Histone H5 in nucleated erythrocytes of fish as determined by radioimmunoassay

236

Biochimica et Biophysica Acta, 517 ( 1 9 7 8 ) 2 3 6 - - 2 4 5 © E l s e v i e r ] N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 99065

HISTONE H5 IN NUCLEATED ERYTHROCYTES OF FISH AS DETERMINED BY RADIOIMMUNOASSAY

G E O R G E G O E T Z , A R D A L A N K. E S M A I L Z A D E H a n d P.C. H U A N G *

Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Md. 21205 (U.S.A.) (Received July 4th, 1977)

Summary Tissue-specific histone H5 in the nucleated erythrocytes of dogfish, scup, skate, tautog, sea robin and toad fish was studied. The presence of this histone was inferred by its electrophoretic mobility on polyacrylamide gels containing either acid-urea or sodium dodecyl sulfate. By radioimmunoprecipitation assays, cross reaction was observed between fish histones and an anti'H5chicken antibody. The antibody was specific to chicken histone H5; purified chicken histone H1 and calf t h y m u s total histones did n o t cross react. It is concluded that fish histone H5 shares c o m m o n antigenic determinants with the chicken H5 histone.

Introduction Avian erythrocytes contain a certain tissue-specific histone [ 1]. This histone, formerly known as histone f2c or KSA, is now more generally designated H5 [2]. Interest in this histone is derived from the observation that it is accumulated in the erythroid cells as the cells mature in developing chick embryos [3, 4] attaining some 17% of the total histone in the transcriptionally inactive nucleus of the mature erythrocyte [5--7]. By t h e r m o d y n a m i c analysis, it is calculated that histone H5 from chick has a greater affinity for DNA than any other histone [8]. Following changes in histone H5 during erythropoiesis, [9--11] it has been shown that H5 is phosphorylated in immature chick red blood cells and that the phosphorylated histone H5 has a weak binding to DNA; as the cells mature, the histones undergo dephosphorylation while the chromatin condenses. This finding is consistent with results obtained by indirect immunofluorescence technique which has demonstrated t h a t the pattern of staining specific * To whom inquiries should be addressed.

237 to anti'H5chicken antibody in the nucleus changes during development of the avian red blood cells [12]. These observations are compatible with the notion that histone H5 as a lysine-rich histone, like histone H1, serves as a generalized repressor by condensing and altering the conformation of the chromatin, preventing the genomic DNA from transcription and replication. Such an argument would be strengthened if this class of histone is present in all species of animals maintaining nucleated mature erythrocytes. The lower vertebrates retain the nuclei in their mature erythrocytes and their histones have been examined in carp, brochea, river trout [13], turtle [14], rainb o w t r o u t [ 15] and a marine invertebrate, Sipunculus nudans [ 16]. The presence of H5-1ike histones in these species was suggested by the criteria of electrophoretic mobility, amino acid composition, and peptide maps identical to that of the avian species. However, uncertainty remains regarding the relatedness of these H5-1ike histones since amino acid sequences data were absent. A possibility that histone H5 is a degradation p r o d u c t from histone H I during stress of the animal was raised [17]. Immunological cross reaction has been shown to be useful in the detection of relatedness among proteins of similar functions. With this reaction Bustin and Stollar [18], for instance, were able to determine that all histone H I subfractions in rat liver are qualitatively different, albeit chemically identical, witho u t having to resort to sequence analysis. Antibodies for histones are specific for a given histone species [19]. This prompted the use of the immunoreactivity in this study to detect the presence of histone H5 in fish erythrocytes that are related to histone H5 from chicken. Materials and Methods Avian erythrocyte nuclei were obtained as described earlier [20]. Fish blood samples were obtained by cardiac puncture from healthy living fish at the Marine Biological Laboratory, Woods Hole, Ma. Elasmobrach Ringer's solution was used to resuspend dogfish red blood cells so that the high osmolarity due to high urea content of the blood could be maintained [21]. The blood of the other species was mixed with 0.1 vol. 10% (w/v) sodium citrate in 0.15 M NaC1 (pH 6.8--7.0) and filtered through four layers of cheesecloth. The blood was centrifuged at 1800 rev./min for 30 min in a Sorvall SS 34 rotor. The red blood cells were washed twice with'the same solution. The cells were resuspended in 0.6% (w/v) saponin in 0.15 M NaC1/0.015 M sodium citrate, pH 6.8 and were stirred at 4"C until lysis occurred. After lysis, the nuclear pellet was washed with this buffer until colorless. The nuclei were disrupted using a teflon homogenizer and the crude chromatin was washed five times with 0.01 M Tris, pH 7.6/1 mM EDTA. Total histones were extracted by adding dropwise with stirring an equal volume of 1 N H2SO4 to the chromatin suspension. The histones were then precipitated with four volumes of 90% ethanol. The precipitate was washed three times with ethanol. It was then dissolved in distilled H20 and lyophilyzed. Polyacrylamide gels containing sodium dodecyl sulfate (SDS) were run according to the m e t h o d of Thomas and Furber [22]. Acid-urea polyacrylamide gels were run according to the method of Ruderman and Gross [23].

