Histone phosphorylation during liver regeneration

Vol. 70, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

HISTONE PHOSPHORYLATION DURING LIVER REGENERATION William

T. Garrard,*

*

Received

March

George

19,1976

studies

at acid-stable

sites

investigations

have

phorylated

during

more extensively

have been performed during shown

S phase

show a rather

while

the phosphorylation

series

of complex

appear

to be related One additional

possible

role

sation.

The system

1

that (l-3),

to

DNA synthesis

initiates

Inc. reserved.

Hl is

throughout for

are phos-

phosphorylated

have

other

the cycle

mitosis

in histone

(2,7).

(1,2),

Recently,

been discovered

suited

proliferate about Harvard

1219

phosphorylation

to chromatin

is well

can be stimulated

0 1976 by Academic Press, of reproduction in any form

These

a

which

(8,9).

as opposed

regeneration

cells.

HZA and H4, on the

modifications

to be considered

Present address: Biological Laboratories, Cambridge, Massachusetts 02138

Copyright Ail rights

Histones

phosphorylation

Hl molecules

of mitosis

specific

assembly

activation",

of liver

In the rat,

H3 is histone

to chromatin

in "gene

at the onset (4-6).

animal

histone

of phosphorylation

of histone

of histone

in cultured

and "new"

residues level

parameter

on the pattern

"old"

while

and short-lived

at acid-stable, alkali-labile sites of liver regeneration, namely at only Hl shows an increase in the end of the period of chromatin temporal correlation between RNA synthesis. The relative levels the change in Hl phosphorylation the patterns exhibited by cultured cycle as described by other investi-

and mitosis

both

constant

hepatocytes

the organ.

growth

and at unique

hand,

Quiescent

**

and James Bonner

Department of Biochemistry, University of Texas Health Science Center, Dallas, Texas 75235 and **Division of Biology, California Institute of Technology, Pasadena, California 91109

of histone phosphorylation SUMMARY : The pattern has been examined throughout the early stages times of "gene activation". Among the histones, phosphorylation. This increase initiates near template activation. Thus, there is no obvious increased histone phosphorylation and increased of phosphorylation of the various histones and observed in the liver system closely parallel animal cells during the Gl and S phases of the gators. Extensive

** 1

I-I. Kidd,

is

replication to address

by removal 18 hours University,

and condenthis

issue.

of two-thirds

after

its

of

partial

16 Divinity

Avenue,

BIOCHEMICAL

Vol. 70, No. 4, 1976

hepatectomy, onset

and mitosis

follows

of DNA synthesis

activity

which

early

pertinent

time

report periods

aside

from histone

sites

does not

exhibited

Hl,

8 hours

a considerable

increase

to a loss we have

of histone

examined partial

of chromatin

significantly observed

by cultured

during

cells

during

(10).

Prior

in chromatin

in histone

hepatectomy;

to the

template

activation.

the first system

phosphorylation

namely,

of histones

in the liver

animal

later

(11).

changes

template

the phosphorylation

change

the pattern

approximately

following

to the question

In fact,

is

may be related

In the present during

there

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

during

intervals

We have found at acid-stable,

25 hours closely

the Gl and S phases

alkali-labile

of liver

parallels

that,

regeneration. the pattern

of the cycle

(1,2).

METHODS: Male albino Sprague-Dawley rats (200-250 g) were partially hepatectomized under ether anesthesia by excision of the left lateral and median lobes (12). To minimize circadian effects (13), all animals were sacrificed from 22.00 to 24.00 hours by cervical dislocation. Nonoperated "etherized" animals served as O-time (normal) controls. At different times, groups of animals (3-5) were injected intraperitoneally with carrier free Na2H 32P04 (3-5 mCi per rat) under ether anesthesia and animals were sacrificed 1.5 hours later. Livers were excised quickly, frozen in liquid N2, and stored at -8O'C until used. Chromatin was prepared as described (ll), however, aliquots were taken from the initial total homogenate for the determination of the specific activity of the inorganic phosphate pool (14). This value has been shown previously (15) not to differ significantly from the specific activity of acid-labile phosphate of liver nucleotides. Histones were extracted from sheared chromatin with 0.4 N H2S04, ethanol precipitated, and dissolved in 0.01 N HCl-5 M urea-l% $-mercaptoethanol at ca. 10 mg per ml final concentration (11). It has been shown earlier that histones prepared as described above are >95% pure (11). Approximately 95% of the radioactivity associated with the resulting histones was released upon alkaline hydrolysis as inorganic phosphate (16). About 2 mg of histone protein, as determined by turbidity measurements at 400 nm in 1.1 M trichloroacetic acid (ll), was applied to a Bio-Gel P-60 column (1.2 x 110 cm) equilibrated with and eluted by 0.01 N HCl, and 1.1 ml fractions were collected. Aliquots of fractions were measured for protein by turbidity as above, and radioactivity by counting in "Aquasol" scintillation fluid using a Beckman LS-200 counter. Recovery of both was quantitative. Assignment of individual histone species to peaks was performed by gel electrophoresis (17). Radioactivity present in DNA was determined in the residues remaining after histone extraction as follows. Phospholipids were extracThe pellet was ted stepwise with 10% HCl-n-butanol (l:l), methanol, and ether. dissolved in 1N NaOH, incubated 15 min at lOO'C, and radioactivity precipitable in cold 10% trichloroacetic acid was measured.

