Proc. Nati. Acad. Sci. USAVol. 86, pp. 9308-9312, December 1989Cell Biology

Histone H1 in two subspecies of Chironomus thummi with differentgenome sizes: Homologous chromosome sites differ largely intheir content of a specific H1 variant

(chromatin structure/Hi heterogeneity/giant chromosomes/centromere/immunofluorescence)

EVITA MOHR*, LOTHAR TRIESCHMANN, AND ULRICH GROSSBACHtIII. Zoologisches Institut-Entwicklungsbiologie, University of Gottingen, Berliner Strasse 28, D-3400 Gottingen, Federal Republic of Germany

Communicated by W. Beermann, August 28, 1989

ABSTRACT Chromatin of Chironomus thummi (Diptera)contains seven sequence variants of histone Hi. A structurallydivergent Hi, variant I-1, accounts for about 20% of the totalHi in C. th. piger and for about 30% in C. th. thummi.Monoclonal antibodies against this protein have been inducedand have revealed its restriction to the centromeres and to alimited number of other bands in the salivary gland chromo-somes. Indirect inmunofluorescence of the somatically pairedhomologous chromosomes of F1 hybrids indicates that thedifference between the two subspecies in Hi I-1 content largelydepends on differences situated at a number of distinct homol-ogous chromosome bands. These bands were intensely deco-rated by antibodies against Hi I-1 in C. th. thummi butappeared virtually black in C. th. piger. The same bands,however, were decorated equally in both subspecies by anantibody that reacts with other Hi variants but does notrecognize Hi I-1 and by a polyclonal anti-Hi antibody. Theresults suggest that Hi variant I-1 is characteristic of a specifictype of chromatin that is confined to distinct chromosomesegments and that is more frequent in the subspecies C. th.thummi, which has a 27% larger genome.

reported earlier, larvae of C. thummi contain seven differentsequence variants of histone H1 (12-14). Salivary glandnuclei contain several different and possibly all seven H1variants. Antibodies that recognize either a subset of the H1complement or one single H1 variant have been induced andhave been used in the present study to decorate larvalsalivary gland chromosomes. We find that a specific H1variant, I-1, is abundant in a limited number of chromosomebands, while it is rare or virtually absent in the chromatin ofother chromosome regions. A comparison oftwo subspecies,C. th. thummi and C. th. piger, which differ remarkably intheir total content of H1 variant I-1, revealed that thisdifference is largely confined to differences between specifichomologous chromosome bands. Many of the C. th. thummichromosome regions with a high content ofH1 I-1 differ fromtheir homologous C. th. piger counterparts also in that theycontain more and repetitive DNA (15, 16), replicate late in Sphase (17), and/or react positively in a C-banding stainingprocedure (18). It appears that a high percentage of H1variant I-1 is characteristic of a specific type of chromatin.

In the periodic structural element of chromatin, the nucleo-some, DNA is associated with octameric protein complexescontaining pairs of molecules of each of four types of his-tones. The folding of this chain ofnucleosomes into the 30-nmfiber of eukaryote chromatin is mediated by the binding ofanother type ofhistone, H1 (1). Numerous organisms and celltypes have been found to contain several different subtypesof H1 (for reviews, see refs. 2, 3), but the functional role ofthis heterogeneity is not known.

In the course of spermiohistogenesis in mammals (4-6) anderythrocyte maturation (7-9), most of the nuclear H1 mole-cules are replaced by other members of the H1 family, Hltand H5, respectively. The transcriptionally active macronu-cleus of the ciliated protozoan, Tetrahymena thermophila,contains an H1 histone that is not found in the transcription-ally silent germinal micronucleus in the same cell (10);instead, micronuclei contain a group of other linker histones(11). In vertebrates as well as in the protozoon, thesedifferences in histone composition are correlated with differ-ences in chromatin function.We have been interested in determining whether specific

structures within one nucleus also differ in their content ofH1subtypes, because a distinct intrachromosomal distributionshould be a prerequisite for, or expression of, possiblefunctional differences between different members of thishistone class. We describe here the localization of H1 se-quence variants in the giant chromosomes of the midge,Chironomus thummi, by means ofmonoclonal antibodies. As

