Chromatin organization of the 87A7 heat shock locus of Drosophila melanogaster

J. Mol. Biol. (1984) 172, 385-403

Chromatin Organization of the 87A7 Heat Shock I,ocus of Drosophila melanogaster ANDOR UDVARDYt AND PAUL SCHEDL

Department of Biology Princeton University Princeton, N.J. 08544, U.S.A. (Received 15 September 1983, and in revised form 7 October 1983) We have examined the chromatin structure of the hsp 70 gene complex at the 87A7 heat shock locus of Dro.s'ophila melanogaster. Our results indicate that this locus has a complex chromatin organization. Heat induction causes highly specific alterations in the chromatin throughout the locus. There are major changes within the heat shock gene transcription units, and in both the upstream and downstream flanking spacers.

1. I n t r o d u c t i o n

The utilization of genetic information in eukaryotes m a y be intimately connected with chromatin structure. We have reported experiments on the histone gene repeat unit of Drosophila melanogaster consistent with this possibility (Samal et al., 1981; Worcel et al., 1983). We found t h a t each DNA segment of the repeat is packaged into a distinct and characteristic chromatin arrangement; the sequences around the 5' ends of all five histone genes are in an exposed configuration and are highly sensitive to nuclease attack; the non-transcribed spacers are assembled into ordered nucleosome arrays with the core particles aligned with respect to the DNA sequence; the histone genes have an " a l t e r e d " chromatin structure probably reflecting a multiphasic distribution of nucleosomes. Although these results point to a relationship between chromatin and the underlying functional organization of the I)NA, it could be argued t h a t these nucleoprotein structures are a peculiar feature of repeated gene complexes. Clearly, it would be of interest to determine if unique (or moderately repeated) RNA polymerase II transcription units plus flanking spacers display a similar sort of chromatin organization. To explore this question we have examined the chromatin organization of a single locus, 87A7, on the right arm of the third chromosome. This locus contains two of tile five 70,000 M r heat shock protein genes. Three other hsp 70 genes plus an assortment of aft heat-inducible transcription units are present at a second t Permanent address: Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, 6701 Szeged, Hungary. 385 (}{)22-2836/84/040385-19 $03.00]0 © 1984 Academic Press Inc. (London) Ltd.

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('ytogenetic locus 87C1 (Ashburner & Bonner, 1979; Mirault et al., 1979; IshHorowicz et at., 19791. As illustrated in Figure 1, t h e 87A7 hsp 70 genes are transcribed in opposite orientation and are separated by an A + T - r i c h -,~ 1.6 k b t intergenic spacer which exhibits some s t r a i n - d e p e n d e n t length variation. Each transcription unit is ~-2"0 kb and is flanked on the 5' side by a conserved 340 bp I)NA s e g m e n t Z,¢ (Moran et al., 19791. The Z,¢ sequence element is also found at the 5' ends of the 87C1 hsp 70 and ctfl genes (Moran et al., 1979; Lis et al., 19811 and a n u m b e r of studies indicate that, Z,~ contains the sequences required for heat-inducihle transcription (Pelham, 1982). Finally, the 3' ends of the 87A7 hsp 70 transcription units are flanked by spacer sequences unique to this cytogenetic ]OCllS. In the studies reported here, we have extended the initial work of Wu (1980) by taking a d v a n t a g e of single copy DNA l o c a ~ d just d o w n s t r e a m fi'om the 3' ends of the two hsp 70 genes. This has p e r m i t t e d a detailed analysis of the c h r o m a t i n s t r u c t u r e of the 87A7 locus both before and after h e a t induction. We have also compared the chromatin of the wild-type locus with t h a t of the deletion m u t a n t Sze-13 which lacks t h e 5' half of the proximal gene and most of the intergenic spacer (see Fig. 1) ( U d v a r d y et al., 1982).

2. Experimental Procedures (a) Materials Mieroeoeeal nuclease and DNase [ were obtained from Worthington or Sigma; the restriction enzyme EcoRI was a kind gift from I)r Paul Modrich. l)epartment of Biochemistry. Duke University: 32p-/abeled nucleotide triphosphates were obtained from New England Nuclear and Amersham: nitrocellulose filters were from 8ch)eieher and Schuell. Recombinant subclones from the 87A7 heat shock locus were constructed from the plasmid 56H8 (Moran et al., I979) and 122 (Goldschmidt-Clermont, 1980).

(b) Methods Isolation of nuclei from frozen embryos: about 30 g of embryos stored at - 7 0 were thawed m] ice. 70 ml of ice-cold buffer A (60 m.~I-KCI, 15 m.~l-NaCI, 15 m,~i-Tris. HCI, pH 7-5. 0-3 m.~l-spe,rmine, ! m,~1-spermhline, i m.~l-dithiothreitol, I m,~l-dithiothreitol. (}.2 m.~1-phcn,vlmethylsulfonyl tluoride, 1 m.~I-EDTA, 0.1 m,~I-EGTA, 0.25 ~I-sucrose) were added and the embryos homogenized in a Teflon Potter homogenizer at, 0°C. The homogenate was filtered through 2 layers of Miraeloth (Calbiochem), centrifuged for 7 rain at 2(~) revs/min t~ remove debris, and the supernatant mixed with NP40 (BDH: 0.2% (v/v) final concentration). After a short vortexing, nuclei were pelleted at 5000 revs/min for I(}mirJ, The. SUl)ernatant was removed anti the pellet rinsed with buffer and then resuspended in 1.5 ml of Imffer A* (buffer A without EDTA and EGTA supplemented with I m.~l-Ca('12). A portion of the nuclear suspension was lysed in 1% (v]v) Sarkosyl {Sigma) and the optical density measured at 260 and 320 nm. (el l~olation oj nuclei from KC tissue culture cells

Tissue culture cells were harvested by eentrifugation and resuspended in buffer A. NP40 was added to 0.2% final concentration, and the cells disrupted by pipetting. Nuclei were then isolated as described above. In the heat shock experiments, KC cells were transferred to 37°C for 45 min, the cells harvested, and nuclei isolated as described above. t Abbreviations used: kh, 103 ha~.~ or b~e-pairs; bp, base-pairs.