RESULTS AND DISCUSSION: histones ating indicated

isolated liver

from

chromatin times).

Figure O-hour samples

Relative

1 shows (normal),

Bio-Gel

P-60

and 3-,

8-,

(Na2H32P0

chromatographic 14-,

was administered 4 to the incorporation exhibited

1220

20-,

profiles

and 25-hour

1.5 hours by histone

prior

of regenerto the

H2A, which

is

BIOCHEMICAL

Vol. 70, No. 4, 1976

-1

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I’ ’’ ’

0 hr

3,600

$

Y

FRACTION Figure

1.

Bio-Gel P-60 at different Carrier times.

Figure

2.

g

4.0

5;

3.0

DNA

REGENERATION TIME (HR)

NUMBER chromatography of histones isolated times after partial hepatectomy.

free Na H32P0 was administered Protein'( -O-4): CPM ( -0- ).

Relative rates of DNA synthesis times after partial hepatectomy.

5.0

1.5 hours

and histone

from rat prior

phosphorylation

liver to the

chromatin indicated

at different

Data of Figure 1 were normalized to cpm at the time of injection by considering the times expired prior to and during the counting of samples. A computer program was used to determine the cpm/mg of each histone species Data were then divided by the following pool specific activities to yield relative umoles phosphate incorporated per mg histone species (decay corrected pool values expressed as cpm per pmole inorganic phosphate: 0 hr, 4.2 x 106. 3 hr, 1.6 x 106; 8 hr, 2.7 x 106; 14 hr, 4.3 x 106; this to adjust for 20 hr, 2.2 x 10 b ; and 25 hr, 1.8 x 106); we consider differences in the leakage of injected isotope through the surgical differences in isotope uptake, incisions, differences in amounts injected, differences in inorganic phosphate content of liver among animals, etc. Finally, histone molecular weights (19) were used to normalize to relaIn the case of DNA, relative mole per tive mole per cent phosphorylation. cent synthesis refers to the umoles of inorganic phosphate incorporated (based on the above pool values) per umole of DNA phosphate times 100. (111

l

1221

Vol. 70, No. 4, 1976

the major

labeled

species,

phosphorylation histone

BIOCHEMICAL

the only

of histone

For comparative relative izing

for

decays,

the relative

rates

which

the histones

had been

here,

the rate

phosphorylation

occurred

activation

correlation

H2A>Hl>H4>H2B

the early

only

change

2).

A similar

change

stages

cedes between It histones

this

situation

two quite

different

important

have been

isolated

sites

that

phosphorylation

during

DNA synthesis

the course

the first

(20).

(1,2).

Thus,

a time

when chromatin

there

histones closely (1,21).

to the onset cells,

is

in Hl

no obvious

is

in normal

liver

parallels

that

Furthermore,

dur-

of Hl is

the

of DNA synthesis namely

from Gl into of this

there

Hl

Hl

increase

phosphorylation

traversing intiation

histone

noticeable

pattern

animal

and the

only

activation".

increased

in cells

from

of the experiments

while

Thus,

of the cycle

prior

shown in Figure

among the histones,

by 77-fold,

This

in synchronized

however, at low

at acid-labile increased

2).