EXPERIMENTAL PROCEDURESAnimals. Active larvae that were able to reach a water

reservoir through a net were collected from large quantitiesof C. thummi purchased from a fish food supplier. Inbredstrains of C. thummi thummi, C. thummi piger, and their F1hybrids were bred in the laboratory by standard methods. C.th. piger was a gift of H.-G. Keyl (Bochum).Monoclonal Antibodies. Female BALB/c mice were intra-

peritoneally injected with C. thummi histone H1 complexedwith yeast RNA (19). Forty micrograms of H1, dissolved in120 Al of 150 mM NaCI/10 mM sodium phosphate, pH 7.2,was given three times every 10th day, emulsified in an equalvolume of complete Freund's adjuvant at the first injectionand in incomplete adjuvant the following times. After another10 days, 40 pug of H1 in 240 .l of the same solution wasadministered four times in daily intervals. The next day thespleen was isolated, and spleen cells were fused with BALB/c myeloma cells (20) of line P3-X63-Ag8.653 (21) by addingpolyethylene glycol 4000 (22). Hybridomas were grown inRPMI medium (Boehringer Mannheim) with 10% bovineserum and HAT (selection medium, 0.1 mM hypoxanthine/0.4 ,M aminopterine/16 ,uM thymidine) on tissue cultureplates at 370C in a 5% CO2 in air atmosphere. The superna-tants of the hybridoma cultures were screened by an enzyme-linked immunosorbent assay initially described by Engvalland Perlmann (23). Culture supernatants were diluted 1:10with 150 mM NaCI/10 mM sodium phosphate, pH 7.2/0.05%

*Present address: Institut fur Zellbiochemie und Klinische Neuro-biologie, Martinistrasse 52, University of Hamburg, D-2000 Ham-burg 20, Federal Republic of Germany.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Tween 20/1% bovine serum albumin and pipetted ontoimmunoplates that had been coated with 500 ng of a mixtureof nearly equal amounts of the seven electrophoreticallypurified variants of C. thummi histone H1. Antibody bindingwas visualized by horseradish peroxidase coupled to rabbitanti-mouse immunoglobulin (Dakopatts, Hamburg); the cou-pled enzyme catalyzed the formation ofa black precipitate byreaction with 2,2'-azino-di[3-ethylbenzthiazoline sulfonate(6)] (24, 25). Positively reacting clones were then testedseparately with 100-200 ng of each of the individual H1sequence variants. The specificity of the antibodies was alsotested on immunoblots (see below) of crude preparations oftotal histone H1. Immune serum, culture medium, serum ofa nonimmunized BALB/c mouse, and supernatant of anIgG-secreting negative clone were used as controls.Immunoblots. Total histone H1 or H1 fractions obtained by

preparative electrophoresis in acetic acid/urea (14) wereseparated in NaDodSO4/15% polyacrylamide (26). Transferof the proteins to nitrocellulose filters (BA85, Schleicher &Schuell) was performed as described by Towbin et al. (27).Protein transfer from acetic acid/urea gels was made in 0.7%acetic acid. The filters were preincubated in 150mM NaCl/10mM sodium phosphate, pH 7.2/1% bovine serum albumin for1 hr at 37°C and washed in 150 mM NaCl/10 mM sodiumphosphate, pH 7.2. They were incubated for 2 hr at 37°C withhybridoma culture supernatant diluted 1:10 with 150 mMNaCl/10 mM sodium phosphate, pH 7.2/1% bovine serumalbumin/0.05% Tween 20 (23). Ascites fluids were used atdilutions between 1:1000 and 1:100,000. The filters werewashed and incubated with horseradish peroxidase-coupledrabbit anti-mouse immunoglobulin (Dakopatts, Hamburg) ata dilution of 1:2000 for 2 hr at 37°C. Binding of the secondantibody was visualized by 4-chloro-1-naphthol (28).

