Chicken histone H5 inhibits transcription and replication when introduced

into proliferating cells by microinjection

MATHIAS G. BERGMAN1, EDGAR WAWRA2 and MARTEN WINGE1

^Department of Medical Cell Genetics, Medical Nobel Institute, Karolinska Institittet, Box 60400, S-104 01 Stockholm, Sweden2 Institut fur Molehularbiologie der Universita't Wien, Wasagasse 9, A-1090 Wien, Austria

Summary

Chicken erythrocyte histone H5 has been suggestedrepeatedly to be a general suppressor of transcrip-tion and replication. Therefore, the biologicalfunctions of H5 were investigated and comparedwith those of HI (Hla + Hlb) by microinjection ofthe purified proteins into proliferating L6 rat myo-blasts. By pulse-labelling of the injected cells with[3H]uridine and [3H]thymidine it was shown thatH5 blocked both transcription and replication sub-stantially, and that the chromatin of the injectedcells became densely compacted. HI also sup-pressed these functions, but to a much lesser de-gree. The effects were specific and not caused bychange in intracellular pH caused by introduction

of the very basic H5, or its non-specific interactionwith nucleic acid, since injection of protamine orlysozyme did not affect the cells. The migration andlocalization of injected H5 was monitored at differ-ent times after injection by immunofluorescence,which revealed that H5 was efficiently and stablyconcentrated in the nucleus.

The results indicate that H5 indeed might func-tion as an inactivator of the erythroid genome in itsnatural environment, probably by keeping thechromatin in a very condensed state.

Key words: microinjection, histone, transcription,replication, myoblast, chicken, inhibition.

Introduction

Most of the eukaryotic chromatin is compacted into a30 nm fibre, the structure of which is known in consider-able detail. The fibre is made up of a helical array ofnucleosomes, probably held together by the linker his-tone HI (for reviews, see Felsenfeld & McGhee, 1986;Thomas, 1984). This structure must form a significantbarrier to RNA polymerase as it moves along the DNA;therefore, the 30 nm fibre is thought to unravel to someextent in transcriptionally active regions. Indeed, thechromatin structure as a whole and the nucleosomestructure of active genes both differ considerably fromthose of inactive genes (reviewed by Mathis et al. 1980;Weisbrod, 1982).

Since HI, or some modified form of it, is considered tokeep the 30 nm fibre intact (and inactive) (Weintraub,1984), and since its removal increases the micrococcalnuclease sensitivity of chromatin (e.g. see Noll &Kornberg, 1977), HI seems to play a crucial role indetermining the state of activity of the chromatin. Recentexperiments indicate that HI possibly acts as a 'crude'general gene repressor, keeping the chromatin in aninactive ground state (Hannon et al. 1984; Schlissel &Brown, 1984; Weintraub, 1984; reviewed by Weintraub,1985).

Two striking examples of special linker histone

Journal of Cell Science 91, 201-209 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

variants associated with a decrease in gene activity areHl° and H5. Hl° accumulates in contact-inhibitedcultured cells (Pehrson & Cole, 1980), and in murineerythroleukaemia cells after induction of erythroid differ-entiation (Osborne & Chabanas, 1984). H5 accumulatesduring maturation in the nucleated erythrocytes of birds,fish and amphibia; the cells become transcriptionallyinactive and enter Go- In chicken erythrocytes thishistone variant accumulates in the chromatin, causing a70% net increase in linker histone content. During thisprocess the nucleosomal DNA repeat length increases(Moss et al. 1973; Appels & Wells, 1972; Billett &Hindley, 1972; Thomas, 1984; Affolter et al. 1987). H5and H I 0 resemble each other at both the nucleotide andamino acid levels (Doenecke & Tonjes, 1986; Pehrson &Cole, 1981), and bind to the same region of the nucleo-some as HI , i.e. at the positions where the DNA entersthe core histone octamer and leaves it after making twoturns around it (Allan et al. 1980; Smith & Johns, 1980;Stein & Kiinzler, 1983).

