensitive sites. We have used the method of Nelson et al. (1981) to reconstitute chromatin in condit,ions known to produce nucleoprotein

complexes with a physiological number of super- coils in the DNA, and which produce normal micrococcal nuclease digestion patterns. The method consists in mixing plasmid DNA (form Tr, or covalently closed relaxed circles) with histones at a 1 ; 1 ratio in the presence of an assembly factor (polyglutamic acid) and topoisomerase I. The purified DNA recovered after reassembly shows that a large majority of the DNA molecules have a substantial increase in their superhelical density; this is evidence of good reassembly. DNA of pEBl.95 was reassembled with histones into chromatin-like material and then trea,ted with S, for increasing lengths of time. The DNA was purified, cut with BamHI and electrophoresed through agarose. Blotting onto nitrocellulose and hybridization to the 485 bp PvuII-BamHI fragment show that, as expected, in naked DNA cut. with two


different batches of S1, the probe hybridizes to the 0.88 kb Xi-BamHI fragment (Fig. 6, lanes 1 and 2). When pEB1.95 is converted to form Ir, it no longer has a specific target for S, and very little of the 0.88 kb fragment is present (lane 3). Upon reconstitution (lanes 4 to ll), no preferred S1 site is readily available. As the time of digestion with S, is increased, diffuse bands (between 2 kb and 5 kb) begin to appear. After 15 minutes, a ladder of bands is established with sizes of 0.7 kb, 0.9 kb, 1.2 kb, 1.6 kb and 2.1 kb. These results show that the enzyme does not preferentially recognize the S, site found at -400 in the purified plasmid, but rather S, cleaves at a series of sites whose position could be a reflection of the nucleosomal arrangement in this chromatin-like material (roughly multiples of 200 bp). Equally apparent is the absence of the in viva HS at - 100; cleavage here would have produced a band of 0.58 kb. Therefore, reconstituting the plasmid DNA into a chromatin- like complex masks the -400 HS (as is the case for

In vitro

Reconstituted Chromatin

pEB pEBlr using pEBl.95 Ir

,s, ,s, ,SI

XB -60 TimeofS,

digestion

(min)

- 2.1

- 1.6

- 1.2

- 0.9

- 0.7

505 -

080 -

,23456?09 10 11

T----F? -Probe

Figure 6. Effect of Si on chromatin reconstituted ilz vitro. Lanes 1 and 2 are control lanes in which pEB1.95 (form I) was digested with 2 different S, preparations and then with BarnHI. Lane 3 is also a control lane in which form Ir of pEB1.95 (the starting material in the reconstitution assay) was digested with Si and BarnHI (lane 3). Lanes 4 to 11 correspond to pEB1.95 reconstituted with histones into chromatin and digested with S, for OS, 15s 30s 1 min, 5min, 15min, 30min and 60 min, respectively. The isolated DNA from each sample was cut with BamHI and loaded on a 1% agarose gel. The PvuII-BamHI fragment shown in the diagram below the autoradiogram was used as a hybridization probe. (E) EcoRI, (P) PvuII, (B) BumHI. Sizes of the fragments (in kb) are indicated on the sides. The hybridization around 2.2 kb in lanes 1, 2 and 3 corresponds to the band already addressed in the legend to Fig. 5.

chromatin in nuclei) but it is not sufficient to generate the - 100 HS seen in nuclei.

(f) Nature of the S, site in supercoiled plasmid

(i) Different conformations of the (C-T),,-containing RsaI fragment

The 282 bp haI fragment can exist in three different structures that can be resolved electrophoretically. Digestion of pEB1.95 with R8aI generates five fragments of 2596 bp, 1565 bp, 883 bp, 581 bp and 282 bp, which account for a total of 5907 bp. This value is in close agreement with the molecular weight deduced from electrophoretic migration on an agarose gel (5940 bp). However, careful examination of such a digest on a polyacrylamide gel (Fig. 7(a)) shows three bands in the 282 bp region. All three move slower than expected for a 282 bp fragment. Their electrophoretic mobility corresponds to apparent molecular weights of about 300 bp, 325 bp and 335 bp for bands 1, 2 and 3, respectively. The weaker bands 1 and 2 cannot result from incomplete digestion of the plasmid, since the EcoRI-BamHI insert in this plasmid has been sequenced completely (data not shown) and no other R8aI site is present that would generate partial digest products of 325 bp and 335 bp. The DNA in these extra bands was isolated, denatured and run through a strand-separating gel. Figure 7(b) shows that, under these conditions, the two strands in lane 1 clearly migrate slower than either strand in lane 2, which move slightly slower than those in lane 3. However, sequencing of these three fragments showed that bands 1 and 2 have a sequence identical to the sequence of band 3, so the different mobilities cannot result from different primary structures. Abnormal electrophoretic migration of the region containing the (C-T),, stretch has been observed using other restriction endonucleases.