238 TABLE I FISH SPECIES USED IN THIS STUDY M u s l e l u s canis (dogfish)

Chondrichthyes

(primitive)

Raja erinacea (skate)

Chondrichthyes

(primitive)

S t e n o t o n a u s c h r y s o p s (scup)

Osteichthyes

(relatively primitive)

Tantoga onitis (tautog)

Osteichthyes

(relatively primitive)

P r e o n o t u s carolinas (sea robin)

Osteichthyes

(intermediate primitive)

O p s a n u s tao (toad fish)

Osteichthyes

(relatively advanced)

Acrylamide and N,N,N',N'-tetramethylethylenediamine (TEMED) were obtained from Eastman Chemical Co. and ~2sI, carrier-free ( 2 . 1 . 1 0 3 Ci/mmol) from Amersham/Searle. Total histones from chicken erythrocyte were iodinated by the following procedures: the incubation mixture contained 100 pl total chicken e r y t h r o c y t e histones (1 mg/ml), 10 pl ~25I (200 pCi/ml), 5 pl 0.12 mM KI, 5 ~l 1 mM tyrosine and 10 pl 7 mM chloramine T. The reaction mixture was incubated for 15 min on ice. The reaction was stopped by the addition of 10 ~l of 21 mM sodium metabisulfate. The free iodine was separated from iodinated histones by extensive dialysis, and the iodinated histones were then lyophilyzed in siliconized vessels. Antiserum against chicken histone H5 was prepared in guinea pigs as previously described [20]. A n t i b o d y c o n t e n t was determined using a precipitation assay [24]. The incubation mixtures each consisted of 125 ~l undiluted antiserum, different amounts of chicken histones (1 mg/ml) and a sufficient volume of saline to give a final concentration of 1% NaC1 in a final volume of 1 ml. The mixtures were incubated at 37°C for 30 rain and further incubated at 4°C for 1 day. The precipitates were centrifuged using a clinical centrifuge; each precipitate was washed three times with 1% cold saline. The protein contents of the precipitate were determined by the m e t h o d of Lowry et al. [25]. A radioimmunoassay was employed to determine if nonradioactive fish histones would compete with labeled chicken e r y t h r o c y t e histones for the antiH5ehicke n antibody. The following procedure was used: 125 gl of undiluted guinea pig antiserum containing anti-H5ehieken antibody was added to 10 pl of labeled chicken erythrocyte total histones (1000 c p m / p g ) a n d various amounts of nonradioactive fish histones were added. The mixture was incubated and processed as described above. The a m o u n t of radioactivity in the precipitate was determined by counting in a Packard gamma counter. The fish species included in this study are listed in Table I. Results

Gel electrophoresis The c o n t e n t of histones in the fish was first examined by gel electrophoresis. The overall patterns of the gels of the fish histones were comparable to those of the chick on two different electrophoretic systems. On these gels, a protein

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Fig. 1. A c i d - u r e a gels of h i s t o n e s o f v a r i o u s species. A c i d - u r e a gels w e r e run as d e s c r i b e d in Materials a n d M e t h o d s . T h e gels w e r e s t a i n e d w i t h 0 . 2 5 % C o o m a s s i e b l u e in 12.5% t r i c h l o r o a c e t i c acid and d e s t a i n e d in 20% m e t h a n o l . A, sea r o b i n t o t a l h i s t o n e s , 20 pg; B, s k a t e t o t a l h i s t o n e s , 20 ~Lg; C, c h i c k e n t o t a l histories, 20 pg; D. t o a d fish t o t a l histories, 20 pg; E, scup t o t a l h i s t o n e s , 20 pg; F, t a u t o g t o t a l h i s t o n e s , 1 0 pg; G, c h i c k e n h i s t o n e H1 a n d H 5 m a r k e r s , 20 pg.

