Increased histone mRNA levels during inhibition of protein synthesis

Vol. 114, No. 1, 1983 July 18, 1983

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

Pages

131-137

INCREASEDHISTONEmRNA LEVELSDURING INHIBITION OF PROTEINSYNTHESIS Eva Stimac,'

Vincent

2

E. Groppi,

and Philip

Coffino"3

Departments of Microbiology and Immunology,' Pharmacology,2 and Medicine 3 University of California, San Francisco, San Francisco, California 94143 Received May 31, 1983 Inhibition of protein synthesis by cycloheximide or puromycin specifically increases the amount of translatable histone mRNAin exponentially growing and in synchronous G, HeLa cells by 5-fold in 3 hours. In this case histone gene expression is uncoupled from DNA replication. We conclude that the level of histone mRNAis regulated by a labile protein and is only indirectly dependent on DNA synthesis.

The major nuclear most proteins cell

(1,2),

cycle.

rate of histone

protein

are the histones.

their

separates

cell

of DNA synthesis

synthesis

and the level

increased

is at least

of histone

Unlike

rate throughout

the bulk of histone

(S phase);

or at the start

than in S phase cells, extent

cells,

the gap (G, phase) that

from DNA replication,

cells

are not made at a uniform

culture

with DNA replication

late during

relative

of eukaryotic

the histones

In HeLa tissue

concurrent either

proteins

synthesis

synthesis division

is

begins (mitosis)

(S phase) (3). lo-fold

the

The

lower in early

G,

mRNAis reduced to a similar

(4,5). When DNA synthesis

hydroxyurea, mRNA fall concurrent

is inhibited

in S phase HeLa cells

both the rate of histone

to that

found in G, cells

treatment

an inhibitor

that

(6,7).

of S phase cells

of protein

They suggested

protein

synthesis,

the histones

synthesis.

If so, by inhibiting

or partially

uncouple histone

of histone

mRNAin G, cells

observed,

but the experiments

synthesis Butler

the decline

may participate protein

and the level

and Mueller

with hydroxyurea

prevents

by a drug such as of histone

found that

and with cycloheximide, of histone

in regulating

mRNA (8).

their

own

synthesis , one should be able to fully

mRNA from DNA synthesis to increase.

and thus cause the amount

Such an increase

were limited

to brief

was not previously

treatments

of S phase tells(8). 0006-291X/83

131

$1.50

Copyright 0 I983 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 114, No. 1, 1983

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

METHODS Culture and Synchronization HeLa S cells were grown in spinner flasks in Swim's 77 medium containing 10% calf se ;ium. G, cells were obtained by loading the elutriator rotor (5,q) with exponentially growing cells at 2200 rpm, pump speed lOml/min, and then increasing the pump speed in 7.5 ml/min increments until the first fraction containing cells free from debris was collected. For autoradiography after incorporation of [3Hlthymidine (Amersham), cells fixed on glass slides were coated with Kodak NTH-2 emulsion. Cycloheximide and puromycin were obtained from Sigma. Cell

RNA Analysis Total cell RNA was prepared by lysing the cells in guanidinium thiocyanide and centrifuging the lysate through 5.7 M CsCl (14). The RNAwas spotted directly onto nitrocellulose filter paper (Schleicher & Schuell) (21) in a 1:3 dilution series for each sample , so that the amount of a specific mRNA/pg in each sample could be compared quantitatively. Alternatively, the RNA was fractionated by electrophoresis through a 7M urea 7% acrylamide gel and transferred to diazobenzyloxymethyl paper (Schleicher & Schuell) (17). 3;5n both cases the immobilized RNA was hybridized to DNA probes (labelled with [ PI by nick-translation) for 18h in 10s dextran sulphate, 50% formamide, 450 mM NaCl, 45mM Na Citrate, pH7.0, 5x Denhardt's solution at 42'; followed by washing in The dried 15mM NaCl, 1.5mM NaCitrate,O.l% sodium dodecyl sulphate at 50°C. papers were placed under Kodak XAR-5 film with DuPont Lightning Plus intensifying screens at -7OOC. The autoradiograms were scanned with a Zeineh Soft Laser Scanning Densitometer to measure the amount of probe bound to each band. Histone mRNAwas also measu5sd by translating total cell RNA using a rabbit reticulocyte lysate containing [ Sl methionine (10) and analysing aliquots containing equal amounts of acid- precipitable radioactivity by NEPHGEtwo dimensional gel electrophoresis (13). The gels were impregnated with Enhance (New England Nuclear), dried , and placed under Kodak XAR-5 film at -7O'C. The autoradiograms were scanned with a Joyce-Loebl densitometer to measure the relative intensity of specific spots after each treatment. Synchronously elutriation