Since the accumulation of H5 in chicken erythrocytesparallels the inactivation of the nucleus, the protein hasbeen suggested to be a general chromatin repressor(Appels & Wells, 1972; Billett & Hindley, 1972). Also, wehave reported earlier that replication in L6 rat myoblastsand quail myoblasts was suppressed when these cellswere fused with mature chicken erythrocytes to form

201

heterokaryons. In the process H5 is taken up by the ratand quail nuclei to some extent, indicating that H5 couldfunction as a repressor in this system (Ringertz et al.1985).

In the present report we compare the effects of H5 andHI on transcription and replication by microinjection ofthe purified proteins into active cells.

Materials and methods

CellsL6J1 rat myoblasts (Ringertz et al. 1978) were grown at 37°C in5% CO2 in Dulbecco's modified Eagle's medium (DMEM)supplemented with 5% foetal calf serum (FCS). Two hoursbefore the beginning of an injection experiment, the mediumwas chjanged to DMEM containing 20 mM-Hepes buffer(pH 7-4) instead of bicarbonate, and the cells were kept at 37CC,without CO2, thereafter.

Histone extractionChicken erythrocytes (CE) were collected by bleeding 18-day-old embryos. The cells were washed twice in phosphate-buffered saline (PBS) and nuclei were prepared by lysing thecells in reticulocyte standard buffer (RSB; 10mM-Tris- HC1,pH7-4, 10mM-NaCl, 3 mM-MgCl2) with 0-5% NP-40. Afterwashing three to four times in RSB/0-005% NP-40 the pelletwas resuspended in 3 pellet vol. of distilled water. A crudelinker histone preparation was obtained by sonicating thepartially lysed nuclei to a homogeneous chromatin suspension,which was extracted with 3 % perchloric acid for 15min on amagnetic stirrer. The linker histones were extracted from thechromatin for IS min at +4°C. After centrifugation, theextracted proteins in the supernatant were precipitated byaddition of 0-1-0-5 vol. of 100 % trichloroacetic acid. To purifyH5 and HI the following steps were performed as described bySpring & Cole (1977). The precipitate was dissolved in aminimum volume of sample buffer (8 M-urea, 1 % /3-mercapto-ethanol, 0 1 mM-phenylmethylsulphonyl fluoride (PMSF)),typically 70-110mg/2-4ml, and loaded onto a Sephadex G-75column equilibrated at pH 1-7. Fractions (3 ml) were collectedand monitored at 230 nm absorbance, dialysed against distilledwater, lyophilized and analysed on SDS-polyacrylamide gels.All steps until the lyophilization were performed at -f 4°C. Onepreparation, starting with 1010 nuclei, gave 20-30 mg of pureH5 and 10-15 mg of pure HI. The HI fraction was not furtherfractionated, but used as an Hla + Hlb mixture. The amountof protein was determined by the Lowry technique (Lowryetal. 1951).

MicroinjectionOn the day of the experiment, the chicken linker histones andthe control proteins were dissolved in sterile, filtered water andcentrifuged for 20 min at 27 000 £ to remove all particles thatcould otherwise plug the injection capillaries. The procedurewas carried out according to Graessmann et al. (1980), withminor modifications. Grids of 1 mm2 has been etched into glassslides; the cells were grown to about 20% confluence andinjected on these slides. For each time point and proteinconcentration, all cells in one single field had their cytoplasmsinjected (50-200 cells). The injected volume was calculated byGraessmann et al. (1980) to be approx. 5 Xl0~' ml per cell.Injection of this volume into the cells gave approx. 0-3 X 106,1-5 X 106 and 15 x 106 exogenous molecules of H5 or HI percell at protein concentrations of 0-2, 1 and 10mgml~ ,respectively.

As control for possible effects of the actual protein injection,FITC-coupled anti-mouse IgG (0-6mgml~') and ovalbumin(Sigma; 1 and lOmgml" ) were used. To control for effects ofinjection of basic proteins, protamine (Salmine; Serva) andchicken egg white lysozyme (Merck) were injected at 1 orlOmgml .

After injection the cells were incubated at 37°C for differentperiods of time and then pulse-labelled with either [3H]uridineor [3H]thymidine.