Indirect Immunofluorescence. Salivary glands of late fourthinstar larvae were dissected in Robert's Ringer solution (29),fixed in formaldehyde, and treated as described earlier (12,30). Hybridoma culture supernatants were diluted 1:10,whereas ascites fluids were applied at dilutions between 1:1000and 1:50,000. Fluorescein isothiocyanate-conjugated rabbitanti-mouse IgG and IgM or goat anti-mouse IgG (Medag,Hamburg), at dilutions in Robert's Ringer solution of 1:50 or1:100, were used as second antibodies. In some of the exper-iments, a solution of0.15M NaCI/0.02% sodium azide/10mMTris, pH 7.2 (TBS) was used instead of the Ringer solution forthe antibodies and the washing steps. After incubation with thesecond antibody, the preparations were stained for DNA in adrop of 4 ,uM Hoechst 33342 fluorescent dye for 10 min. Afterextensive washing in Robert's Ringer solution or in TBScontaining 0.02% Tween 20 and a final wash in TBS, thepreparations were mounted in Robert's Ringer solution/glycerol (1:9) containing 0.1% p-phenylenediamine.

Determination of H1 Subtypes. The gels were fixed for 1 hrin 20o trichloroacetic acid, stained for 12 hr in 0.57% procionbrilliant blue (ICI)/45% methanol/9% acetic acid (31), anddestained in 45% methanol/9% acetic acid. For evaluation ofrelative protein concentrations the gels were scanned in amicrodensitometer (Joyce-Loebl).

RESULTSMonoclonal Antibodies Against H1 Variants. The larvae of

C. thummi contain seven sequence variants of histone H1that have been designated I-1, II-1, 11-2, 11-3, III-1, 111-2, andI11-3 according to the order of migration in two steps ofelectrophoresis (12-14). A series of monoclonal antibodieswas raised against purified total H1 and was screened with amixture of nearly equal amounts of the H1 variants in anenzyme-linked immunosorbent assay (23, 25). Thirty-fivehybridoma clones were identified that secreted antibodyagainst C. thummi H1. The cell culture supernatants were

then screened in the immunosorbent assay and on immuno-blots for reaction with the individual H1 variants.Many of the antibodies were found to react with all or with

the great majority of the H1 variants. Several antibodies thatrecognize different subsets of H1 histones on immunoblotswere applied to localize these subsets within the giant chro-mosomes. The results (not shown) revealed that all easilyidentified chromosome bands as a rule contain more than oneH1 variant.The intrachromosomal distribution of variant H1 I-1 was

studied by means of two antibodies that showed a highpreference for this variant.

Identification of H1 Variant I-1 by Antibodies. Variant I-1differs from the other H1 variants of C. thummi by a specifictyrosine residue and several thrombin cleavage sites in theglobular domain (14). It is also the only variant that could beisolated from all H1 proteins by a single step of electropho-resis (refs. 12-14; see below). In our series of anti-H1antibodies, one is specific for I-1 and three others react withit much more strongly than with any other H1 variant. Twoof these antibodies, including the one specific for I-1, did notrecognize H1 in chromosomes, but the two remaining ones(clones 5/C2 and 8/A7) could be applied to the intrachro-mosomal localization of H1 variant I-1. A third antibody(clone 7/E5), which on blots did not recognize H1 I-1 butreacted with the other H1 variants, was used as a control.At high and intermediate antibody concentrations, the

antibodies of clones 5/C2 and 8/A7 exhibited a very weakreaction with other H1 variants on immunoblots. This reac-tion was not detectable on further antibody dilution. Fig. 1,lane 1, shows the separation of C. thummi H1 into the threefractions, I, II, and III. Fraction I represents variant I-1,whereas fractions II and III comprise the other variants thatcan be separated by a second electrophoresis step onNaDodSO4 gels (compare Fig. 2a). Lanes 2-6 show differentimmunoblots of the same sample prepared with ascites fluidat dilution ratios between 1:100,000 and 1:1000. The asciteswas induced by cells of clone 5/C2. At all concentrations,fraction I, which represents H1 variant I-1, was stained by theantibody reaction. A very weak reaction with the H1 proteinsof fraction II was visible at dilution ratios of 1:1000 (lane 6)and 1:5000 (lane 5) but disappeared at higher antibodydilutions (lanes 2-4). For indirect immunofluorescence, con-centrations of the 5/C2 antibody in the range of 1:10,000 orlower were therefore applied to reveal specifically the dis-