These data show that in a supercoiled plasmid, a sequence within the 282 bp RsaI fragment (presumably the repeating C-T dinucleotide) adopt’s at least three different structures. These are stable. since they can be detected as bands of altered electrophoretic mobility after cleavage with RsaI and they show a different mobility after denaturation and electrophoresis on a strand- separation gel. These latter two observations argue against the possibility that the different mobilities result from different secondary structures (e.g. different amounts of denaturation), because the different secondary structures should relax to a full duplex upon linearization and the secondary structure differences would be lost after denaturation. However, the different mobilities could result from the presence of a segment of DNA in an unusual conformation, such as a bend or a kink. This unusual conformation could be stable to linearization of the plasmid and appears to persist even after denaturation. Alternatively, the slower

Nucleate Studies of Rabbit Globin Gene fil 203

1565

581

1 Y-1

282 -2 ‘3

Figure 7. Different electrophoretic mobilities of the 282 bp RsuI fragment. (a) The plasmid pEB1.95 was cut with RsaI, end-labeled and electrophoresed on a 5% polyacrylamide gel. The resulting autoradiogram is shown. The unlabeled band at the top marks the position of the well. Bands 1, 2 and 3 correspond to 3 different mobilities of the same 282 bp restriction fragment. (b) Bands 1, 2 and 3 were cut from the gel, electroeluted and run on a strand- separating polyacrylamide gel according to Maniatis et aE. (1982). A negative print of the autoradiogram is shown. which accounts for the light bands on a black background.

mobility of the single strands from bands 1 and 2 could result from some type of covalent modification, although this hypothesis would require that. some fraction of the DNA molecules be modified to different extents.

(ii) Strand preference of S, in the nicking of pEBl.95

The DNA structure actually recognized by nuclease S, in these assays is unknown. Various models, including slippage to produce single- stranded regions (Hentschel, 1982) or adoption of a non-B-form DNA (Schon et al., 1983), have been proposed. The strand slippage model would predict. that both strands should be equally susceptible to nicking by S,, so we investigated the possibility t.hat one strand would be nicked preferentially in the early stages of the reaction. The approach taken is illustrated by Figure 8. Briefly, the supercoiled pEB1.95 was lightly digested with S, to create a majority of nicked molecules (see Materials and Methods). The DNA was digested with a restriction endonuclease cutting on both sides of the nick but at different distances. The fragments were labeled

at their 5’ or 3’ ends and separated by polyaeryl- amide gel electrophoresis with or without secondary restriction endonuclease digestion. The rationale of this strategy is to monitor the behavior of only nicked molecules that were end-labeled either upstream or downstream from the nick.

Procedure I (Fig. 8) resulted in the isolation of four types of molecules, which were run on a sequencing gel (Fig. 9(a)). Figure 9(a), la,ne 2. shows that a light treatment with S1 results in nicking of the intact AvaII fragment with production of bands around 300 nt and 700 nt; no specific nicking was detected in the absence of S, (Fig. 9(a), lane 1). For clarity, we will refer to 300 nt. and 700 nt, bands, even though S1 does not nick at a specific site but rather within a cluster of 10 to 12 nt. The most striking feature is that the 300 nt band is much stronger than the 700 nt band. If the label were present only at the AvaII sites at the ends of the fragment, this result would show that the bottom strand (mRNA-complementary) is nicked to produce a 300 nt fragment much more frequently than the top strand (mRNA-synonymous), which produces a 700 nt fragment. However, as will be


(C-T) ,z r 1 I I I I

ALaIt DroI RsaI Bg/II BsfNl RsoI NC01 &alI

pEB 1.95 pEB 1.95

Fragment

isolated : ‘A-A’

bp: 994

FMlane: Al

*A- A’ D-A’ ‘A-N ‘R-R’ ‘R-as Bg-R’ ‘R-R’ ‘R-Bs Bg-R’