Pulse-labelling, autoradiography and immune stainingFor autoradiography (ARG) the cells were labelled with 20-60,uCi of [3H]uridine (spec, act., 29 Ci ramol"1) per 15 ml culturemedium for 1-14 h for the shorter, and 23 h for the longertranscription experiments. The [3H]thymidine incorporationexperiments were done with 20-60 juCi of [3H]thymidine/l5 mlmedium (spec, act., 20Cimmol~') for 0-5-20-5h in theshorter, and 23 h in the longer experiments.

After labelling, the cells were fixed in absolute ethanol:acetone, 1: 1 (v/v). In the case of [3H]uridine, the slides werehydrated, washed in 5% cold trichloroacetic acid, and thenrinsed in water and overlaid with film (Kodak). The film wasexposed 4-5 days at +4°C, developed and photographs weretaken. The film was then removed and the slides were stainedwith a rabbit anti-H5 antiserum (a generous gift from T. Graf(Beug et al. 1979)), and then with a FITC-coupled secondaryanti-rabbit antibody (Kappel).

In the case of thymidine the slides were fixed, stained withantibody and then covered with film and exposed as above. Thecells injected with control protein were treated the same as thehistone-injected cells.

Results

Morphology of injected cells and localization of H5 aftermicroinjection

Cells injected with 1 mg ml~ H5 into the cytoplasm wereincubated for 0-10 h, fixed and then stained with anti-H5antiserum in order to study the migration, localizationand visible effects of introduction of a protein specific forinactive cells.

At Oh (fixation immediately after injection) themorphology of the cells appeared normal and not dis-turbed by the injection (Fig. 1A). All the antigen wasspread throughout the whole cytoplasm, with none foundin the nucleus (Fig. IB). After 1-5 h all the H5 antigenwas concentrated in the nucleus, appearing to accumulatein dots and around the nuclear periphery. Many of thecells had decreased in size, and had vacuolated cyto-plasms and compacted nuclei (Fig. 1C,D). By 3-5 h themyoblasts had lost their characteristic shape, and bothcells and nuclei were small and compact. H5 was concen-trated in the nucleus (Fig. 1E,F). After a 10-h incu-bation, the volume of the cytoplasm of most of theinjected cells was strikingly reduced, and the small nucleiwere very compact, resembling chicken erythrocyte nu-clei. H5 was highly concentrated in the nuclei(Fig. 1G,H). Cells injected with IgG or ovalbumin(Fig. 8), lysozyme or protamine (not shown) appearednormal.

202 M. G. Bergman et al.

1A

• -v?r' i

Fig. 1. L6J1 cells were microinjected into the cytoplasm with 1 mgml ' HS. The cells were fixed after various periods of timeand stained with H5 antiserum. X2S0. A. At Oh, fixation directly after injection; phase-contrast. Note that the injected cells donot differ morphologically from the uninjected ones. Half of the field was injected. B. At Oh, same field as A;immunofluorescence. H5 is spread in the cytoplasm and has not entered the nucleus. The antigen is not transported across thecell membrane from an injected cell to an uninjected cell. C. At 1-5 h after injection; phase-contrast. All cells injected. The cellsare vacuolated and shrunken. D. Same field as C; immunofluorescence. H5 is concentrated in the nucleus. Some nuclei arecompacted. E. At 3-5 h after injection; phase-contrast. The myoblasts are losing their shape. F. Same field as E; .immunofluorescence. H5 is in the nuclei, which are increasingly compacted. G. At 10h after injection; phase-contrast. The cellsare small and non-myoblast-like. H. Same field as G; immunofluorescence. The nuclei are very compact, almost resembling CEnuclei.