1

Is

II- loIII'

2 3 4 5 6

I

Ii_ -ii o

i IA._ w _

FIG. 1. Immunoblots of a crude extract of histone H1 of C. th.thummi after electrophoresis in 5% acetic acid/4 M urea/6 mMTriton X-100. Lane 1, filter stained with amido black. Blots 2-6,antibody ofclone 5/C2 (ascites fluid) applied at dilutions of 1:100,000(blot 2), 1:50,000 (blot 3), 1:10,000 (blot 4), 1:5000 (blot 5), and 1:1000(blot 6). The ascites fluid reacted with fraction I at all concentrationsand showed a very weak reaction with fraction II at dilutions of1:1000 and 1:5000.

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tribution of variant I-1. The antibody of clone 7/E5 was usedfor comparison to show the distribution of the other H1variants. This antibody has a broad reactivity with the H1histones of C. thummi on blots (Fig. 2, lanes 3 and 4) but doesnot recognize H1 variant I-1 (Fig. 2, lanes 2).H1 Variant I-1 Is Located in Specific Bands. Fig. 3 shows the

decoration of the right arm of chromosome III from thesalivary gland of C. th. thummi with the antibodies of clone5/C2 (Fig. 3a) and 7/ES (Fig. 3b), respectively. Although thelatter yielded a rather uniform decoration ofthe chromosomebands in this (and other) chromosome(s), the 5/C2 antibodystained the same structures in a differential way. A numberof bands exhibited intense immunofluorescence, whereasothers appeared almost or even completely dark. Theseobservations suggest that either the concentration of H1 I-1or its accessibility to the antibody differs widely betweendifferent chromosome loci.For a further characterization of the bands that are in-

tensely decorated by the 5/C2 antibody, advantage was takenof the fact that the two subspecies of C. thummi, C. th.thummi and C. th. piger, contain very different relativeamounts of H1 variant I-1. When H1 was extracted fromlarvae and separated on acetic acid/urea/Triton X-100 gels,fraction I (compare Fig. 1, lane 1), which is identical withvariant I-1 (Fig. 2), was found to account for about 20% ofthetotal H1 in C. th. piger and for about 30%o in C. th. thummi(Table 1). Larvae of F1 hybrids contained intermediate-rel-ative amounts of H1 I-1, between 23% and 28% (Table 1).

Variant I-1 was localized by indirect immunofluorescencein the chromosomes of both subspecies. For a direct com-parison of homologous chromosome regions, preparations ofthe salivary gland chromosomes of F1 hybrids of C. th. pigerand C. th. thummi were made in which the homologues aresomatically paired over part oftheir length and lie side by sidein the unpaired sections (33). Fig. 4 shows chromosome II ofa hybrid that was incubated with the antibody of clone 5/C2diluted 1:16,000. The centromere regions (arrows) and alimited number of other bands exhibited a high level ofimmunofluorescence; on the other hand, many regions of thechromosome appeared completely dark. Correspondinglydistinct patterns of immunofluorescence were found in theother three chromosomes of the complement (not shown).Most striking, however, was a remarkable difference be-tween C. th. thummi and C. th. piger in the unpaired centralregions of chromosomes I-III. A number of chromosome

1 2 3 4 1 2 3 4

II.'uw w rNo

bFIG. 2. Immunoblot of total histone H1 and the H1 variants with

the antibody of clone 7/E5. Total histone H1 (lanes 1) and fractionsI (lanes 2), II (lanes 3), and III (lanes 4), respectively, from an aceticacid/urea/Triton X-100 gel (such as shown in Fig. 1, lane 1) wereseparated by NaDodSO4 gel electrophoresis and yielded H1 variantsI-1 (lanes 2), II-1, 11-2, and 11-3 (lanes 3), and 11-1, 111-2, and 111-3(lanes 4), respectively. (a) Filter stained with amido black. (b)Immunoblot. The 7/E5 antibody did not recognize H1 I-1 (lane 2 inb).

