A protein released by DNAase I digestion of drosophila nuclei is preferentially associated with puffs

Cell, Vol. 14, 539-544,

July

1978, Copyright

0 1978 by MIT

A Protein Released by DNAase I Digestion of Drosophila Nuclei Is Preferentially Associated with Puffs J. E. Mayfield,’ S. C. R. Eigin

L. A. Serunian, L. M. Silvert and

The Biological Laboratories Harvard University 16 Divinity Avenue Cambridge, Massachusetts

02138

Summary Antisera have been produced against five moiecuiar weight subfractions of the Drosophila proteins readily extracted from nuclei following iimited DNAase I digestion. lmmunofiuorescence staining techniques were used to assess the distributions of these proteins in the poiytene chromosomes of Drosophila. in three cases, the antigens were widely distributed; in one case, the antigens appeared to be slightly more concentrated at active loci; and in one case, the antigens were strongly concentrated at a defined set of loci, including puffs and most of the loci which are active (puffed) at some time during third instar larval and prepupai development. The latter distribution pattern differs from that of RNA poiymerase. Nonhistone chromosomai proteins of this type may have a key role in establishing and/or maintaining the altered chromatin structure characteristic of the active state. Introduction in essentially all eucaryotes, the DNA of the genome is found in association with the histones and nonhistone chromosomal proteins (NHC proteins) in a stable complex referred to as chromatin. it is reasonable to suppose that the limited and tissuespecific pattern of transcription observed to occur in vivo is a consequence, to some degree, of the pattern of association of these proteins with specific DNA sequences. In the last few years, the nucleosome or “beads-on-a-string” model of chromatin fiber structure has been verified. Eight molecules of the four smaller histones (H2A, H2B, H3, H4), probably two of each, are associated with each other in subunits; - 160 base pairs of DNA are wrapped around the outside of each histone “bead.” Histone I is apparently associated with the DNA spacer between the subunits. The NHC proteins may occupy a similar position and/or be associated with the DNA and portions of histones on the outer surface of the nucleosome beads (for a review of the evidence leading to the nucleosome * Present address: Department of Zoology, Iowa State Ames, Iowa 50011, t Present address: Sloan-Kettering Memorial Cancer New York, New York 10021.

University, Institute,

model and a discussion of issues remaining to be resolved, see Elgin and Weintraub, 1975; Kornberg, 1977). Interestingly, analysis of the DNA sequences associated with histones in isolated nucleosomes indicates that all DNA sequences, whether or not they are being actively transcribed, are organized in this manner (Lacy and Axel, 1975; Reeves, 1976; Kuo, Sahasrabuddhe and Saunders, 1976). There is, nonetheless, a difference in chromatin structure between active and inactive regions. This is demonstrated by the preferential DNAase I sensitivity of active sequences originally observed by Weintraub and Groudine (1976) and subsequently by other investigators (Garel and Axel, 1976; Levy and Dixon, 1977; Bellard, Gannon and Chambon, 1978). This DNAase I sensitivity appears not to be a consequence of the actual transcription process, since sequences transcribed at very different rates are equally sensitive (Garel, Zolan and Axel, 1977), and sequences which are apparently no longer being transcribed maintain their sensitivity at least for a time (Weintraub and Groudine, 1976; Palmiter et al., 1978). It may be suggested that the DNAase I sensitivity indicates a developmentally dependent shift in the underlying chromatin structure which is necessary, but not sufficient, for gene activity. The different chromatin structures revealed by differential DNAase I sensitivity may be a consequence of differences in histone/DNA interactions and/or of differences in higher order packaging of the chromatin fiber. NHC proteins could have important roles affecting these aspects of chromatin structure. Recent studies in this and other laboratories using an immunofluorescence technique for “staining” the polytene chromosomes of third instar Drosophila larvae have demonstrated that different subfractions of the NHC proteins show different distribution patterns along the chromosomes in vivo (Alfageme, Rudkin and Cohen, 1976; Silver and Elgin, 1977, 1978). In particular, a highly selective pattern is obtained using an antiserum against a molecular weight subfraction (centered around 100,000 daltons) of the NHC proteins. A defined subset of bands, which includes all visible puffs, is stained using this serum. A careful analysis of chromosome arms 3L and 3R (-40% of the euchromatin) indicated a 90% correlation between the set of loci dominantly and consistently stained and the set of loci identified by Ashburner (1972) as puffing at some time during the third instar larval or prepupal periods. Heat shock treatment of Drosophila melanogaster induces new RNA synthesis at a set of nine loci, including 87A and 87B-Cl, which do not normally puff during this period. In the chromosomes from larvae maintained at normal temperature, these loci stain at very low levels