Effects of microinjection of H5 and HI on transcriptionin proliferating cellsThe experiments presented in Fig. 1 showed that sometime was needed for H5 to reach the nucleus, but gave noindication of when effects on transcription and repli-cation would occur. Also, no data of what proteinconcentrations would give such effects could be obtainedfrom these experiments. We therefore tested various timeintervals between injection and pulse-labelling, with aprotein concentration of 10mgml"1 histone in the sol-ution to be injected. This concentration was reducedduring subsequent work; most experiments were donewith lmgml" 1 . Table 1 shows the results of separateinjection experiments using different histone concen-trations and incubation times. There was an obviousconcentration dependence of the inhibition caused by theinjected histones, but the effect of varying the timeinterval between injection and pulse-labelling was notpronounced, being more or less obscured by statisticalerrors caused by the low number of cells per field (seeMaterials and methods and Discussion).

Therefore, all data were pooled (as mean values fromthe different experiments) into two groups, in order toobtain a statistically satisfying number of cells per group,

These groups were short and long studies: 1-25 h and25-42h incubations for the [3H]uridine experiments(incubation including labelling; see Materials andmethods). In the transcription experiments, all unin-jected cells were labelled with [3H]uridine, as were allcells injected with control protein (see Figs 6, 7 and 8,below), so that the effect of H5 and HI could be simplycalculated as the decrease in the number of labelled cells.Only totally unlabelled (no silver grains at all on theautoradiogram (ARG)) cells were counted as negative,the rest as positive (see Discussion).

The micrographs in Fig. 2A-C show the effects ofmicroinjection of H5 ( lmgml"1) on transcription inproliferating rat myoblasts. The cells became compactedand unable to incorporate [3H]uridine (Fig. 2C). Fig. 3groups the results from the different experimentsmeasuring transcription. Three histone concentrationswere used: 10, 1 and 0-2 mgml"1. For H5 both short andlong studies were done, for HI only the short study. In allthe above categories (Fig. 3) H5 inhibited transcriptionto a greater extent than HI . Clearly, the effect wasconcentration-dependent, and the amount of proteinneeded to obtain inhibition was less for H5 than for HI .At the longer incubation time, 25-42h, the inhibitory

Chicken erythrocyte histone H5 203

Table 1. Examples of microinjection of histories: undine incorporation

Protein (concn)Pulse length

(h)Total incubation

time (h)Label ' Positive

cells

HS (lmgml-1)

HI (lmgmr1)

HS(10mgmrI)

HI (lOmgrnP1)

1,1I1-511

1414

11IS31

141

141-51-51-5

1-51-51-5

12-54-56

JO16-51925144-57*5

101718-523-5

4

11

2-5S8-5

14911811310

166112S344

1981695025

19392

23090

*«

14

16

11711911250

106441830

23622311145

174

111926

199

19

5650SO1761727560

9073687093959396

2117ttM3146

m

Fig. 2. Examples of transcriptional and replicationalinhibition by HS. X2S0. A. Phase-contrast. The two cells inthe upper right corner are uninjected (arrows). Injection withl m g m P 1 HS, lOh incubation. B. Immunofluorescence.C. Autoradiogram; [3H]uridine incorporation. Only theuninjected cells are labelled. D. Inhibition of replication byH5. The cells were injected with OZmgrnl"1 H5 andincubated 45 h before fixation. Phase-contrast. Note thedistorted shape of the cell (arrow). E. Immunofluorescence.The nucleus of the injected cell is compact and brightlystained, those of the uninjected cells are not stained.F. A R C No [3H]thymidine uptake at all is seen in theinjected cell, while the uninjected cells are strongly labelled.

effect of H5 decreased (Fig. 3C).To obtain more detailed information about the effects

of H5 on transcription, a kinetic study was done thatincluded time points soon after injection. The inhibitoryeffects are plotted against time in Fig. 4. Transcription

loo-

's 60-

&

1-25 h 1-25 h 25-42 h

Fig. 3. Graphic presentation of the suppressing effects of H5and HI: effects on [ HJuridine incorporation. The barsrepresent the percentage of labelled cells, using differentconcentrations of injected protein as indicated. Only cellstotally lacking silver grains were counted as negative.A. Effect of H5 during the short study (lOmgmr1, 10%positive cells; 1 mgral"1, 55%; O^mgrnl"1, 87%). B. Effectof HI during short study (lOmgml"1, 33% positive cells;lmgmr 1 , 87%; 0-2mgmr', 100%). C. Effect of H5during long study (1 mgml"0'ZmgmP, 91%).