994 910 990 282 217 209 282 217 209

A2 A3 A4 Bl 83 B5 82 B4 E6

Figure 8. Labeling strategy for mapping the Si nick 5’ to 81. The top line is a restriction map of the area under study. The hatched boxes represent exon 1, and part of exon 2 interrupted by intron 1 (open box). Below, procedures I and II are illustrated. In procedure I, the supercoiled plasmid was nicked with S1 and cut with AvaII (A). The restriction fragments were 3’ end-labeled and either loaded on a 5% acrylamide gel or cut with DraI (D) or NcoI (N) before loadin After autoradiography, the DNA bands with the molecular weight indicated at the bottom of the Figure were excise f from the gel and electroeluted. The reference (Ref.) lanes correspond to the lanes in Fig. 9 where the fragments were loaded. Procedure II is almost identical, except that the plasmid was cut with RsaI (R) and the restriction fragments were 5’ labeled. The reaction mixture was either loaded on a 5% polyacrylamide gel or cut with B&N1 (Bs) or BgZII (Bg) before loading. In both procedures, a control was included in which the plasmid was incubated in the S, digestion buffer without enzyme.

shown below, some label is incorporated at the nick on the bottom strand, so the intensity of the 700 nt band is an overestimate of the frequency of the top strand nicking, and consequently the bottom strand nicking preference by S, is even more pronounced than the results in Figure 9(a), lane 2, indicate. Removal of the leftward AvaII label by cutting with DraI gives a DraI-AvaII fragment of 910 bp. Figure 9(a), lane 3, shows that only a 700 nt band is present besides the 910 nt DraI-AvaII fragment. Therefore, the label of the 300 nt band comes from the AvaII site (complete disappearance of the 300 nt band) and is not due to 3’ labeling of the top strand at the nick (no 220 nt band that would correspond to a DraI-S, fragment is present in Fig. 9(a), lane 3). The label associated with the 700 nt band can come from the 3’ end at the rightward AvaII site (top strand) or from the 3’ end at the S, site on the bottom strand. Removal of the rightward label by cutting with NcoI gives the pattern shown in Figure 9(a), lane 4. Both 300 nt’ and 700 nt bands are present besides the intact

AvaII-NcoI fragment. The presence of the 700 nt band means that the nick on the bottom strand is labeled, presumably by a repair reaction using [a-32P]dGTP to fill-in at the G residues in the G-A)12 segment. Therefore, the intensity of the 700 nt band is the sum of label coming from the rightward AvaII site (top strand nick) and label coming from the nick (bottom strand nick). To determine the relative contributions of each label site to the intensity of the 700 nt band, the 910 bp DraI-AvaII fragment was digested with Real and run on a sequencing gel. We detected two labeled bands of 200 nt and 500 nt corresponding, respectively, to the S,-RsaI and RsaI-AvaII fragments (data not shown). The distribution of the label between these two bands indicates that about SOY/, of the label in the 700 nt band (Fig. 9(a). lane 2) is coming from the label at the rightward AvaII site and 40% is from incorporation at the bottom strand nick. Thus, comparison in Figure 9(a) between the intensity of the 300 nt band and the readjusted intensity of the 790 nt

Nuclease Studies of Rabbit Globin Gene pl PO5

Ml234 Ml23456

310 -08

1125 272

853 242

215 7001

1901

557 cnll ; I .

151

b

406 mm /

396 Q*

300f

282 0-e 73

67

227-6

221

(4 (b) Figure 9. Strand preference in 8, nicking of pEB1.95. The various DNA fragments obtained according to the labeling

scheme in Fig. 8 were isolated and run on a 5% sequencing gel along with end-labeled molecular weight markers (M), which were pBR322 cut with HphI ((a) lane M) or 1 cut with AvaII ((b) lane M). A pEB1.95 sequencing ladder (not shown) was run simultaneously with the samples in (b). (a) 3’ End-labeled fragment,s: (b) 5’ end-labeled fragments. The identity of each lane is indicated at the bottom of Fig. 8.

band (about SOoi, of the intensity in Fig. 9(a), lane 2), which is a reflection of how frequently S, nicks the bottom strand versus the top strand, clearly indicates that S, preferentially nicks the K-Ah2 stretch in the bottom strand.