Cell 540

if at all, but they stain brightly in the chromosomes from larvae subjected to heat shock using the p antiserum. The staining of the heat shock puffs appears to be superimposed upon the usual p pattern, described above, which remains (Silver and Elgin, 1977). This result contrasts with that obtained using anti-RNA polymerase II serum in similar immunofluorescence staining experiments. Using chromosomes from a larva subjected to heat shock for 20 min, one observes that only the heat shock loci are brightly stained with this serum; staining at the developmentally active loci (such as 74EF and 758, which still look puffed but show very low levels of uridine incorporation) is much reduced (Jamrich, Greenleaf and Bautz, 1977; Elgin, Serunian and Silver, 1978; Silver and Elgin, 1978). The staining pattern obtained using the p antiserum is thus similar to the DNAase I sensitivity pattern in that it appears indicative of a chromatin state that is necessary but not sufficient for transcription, and in that it is independent of the distribution (activity) of RNA polymerase II. The specific chromatin structure defined by high levels of p staining could be the consequence of a limited distribution of p antigenic determinants (at potentially active sites) or a limited accessibility (restricted to these sites) of p antigenic determinants which are more widely distributed. The former interpretation is supported by the fact that the heat shock-specific loci, 87A and 87B-Cl, which are relatively unstained in the unpuffed state with the use of anti-p serum, can be stained in this state using anti-histone 3 serum (Silver and Elgin, 1977). One might infer that p antigens should be accessible, if present, at those loci at which a chromatin core histone (such as H3) is accessible. Intense staining of chromosomes with anti-H3 serum is much more extensive than is staining with anti-p serum. It has recently been observed that certain NHC proteins are preferentially released following DNAase I digestion of nuclei or chromatin, and it has consequently been suggested that these proteins might be associated with the DNAase I-sensitive (active) loci in vivo (Levy, Wong and Dixon, 1977; Vidali, Boffa and Allfrey, 1977). In these cases (work with trout testis and avian erthrocytes, respectively), the major proteins released appeared related to the HMG proteins (lysine-rich NHC proteins) studied extensively by Johns et al. (1975) and Goodwin, Walker and Johns (1978). It is obviously of interest to use the Drosophila system in an attempt to correlate these observations. Accordingly, we have examined (by immunofluorescence) the in situ distribution patterns for several molecular weight fractions of nonhistone proteins released by partial DNAase I digestion of Drosophila nuclei.

Results A comparative analysis on SDS-polyacrylamide gels of the protein fractions obtained by EDTA extraction of nuclei which were and were not treated with DNAase I is presented in Figure 1. The digestion reaction was stopped at a point where 510% of the DNA and no histones are released from nuclei washed without EDTA. This is equivalent to digestion to acid solubility of 5-10% of the DNA (Weintraub and Groudine, 1976). Separate experiments have demonstrated that following heat shock of the Drosophila embryos, preferential digestion by DNAase I of the sequences encoding the major heat shock-induced protein (that of

N BI SI B2 S2 >5 >4

129,

>3 55,

>2

45=

;>I

25w

17, Figure 1. SDS-Polyacrylamide clear Proteins

Gels

of

Drosophila

Embryo

Nu-

(N) proteins of whole nuclei: (Bl, 82) first and second EDTA extracts of nuclei incubated in the absence of DNAase I; (Sl, S2) first and second EDTA extracts of nuclei incubated in the presence of DNAase I. In each case, 20 pl of 10 fold concentrated nuclear extract were loaded onto the gel. The position of molecular weight markers is indicated on the left (myoglobin, 17,000 daltons; ovalbumin. 45,000 daltons; gammaglobulin, 25,000 and 55,000 daltons; and /3-galactosidase, 129,000 daltons). The positions of bands I-5 used as immunogens and of HI are indicated on the right.