, 64% positive cells;

was inhibited both faster and more efficiently with H5than with HI . For H5, a near-maximum effect wasalready seen 1 h after injection, whereas at the same timepoint the value for HI was 1/3 of its maximum. Themaximum effects for H5 for HI at 4-5 h after injectiondiffered by approx. 20 % (50 % positive cells for H5, 73 %for HI; Fig. 4). The inhibition caused by H5 was more

204 M. G. Bergman et al.

0 5 10 15Time after injection (h)

Fig. 4. Kinetics of transcriptional inhibition by H5 and HIat time points early after injection. The experiments weredone at a protein concentration of 1 mgml"1.

persistent than that caused by HI; 10 h after injection theHl-injected cells had almost completely recovered. De-spite the differences in kinetics and degree of inhibition,the maximum effects occurred around the same time(4-5 h), and the time curve showed similar patterns forboth proteins.

Injection of control proteins (IgG, ovalbumin, lyso-zyme or protamine) did not to any significant degreeaffect the ability of the cells to incorporate [3H]uridine.Figs 6 and 7 show the results of injection of the differentcontrol proteins, the various concentrations and incu-bation classes. The data were collected, as in the histoneexperiments, as mean values of the ARG counts in thedifferent groups. The bars (striped) in Fig. 6 indicatethat injection of IgG or ovalbumin, even at high concen-trations, did not affect the ability of the myoblasts toincorporate [3H]uridine, regardless of duration of incu-bation. The morphology of the cells was likewise notaffected, and the FITC-labelled IgG did not enter thenucleus (Fig. 8B). These results show that injection of aprotein per se does not affect transcription. Since his-tones are very basic (pi for H5 is approx. 12), the effectsseen after injection could be those of a sudden change inintracellular pH or of non-specific interactions with thenucleic acids. To rule out these possibilities lysozyme and

protamine were injected. Fig. 7A,B (dark, striped pat-tern) and 7C,D (black) show that the basic proteinsinitially disturbed transcription to some degree (96 %positive cells for lysozyme; 67% for protamine in theshort studies) at lOmgml . At later time points therewas no effect (Fig. 7B,D), and at no time did they affectcell morphology (not shown).

Effects ofH5 and HI on DNA replicationIn the [ H]thymidine incorporation experiments, theshort study was 0-5-26-5 h (H5) or 1-5-26-5 h (HI), andthe long study was 26-5-46 h (incubation includinglabelling). The labelling index of the uninjected cellsvaried from slide to slide, with the degree of myogenicdifferentiation of the L6 myoblasts and with the length ofthe labelling period, between 95% and 27% positivecells. Therefore, for each slide, the index obtained fromthe uninjected cells was set as 100%, and the effects ofinjection were calculated relative to that value.

The results of the individual injection experiments areshown in Table 2, the morphological effects of H5 areshown in Fig. 2D-F, and the results of the differentexperimental groups are collected in Fig. 5.

H5 inhibited replication more efficiently than HI inboth short and long experiments, and the effects wereagain concentration-dependent. Also, replication seemedmore sensitive to H5 than transcription, and the sup-pression was more persistent (compare Figs 3A,C and5A,C). HI also inhibited replication of the rat myoblasts,but to a much lesser extent: at 10 mg ml~' H1 in the shortstudy, 22% of the cells were positive; in the othercategories the inhibition was negligible.

As a whole, the replicative mechanisms of the cellsappeared more sensitive to microinjection than the tran-scriptional processes, since injection of the neutral con-trol proteins slightly lowered the percentage of labelledcells (Fig. 6A-C, dotted bars). However, the absolutemajority of the cells injected with ovalbumin or IgG wereable to incorporate [3H]thymidine, as shown in Fig. 8F.Of the basic control proteins only lysozyme at 10 mg ml"1

affected replication slightly (Fig. 7A, striped bar: 74%positive cells; B, striped bar: 80% positive cells).