87A7 HEAT SHOCK L()(!US OF D. melal, offa.~'ter

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(d) Micrococcal nuclease digests Microeoecal nuclease (0.02 unit) was added/! o.m unit (260-:~20) and incubated at 25°C for increasing times (1 to l0 min). The digestion was stopped with EDTA/sodium dodecyl sulfate (10 m,~i and l°/u final concentrations, respectively) and incubated overnight at 37°C with 100/~g proteinase K/m]. After repeated phenol and phenol/chloroform extractions the DNA was precipitated 3 times with ethanol and resuspended in TE buffer (10m~1Tris. HCI, pH 7.5, I m,~I-EDTA).

(e) DNase I digests The nuclear pellet was resuspended into buffer A and divided into equal portions. The digested with increasing concentrations of DNase I (3 to 50 units/ml for 5 to l0 min). The reaction was terminated as described above. (f) Salt extractions The nuclear pellet was resuspended into buffer A* and divided into equal portions. The salt concentration was adjusted to 0.35 M-NaCI and after gentle mixing the suspension was incubated on ice for l0 rain. The viscous nuclear suspension was then centrifuged for l0 min at 4000 revs/min, the supernatant was carefully removed and the nuclear pellet resuspended in buffer A* s~pplemented with I m~i-CaC!2. (g) Naked DNA digests Total genomic DNA was purified from either embryos or tissue culture cells. Digestion was performed at 25°C under the same conditions as used for nuclei. (h) Processing of the DNA samples The various DNA samples were digested with appropriate restriction enzymes, electrophoresed on 45 cm (or 25 cm) fiat bed agarose gels in glycine buffer (Samal el al., 1981), transferred to nitrocellulose, and then probed with nick-translated subcloned fragments from 87A7.

3. Results (a) A n overview of the 87A7 locus To e x a m i n e the c h r o m a t i n organization of t h e 87A7 h e a t shock locus, we used the indirect end-labeling technique (Nedospasov & Georgiev, 1980; Wu, 1980). The 87A7 locus has c o n v e n i e n t l y placed EcoRI restriction sites for secondary cleavage a n d end-labeling (Fig. 1). One site is located a p p r o x i m a t e l y 2 . S k b b e y o n d t h e 3' end of the proximal hsp 70 gene, while t h e other is a b o u t 2.0 kb from the distal gene. To analyze t h e e h r o m a t i n of t h e proximal gene we can probe with an EcoRI-SalI subcloned fragment, while the distal transcription u n i t can be displayed using an EcoRI-BglII f r a g m e n t . Shown in Figure 2(a) is the distribution of micrococcal nuclease cleavage p r o d u c t s reading from the left EeoI~I site t o w a r d s t h e proximal hsp 70 gene. The c h r o m a t i n digests in these e x p e r i m e n t s were from n o n - h e a t shocked 18-hour e m b r y o s (a mix p o p u l a t i o n of Or5 and Sze-13) (lane 3) and KC cells (lane 5). As a control, total deproteinized genomic DNA was also partially digested with micrococcal nuclease (lanes 2 a n d 4).

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Fro. 1. Sequence organization of the 87A7 heat shock locus. The restriction map of the wild-type 0,WI') (KC (~lls or Or5 embryo) 87A7 heat shock locus. Except for some minor differences which are descrit~ed in the text, the DNA ~quenee organization of the 87A7 heat shock locus in KC cells and in the Or5 fly stock are nearly identical. Lines above the restriction map indicate the positions of the sulJelon£~l fragments u ~ d to probe the chroma¢in structure of the 87A7 locus and the flanking regions. Also shown is the structure of the 87A7 locus in tim deletion variant 8ze-13. This mutant is described in detail by Udvardy el al. (1982).

(i) 3' Flanking spacer In the spacer downstream from the proximal hsp 70 gene many of the preferred micrococcal nuclease cleavage sites in naked DNA are exposed in chromatin. This result contrasts with that obtained for the spacer DNA of the Drosophila histone repeat. In this case, most preferred naked DNA sites are protected and the relative yield and distribution of chromatin cleavage products differs substantially from naked DNA (Samal et al., 1981; Worcei et al., 1983). These findings would suggest that the proximal hsp 70 spacer does not have a "static" nucleosome arrangement like that of the histone repeat spacers. This is probably also true of the spacer flanking the 3' end of the distal hsp 70 gene (see Fig. 3).

Fro. 2. ('hromatin structure of the 87A7 heat shock locus from the proximal edge. DNA isolated from chromatin dig :sts of nuclei prepared from embryos, and KC cells (either before or after heat shock) was n..'stricted with Ecol{I~ The I)NA was electrol)horesed on a 0"8% (w/v) agarose gel, hlutted and then l:robed with the EcoRI-SMI subeloned fragment from the proximal side of the locus (see Fig. 1). (a) The distribution of micrococeal nuclea.~e cleavage products in chromatin and naked DNA reading fr(,n the EcoRl cleavage site on the proximal side of the locus towards the hsp 70 genes. EcoRl was used for .~.,condary elea~'age and the EcoRl-,%ll subcloned fragment fsee Fig. 1) was used as a prol~,. Lane I, pBR322 marker digests; hme 2, cleavage products from a microcoeeal nuclease digest of deproteinized genomic DNA prepared from Or5 embryos; lane 3, cleavage products from a mieroeoccal nuelea.~e digest of nuclei prepared from a mixed population of Or5 and 8ze-13 embryos: lane 4, cleavage products from a micrococcal digest of deproteinized genomie I)NA prepared from KC cells; lane 5. cleavage products from a micrococeal nuclease digest of nuclei prepared from KC cells. The diagram indicated the lsmitioning of the "ehromatin-speeifie" microcoecal nuelease fragments in the 3' spaoPr. {h) The ~econdary cleavage was the EcoRl and shows the ehromatin organization of the two 87A7 hsp70 genes from the proximal EroRI restriction site. Lanes 1 to 7 are microeoeeal nuclease digests, lane 8 a DNase I digest. Lane l, Or5 ehromatin; lane 2, Sze.13 ehromatin; lane 3, naked DNA control (KC cell genomic DNA); lane 4, KC cell ehromatin before heat shock: lane 5, KC cell chromatin after a 45-rain heat shock: lane 6, KC cell chromatin before heat shock from nuclei washed with 0.35 MNaCl: lane 7, KC cell chromatin after a 45-rain heat shock from nuclei w&~hed with 0.35 M-NaCI; lane 8, KC exdt ehromatin from nuclei digested with I)Nase I. Lane 9 has pBR322 marker fragments.