Nonhistone 541

Chromosomal

Proteins

70,000 daltons) is observed (Wu, Livak and Elgin, 1978). Some variability was noted in the effectiveness of the protein extraction; it was apparently dependent upon the final concentration of divalent cations in the nuclei. Consequently, extracts 1 and/or 2 from different nuclear preparations were used for antibody preparation. The five most prominent molecular weight bands were used for antibody preparation (one rabbit each). In three of the five cases (bands 1, 3 and 5), the sera obtained caused staining of the polytene chromosomes over background with a very general, nondistinctive pattern. Antiserum prepared against band 4 caused positive but rather low level staining of the chromosomes. Puffs were stained slightly above the remainder of the genome. In the remaining case (antiserum prepared against band 2), a selective staining pattern was obtained on formaldehyde-fixed polytene chromosomes, including brilliant staining of the puffs.as shown in Figure 2. Careful analysis of chromosome arms 3L and 3R established that the staining pattern is very similar to that observed using the p antiserum. Not only actual puffs, but many loci known to puff at other times during the third instar larval and prepupal stages of development are stained (for detailed analysis and discussion of the p staining pattern, see Silver and Elgin, 1977). The band 2 antiserum produced the specific pattern with less secondary chromosomal staining than did the p antiserum. A few specific differences were noted. For example, 68C (a locus which puffs during developmental stage l), which is consistently stained using the p antiserum, is not stained in chromosomes from animals at late puffing stages using band 2 antiserum. Following heat shock, all the heat shock puffs on chromosome 3 are stained using band 2 antiserum; the staining intensity, however, is less than that in the major developmental puffs, as shown in Figure 3. As in the p case, intense staining of most developmentally active loci is maintained following heat shock, even though these chromomeres are no longer major sites of uridine incorporation. A few of the developmentally active loci (for example, 61A and 758) show some reduction in the staining using anti-band 2 serum following heat shock. Discussion The results suggest that at least some of the proteins released by DNAase I are preferentially associated with a set of active and potentially active loci. The results obtained with three of the five protein bands indicating broad distribution patterns suggest that some general NHC proteins are released by DNase I, but do not preclude the possibility that proteins of these molecular weights

preferentially associated with DNAase i-susceptible sites will be detected in the future. It remains a major goal to determine the identity of the band 2 immunogen. This protein band is of 63,000 dalton molecular weight. Successful antibody staining using this serum requires formaldehyde-fixed polytene chromosomes. Staining of the chromosomes is greatly reduced if the salivary glands are squashed in 45% acetic acid without prior formaldehyde fixation (data not shown). This effect is most pronounced when chromosomes from larvae that have been heat-shocked are used. A similar reduction in staining of chromosomes squashed directly in acetic acid has been observed in experiments with antisera directed against Drosophila histone 1 and calf thymus HMGl and HMG2 proteins (Silver, 1978; L. A. Serunian, K. Javaherian and S. C. R. Elgin, unpublished observations). This suggests that that band 2 antigen may be lysinerich, as are these proteins. HMG proteins characterized to date (all from vertebrates), however, have molecular weights of -10,000 and -30,000 daltons (Johns et al., 1975; Goodwin et al., 1978). No small lysine-rich proteins from Drosophila chromatin can be selectively obtained by extraction with 0.3 NaCI, a technique effective in other cases (F. Wu and S. C. R. Elgin, unpublished observations). Further analysis of the Drosophila chromosomal proteins is being undertaken. The relationship between the p antigens and the band 2 antigens has not been fully resolved; one may suggest, however, that the band 2 antigens are a subset of the p antigens. Analysis of the p serum by immunofluorescent staining of total NHC proteins on SDS gels has indicated that it includes an activity against protein of the 55,000-70,000 dalton class, as well as of the 80,000-110,000 dalton class (Elgin, Silver and Wu, 1977; Silver, Wu and Elgin, 1978). While the bulk of the p staining pattern remains the same for formaldehyde-fixed and acetic acid chromosome squashes, the chromosome staining is considerably weaker in the latter case. The intense staining obtained at the heat shock-induced loci using the p antiserum is, for the most part, however, observed only in formaldehyde-fixed preparations. In conventionally prepared squashes of chromosomes from heatshocked larvae, staining with the p antiserum is observed at the developmental loci and at 93D, but not at 87A and 87B-Cl (Silver, 1978). One may suggest from the above data that there are at least two (and possibly more) NHC proteins preferentially associated with a set of potentially active loci in Drosophila. At least one of these, apparently an acid-extractable protein, is also associated with active sites induced by heat shock. The latter appears to be preferentially extracted