_S~~~~~~~~

b

FIG. 3. Immunofluorescent images ofchromosome III (right arm)from salivary gland cells decorated with antibodies against H1 of C.th. thummi. (a) Ascites fluid of the antibody of clone 5/C2 diluted1:10,000. (b) Antibody of clone 7/E5. Letters indicate segments andbands of the chromosome map (32); arrowheads indicate the cen-tromere regions. (Bars = 20 ,um.)

bands in these regions were intensely decorated by theantibody in C. th. thummi, but appeared virtually black in C.th. piger. As shown here for chromosome II (Fig. 4), theantibody decoration in the unpaired central region of the C.th. piger homologue (above) was largely limited to thecentromere (arrowhead). Part of this region of chromosomeII from another preparation is shown in Fig. 5 (above) in moredetail. A view of the same chromosome segment stained forDNA (below) makes it possible to identify the individual lociand demonstrates that the difference in antibody decorationbetween homologous bands in C. th. piger and C. th. thummiis not simply due to differences in chromatin concentration.Under the conditions applied, the antibody of clone 5/C2

detected exclusively the H1 variant I-i (compare Fig. 1). Thelarge difference in the H1 I-1 content between the two sub-species (Table 1) appears to be mirrored in the antibodydecoration of their chromosomes. The results, rather thanrevealing differences in accessibility, show that a limitednumber of chromosome bands, including the centromere re-gions, contain an especially high amount of this H1 variant,whereas its concentration is below the limits of detection inmany other chromosome sites. This difference in H1 I-1concentration exists also between certain homologous bandsof the two subspecies in chromosome II (Figs. 4 and 5) and inthe other two large chromosomes (not shown). Correspondingresults were obtained with the antibody of clone 8/A7.Chromosome bands that were not decorated with one of the

antibodies against H1 variant I-i were found to react with theantibody of clone 7/ES. As shown in Fig. 6 for the unpaired

Table 1. Quantities of the histone H1 fractions in larvae of C.thummi subspecies and their F1 hybrids

% of total H1

C. th. C. th. C. th. thummiH1 thummi piger x C. th. piger

fraction (n = 9) (n = 21) (n = 53)I 31.3 ± 1.2 21.1 ± 2.2 25.9 ± 2.7II 60.3 ± 2.4 59.2 ± 3.7 58.2 ± 2.5III 8.4 + 2.2 19.7 ± 3.1 15.8 ± 3.0Values are expressed as mean ± SD of several independently

isolated samples. F1 hybrids include crosses in both directions. Gelswere scanned after staining with the quantitative protein dye procionbrilliant blue (31).

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p~~~~~~~~~~~~~~~~~~.thN'P'

uS~~~~f¼ ~ ~

K~~~~~.IC~~~~

a bFIG. 4. Chromosome II ofan F1 hybrid of C. th. piger x C. th. thummi with partial somatic pairing ofthe piger (p, top) and thummi (th, bottom)

homologues. (a) Phase contrast. (b) Immunofluorescence of histone H1 variant I-1. Ascites fluid of the antibody of clone 5/C2 was applied at adilution of 1:16,000. The positions of the chromosome bands that are heavily decorated by the antibody in C. th. thummi but totally or virtuallydark in the C. th. piger homologue are marked with short bars. Arrows and arrowheads indicate the centromere regions. (Bars = 20 Azm.)central region of chromosome II, the level of immunofluores-cence ofC. th. piger chromosomes with this antibody was very

similar to that of C. th. thummi chromosomes and corre-sponded to the pattern ofDNA staining (Fig. 5). Homologousbands of C. th. piger and C. th. thummi chromosomes withstriking differences in their H1 I-1 content therefore probablycontain similar amounts of other H1 variants.