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87A7 HEAT SHOCK LO(?US OF D. melanoffaster

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Though our d a t a argue against a stable array of aligned nucleosomes in the 87A7 3' spacers, the organization may not be completely random. A careful analysis of the chromatin digests reveals cleavage products not readily detected in the control. In the proximal spacer, for example, there is a set of "chromatinspecific" cleavage products (a) distributed at nucleosome-like intervals of 180 to 210 bp (see Fig. 2(a)). While these chromatin fragments are often present in quite low yield, their occurrence may indicate t h a t the 3' spacers are assembled into an unstable, but nevertheless ordered structure. At present, it is unclear if there is a shifting in vivo between the set (a) nucleosome format and some other distribution of core particles, or if the general predominance of naked DNA cleavage products l'eftects a disruption of the chromatin by our nuclei isolation procedure (cf. Worcel el al., 1983). (ii) 3' End of the hsp 70 gene The 3' end of the inactive hsp 70 gene is punctuated by a series of prominent fl'agments offset from the naked DNA bands. The first "chromatin-specific" 3' fragment of the proximal hsp 70 gene, fragment 8, (Fig. 2(a)) is aligned with the (a) phase ladder, and maps about 200 bp from fragment 7. Band 9 is a doublet consisting of one chromatin-specific fragment plus a second closely spaced fragment t h a t is also present in naked DNA. This doublet is followed by a more widely spaced doublet, fragments l0 and I 1 which map 160 bp and about 215 bp from fragment 9. This distribution of micrococcal nuclease bands would be consistent with one "static" core particle between fragments 8 and 9, and perhaps a second between 9 and 10-11. The 10-11 doublet is unusual, and could represent a peculiar nucleoprotein structure close to the 3' boundary of the hsp 70 transcription unit (see Levy & Noil, 1981). (iii) The inactive hsp 70 gene The micrococcal nuclease cleavage products from gene chromatin are generally less prominent (see lane 5 in Fig. 2(a)). While some bands are separated by about 180 bp (17 and 18), others are less than 70 bp apart (13 and 14), and there does not appear to be a !adder of fragments consistently spaced at nucleosomal length intervals. Moreover, throughout most of the transcription unit (see, however, below) the nuclease cleavage sites and the relative yield of fragments are essentially the same in chromatin and naked DNA (compare lanes 5 and 4). This finding is similar to t h a t obtained for the Drosophila histone genes (Samai et al.~ 1981; Worcel et al., 1983), and we suggested t h a t this could be explained by a multiphasic nucleosome organization which on average exposes all preferred naked I)NA cleavage sites. This interpretation is consistent with our d a t a on the repressed hsp 70 gene, and would argue t h a t a multiphasic structure m a y occur

a I)Nase I digest. Lane I, Or5 chromatin; lane 2, 8ze-13 chromatin; lane 3, naked DNA control (micrococcal nuclease-treated KC cell DNA); lane 4, KC cell chromatin before heat shock; lane 5, KC cell chromatin after a 45-rain heat shock; lane 6, KC cell ehromatin before heat shock from nuclei washed with 0':35 M-NaCI;lane 7, KC cell ehromatin after a 45-min heat shock from nuclei washed with 0.35 M-Nagl; lane 8, KC cell chromatin treated with DNase I. Lane 9 has pBR322 markers.

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nuelei prep,~red from e m b r y o s or KCJ cells (before and tilter heat shoek) wa.~ cleaved with Byll I. After gel eleetrophoresin +rod blotting, the digest8 were probed with the ,~o[l-Bflll | subehmed fr+~gment from j u , t lwyond the a' end of the dista| }rap 70 gene. (a) The distribution o£ mic.roeoeeal nuelease ele,tvagt:

87A7 HEAT SHO(!K I,()(~US OF D. melanogaster

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even in the absence of transcription. I t should be p o i n t e d o u t t h a t because of the high degree of sequence specificity of micrococcal nuclease (Dingwall et al., 1981; Horz & Aitenburger, 1981) there is a considerable bias in favor of cleavage at talked i)NA hypersensitive sites in c h r o m a t i n digests. Moreover, this problem is particularly acute in the very mild digests used for indirect end-labeling e x p e r i m e n t s (Worcei et al., 1983). Hence, it is difficult to conclusively exclude a i)refer'ential b u t unstable a r r a n g e m e n t of gene nucleosomes (Levy & Noll, 1980) wilich is particularly susceptible to disruption d u r i n g nuclei isolation. This caveat would also apply to the 3' flanking spacers of the hsp 70 genes (see above). (b) Chromatin fine structure at the 5' end of the hsp 70 9ene To analyze the c h r o m a t i n at the beginning of the hsp 70 genes and in the intergenic spacer, we took a d v a n t a g e of the Bff/I] restriction site located d o w n s t r e a m from the distal transcription unit (see Fig. 1). The distribution of cleavage products reading t h r o u g h the 5' half of the distal gene and into the intergenie spacer is shown in Figure 4(a) for Or5 e m b r y o s a n d in (b) for KC cells, and these results are schematically s u m m a r i z e d in Figure 5. (i) Beginninff of the transcription unit In t h e region just d o w n s t r e a m from the transcription s t a r t site we find four p r o m i n e n t c h r o m a t i n bands. The first is a b o u t 550 to 570 bp from the 5' end, while the fourth overlaps the beginning of the gene. T h o u g h these f r a g m e n t s are spaced Irt mwleosomal i,,Lervals of 190 bp (1 to 2), 170 bp (2 to 3) and 200 bp (3 to 4), bands 1 and 2 are found in both c h r o m a t i n and n a k e d DNA and only 3 a n d 4 are "chromatin-specific" (compare lanes I a n d 3 in Fig. 4(a) and 2 and 1 in Fig. 4(b)). There is a similar set of f r a g m e n t s for ~he proximal gene (Figs 4 and 2). These results suggest t h a t the 5' leader s e g m e n t of the inactive hsp 70 gene has at least one aligned nucleosome (see L e v y & Noll, 1980). (ii) 5' End of the gene The 5' Z.~ element of the distal hsp 70 gene is m a r k e d by two heavily labeled micrococcal nuclease cleavage p r o d u c t s {bands 4 and 5) t h a t are absent in equivalent digests of n a k e d DNA. B a n d 4 m a p s to t h e DNA s e g m e n t 20 to 35 bp d o w n s t r e a m |'toni the T-A-T-A box and spans the s t a r t of transcription, and the first nucleotides (if the messenger RNA leader sequence. Band 5, which appears to lie a closely spaced d o u b l e t (see lane 2 in Fig. 4(1))) m a p s 175 bp away near t h e