Figure

2. Staining

Pattern

Obtained

Using

Anti-Band

2 Serum

Glands were obtained from a late third instar larva grown at 25°C and processed through serum was used for staining at a 1 :lO dilution. (a) Phase-contrast; (b) fluorescence chromosome arm, 3L is split from band 64C to the chromocenter.

the formaldehyde fixation technique. Anti -band 2 micrographs. Note that in this particular squash

f$histone

Figure

Chromosomal

3. Staining

Pattern

Proteins

Obtained

following

Heat-Shock

Using

Anti-Band

2 Serum

Parameters as in Figure 2, except that the larva was maintained at 3PC for 20 min prior 950 can be seen in this partial chromosome set. Other labeled loci are developmentally

from nuclei following DNAase I digestion. It will be of considerable interest to study the role of these proteins in determining chromatin structure and to explore the functional consequences of any structural alterations induced in chromatin by these proteins. Experimental Isolation

Procedures of Nuclei

Crude nuclei were obtained as follows: 6-16 hr Drosophila melanogaster (Oregon R) embryos were dechorinated and subsequently disrupted in a glass-teflon homogenizer in buffer A [l M sucrose, 60 mM KCI, 15 mM NaCI, 0.15 mM spermine, 0.5 mM spermidine, 15 mM Tris-HCI (pH 7.4), 0.5 mM dithiothreitol (DTT), 0.1 mM phenylmethyl sulfonyl fluoride (PMSF), 1 mM disodium ethylenediamine tetraacetic acid (Na*EDTA), 0.1 mM ethyleneglycol-bis @-aminoethyl ether)-N,N’-tetraacetic acid (EGTA)] (Hewisch and Burgoyne, 1973). Debris was removed by filtration through Miracloth. followed by centrifugation at -500 x g for 10 min. The supernatant was made 0.2% in Nonidet P-40, and the nuclei were collected by centrifugation at 5000 x g for 10 min. Nuclei were further purified by centrifugation (20,000 x g for 20 min) through a step gradient of sucrose (final concentration 1.9 M) buffered as above omitting the PMSF, EDTA and EGTA.

to dissection. Heat shock active sites.

loci 67A. 67B-Cl,

93D and

DNAase I Digestion Nuclei were washed twice, resuspended at -1 .O mg DNA per ml in buffer B [0.25 M sucrose, 10 mM Tris-HCI (pH 7.5), 10 mM NaCI, 1 mM CaCI,, 3 mM MgCI,) (Weintraub and Groudine, 1976) and digested with 25 units per ml of DNAase I (Worthington) at 3PC for 2.5 min. Equal size control samples were incubated in this way in the absence of DNAase I. The reaction was stopped by the addition of EDTA to a final concentration of 5 mM. and the nuclei were collected by centrifugation at 12,000 x g for 5 min. The nuclei were then extracted twice at -1 .O mg DNA per ml with 0.2 mM EDTA. Analysis and Isolation of Protelns The nuclear extracts were made 0.1% in SDS, dialyzed against 1 mM Tris-HCI (pH 7.5) for 3-4 hr and lyophilized. They were subsequently analyzed by SDS-polyacrylamide gel electrophoresis using the method of Laemmli (1970). Rabbits were immunized with the lyophilized SDS-polyacrylamide gel strips containing the molecular weight subfractions of interest using the method of Tjian, Stinchcomb and Losick (1974) with minor modifications (Silver et al., 1976). 100-400 Fg protein were used for each of two injections. Immunofluorescence Analysis of Proteln Distribution Sera prepared by the above procedure were used in the indirect immunofluorescence staining assay developed in this laboratory

Cdl 544

(Silver and Elgin, 1976, 1977; Silver et al., 1978) to determine the distribution patterns of these protein subfractions in the polytene chromosomes of Drosophila melanogaster (Oregon R).

Acknowledgments This work was supported by grants from the NIH and from the American Cancer Society to S.C.R.E., and a grant from the NIH to J.E.M. S.C.R.E. is supported by an NIH Research Career Development Award. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 U.S.C. Section 1734 solely to indicate this fact. Received

March

24. 1976

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