DISCUSSIONWe have addressed the functional role of histone H1 heter-ogeneity by studying the distribution of a specific H1 variantin the giant chromosomes of C. thummi. Monoclonal anti-bodies have been induced that recognize H1 variant I-1, an

I:.

FIG. 5. Central unpaired region ofchromosome II ofan F1 hybridof C. th. piger x C. th. thummi stained for H1 variant I-1 by theantibody of clone 5/C2 (Upper) and for DNA by Hoechst 33342fluorescent dye (Lower). The positions of the fluorescent bands ofthe C. th. thummi homologue are marked on the image stained forDNA. Arrowheads indicate the centromere regions. Long bars showthe boundaries of the indicated segments of the chromosome map(32). Dilution of ascites fluid, 1:10,000. (Bar = 10 ,um.)

FIG. 6. Central unpaired region of chromosome II of a hybrid ofC. th. piger x C. th. thummi stained by the antibody of clone 7/E5to show the similar distribution of H1 variants other than H1 I-1 inthe corresponding patterns of bands of the C. th. piger (top) and C.th. thummi (bottom) homologues. Arrowheads indicate the cen-tromere regions. Long bars show the boundaries of the indicatedchromosome map segments. (Bar = 20 gm.)

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H1 histone with structural properties divergent from theother, more closely related, H1 sequence variants of thisorganism (14). The specific pattern of indirect immunofluo-rescence (Figs. 3-5) suggests that H1 variant I-1 is not evenlydistributed but is restricted to the centromere regions and alimited number of other chromosome bands. An alternativeinterpretation would be that the antigen is not accessible forantibody binding in most chromosome sites. This possibilitycan be excluded, however, because extracts of total H1 fromlarvae of the two subspecies and of F1 hybrids showeddifferences in their content of H1 variant I-1 that corre-sponded to the differences in the level of antibody decora-tion.

Morphological and biochemical differences between spe-cific homologous bands or segments in the chromosomes ofthe two subspecies have been described earlier (15-18, 33).It is interesting to note that all bands that have been found tocontain strikingly different amounts of H1 variant I-1 in thetwo subspecies also show morphological differences (15, 33).These are not easily seen after formaldehyde fixation (Fig.4a) but become apparent after ethanol/acetic acid fixationand staining (15, 33). Most, but not all, of the bands with ahigh H1 I-1 content (bars in Figs. 4 and 5) differ from thehomologous C. th. piger bands also by one or more of thefollowing features: (i) they contain repeats of a specific classof (A+T)-rich sequences (ref. 16; E. Schmidt, personalcommunication); (ii) they stain in a C-banding procedure (18);and (iii) they contain a higher amount of DNA (15). Thesecorrelations also apply to the bands with high H1 I-1 contentsin chromosomes I and III-e.g., to the centromere and otherconspicuous bands in segment b (bl,13; b3,11; b3,17; seechromosome map in ref. 32) ofchromosome III shown in Fig.3.

It thus appears that the presence of H1 variant I-1 ischaracteristic of a class of chromosome bands that also haveother features in common. The genome of C. th. thummicontains about 27% more DNA than that of C. th. piger (15).Part of the excess DNA in C. th. thummi appears to beorganized in a specific type of chromatin that contains a highamount of H1 variant I-1 in addition to the other H1 histonesthat are present in the C. th. thummi and the homologous C.th. piger bands. Subtypes of bovine H1 differ in their abilityto condense DNA and dinucleosomes in vitro (2). The twosubspecies of C. thummi and their hybrid appear to be asuitable system for studying, with quantitative methods,whether a particular H1 variant, I-1, is involved in theassembly of a specific chromatin architecture and how it istargeted to specific chromosome sites.

We are grateful to Professor J. Hinrich Peters for helpful adviceand stimulating discussions and for making possible the productionof monoclonal antibodies in his laboratory. We thank Ms. SabinePitzel and Ms. Ingeborg Streichhan for expert assistance and Ms.Heidegred Kirchberg for typing the manuscript. This work was

supported by grants from the Stiftung Volkswagenwerk and theDeutsche Forschungsgemeinschaft to U.G., which are gratefullyacknowledged.

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