prc~ducts at the 5' ends of the hsp 70 genes and in the intergenie spacer of Or5 and 8ze-13 embryos. I,ane I, Or5 chromatin; lane 2, 8ze.13 ehromatin; lane 3, embryo naked DNA control. Numbers fefel"to bands discussed ill tile lext and shown ill Fig. 5. (b) The mierococcal nuclease (lanes 1 to 5) and I)Nasc I (hme6) cleavage products from the 5' ends of the hsp 70 genes and in the intergenic spacer from KC cells either before or after beat shock. Lane l, KC naked DNA control; lane 2, KC cell chrtit;~atin betbre hcat shock: lane 3, l(.C cell chromatill after a 45-rain heat shock; lane 4, KC cell ~.hr,)malin from nuclei washed with 0.35 .~I-NaCI;hme 5, KC cell ebromatin after a 45-rain keat shock I'mm aut,h,i washed wilh 0.35 at-Na(Yl: lane fi, l)Nase l-treated KC cell cbromatin. The numbers refer h) hands discussed in the text and shown in Fig. 5.

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i"l('. 5. f ! h r o m a t i n org~ufization at t h e 5' end of the hsp 70 genes and in the intergenic spacer. ']'ht The arra~gemet~t ~Jf mieroeoeeai nuelea,~e eteavage sites at the 5' end of the hsp 70 genies and in th~ the intoriLtmiv spacer of both Or,5 and KC cells. See Fig. 4 and the text. for details.

mid(tie of Z,:. The same Imnding pattern is repeated in the proximal Z~¢ element (bands 15 and l~i; see also Fig. 2). Although mierocoeeal nuetease does not cleave within the 175 bp sequence separating the Z,, hypersensitive sites, much of this segment (and perhaps sequences further upstream; see below) is probably in an exposed configuration in the inaetive hsp 70 gene. This is suggested by the DNase I experiment in lane 6 of Figure4(b) (see also Figs 2(b) and 3). We find one major DNase ] cleavage product near the center of the 175 bp Z,~ segment., plus a number of minor fragments to either side, and together, these cover most of the region between the mieroeoecat nuelease hypersensitive sites. A similar set of DNase I cleavage products was observed by Wu (1980). Thus, this 175 bp sequence is probably not coiled about a nueleosome core particle, but rather may be a gapped region (perhaps containing other sorts of proteins) similar to the open segments found at the 5' ends of many genes (McGhee et al.. 1981; Keene et al., I981; Worcel et al., 1983). (iii) Chromatin structure of the intergenic .spacer; Or5 The arrangement of "chromatin-speeifie" microcoeeal nuciease fi'agments in the remainder of Z.c and the adjacent intergenie spacer has some unusual features. As shown fi)r Or5 (Fig. 4(a)) the first fragment for the distal gene is hand (i and the equivalent cleavage products for the proxima[ gene is band 14. Band 6 maps about 80 to 85 bp upstream from band 5, just beyond the Xbal site in Z,c (see Fig. 5). It is only weakly labeled in the distal Or,5 Z.:, while the corresponding proximal cleavage product (band 14) is considerably more prominent, This difference appears to be due to a variation in the Z.: sequence, In some third

87A7 H E A T S H ( ) ( ! K IA)('UN ()F D. melanofp, ster

395

chromosome one or the other 87A7 Z,c element may have a 15 nueleotide insertion 10 bp upstream from the XbaI site (Mason et al., 1982; Udvardy et al., 1982). Although the Or5 spacer has not been sequenced, studies on the ehromatin of sequenced variants (see below and unpublished data) indicate that (in the inactive state) the more prominent fragment is generated when the insertion is present. The next, microeoecal nuelease fragments for the distal (band 7) and proximal (band 13) genes map outside of Z,c in the intergenic spacer (see Fig. 4). Fragments 7 and 6 (distal) are about 190 bp apart while 13 and 14 (proximal) are separated by 180 bp, and this nueleotide chain length would be suffieient for a nueleosome core particle on the outside edge of Z,~. In contrast, the distance between bands 6 and 5 or 14 and 15 (less than 100 bp) is too short for a core. Hence, there may be only a single nucleosome in the entire interval between the eleavage site in the intergenie spacer (band 7 or 13) and the prominent band in the Z,~ gap (band 5 or ! 5). Curiously, this DNA segment would be ~ 270 bp (see Fig. 5), which is rather large for one nucleosome. This may indicate that the Z,~ gap is larger than 175 bp and extends to fragments 6 and 14, respectively. This suggestion would be consistent with the position of minor DNase I cleavage products in Z,¢ (see Fig. 4(b)). In the remainder of the Or5 intergenic spacer, there are a series of prominent micrococcal nuclease cleavage products which in general differ from those in control naked I)NA digests. Size estimates indicate that the chromatin cleavage products are spaced at nucleosome-like intervals and would be consistent with some type of ordered chromatin structure in the Or5 intergenic spacer perhaps like that diagrammed in Figure 5. Though such a structure would be analogous to that found in the histone repeat spacers (Samal et al., 1981), one major difference should be noted. In the histone repeat, most "linker-to-linker" distances are -~20(1 bp, (.lose to the average size o f a Drosop/lila nucleosomal unit (core+ linker I)NA). In contrast the Or5 intergenic spacer appears to have a wider range of" linker-to-linker intervals with two unusually large units flanking two normal size units. A number of factors could account for this result. First, the "center" of a linker need not correspond to the nuclease cleavage site. Since micrococcal nuclease exhibits a rather pronounced sequence specificity (Dingwall et al., 1981; Horz & Altenburger, 1981 ), it is probable that some linker-to-linker intervals may be overestimated (e.g. bands 9 and 7) or underestimated (bands l0 and l l ) ac(.ording to the position of preferred cleavage sites. Second, because of variations' in linker length, the size of nucleosomal units may not be constant. In this regard, it may be of interest that both large "units" have internal microcoecal nuclease cleavage products (bands 8 and 12, respectively). Band 8 is heavily labeled in control digests (see lane 4), and is probably derived from micrococcal nuclease attack at a naked I)NA hypersensitive site which can sometimes be exposed in ehromatin. There is also a naked DNA cleavage site corresponding to band 12, though it is considerably less sensitive than band 8. Micrococcal nuclease cleavage at these naked DNA sites could indicate that the core particles of the larger units are not precisely fixed, but occupy a number of different positions. Finally, nonhistone proteins might alter nueleosome spacing. For example Jack et al. (1981)