of an exponentially

by autoradiography smallest

RESULTS growing Cl HeLa cells were obtained

cells,

phase cells

after

after

elutriated

G, populations

replicated

their

were treated

Inhibition

of protein

the reduction prevents Total cell

in the first fractionation.

fraction,

The population contained

2-10% of the cells 4 hours of growth,

G, cells

or asynchronous

3.5 hours with cycloheximide synthesis

inhibits

of c3HlTdR incorporation

the progression

Synchrony was evaluated of

less than 4%.S in such

but 98% had

24 hours.

synthesis,

for

(5,q).

of C3H]thymidine.

were in S after

DNA after

To block protein cells

incorporation

those collected

immediately

growing culture

by centrifugal

of G, cells

exponentially

or with puromycin.

DNA synthesis

(11);

this

seen by autoradiography. into

RNA was prepared and translated 132

S phase during in vitro --

growing

was evident It also

the treatment

using a rabbit

(13).

reticulocyte

in

Vol.

114, No. 1, 1983

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

Figure 1. In vitro translation of RNA from a) exponentially growing HeLa cells, bl these cells treated for 3.5 h with 100 pg/ml puromycin, cl synchronously growing C, HeLa cells after 3.5 h growth at 37”C, d) C, Hela cells treated for 3.5 h with puromycin. 10 pg of RNA was translated in a rabbit retlculocyte system (10) and 11-20 ul containing 222,000 cpm were analysed by NEPHGE two-dimensional gel electrophoresls (131. Only the basic half of each gel is shown.

system

(10)

containing

NEPHCE

two-dimensional

c3* Slmethionine, gel

and

electrophoresis

the (12)

133

reaction (Fig.

mixture 1).

The

was relative

analyzed amounts

by

Vol. 114, No. 1, 1983

of proteins

BIOCHEMICAL

synthesized

AND

BIOPHYSICAL

were quantitated

RESEARCH COMMUNICATIONS

by scanning autoradiograms

with a

densitometer. It can detectable

seen that

be

because it

in the reaction

directed

Treatment

of either

3-4 fold,

while

(results

by

cell

treatment

inhibits

and

ribosomes

causes

is transferred translatable must

to dissociate

to the inhibitor

from inhibition

than in the one directed

by

were in S phase). the histone

of translation,

from the mRNAafter (16);

in this

effect

of ribosomes so that

ribosomes tRNA analogue

the nascent polypeptide

case there is no protection

Therefore,

of protein

mRNAby

proteins

produced a similar

however, is an aminoacyl

message by polyribosomes.

result

effect

Puromycin,

synthesis

not

less

the translocation

along messenger RNA, but not the initiation

is

Hl

S-fold

of mRNAsencoding most of the other

Cycloheximide

on the mRNA (15).

(human

was approximately

with puromycin increased

the level

accumulate

histones

(25% of the latter

Cycloheximide

shown).

methionine)

from C, phase cells

population

unchanged.

not

RNA

growing cells

leaving

of nucleosomal

not contain

does

RNA from exponentially

we visualized

the synthesis

the increase rather

of

in histone

mRNA

than from some other

of these agents. To further

histone

evaluate

mRNA levels,

electrophoresis The immobilized mouse histone

histone

cell

of inhibition

of protein

RNA was fractionated

synthesis

by polyacryamide

on gel

to diazobenzyloxymethyl paper (17). 32 RNAs were annealed with a [ PI labelled DNA cloned from the

transferred

H3.2 gene (181, and the amount of hybridization The results

are shown in Figure 2.