Table 2. Examples of microinjection ofhistones: thymidine incorporation

Protein (concn)

H5 (lmgml"1)

HI (lmgmr1)

H5 (lOrngmP1)

HI (lOmgml"')

* The values are corrected for the

Pulse length(h)

0-51414

141420-5

0-50-50-5

0-50-5

labelling index of

Total incubationtime (h)

2-51824-5

16-52324-5

0-53-57-5

1-54-5

the uninjected control.

+

01512

262126

102

03

Label-

345843

556417

181136

3215

% Positivecells*

04952

775967

190

19

062

Chicken erythrocyte histone H5 205

0-5-26-5 h 26-5-46 h 26-5-46 h

Fig. 5. Graphic presentation of the effects of HS and HI onreplication. The bars represent the frequency of[3H]thymidine-labelled cells, adjusted for the labelling indexof uninjected cells (= 100%). A. Effect of H5 during shortstudy (lOmgml"1, 16% positive cells; lmgml" 1 , 3 1 % ;0-2mgml*', 51 %). B. Effect of HI, short study. There is noinhibitory effect of the injection of 0'2mgml~ HI (compareFig. 6A,B) ( lOmgmr 1 , 22% positive cells; lmgrnl"1, 68%;O'Zmgmr1, 89%). C. Effect of H5, long study ( l m g n i r 1 ,22% positive cells; 0-2, 45%). D. Effect of HI, long study(75 % positive cells).

Discussion

In order to study the biological functions and effects ofthe erythrospecific linker histone H5 in living cells, ascompared to those of HI, we have microinjected theseproteins into proliferating rat myoblasts. Since H5 ac-cumulates in the chicken erythrocyte (CE) nucleusduring erythroid maturation concomitantly with a com-paction of the chromatin, while both transcription andreplication are drastically reduced, we have studied theseparameters in myoblasts after injection of H5. Theresults show that H5 blocks both transcription andreplication to a higher degree than HI, and that themyoblast nuclei become compacted. It should be pointedout that many of the H5-injected cells were detachedfrom the dish and lost their shape as they becameinactivated after subsequent incubation (e.g. in a uridineexperiment approx. 35% of the cells were lost; in athymidine experiment approx. 25%; not shown). Thisgave a bias to the results. Also, many H5-injected cellsthat were counted as positive here showed a reduced labelcompared to control cells, in both uridine and thymidineexperiments. None of these phenomena occurred whencontrol proteins were injected. These facts indicate aneven greater effect of H5 than that reported numericallyabove.

The effects occurred quite rapidly: already after onehour of incubation, transcription was reduced by almost50% at 1 mgrnl" of H5 (Fig. 4), and replication in oneexperiment by 80% at lOmgml"1 after half an hour(Table 2). Einck & Bustin (1983) have shown thatmicroinjected antibodies against HMG 17 and histones

H2A, H2B and H3 inhibited transcription, when concen-trated into the nucleus. This points to the fact that thecore histones may also be involved in determining theactivity of the chromatin. To investigate this, we tried toinject core histones (our own preparations from CE) andhistone H4 (Boehringer) as controls. However, thesepreparations were extremely toxic to the cells, even at lowconcentrations (O'lrngmP ), causing massive cell de-tachment and death (not shown). This might be due tocontamination by non-histone chromosomal proteins inthe preparations. It should be stressed, however, that theaim of this study was to compare the effects of the linkerhistones H5 and HI .