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A, I ' I ) V A I I I)Y A N D P. S('H I.:I)L

recenll,v identified a pz'l,tein which I)inds tl)a spe(.ific sequence in the 87A7 intergenic SlmCer. Additionally. since this spacer is very A + T - r i c h (Mason et ul., 19S2) it s]muld contain many sites f()r proteins like i)1 which bind A + T slrel(']les (l/odz'iguez-Alt'ageme c/u/.. I(.l'SI): i.,evinger & Varshavsky. 1982). have

(iv) ('hromt)tin structure of the inlerfp'nic Slmcer: K(' cells The chromatin structure at the end of gn~ and in the intergenic sl)acer ()|" K(? cells (lifters in several respects from OrS. First, the distal rather than the proximal Z,¢ ehmu'nt has the I)Z'ominenl band n e a r the Xbul restriction site (see I)and 6, lane 2. Fig. 4(t))). (The (,orresplm(ling fragment of the, proximal gene is located jus| belmv hand 13 and can I)arely he detected.) As suggested al)ove, this is likely t() reth, cl tim presence (in the distal Z,c ) or al)sence (in the proximal Z.,c) of the 15 hp insert. S(q,ond, though the distribution of microeoccal nuelease fl'agments in the K(' inlet'genie spacer al)pears to parallel Or5 (compare Fig. 4(a) and (h)), there is a general (.onlz'acli()n ill (listances I)etween cleawtge sites. This is prohal)ly (lue t() a (lilli,rence in the size ()t" the intergenic spacers. In OrS, the 5' ends of the hsp7l) lranscriptilm t,nits are separated by about i.75kb. In contrast, this se,,,men! is I.(14 kb in K(~ cells and our genomie restriction mapping d a t a (not shmvn) indicate that trill, sequence organization is quite similar to the recombinant 122 ((:olds('hmidt-('lerm(mt, 1980: Mason et al., 1982). (The 15 I)1) insert of ]22 is in the distal Z.¢.) In spite of the size difference, the KC cleavage l)roduets arc typicall.v sl)a('e(l at nuvleosome-like intervals. Reading from the "'bomuiary" t>f the nuvh,()sl,me-frec gap (I)an
The S7A7 heal shock locus cff Sze-13 has a small deletion which removes the 5' half of the proximal hsp 70 gene and most of the intergenic spacer. As shown in I"igur'p I, lhe Jeff hreaklmint maps some 3(t bp upstream from the B a m H l site in the middle of the proximal transcription unit. and the right breakpoint is 464 bp uj)stream from the heginning of t h e distal gene. In previous studies (Udvardy el al.. 1982) we have shown that the deletion inactivates the proximal hsp 70 gene, whih' the distal gene is still heat inducible (though the kinetics of induction may not be identi(.al to a wild-type 87A7 locus). We have examined the chromatin of the A':,,-/3 heat slmck Io('us and these results arc presented in Figure 2(I)) (reading I'z'mn the proximal EcoRl site: lane 2), Figure 3 {from the distal Eco[~l site: hum 2) ~.nd Figure 4(a) (from the Bglll site: lane 3). Even though the deleted proximal hsp 70 gene is ina(:tivated, the pattern of micro(.occal nucleas(, cleavage l)rodu(-ts in (.hromatin closely resembles that observed over the sltme region in nake(i I)NA (compare lanes 2 and 3 in Fig. 2(1))). Moreover. the yield and distribution of fragments across the deleted gene is quite similar to t h a t of the corresponding regions of the repressed Or5 and KC cell genes

$7A7 H I ' ] A T , ~ H { ) ( ' K L ( ) ( ' U N ( I F D. t~elrt~tr~g~,~'lr'r

:~9"~

(la.ncs 1 and 4 in Fig. 2(b)). This result would suggest that a multiphasic (or unstable) organization of gene chromatin can be present even when there is no transcription. In ~ddition, it would appear to rule out an alternative explanatioa ['~n' the Or5 and KC results; namely, that a low level of uninduced expression of wild-type genes clisrupts a normally ordered arrangement of nucleosomes. In the intact distal Sze-13 gene, the chromatin is similar but not identicaJ to the wild-type Or.5 (lane 2, Fig. 3 and lane 3, Fig. 4(a)). First, deletion of the intergenic spacer at - 4 6 4 bp does not appear to alter the special structure at, the beginning of Z,~, and the exposed 175 bp segment, is still present. Second, like Or(J, band 6 is only weakly labeled in the Sze-13 Z.¢ element. Sequence studies (Udw~rcly el al., 1982) have shown that the 15 bp insertion is missing in the £'ze-13 Z,¢ element. Finally, fragment 7 which is readily detected in the Or.~ digest, is present in much lower yield in Sze-13. Interestingly, this chromatin fragment maps ---20 bp duwnstrcam from the breakpoint, and hence, the "linker" cleavage site shmthl still be present. One plausible explanation for the apparei~t loss of this linker is that the close juxtapositioning of gene and spacer sequences in the mutant may partially destabilize the chromatin ~t the edge of Z,~.