The results using 5’ labeled fragments are displayed in Figure 9(b). A smaller initial fragment was used in this case, both as a confirmation of the above results and to obtain a precise location of the nick (see Fig. 8, procedure II; and Fig. 9(b)). The plasmid was nicked with S, and cut with Rsal. Figure 9(b), lane 2, shows that S, nicking of pEB1.95 followed by isolation of the 282 bp RsaI fragment generates a very intense 190 nt band and a weak

90 nt band. A small amount of nicking at the S, site was detected in the absence of nuclease S, (Fig. 9(b), lane 1) presumably from partial de- purination in the S, buffer. Removal of the rightward RsaI label by cut,ting with BstN (Fig. 9(b). lane 4) totally removes the 190 nt band without appearance of a band around 125 nt. A band of 125 nt would correspond to an 8,-BstKI fragment labeled at the S, site if t)here were 5,’ labeling of the top strand at the nick. Therefore, the label in the 190 nt band comes from the rightward (closer to the gene) RsaI site only, and its intensity is a reflection of the frequency of nicking on the bottom strand. Figure 9(b). lane 6. shows that


cutting with BglII generates an intense band at 190nt but the weak band at 90 nt is absent. Instead, a series of low molecular weight bands (11 nt, 13 nt, 15 nt, 17 nt and 19 nt) can be seen when the mat’erial loaded in Figure 9(b), lane 6. is run for only a short distance (results not shown). This means that part of the label in the 90 nt band is due to internal label at the nick of the lower strand. We estimate that about half the intensity of the 90 nt band is due to the external label (at the leftward RsaI site, top strand) and about half is from label at the bottom strand nick. A comparison of t,he intensity in Fig. 9(b), lane 2) is a comparison readjusted intensity of the 90 nt band (about 5Oqh of the intensity in Fig. 9(b) lane 2) is a comparison of the frequency of S, nicking of the bottom strand versus the frequency of Si nicking of the top strand. A dramatic preference for nicking the bottom strand is again observed here, as shown in Figure 9(b) by a very intense 190 nt band in lane 2 or 6 versus a very weak 90 nt band in lane 2 or 4.

The precise location of the nicks was determined from the 190 nt’ band run alongside a sequencing ladder. A lighter exposure of the autoradiogram in Figure 9(b) was used to visualize every nucleotide in this region. The locations of the nicks are indicated in Figure 10, along with the DNase 1 and S, HSs mapped in nuclei. The positions of t’he nicks cover the right half (closer to the gene) of the (G-A),, stretch. Furthermore, in that half. every other phosphodiester bond is cut preferentially. As shown above, when pEBl.95 is linearized with S, and 5’ labeled at the cut, every nucleotide in the S, site is labeled (Fig. 4). Thus, the S, cleavage pattern differs between the early nicking and subsequent double-strand cutting. During the initial nicking, S, is able to recognize specific phosphodiester bonds in the (G-A)i2 stret’ch. presumably due t’o some unusual DNA structure in this region. Upon further nicking, S, cleaves across from t’he primary nicks. The ends of the resultant linear molecule are “breathing” and can be nibbled by the enzyme (Shenk et al., 1975). The nibbling is probably not specific so that, upon 5’ labeling, all bonds within the S, cutting site appear to be cut.

4. Discussion

Our results show that a specific region of the chromatin 5’ to the active /?l globin gene of rabbits is sensitive to digestion by DNase I in bone marrow. This HS maps 100 bp 5’ to the cap site in a region shown to be required for efficient transcription of j31 (Dierks et al., 1983). This region consists of an imperfect tandem repeat of 14 bp and 15 bp. Based on sequence comparison between the P-like globin genes of mouse, human, goat and rabbit, these authors were able to define a consensus sequence Pu-Pu-C-Py-N-C-A-C-C-C located about nine or 23 to 25 bp upstream from the C-C-A-A-T sequence. It is interesting to note that, in chickens, this same consensus sequence is found within the DNase I HS (McGhee et al.. 1981).