corresponding to chick H5 histone can be seen in all fish examined, although the a m o u n t and exact mobility varied. Since the proteins corresponding to histone H3 dimer and histone H4 are expected to be more conserved evolutionarily these are used as the markers. By such a criterion, the mobilities of histone H I in the species examined are comparable in all cases, so are the corresponding histones H2a and H2b (Fig. 1). Histones H5 are assumed to move immediately ahead of histone H I in the gel systems used. In this acid urea system there is much heterogeneity in the bands assumed to be histones H1 and H5. This limits the usefulness of this system in identification. Tautog histones did not show bands corresponding to histone H1. Likewise, histone H1 content of scup is small as compared to other histones. Our further work utilizes the SDS gel system exclusively. An a t t e m p t to quantitate the a m o u n t of this histone H5-1ike protein in proportion to other histones was made. As shown in Fig. 2 and Table II, it was most prominent in scup and skate, and less prominent in the others. The density of the H1 band varied also, generally being darker in the fish w i t h o u t a prominent H5 band, such as toad fish and tautog, and lighter in fish with a prominent H5 band. Scup has the darkest H5-1ike band on SDS gels.

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F i g . 2. D e n s i t o m e t e r t r a c i n g s o f S D S gels. S D S g e l s w e r e m a d e a n d r u n a s d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . T h e g e l s w e r e s t a i n e d w i t h 0 . 1 % A m i d o B l a c k i n 2 0 % e t h a n o l a n d "/% a c e t i c a c i d . T h e g e l s w e r e destained in 0.9 M acetic acid/5% methanol. Positive photographic transparencies were made and these were scanned with a B & L speetrophotometcr. The bottom of the sample well was taken as the origin for all s c a n s . A , c h i c k e n H 1 a n d H 5 h i s t n n e s ; B, s c u p t o t a l h i s t o n e s . 12, t o a d f i s h t o t a l h i s t o n e s ; D , s e a r o b i n total histories; E, skate total histones; F, tautog total histones; G, chicken total histones; H, dogfish shark total histories.

TABLE

lI

RELATIVE

AMOUNTS

OF H1 AND H5 HISTONES

1N F I S H

Peaks of various histones were cut from the densitometer tracings of the SDS gel and weighed. The H1 and H5 peaks were the ones which approximately c o r r e s p o n d t o t h e c h i c k H 1 a n d H 5 p e a k s in m o b i l i t y . T h e f i r s t h i s t o n e b a n d o n t h e S D S g e l is a s s u m e d t o b e h i s t o r i c H 1 a n d t h e s e c o n d is a s s u m e d t o b e histone HS.

Sea robin Seup Tautog Skate Toad fish Dogfish shark

H1 (%)

H5 (%)

H1 + H5 (%)

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17.1 24.8 18.3 24.2 19.9 9.9

* The dogfish shark erythroeyte t h e gel.

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TOTAL CHICKEN ERYTHROCYTE HISTONES ADDED(in ~g) Fig. 3. D e t e r m i n a t i o n o f a n t i b o d y titer b y i m m u n o p r e e i p i t a t i o n . T h e r e a c t i o n m i x t u r e c o n t a i n e d differe n t c o n c e n t x a t i o n s o f c h i c k e r y t h r o c y t e h i s t o n e s and a g i v e n a m o u n t ( 1 2 5 pl) o f g u i n e a - p i g a n t i s e r u m in a f i n a l v o l u m e o f 1 m l o f 1% saline. T h e m i x t u r e w a s first i n c u b a t e d at 3 7 ° C f o r 3 0 m i n a n d t h e n f u r t h e r i n c u b a t e d a t 4°C f o r 1 d a y . T h e a m o u n t o f a n t i b o d y • a n t i g e n c o m p l e x p r o t e i n in e a c h p r e c i p i t a t e w a s d e t e r m i n e d b y m e t h o d o f L o w r y e t al. [ 2 5 ] . A b , a n t i b o d y ; A g , a n t i g e n .