(d) Cha'n~je, in the chromalin slr~,clure of the 87,4 7 locu,s after heal induction Heat induction has a rather dramatic effect on the chromatin organization of" the KC c:ell 87A7 locus. The pattern of micrococcai nuc]ease fragments in nuclei from heat shocked eeils is presented in Figure 2(b) {reading from the proximal EcoR| site: lane 5), Figure 3 (from the distal EcoR| site: lane 5) and Figure 4(b) (from the BglII site: lane 3). (i) 3' Flanking spacers SurprisingLy, heat shock alters the chromatin of the 3' spacers. We find that a region extending approximately 1.0 kb beyond the end of the hsp 70 genes becomes more sensitive to microeocca] nuclease, though in most, but not all cases, the primary cleavage sites correspond to preferred sites i n n a k e d DNA. Several factors might account for this transition. Since transcription disrupts the chromatin organization of the hsp 70 genes (see below) this structural alteration might be propagated into adjacent spacer I)NA. Such an effect could be direct, ~nd caused by occ'asional readthrough into spacer sequences, or it might be inctircct, and result from an upstream shift in the position of nueteosomes (e.g. at the 3' end of the gent). Alternatively, induction may be accompanied by a transition in the three-dimensional architecture of the locus, e.g. from a compacted configuration to some type of unfolded organization. This possit~ility would be consistent with cytological studies on polytene chromosomes which indicate that a relatively large DNA segment participates in heat-induced puffing. F'inally, there is at least one "chromatin-specific" cleavage product downstream from each gene which becomes more promJnent in heat-shocked nuclei. In the proximal spacer, this heat-induced micrococcal nuetease cleavage site is 7' while the "new" cleavage product in the distal spacer is indicated in Figure 3. In both

;19,~

A. U I ) V A R I ) Y A N D P. S C H E D L

eases, the "new" mieroeoceal fragment maps ,,-400 bp downstream fl'om the putative 3' f~.ntl of the hsp 70 transcription unit (Torok & Karch, 1980), and could represent either some spet'ialized chromatin structure wtlich facilitates upstream transcription (e.g. the unwinding of the helix) or an alternative nucleosome format. (ii) 3' End of the gene Tile ordered ehromatin structure at the 3' end of the hsp 70 genes is disrupted I)y heat shock, and this entire segment becomes more sensitive to micrococcal nuelease. Some of the new fragments are generated by nuclease attack at prei~rred I)NA cleavage sites, while others may arise from new "chromatinspecific" cleavage sites. In the region close to the Sail site, these changes are presumably due to transversing RNA polymerase molecules, and perhaps also to the pausing of polymerase complexes during termination (see Figs 2 and 3). Further downstream these new sites could reflect some type of concerted displacement of core particles. (iii) The h~p 70 ffene As has been reported by other workers (Wu et al., 1979; Wu, 1980; Levy & Noil, 1981) tile overall nuclease sensitivity of the hspT0 genes appears to be much enhanced by heat induction, and the yield of micrococcal fragments throughout the gene is considerably greater than in the non-heat-shocked control (compare lanes 4 and 5 in Figs 2(b) and 3). In fact, gene sequences appear to be more sensitive in chromatin from heat-shocked cells than as naked DNA (compare lanes 3 and 5 in Figs 2(b) and 3). On the other hand, the actual nuclease cleavage sites in heat-sho(.ked chromatin are virtually identical to those in naked DNA (or in the chromatin of the repressed gene). (iv) The 5' e~ul and intergenic spacer Perhaps the most interesting alterations occur at the beginning of the hsp 70 gene and in the intergeni(, spacer (see Figs 2 to 4). Not only are some ehromatinspecific micrococcal nuclease fragments lost or significantly reduced in yield, we also find new bands. (l) Prior to induction, there is probably an aligned nucleosome core spanning the leader sequence of the mRNA. The upstream "linker" is defined by the 5' mierococcal nu('iease hypersensitive site (fragment 4), while the other linker is fragment 3 210 bp downstream. After heat shock, both are only weakly labeled (compare lanes 2 and 3 in Fig. 4(b)), and other bands within the gene are considerably more prominent. This change probably reflects a transcriptionmediated disruption or displacement of the leader core particle. Curiously, this disruption may not be complete as band 3 (and also band 4) can still be detected in heat-shocked nuclei. Presumably either some KC cells were not fully induced 6r this nucleosome may return to its original position (or structure) after each polymera~se complex transcribes the leader sequence. (2) The chromatin organization of Z~¢ and the upstream intergenic spacer is altered l)y heat induction. First, both micrococcal nuclease hypersensitive sites in

~17A7 H E A T

,";H(}C:K LO(!US Ol," D . mela~toffa.~ler

:~l~!t

Z.~ become considerably less prominent (compare lanes 2 and 3 in Fig. 4(b)). This change is Imrticularly evident in the ease of 5' Z.¢ fragments (bands 4 and 14), while it is less dr~tmatic for the upstream cleavage sites (bands fi and 13). A similar phenomenon is observed with l)Nase I (see also Wu, 1980). Figure 6 shows the I)Nase I ele~v~Lge products reading fl'om t!;~ distal EcoRl site either before or after heat shock. As evident from the ~,ut,oradiograph, the major l)Nase I fragments fl'om each Z.~ element are only weakly i~beled after heat shock, and just +t residual band remains. A number of different factors could account for the reduced nuelease sensitivity. The hypersensitive sequences might be protected by either RNA polymerase or other 1)NA binding proteins. Alternatively, 1

2

k X':.x :~ : . :~..:.,..,

•

":

¢, , + ¢ ' . :

. ,:.~ ~.'~:." .

(:

:...:-: ;:!:.-:"-+ .: Fro. 6. l)Nase I cleavage products before and after heat shock. The autoradiograph shows the DNasc l 5' hylversensitive sites in KC cells either before {lane 2) or after heat shock (lane l) reading from tile E c o R l site on the distal edge of the locus.