In humans, the DNase I HS 5’ to /I maps roughly within the region containing the consensus sequence (Groudine et al., 1983). A tandem repeat of C-C-C-C-A-C-C-C-T coincides with the DNase I HS associated with the Pl promoter 5’ to the myc gene (Siebenlist et al., 1984). Also. the region of the HSV tic gene between - 109 and -95 that governs the frequency of transcriptional initiation of the gene contains a HS located within a sequence homologous (12 out of 14 nt) t’o t,he consensus sequence (Sweet, et al., 1982). However, this apparent’ correlation between DNase T hypersensitivity and t)he consensus sequence is probably not due to the sequence per se but. rather is a reflection of the chromatin associated wit,h structures that direct or influence transcription. The special feature of active chromatin that is recognized by DNase I could be a protein-depleted region. perhaps one that is nucleosome-free (Jakobovits et al., 1980; Saragosti it al., 1980: McGhee et al.. 1981). Binding of part,icular protein factors may be required t’o maintain this nucleosome-free region (Emerson & Felsenfeld, 1984) and it is possible that these factors may recognize the consensus sequence mentioned above. The accessibility of the - 100 region of the rabbit) /?l gene t’o both DNase I and PstI is consistent with this region being protein-depleted.

Our results concerning 8, sites in nuclei agree in most respects with the data of Larsen & Weintraub (1982). We find one S, hypersensitive region about 100 bp upstream from the cap site of /Il. This region corresponds to the position of one of the sites found 5’ to the chicken PA gene (Larsen & Weintraub, 1982). On the other hand, these authors detect’ a secondary site 200 bp upst,ream from the cap site of /I”, mapping in a, (G)i6 string. Such a purine-rich region is found 400 bp 5’ to fil but no corresponding ill r:i~o HS was found, although we probably would not have been able to detect, minor HSs in our assay.

The close proximity between S1 and DNase I HSs 5’ to fll was found also for t’he a and /I globin genes of chickens (Larsen & W’eintraub. 1982). This suggests that these enzymes recognize either neighboring chromatin features or different aspects of the same feature. Our in vivo data define a region of t,he chromat,in 5’ to /I1 that is particularly sensit’ive t’o a variety of nucleases. The corresponding sensitivity is not observed 5’ to 84 (a gene not expressed in bone marrow). indicating a correlation bet’ween hypersensitivity and the active t,ranscript,ion of Bl. Similarly, no S, HS has been found at t,he 5’ end of the fetal Gy and *y globin genes in adult human bone marrow (Groudine et al., 1983). In recent) studies with murine erythro- leukemia cells, Sheffery et al. (1984) have shown that the DNase I and Si HSs 5’ to the ~rl globin gene are overlapping. Upon induction, these sites remain sensitive but new sites appear. The new DNase I and S1 HSs, which are associated with the actively transcribed gene, are separated by about 300 hp. This suggests that at least in some cases

Nuclease Studies of Rabbit Globin Gene /31

Mnl I

A Rsa I

Figure 10. Summary of DNase I, Si and PatI HSs in the 5’ flank of rabbit fil globin gene. The 440 nt preceding the cap site of allele 2 of 81 are shown. The overlapping S,, Pat1 and DNase I HSs mapped in nuclei are indicated as broad arrows (double-strand cuts). The mapping accuracy of these sites (except for PstI) is within 30 to 50 bp. The S, HS in plasmids is indicated by filled arrows and arrowheads. The accuracy of the position of the S, nicks in the (G-A)12 stretch (message-complementary strand) is within 1 nt. The nicking pattern displayed corresponds to the sizes of the singlr- stranded S,-RsaI molecules, labeled at their RsaI end. In the nick, the length of the arrows reflects the frequency of cutting in the (G-A)r2 stretch and arrowheads indicate that only minimal nicking was observed. The location of the in vitro double-strand cut described in Fig. 4 is not shown here but does map exactly opposite the thin arrows. The 3 boxed sequences correspond to the - 100, C-C-A-A-T and ATA boxes defined by Dierks et al. (1983). The arrowheads at positions -316 and -213 indicate the position of cuts by the restriction endonucleases MnlI and KsnI. respectively. Sequence hyphens have been omitted for clarity.

these two enzymes apparently are able to recognize different structures. In the 5’ flank of the heat- inducible hap70 genes in the 87A7 locus of Drosophila melanogaster, there is an approximately 175 bp “nucleosome-free gap” that contains multiple cleavage sites for DNase I and micrococcal nuclease (Udvardy & Schedl, 1984) as well as sites for Beurospora cmssa and nuclease S, (Han et al., 1984). In this case as well, the different cleavage sites are closely linked but, distinct.