Immunological cross reactivity In order to show similarity between histone H5 of chick and the " H 5 " of fish by immunological cross reactivity, it is first necessary to determine the titer of the guinea pig anti-H5chicke n antibody in the serum by the quantitative precipitation reaction. In Fig. 3, the results show that increasing the a m o u n t of chicken e r y t h r o c y t e histone from 0 to 60 pg causes the amount of precipitate to increase. Precipitation is maximum between 60 and 100 pg of added histones and then decreases as the a m o u n t of added histones is increased above 100 pg. A radioimmunoassay was then developed to make this test more convenient. Total chick histone which contained 17% histone H5 was labeled in vitro with '2sI and reacted with the anti-H5cbicken serum. The amount of radioactivity present in the precipitate was used to measure the extent of crossreaction. When a fixed level of antigen (i.e. 100 pg) was used, the a m o u n t of radioactivity in the precipitate was directly proportional to the specific activity (Fig. 4), indicating the homologous proteins will compete for the reaction and that iodinated histones have the same affinity for anti-H5 as unlabeled histones. As a control, identical aliquots of sera from guinea pigs without prior exposure to chicken H5 histone were used. There was only background level of precipitation detectable (approx. 7% of input). That the radio-immunoprecipitation assay was reflective of specific interaction between the antigen (H5) and antibody (anti-H5) was further examined.

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CHICK ERYTHROCYTE HISTONE (in ug) Fig. 4. I s o t o p i c d i l u t i o n f o r l a b e l e d h i s t o n e s in i m m u n o p r e c i p i t a t i o n r e a c t i o n . T h e basic r e a c t i o n c o n d i t i o n w a s t h e s a m e as d e s c r i b e d in Fig. 3. T h e t o t a l c o n c e n t r a t i o n o f c h i c k e r y t h r o e y t e h i s t o n e in all m i x t u r e s w a s k e p t c o n s t a n t at lOO p g ; o n l y t h e a m o u n t o f l a b e l e d t o t a l histories f r o m c h i c k e r y t h r o e y t e varied. T h e r a d i o a c t i v i t y in e a c h p r e c i p i t a t e w a s d e t e c t e d as d e s c r i b e d in Materials a n d M e t h o d s , Fig. 5. C o m p e t i t i o n b e t w e e n 1 2 5 i . l a b e l e d c h i c k e n e r y t h r o c y t e h i s t o n e s w i t h t o n e s p u r i f i e d f r o m c h i c k e n e r y t h r o c y t e s . T h e basic i n c u b a t i o n m i x t u r e w a s Fig. 3, e x c e p t t h a t u n l a b e l e d c h i c k e n H 5 or H1 h i s t o n e w a s a d d e d , rJ h i s t o n e p g h i s t o n e H 5 p l u s 80 p g t o t a l h i s t o n e s f r o m c a l f t h y m u s . T h e v a l u e for z e r o 3200 cpm.

u n l a b e l e d H 5 a n d H 1 hist h e s a m e as d e s c r i b e d in H 5 ; •, h i s t o n e H 1 ; $, 10 competition was approx.

As shown in Fig. 5, the amount of precipitation decreased as an increasing amount of purified histone H5 was used; a maximum of 80% reduction in the cpm was observed. On the other hand, purified histone H1 did not interfere with this reaction nor did total histones from calf thymus which contains all histories except H5. These tests augmented earlier efforts in the laboratory to determine specificity of anti-H5 chicken antibody for histone H5 using immunodiffusion and microcomplement fixation assays [20]. In results not shown, antigen • antibody precipitate was subjected to polyacrylamide gel electrophoresis containing SDS and shown to give only one band of radioactivity corresponding to histone H5. Taking into consideration the data established with the precipitate curve (Fig. 3), the isotopic dilution experiment (Fig. 4), and the specificity tests (Fig. 5), a series of competition experiments was done. In these experiments, the ability of total histones from various species of animals to compete with radiolabeled chicken total histones in the precipitation by anti-H5eaieken antibody was examined. The results are shown in Fig. 6. All fish total histones tested compete with the radiolabeled chick total histone for precipitation by the anti-H5chicke n antibody. It is interesting to note that two major levels of com-

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~g T O T A L HISTONES ( U N L A B E L E D ) Fig. 6. C o m p e t i t i o n b e t w e e n 12 S I-labeled c h i c k e n e r y t h r o e y t e h i s t o n e s w i t h u n l a b e l e d histones. C h i c k e n e r y t h r o c y t e h i s t o n e s l a b e l e d in v i t r o ( 1 0 ~I o f 1 m g / m l w i t h spec. a c t . 1 0 0 0 c p m l p g ) w e r e a d d e d t o t h e i n c u b a t i o n m i x t u r e c o n t a i n i n g varying a m o u n t s o f p u r i f i e d h i s t o n e s a n d 1 2 5 pl guinea-pig a n t i s e r u m in a final v o l u m e o f 1 m l 1% saline. T h e level o f radioactivity in the precipitate w a s d e t e c t e d as d e s c r i b e d in Materials a n d Methods; t h e value w i t h o u t c o m p e t i t i o n w a s a p p r o x . 1 5 0 0 c p m . o, calf t h y m u s ; e, dogfish s h a r k ; o, c h i c k ; ~, sea r o b i n ; a, t a u t o g ; X, s c u p .