40~t

A. U D V A R D Y

A N D P. S C H E D L

transcription could alter the structural organization of the DNA in Z,¢. In this regard, recent studies by Weintraub {1983) suggest t h a t in 5' D N a s e l hypersensitive regions ]rave an unusual single-stranded, S 1 nuclease-sensitive conformation, which appears to depend upon superhelical strain. If this is the ease for Z,¢, then the toss of nuclease sensitivity may reflect a local alteration in DNA conformation. This could be mediated by a DNA binding protein or may be due to a lessening of supcrhelieat strain by downstream transcription. Second, we find a new heavily labeled micrococcal nuclease band in both Z,¢ elements. Size measurements place this band ~ 2 8 0 to 300 bp from the transcription start site, towards the end of Z,¢. ]n the distal gene, the heat-induced micrococcal nuclease fi'agment is slightly displaced fi'om the cleavage site ['or fragment 6 which is observed when the gene is turned off (compare lanes 2 and 3 of Fig. 4(b)). The appearance of the Z,¢ cleavage products is accompanied by the loss of fragments 7 and 1"2 (see lane 3 in Fig. 4(b)) in the intergenic spacer. These alterations suggest 1hat heat induction causes a significant restructuring of the nucleoprotein organization of the DNA beyond the 5' ends of the hsp 70 genes, including a possibh, displacement of the core particle spanning the outside edge of each Z,¢ (,lemeni. Since we also observe some reduction in the yield of cleavage products in Ihe center of the spa(.cr, these structural alterations may be propagated into this segmenl.

(e) Salt extraction of nuclei Non-histone chromosomal proteins are t h o u g h t to play an i m p o r t a n t role in gene regulation. At 87A7, such proteins could be involved in establishing the overall ehromatin organization of the locus prior to heat induction, and might also mediate the restructuring after induction. In previous studies we have shown that man)" non-historic proteins are preferentially extracted from nuclei by washing with 0.35 ,~-sait (see Worcel et al., 1983). To gain some insights into the possible role of non-histone proteins in the chromatin of the 87A7 locus, we examined the effect of a 0"35 ~t-NaCI extraction on the micrococcal nuclease cleavage pattern. The results of these experiments for nuclei isolated from both control and heat-shocked KC cells are presented in Figures 2(b) (lane 6, control nu(.iei: lane 7, heat-shocked nuclei), 3 (lane fi, control nuclei; lane 7, heat-shocked nuclei) and 4(b) (lane 4, controi nuclei; lane 5, heat-shocked nuclei). The most obvious changes arc fovnd at the 5' ends of the hsp 70 genes. While the very prominent micrococcal nuclease cleavage products in Z,~ are apparently unaltered in salt-washed nuclei from control cells, tim yield of the upstream fragment 6 is reduced (see Fig. 4). This could indicate t h a t some chromosomal protein(s) is required to maintain this particular sequence in a~l exposed configuration. Interestingly, a similar phenomenon occurs at the 5' end of the D. melanofaster rudimeutary gone after salt extraction (unpublished results). The apparent destai)itization by 0'35 ~z-NaCI is even more dramatic in nuclei from heat-shocked cells, and the heavily labeled heat-induced band in Z,~ is much less prominent after salt. extraction. There also appears to be a further reduction in the y i e l d of the two micrococcal nuclea~e hypersensitive sites in the Z,: "nucleosome-free

8 7 A 7 H E A T S H O C K L O C U S O F D. raelanogaster

401

gap" (compare lanes 5 and 7 in Fig. 2(b) or lanes 3 and 5 in Fig. 4(b)). Additional studies will be required to determine if these alterations are due to the loss of specific DNA binding proteins. 4. Conclusion

The studies presented here provide some additional support for the notion that the chromatin organization of DNA may play an important role in the utilization of genetic information in eukaryotes. We find that each segment of the D. melanoffaster 87A7 heat shock locus has a distinct chromatin structure which appears to be related to the underlying functional properties of the DNA. Moreover, there are some interesting similarities as well as differences between tile chromatin of this heat shock locus and that of the D. melanoflaster histone gcne repeat unit (Samal et al., 1981; Worcei et al., 1983). (a) Spacer D N A Our analysis of the histone repeat unit raised the possibility that nontranscribed spacers flanking RNA polymerase II genes might typically have a "static" chromatin organization in which nucleosomes are aligned with respect to the underlying DNA sequence. However, this is apparently not the case; rather, there are probably at least two different classes of spacer chromatin. One class would be exemplified by the spacers adjacent to the 3' ends of the proximal and distal hsp 70 genes. Although there may be some preference for a particular nucleosome format(s), these DNA segments do not appear to have a stable arrangement of "static" core particles. The second class would be the intergenic spacer between the two heat shock transcription units. In this DNA segment, the chromatin organization (see Fig. 5) more closely resembles that of the histone repeat spacers (Samal et al., 1981). Interestingly, we have obtained equivalent results for the 3' and 5' spacers of the rudimentary gene of D. melanogaster (unpublished results), and these findings would indicate that highly organized nucleoprotein structures occur primarily in DNA segments upstream from transcription units. Conceivably, such structures could facilitate the interaction of various non-histone proteins as well as RNA polymerase with specific 5' regulatory DNA sequences. In contrast, this sort of chromatin organization might not he required in most 3' spacers. (b) Gene D N A Prior to heat induction, the distribution of microeoceal nuelease cleavage products across the hsp 70 genes would be consistent with some sort of multiphasic nucleosome arrangement. Hence, it seems likely that the "altered" structure of gene ehromatin noted in our studies on the histone repeat unit (see Samal et al., 1981; Woreel et al., 1983) is present independent of actual transcription. It should be mentioned that this sort of chromatin organization may be a peculiar feature of transcription units, like the histone and heat shock 18