Our results indicate that replication is more sensitive tothe introduction of H5 than is transcription. This may bedue to a disturbance of a variety of mechanisms determin-ing the length and regulation of the cell cycle, comparedto a possible blockage of the movement of RNA polym-erase or of initiation of transcription. From Fig. 5 it canalso be seen that the inhibition by H5 of replication wasmore persistent than that by HI . These results onreplication also support our earlier notion that leakage ofH5 from the chicken erythrocyte nuclei to the active ratnuclei in CE X L6J1 heterokaryons may suppress DNAsynthesis in the rat nuclei (Ringertz et al. 1985). Theinhibitory effect of H5 was clearly concentration-depen-dent, in agreement with the situation in early (immature)polychromatic CE, which contain H5 and are geneticallyactive. They are only inactivated later in the differen-tiation process when H5 has accumulated to largeamounts (Billett & Hindley, 1972; Williams, 1972). Fromthe time curve experiment (Fig. 4) it can be seen that HShad to be present in the nucleus to exert its inhibitoryeffect, indicating that the effects were not mediated byother cytoplasmic factors induced or activated by H5.The protein became detectable exclusively in the nucleuswithin 1-5 h after injection (Fig. ID), suggesting a rapidand efficient translocation mechanism. Moreover, onceconcentrated in the nucleus, H5 remained there and didnot seem to be massively degraded, since the immunoflu-orescence was still intense after 30-40h (not shown).Uptake of proteins into the nucleus has been shown todepend on the size of the molecule, its origin and itscharge (De Robertis, 1983). Paine et al. (1975) haveshown the functional diameter of the nuclear pore to beapprox. 45 A, and proteins below that size can enter thenucleus but do not accumulate unless they are of nuclearorigin. However, nuclear proteins of up to 165 000Mr cantransverse the nuclear membrane, but non-nuclear pro-teins such as IgG (160000Mr) cannot enter at all(Bonner, 1975; Fig. 7B of this paper). Histones haveearlier been shown to be taken up rapidly by isolatednuclei in suspension (Cox, 1982), and HMG 1 and 2 havebeen shown to accumulate in the nucleus after micro-injection (Wu et al. 1981). The faithful transport andlocalization of H5 (20580Mr) suggests that the histonemolecules might contain some sort of transport signal, ashas been shown for nucleoplasmin (Dingwall et al. 1982),SV40 large T antigen (Kalderon et al. 1984) and yeasthistone H2B (Moreland et al. 1987). However, the sevenamino acid stretch that determines the nuclear localiz-

206 M. G. Bergman et al.

100-

1-25 h 25-45 h 1 -25 h 25-45 h

Fig. 7. Effects of basic control proteins ontranscription and replication. A. Lysozyme,short incubation ((^) lOmgmF1, [3H]uridineincorporation; (0) l m g m P 1 , [3H]thymidine;(0) lOmgmr 1 , [3H]thymidine). B. Lysozyme,long incubation; bars as in A. C. Protamine,short incubation ( ( • ) lOmgmP1, [3H]uridine;(ED) lmgml" 1 , [3H]thymidine;(M) lOmgml"1, [3H]thymidine).D. Protamine, long incubation; bars as in C.

1 -25 h

Fig. 6. Graphic presentation of the collected results fordifferent incubation classes, for injection of neutral controlproteins. The bars represent percentage of positive cells (asmean values from the individual experiments) for either[3H]uridine (stripes) or [3H]thymidine (dots) incorporationafter injection of different concentrations of the proteins.A. Mouse igG, short incubation ((0) 0-6 ing ml" ,[3H]uridine incorporation; (02!) 0-6mgml"1, [3H]thymidine).

> , * ,

Fig. 8. Effects of injection of IgG or ovalbumin ontranscription and replication. X250. A. Cells injected withIgG (O^mgrnP1); phase-contrast. Cells incubated 10h, allcells injected. The cells appear normal. B. Same field as A;immunofluorescence. The protein is spread in the cytoplasmand does not enter the nucleus. C. Same field as B, ARG. Allof the injected cells incorporate [3H]uridine. D. Uninjected

B. Ovalbumin, short incubation ((Q) 1 mgrnP1, [3H]uridine; cells; phase-contrast. E. ARG. Ovalbumin (lOmgml ) does(B) lOmgmr 1 , [3H]uridine; (•) 1 mgrnl"1, [3H]thymidine; n o t a f f e c t I H J u r l d l n e incorporation. 3h mcubation. All cells( • ) lOmgmr 1 , [3H]thymidine). C. Ovalbumin, long [TT, Ovalbumin (lOmgml ) does not affectincubation; bars as in B. [ HJthymidine incorporation, 40h incubation. All cells

injected.