of 100 pg/ml inhibited

mRNAlevels

synthesis

total

and subsequently

autoradiography. concentration

the effect

Q-fold

within

was less effective

incorporation 3 h.

measured by

Cycloheximide

of leucine

Less complete inhibition

, as was cycloheximide

treatment

used

at

a

by 96% and raised of protein for shorter

periods. Inhibiting the synthesis found that

protein

synthesis

and processing

incorporation

100 ug/ml cycloheximide.

has the secondary effect

of ribosomal

of uridine To rule

RNA in the nucleolus

declined

by 30-50% after

out the possibility 134

of inhibiting (19,201.

3h treatment

of a non-specific

both We

with

BIOCHEMICAL

Vol. 114, No. 1, 1983

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

Flgure 2. Hlstone mRNA after cycloheximide treatment. 10 ug RNA were fractionated on a 7U urea 7% acrylamide electrophoresis7gel, el&,ctroblotted to diazobenzyloxymethyl paper (17), and hybridized with 10 cpm [ P] labelled mouse histone MH3.2 gene (18). Inhibition of overall protefe synthesis was determined by labeling an aliquot of cells with 5 &l/ml [ Clleuclne for the last 30 min of each treatment and measuring acid-precipitable radioactivity. In,qet: 3 pg of RNA were spotted on nltrocellulose paper (21) and hybridized with [ PI labelled chick actln cDNA probe (22).

Lane 95% Lane Jane Lane Lane Lane Lane Lane lane

min.

enrichment

1: Exponentially growing HeLa cells + 100 inhibition of protein synthesis. 2: Exponentially growing untreated cells untreated 3: Cl cells, I): C, cells + 0.2 ug/ml cycloheximide for 5: G, cells + 2.0 ug/ml cycloheximide for for 6: G, cells + 20 pg/ml cyclohexlmide for 7: Cl cells + 100 pg/ml cycloheximide 8: G, cells + 100 pg/ml cycloheximide for for 9: G, cells + 100 pg/ml cycloheximide

for

hybridizable

in our

experiments,

probes

homologous

contrast

to the

we reprobed to chick

to

little

by inhibition

total

actin

observations

mRNA was found changed

mRNA rather

(Figure

than

a specific

RNA from

the

2 , inset)

made on histone

be essentially

the of

pg/ml

180 180 180 180 120 60

cycloheximide

min, min, min, min, mln, mln,

previous

mRNA, the

level

in histone

experiments

and tubulin

180

inhibition. inhibition. inhibition. inhibition. inhibition. inhibition.

increase

same in G, and S phase

protein

54% 82% 91s 96% 96% 972

for

genes of actin cells

mRNA

with (22).

In

or tubulin

and to be

synthesis.

DISCUSSION We conclude proteins

whose

that synthesis

histone is

synthesis inhibited

is

regulated

in our 135

through

experiments.

one or more As Butler

and Mueller

Vol. 114, No. 1, 1983

suggested

(8),

increase result life

the histones

in histone of either

mRNAduring

synthesis

G, cells

of histone

or cytosine

is not known.

is uncoupled

occurs,

that

In yeast,

histone

blocked in 0, (28). protein(s)

synthesis

control

q RNA levels

from DNA synthesis

'Further

control

investigation

histone

and the relationship

here to the modulation

during

half-life

if the

increase

in rate of

of histone

min

has been estimated

(24,25,26,27);

the half-life

and transcription

in one cell

cycle,

be required

of the pharmacological

is In

have been

and histone

division

mRNAlevels , the molecular

gene expression

to be

15 min when DNA synthesis

the cell

will

half-

a 12

mRNAin HeLa cells

both turnover

may be the

the half-life

by a IO-fold

arabinoside

The

requires

1 h, and to be reduced to about

by hydroxyurea

expression

of histone

proteins.

genes or of increased

models must postulate

The half-life

(23).

RESEARCH COMMUNICATIONS

of protein

we observe in 3 h is to be explained

shown to regulate

that

inhibition

Transcriptional

mRNA.

to be approximately inhibited

BIOPHYSICAL

transcription

For example, kinetic

increase

AND

themselves might be the target

increased

of the histone

short.