4(~2

A. UI)VARDY AND P. SCHEDL

genes, which are potentially active in all cell types. Moreover, both the histone and heat shock genes do not have intervening sequences which might exhibit a different sort of ehromatin architecture. Afler heat induction, there is a transition in the structure of gene chromatin and virtually the entire transcription unit becomes much more sensitive to micrococcal nuclease. Similar findings have been reported by other workers for the hsp 70 gene (Wu et ai., 1979; Levy & Noll, 1981) and for other genes (Bellard et al., 1982). In fact, it could be argued t h a t the transcribed sequences are cleaved more readily by this nuetease in heat-shocked ehromatin than in naked DNA (see Figs 2 to 5). Hence, the purturbations accompanying transcription could involve not only the disruption or displacement of nucleosome core particles, but also structural alterations in the DNA which somehow enhance nuclease sensitivity. In this regard, it may be of interest t h a t in situ hybridization studies to polytene chromosomes (Artavanis-Tsakonas et al., 1979; Henikoff, 1981 ) indicate t h a t after heat induction the hsp 70 gene sequences are in some unusual configuration which permits hybridization even without denaturing the chromosomes. Conceivably, this peculiar phenomenon might be attributed to the transient formation of single-stranded DNA-nucleosome (see Palter el al., 1979) and/or D N A - R N A p o l y m e r a ~ complexes. (c) Boundaries of the genes While there appears to be some sort of multiphasic nucleosome organization through most of the hsp 70 gene, this is not the case for the boundaries of the transcription unit, and we find specialized chromatin structures at both the 5' and 3' ends. The chromatin Organization at the beginning of the transcription unit has several interesting features (see Fig. 5). First, there is probably one (and perhaps three) aligned core particles downstream from the transcription start site. Second, the pattern of micrococeal nuclease and DNase I cleavage products from the 5' end of each gene is consistent with a nucleosome-free gap of at least 175 bp. This gap would span the transcription start site, the T-A-T-A box and the sequences involved in controlling heat induction (see Pelham, 1982), and could contain some specialized non-histone proteins (McGhee et al., 1981). Third, the high level of hsp 70 gene expression accompanying heat shock appears to alter both the structure of this open DNA segment as well as the chromatin further upstream at the outside edge of Z~¢. Fourth, in Z,c there is a micrococeal nuclease cleavage site which disappears after a 0.35,~-KC! salt extraction. This particular open region may be stabilized by some sort of non-histone chromosomal protein (see Worcel et al., 1983). In this regard, it may be of interest t h a t a similar set of structural features arc observed at the beginning of the rudimentary gene (unpublished data). We thank Kati Udvardy and Stella Han for excellent technical assistance. We would like to acknowledge I)r Abe Worcel, Dr Ruth Steward and Dr Kitsos M. Louis for suggestions and stimulating discussions during the course of this work. Support was provided by research grants from the National Institutes of Health, the American Cancer Society, and the Nationa[ Science Foundation. One of us (P.S.) would also like to thank the March of

87A7 HEAT SHOCK LOt'US OF D. mela~ogaster

403

Dimes Birth Defect Foundation for funds from the Basil O'Conner Starter Grant Program, and a research grant. 1%EFERENCES Artavanis-Tsakonas, S. M., Schedl, P., Mirault, M.-E., Moran, L. & Lis, J. (1979). Cell, 17, 9-18. Ashburner, M. & Bonnet, J. J. (I979). Cell, 17, 241-254. Beliard, M., Dretzen, G., Bellard, F., Oudet, P. & Chambon, P. {1982). EMBO J. 1, 223230. Dingwall, C., Lomonossoff, G. P. & Laskey, R. A. (1981). Nucl. Acids Res. 9, 2659-2673. Goldschmidt-Clermont, M. (1980). Nuel. Acids Res. 8, 235-252. Henikoff. S. (1981). Chromosoma, 83,381-393. Horz, W. & Altenburger, W. (1981). Nucl. Acids Res. 9, 2643-2658. |sh-Horowiez, ])., Pinchin, S. M., Sched], P., Artavanis-Isahouas, S. & Mirault, M.-E. (1979}. Cell, 18, 1351-1358. Jack, R., Gehring, W. J. & Braek, C. (1981). Cell, 24, 321-331. Keene, M. A., Corces, V., Lowenhaupt, K. & Elgin, S. C. 1%. (1981). Proc. Nat. Acad. 8Ci., U.S.A. 78, 143-146. Levinger, L. & Varshavsky, A. (1982). Proc. ,Vat. Acad. Sci., ~LS.A. 79, 7152-7156. Levy, A. & Noll, M. (1980). Nucl. Acids Res. 8, 6059-6068. Levy, A. & Noll, M. (1981). Nature (London), 289, 198-203. |,is, J., Neckamayer, W., Mirault, M. E., Artavanis-Tsakonose, S. M., Lafl, P., Martin, G. & Schedl, P. (1981). Develop. Biol. 83,291-300. Mason, P. J., Torok, I., Kiss, I., Karch, F. & Udvardy, A. (1982). J. 3fol. Biol. 155, 21-35. MeOhee, J. D., Wood, W. I., Dolan, M., Engel, J. D. & Felsenfeld, B. (1981). Cell, 27, 4555. Mirault, M. E., Goldschmidt-Clermont, M., Artavanis-Tsakonas, S. M. & Schedl, P. (1979). Proc. Nat. Acad. Sci., U.S.A. 76, 5254-6258. Moran, L., Mirault, M. E., Tissieres, A.. Lis, J., Sehedl. P., Artavanis-Tsakanas, S. M. & Gehring, W.J. (1979). Cell, 17, 1-8. Nedospasov, S. A. & Georgiev, G. P. (1980). Biochem. Biophys. Res. Commun. 92,532-539. Palter, K. B., Foe, V. E. & Aiberts, B. M. (1979). Cell, I8,451-468. Pelham, H. R. ~. (1982). Cell, 30, 517-528. Rodriguez-Alfageme, C., 1%udkin, G. T. & Cohen, L. H. (1980). Chromosoma, 78, |-31. Sama], B., Woree], A., Louis, C. & Schedl, P. (1981). Cell, 23,401-409. Torok, I. & Karch, F. (1980). Nucl. Acid Res. 8, 3105-3123. Udvardy, A., Sumegi, J., Toth, E., Gausz, J., Gyurkovics, H., Schedl, P. & Ish-Horowicz, D. (1982). J. Mol. Biol. 155, 267-280. Weintraub, H. (1983). Cell, 32, 1191-1203. Worcel, A., Gargiu}o, G., Jessee, B., Udvardy, A., Louis, C. & Sehed], P. {1983). Nucl. Acids Res. 11,421-439. Wu, C. (1980). Nature (London), 286, 854-860. Wu, C., Wong, Y.-C. & Elgin, S. C. 1%. (1979). Cell, 16, 807-814. Edited by P. Chambon