ation of the latter histone is not present in H5.The persistent inhibition of transcription and repli-

cation, and the change in nuclear and cellular mor-phology detected after injection of H5 were not caused bysecondary effects of a sudden change in pH or by non-specific base-acid interactions between nucleic acid andbasic proteins. This was shown by injection of lysozymeand protamine, two very basic proteins. They disturbedcell functions only slightly and reversibly, when injectedat high concentrations (Fig. 7A-D). In fact, the initialtranscriptional inhibition of protamine seen in Fig. 7Cwas detected 3 h after injection, and completely overcome

23 h later (Fig. 7D). An even higher degree of inhibitioncould have been expected for protamine, since this small,very basic peptide binds very strongly to DNA ofspermatocytes of higher animals. In a process muchresembling avian erythrocyte maturation and inacti-vation, protamines replace histones concomitantly withan inactivation of the genome (Olson & Busch, 1974).Despite the strong analogy with H5, protamine probablyrequires the milieu of a more mature and spermatocyte-like cell type to exert its function. The weak effect of10 mg lysozyme ml" on replication (Fig. 7A,B) could beexplained by non-specific disturbance of cytoplasmic

Chicken erythrocyte histone H5 207

functions (translation, nucleotide synthesis pathways),since this protein should not enter the nucleus. Takentogether, the basic proteins caused some inhibition onlywhen injected at the highest concentrations.

The results of the histone injections show a cleardifference between the inhibitory effects of H5 and HI.However, HI did initially cause a significant decrease intranscription and replication. Chicken erythrocyte HI(the same Hla + Hlb fraction as used here) has pre-viously been shown to reduce the binding of RNApolymerase to isolated chromatin to about the sameextent as low concentrations of H5. However, at highconcentrations giving about two molecules per nucleo-some (comparable to the situation in mature CE) H5 wasshown to block transcription totally (Hannon et al. 1984).Also, Schlissel & Brown (1984) have shown elegantly thatthe binding of HI to 5 S RNA gene chromatin mXenopusinhibits formation of a transcription complex. In ourexperiments, however, the cells recovered from theeffects of HI with time (Figs 3, 4, 5). Differences intranscriptional and replicational blocking and ability toaccumulate on chromatin could be explained by differ-ences between H5 and HI in affinity for DNA. Kumar &Walker (1980) have determined the standard free energyof dissociation of HI to be 0-5kcalmol~ , and 3-5kcalmol~' for H5, in 1 M-NaCl. We have found the bindingconstant of H5 to be approximately double that of HI bya fluorescence-quenching technique (M. Bergman & F.Watanabe, unpublished).

The exact mechanism of inhibition of transcription andreplication is unknown. The simplest model wouldpresume a steric blocking of RNA and DNA polymerasesby the interaction of H5 with DNA. In conflict with thismodel, however, it has been shown that so-called activechromatin of chicken erythrocytes contains both H5 andHI (Weintraub, 1984), and that H5 is scattered all overthe genome, intermixed with HI (Mazen et al. 1982;Torrez-Martinez & Riuz-Carrillo, 1982). A more compli-cated mode of this blocking, then, would be that H5 has agreater preference than HI to form stable, higher-orderstructures of chromatin, and thus condense it to a higherdegree than chromatin containing only HI (Allan et al.1981; Thomas, 1984; Thomas e* a/. 1985).

In summary, in a comparison of chicken linker histonesH5 and HI, we have shown that H5 efficiently inhibitsboth transcription and replication when introduced intoproliferating rat myoblasts by microinjection. HI shows aclearly smaller degree of inhibition of these functions.

That in turn, indicates that H5 might be an importantfactor in the later stages of inactivation of the erythrocytegenome during avian erythropoesis.

We thank Dr Nils R. Ringertz for supplying facilities andfinancial support (Swedish Medical Research Council (13U-5951), and funds from Karolinska Institute). We thank Mrs E.Mellqvist for excellent photographic work and assistance withthe ARG and immune staining, and Mrs Jennifer Abrahamssonfor assistance with the figures. The stay of E. W. at KarolinskaInstitutet was sponsored by an EMBO short-term fellowship(ASTF 4943).

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(Received 20 February 1988 -Accepted, in revised form, 5 July 1988)

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