BIOCHEMICAL

gene

cycle mutant

to identify

the

mechanisms by which effect

in cycling

demonstrated

cells.

ACKNOWLEDGEMENTS E.S. was supported by an Anna Fuller supported by NSF grant PCM78-07382.

post-doctoral

fellowship,

and this

work was

REFERENCES 1. 2. 3. 4. 5. 6. X: 9. 10. 11. 12. 13. 14. 15.

Milcarek, C. and Zahn, K. (1978) J. Cell Biol. 79, 833-838. Bravo, R., and Celis, J.E. (1980) J. Cell Biol. 84, 795-802. Marashi, F., Baumbach, L., Rickles, R., Sierra, F., Stein, J.L., and Stein, G. (1982) Science 215, 683-685. Rickles, R., Marashi, F., Sierra, F., Clark, S., Wells, J., Stein, J., and Stein, G. (1982) Proc. Natl. Acad. Sci. USA 79, 749-753. Coffino, P., Stimac, E., Groppi, V.E., and Bieber, D. (1983) in Histone Genes and Histone Gene Expresion, Stein, G.S., Stein, J.L., and Marzluff, W-F., eds. (in press). Wu, R.S., and Bonner, W.M. (1981) Cell 27, 321-330. Breindl, M., and Gallwitz, D. (1974) Eur. J. Biochem. 45, 91-97. Butler, W.B., and Mueller, G.C. (1973) Blochim. Biophys. Acta 294, 481-496. Meistrich, M.L., Ueyn, R.E., and Barlogie, B. (1977). Exp. Cell. Res. 105, 169-177. Pelham, H.R.B., and Jackson, R.J.B. (1976) Eur. J. Biochem. 67, 247-256. Stimac, E., Housman, D., and Huberman. J. A. (1977) J. Mol. Biol.115, 485-511. Brooks, R.F. (1977) Cell 12, 311-317. Sanders, M-M., Groppi, V.E., Jr., and Browning, E.T. (1980) Anal. Biochem. 103, 157-165. Chirgwin, J.M., Przbyla, A.E., MacDonald, R.J., and Rutter, W.J. (1979) Biochem. 18, 5z94-5zgg. Lodish, H.F., Housman, D., and Jacobsen, M. (1979) Biochem. 10, 2348-2356. 136

Vol. 114, No. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

16. Nathans, D. (1964) Proc. Natl. Acad. Sci. USA 51, 585-592. 17. Stellwag, E.J., Dahlberg, A.E. (1980) Nucleic Acid Res. 8, 299-317. 18. Sittman, D.S., Chin, I., Pan, C., Cohn, R.H., Kedes, L.H., and Marzluff, W.F. (198 Proc. Natl. Acad. Sci. USA 78, 4078-4082. 19. Willems, M., Penman, M., and Penman, S. (19691 J. Cell. Biol. 41, 177-187. 20. Grummt, I., Smith, V.A., and Grusnut, F. (1976) Cell 7, 439-445. 21. Thomas, P.S. (1980) Proc. Natl. Acad. Sci. USA 77, 5201-5205. 22. Cleveland, D.W., Lopata, M.A., MacDonald, R.J., Cowan, N.J., Rutter, W.J., and Kirschner, M.W. (1980) Cell 20, 95-105. 23. Berlin, C.M., and Schimke, R.T. (1965) Mol. Pharmacol. 1, 149-156. 24. Borun, T.W., Scharff, M.D., and Robbins, E. (1967) Proc. Natl. Acad. Sci. USA 58, 1977-1983. 25. Gallwitz, D. (1975) Nature 257, 247-248. 26. Pederson, T. (1976) in -Protein Synthesis, vol. 2., McConkey, E.H., ed. (Marcel Dekker, N.Y.) p. 69-123. 27. Heinz, N., Sive, H.L., and Roeder, R.G. (19831 Mol. Cell. Biol. 3, 539-550. 28. Hereford, L.M., Bromley, S., and Osley. M.A. (1982) Cell 30, 305-310.

137