We have shown that a plasmid carrying part of /?I and its 5’ flank is highly susceptible to S, but only when supercoiled. The cut is made within a &retch of (C-T) i 2 located 400 bp upstream to the cap site. S, susceptibi1it.y of repeating polypyrimidine/polypurine stret.ches in supercoiled plasmids has been found for several other genes including the chicken pro-a2(2) collagen gene (Finer et al., 1984), the Drosophila heat shock genes (Mace et al., 1983), the human CI globin gene (Shen, 1983) and the chicken B globin gene (Schon et al., 1983; Nick01 & Felsenfeld, 1983). Similar sequences found in the spacer region of the sea urchin histone genes (Hentschel, 1982) and 3’ to the human Ul RNA

genes (Htun et aZ., 1984) are S,-sensitive as well, but are independent of supercoiling. Thus, in the latter cases, the generation of the HS appears to be different in some respects from what we observe. S, is known to recognize and cut DNA within inverted repeats or A+T-rich sequences (Lilley, 1980; Panayotatos & Wells, 1981; Goding & Russell, 1983) and at the junction between B and Z-form DNA (Singleton et al., 1982). Thus, t,he ability of S, to cut within the (C-T),, stretch suggests that either it is partially denatured or it can adopt an unusual conformation. It is interesting to note that not all single-strand-specific nucleases recognize identical structures. In a supercoiled piasmid carrying Drosophila heat shock genes, S, and mung bean nucleases cut upstream of hsp26 and hsp28. whereas Ba131 cleaves 5’ to hsp23 and hdp28. This indicates that at least two classes of DNA perturbations can result from torsional stress (Selleck et al., 1984).

We have shown that the major S1 HSs found in nuclei and in supercoiled plasmids are not identical for rabbit gene pl. They are separated by about 300 bp. Schon et nZ. (1983) have shown in the


chicken PA globin gene that the major site in nuclei corresponds to a secondary site that appears in plasmids only after deletion of the (G),, string (the major Si site in plasmids). We were able to show that deletion of the (C-T),, stretch resulted in the appearance of a secondary site at about - 1300; it does not correspond to the HS seen in nuclei. Upon deletion of this secondary site, no site corresponding to the in vivo site was detected. Besides the different sequences of the genes being studied, a major difference between our assay and that of Schon et al. (1983) is that we used the whole plasmid (pBR322 and insert with or without deletions), whereas they used only a small fragment that had been recircularized in the presence of ethidium bromide. The latter assay eliminates the “noise” coming from S,-sensitive sites in pBR322, but it also prevents the detection of possible secondary sites further upstream.

Han et al. (1984) have compared S1 HSs for the 87A7 hsp70 genes in supercoiled plasmids, naked genomic DNA and nuclei from Drosophila KC cells. The major S, HSs in genomic DNA and linear DNA are located in the A+ T-rich intergenic spacer between the two hsp70 genes, whereas the major S1 HSs in nuclei are located in the 175 bp “nucleosome-free gap” located immediately 5’ to the genes. The HSs in both the intergenic spacer and the immediate 5’ flank are present in supercoiled plasmids. Thus, in this system, supercoiling the DNA produces some S, HSs that correspond to those seen in nuclei; the site at - 127 is flanked by repeating (C-T), segments. The presence of the same S, HSs in nuclei and supercoiled plasmids contrasts with the situation described here for the rabbit /?l gene. However, the S, HSs in the intergenic spacer of the hsp70 genes are refractory to digestion by S, in nuclei. These sites have not been mapped precisely to the nucleotide sequence level, so a direct comparison with the rabbit fll data cannot be made. It could be significant, however, that the far upstream sequences in both the rabbit 81 and Drosophila hsp70 genes are hypersensitive to S, in supercoiled plasmids but not in nuclei. Many of the S1 HSs in plasmids containing repeating polypyrimidine polypurine tracts have not been tested for cleavage in nuclei from transcriptionally active or inactive cells. Such studies for a variety of plasmid HSs are needed to ascertain their possible biological function, if in fact such a function exists.

The chromatin reconstitution experiments show that histone assembly is capable of suppressing the in vitro site at -400, but simple reconstitution is not sufficient to generate the HS observed in nuclei. The discrepancy between our results and those of Weintraub (1983) as to the formation of the in vivo

site upon reconstitution could be attributed to a species-specific or cell-specific factor, since we used calf thymus histones in our assay. Alternatively, the presence of the polyanion polyglutamic acid could prevent formation of the in vivo S1 HS. The results of Emerson & Felsenfeld (1984) provide

another possible explanation. They have isolated a protein fraction that confers DNase I hypersensitivity to the 5’ flank of the chicken /I” gene. Even though a different enzyme is involved, the close association between S, and DNase I HSs would suggest that, as in the DNase I case, non- histone factors are necessary for formation of the in vivo S1 HS.