petition existed. Dogfish competed less well (38%) than the others (67--75%). Total histones from chicken competed at approx. 50% level under the same conditions. Discussion In this study, the presence of a chicken H5-1ike histone from several species of fish erythrocytes is demonstrated by electrophoretic similarity and immunological cross reactivity. If cross reactivity is indicative of sequence relatedness and functional similarity, we can infer that a H5-1ike histone is also a component of the fish erythrocyte. It remains to be established if the existence of this unusual protein in fish chromosomes signifies metabolic inactivation during maturation of the erythrocytes as is the case in avian species. This report demonstrates also the usefulness of radioimmunoassay in the study of histone. As shown in this and other studies using radioimmunoadsorbance principle [26], this approach is rapid, sensitive and informative. In experiments n o t shown here, we n o t e d that little dilution of the chicken histone H5 antisera prepared from guinea-pig was possible. This is in contrast with other antisera whose titer was higher than ours. It may be that histones are n o t strong antigens and, particularly for radioimmunoprecipitation, a higher concentration of specific antibody was necessary than that needed for gel diffusion and microcomplement fixation tests [20]. The low titer, however, apparently did n o t interfere with the precipitation reaction and a very low background was observed (Fig. 5).

244 Our results at the present stage are more qualitatively significant than quantitative, as m a n y factors may affect them. The level of competition, for instance, is subject to correction by at least two parameters. The first is the extent of homologous, immunological determinants; w i t h o u t this information one cann o t extrapolate the percentage of histone H5 in other species normalized against chicken. The second is the e x t e n t of coprecipitation by nonspecific trapping. Non-cross-reacting proteins could induce additional precipitation of Ab-Ag complex. Comparing results presented in Figs. 5 and 6, it can be seen t h a t purified histone H5 can compete to as much as 80%, but the same a m o u n t of histone H5 in the presence of calf t h y m u s histone will reduce the competition of 60%. Without knowing the nature and specificity of this interference, one can only tentatively infer from the competition experiments that dogfish has less histone H5 and other fish examined have more histone H5 than chicken per unit total histones. This inference is compatible with the values derived from polyacrylamide gel electrophoresis, if the a m o u n t of both histone H1 and H5 fractions were summed (Table II). Dogfish is lower in histones H1 and H5 by both criteria. In toad fish there is more cross-reacting material than can be accounted for by the H5 band alone. It is possible that in this case histone " H I " is also reacting. The radioimmunoassay is helpful in estimating the a m o u n t of histone H5 w i t h o u t depending upon the a priori assignment of electrophoretic mobility for each o f these histones. Conceivably, immunofluorescence assay of the gel bands with anti-H5 and anti-H1 sera would further aid the identification and quantitation. Although the absence of competition between H1 and H5 from chicken eryt h r o c y t e for anti-H5 chicken antibody argues against the conversion of one from the other in the chicken, the experiment was n o t designed to rule out the possibility that histone H5 is converted from histone H1 in the fish under unfavourable physiological conditions, as suggested by Panyim et al. [ 17 ]. We did find that when a skate was subject to stress under starvation for two weeks, it did n o t produce an extra quantity of H5-1ike histones, by both SDS-polyacrylamide gel electrophoresis and radioimmunoassays (Esmailzadeh, Huang and Goetz, unpublished data); instead, both the extractable H1 and H5 histones decreased. The relatedness of H5 and H5-1ike histones in various vertebrates can now be surmised. Clearly, those of the fish must share c o m m o n H5-determinants with that of the chicks. Chicken histone H5 in turn shares c o m m o n determinants with pigeon, quail, goose, and duck H5 histones since all of these H5 histones react positively with anti-H5ehicken antiserum in gel diffusion and microcomplement fixation assays (Mura, Huang and Seligy, unpublished results). Recent analysis of the N-terminal sequences of H5 histones from these birds has shown t h a t they differ considerably in the amino acids, although certain segments are exactly the same [27]. It is thus reasonably to predict that fish histone H5 is n o t the same as the chicken but that there is a sufficient degree of homology to be so recognized. In this sense, the genetics of histone H5 is very similar to its sister histone H I in t h a t both are highly vulnerable to mutation. It remains possible that histone H5 is a nonallelic variant of histone H1 in the early evolution of the vertebrate and was selected in the evolution of nucleated erythrocytes.

245

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