The exact nature of the structure in supercoiled plasmids recognized by nuclease S1 has not been elucidated. We have made several observations that give some information about the S1 hypersensitive structure. (1) The sequence of the S, cleavage site is a repeating dinucleotide (C-T),,. It is polypyrimidine on the top (message synonymous) st’rand and polypurine on the bottom (message complementary) strand. (2) Si nicks the bottom strand preferentially in the sequence (G-A)12. (3) The DNA at the nick site can be labeled with reverse transcriptase and [a-j2P]dGTP, which shows that at least some of the molecules actually contain a gap, not just a nick. (4) S1 eventually nicks the top strand to produce a double-strand cleavage. (5) A 282 bp RsaI fragment containing the S,-sensitive site can adopt at least three different, conformations that are stable to cleavage by RsaI and that’ can be detected by their different electrophoretic mobilities. S, cuts several different cloned genes in supercoiled plasmids in regions of simple tandem repeats, and this observation led Hentschel (1982) to propose that strand slippage in the repeated segment allowed denatured regions of DNA to form and that S, cleaved the single- stranded regions. The strand slippage model is consistent with observations (1 ), (3) and (4). However, such a model would produce sym- metrically arranged denatured regions in both strands. Thus, as no sequence specificity has been observed in the mechanism of S, action (Vogt, 1973), this model is not consistent with the pronounced preference of S, for cutting the bottom strand. Schon et al. (1983) reported a strand preference for cleavage of the 5’ flank of the chicken PA in the (G),, string and the ATA box, although symmetrical cleavage in the (G)i6 string was reported by Nick01 & Felsenfeld (1983).

Schon et al. (1983) suggest that S, is cleaving in a region of non-B-form DNA rather than in a single- stranded region. Our observation of stable alternative structures in the 282 bp RsaI fragment is also not readily explained by denatured regions; if the different structures were merely different degrees of denaturation, they should all renature to a full duplex form after cutting with RsaI. This observation is more easily explained by the DNA adopting an unusual conformation. It is unlikely that the unusual DNA conformation is Z-form DNA, since 2 DNA tends to form in segments of alternating purine and purimidine nucleotides. Abnormal electrophoretic mobility has been observed for a few particular DNA fragments. In Salmonella. the sequence integrity of a region upstream of the h&R locus, which is responsible for

Nuclease Studies of Rabbit Globin Gene /I1 209

the unusual electrophoretic properties of the corresponding DNA fragment, is important for promoter function (Bossi & Smith, 1984). Certain restriction fragments derived from trypanosome kinetoplast minicircles exhibit low electrophoretic mobility when compared to a DNA fragment of comparable size (Simpson: 1979). The kinetoplast Dh’A fragments. which are essentSially in the B-form (Marini et al.. 1984), were shown to be bent in solution (Hagerman, 1984: Wu B Crot,hers, 1984). It is possible that a bend is the unusual DNA conformation responsible for the abnormal electrophoretic mobilities of the RsaI fragment.

The strand preference for S, nicking is not determined by t’he purine or the pyrimidine content of t,he segment. S, nicks in the polypyrimidine (C),, string of the chicken /I” (Schon et al.. 1983) but it nicks in the polypurine (U-A),2 segment of the rabbit fll. In both cases. it is the message- complement>ar> st’rand that is being nicked preferentially. Also. the position of the nicks in the chicken PA ((016 segment is similar to t,he position of t,he nicks in the rabbit’ /I1 (C-A),, segment; both cover t>he rightward half (closer to the gene) of the segment, This apparent relationship bet,ween the strand preference of S, cleavage and the position of the gene in the supercoiled plasmid is unexpected, and we do not know if t,his is a general phenomenon. \Yt& are examining this apparent relationship by testing whether the orientation of t,he 282 bp KsuI fragment has an effect on S, strand preference. LUt’ernatively, the strand preference could be determined I;‘? sequences adjacent to the polypurine~polppyrimld~ne tract.

This research was supported by PHS grants AM27635 and AM31961.

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DNase I and nuclease S1 sensitivity of the rabbit Î²1 globin gene in nuclei and in supercoiled...

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