Also

the only S phase

increase

obvious

is

slightly

a remarkable

(Fig.

pre-

similarity

systems.

to note,

are

that

isotope

by the same animals

of the various

initiates

of DNA synthesis

(19).

and "gene

regeneration

of Hl,

activities,

hepatectomy,

the Gl phase

exists

normal-

saturation

(Fig.

change

phosphorylation

is

reached

phosphorylation

the onset

partial

phosphorylation

of liver

detected;

in histone

increased

after

during

required

During

of phosphorylation

cells

representation

clear

However,

2 H3 2 background

animal

ing

8 hours

levels

is

in

2 as

increased

by 6.4-fold.

histone

The relative

It

increase

in Figure

exhibited

in phosphorylation.

has nearly

between

of cultured

isolated.

in the

(18).

specific

DNA synthesis

an increase

1 are presented

This pool

is

A similar

of the histones

of DNA synthesis

increased

samples.

of Figure

weights

observed

previously

phosphate

of nuclear

change

phosphorylation

template

time

values.

in inorganic

2 are

are

the data

and the known molecular

reported

later

phosphorylation

differences

shows a significant

change

has been reported

purposes,

mole percent

striking

Hl in the

Hl phosphorylation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

still

that

in all

pH, and therefore

open questions. of histones

in regenerating

the studies

described

differences

Furthermore,

in phosphorylation recent

Hl and H4 at acid-labile

liver

1222

(22).

above

evidence sites

suggests occurs

Vol. 70, No. 4, 1976

The relative represent data

are

mole

absolute

suggest

molecules

have

partial

cated.

for

phosphorylation

of label

precursor

possible taken

that

values

in 1.5 hours group

-on average,

effects

(13),

studies However,

and possible

to be the

and from

23.5

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

case, H2A

to 25 hours

DNA has been

repli-

by several

fold.

on changes

in histone

in our

studies

differences

we have in the

up by the hepatocytes.

ACKNOWLEDGEMENTS: This work was supported in part by NIH Grant GM 13762 the National Science Foundation (G.K.), and by grants from Damon Runyon, National Foundation, and the American Heart Association (W.G.).

1.

the

19% of the histone

nuclear

, possibly

2 would

to normalize

this

approximately

two earlier (23,24).

used

Assuming

to be overestimates

regeneration

circadian

shown in Figure

5% of the total

with

RESEARCH COMMUNICATIONS

activities

quantities.

in disagreement liver

values

specific

approximately

these

during for

the pool

one phosphate

hepatectomy

are

AND BIOPHYSICAL

phophorylation

example,

incorporated

Our findings

amounts

if

--in vivo

We consider

controlled

percent

values

the actual

the data

after

BIOCHEMICAL

(J.B.), by NIH, The

Marks, D. B., Paik, W. K., and Borun, T. W. (1973) J. Biol. Chem. 284, 5660-5667. Gurley, L. R., Walters, R. A., and Tobey, R. A. (1974) J. Cell Biol. 60, 356-364. R. (1974) BiochemTanphaichitr, N., Balhorn, R., Granner, D., and Chalkley, istry l3, 4249-4254. Lake, R. S. (1973) J. Cell Biol. 58, 317-331. R. (1975) Biochemistry 16, Balhorn, R., Jackson, V., Granner, D., and Chalkley, 2504-2511. L. R. (1975) Biochem. Biophys. Res. Hohmann, P., Tobey, R. A., and Gurley, Commun. 63, 126-133. N. P. (1972) Biochemistry 11, 4817-4826. Lake, R. S., and Salzman, D. K. (1975) J. Biol. Chem. Jackson, V., Shires, A., Chalkley, R., and Granner, 250, 4856-4863. V. G. (1975) Science 190, Ruiz-Carrillo, A., Wangh, L. J., and Allfrey, 117-128. Grisham, J. W. (1962) Cancer Res. 22, 842-849. Garrard, W. T., and Bonner, J. (1974) J. Biol. Chem. 249, 5570-5579. Higgins, G. M., and Anderson, R. M. (1931) Arch. Pathol. l2, 186-202. Letnansky, K., and Reisinger, L. (1972) Biochem. Biophys. Res. Commun. 49, 312-320. Martin, J. B., and Doty, D. M. (1949) Anal. Chem. 2l, 965-967. Langan, T. A. (1969) Proc. Nat. Acad. Sci. USA 64, 1276-1283. Teng, C. S., Teng, C. T., and Allfrey, V. G. (1971) J. Biol. Chem. 246, 3597-3609. Panyim, S., and Chalkley, R. (1969) Arch. Biochem. Biophys. 130, 337-346. Balhorn, R., Rieke, W. O., and Chalkley, R. (1971) Biochemistry l0, 3952-3959. Elgin, S. C. R., and Weintraub, H. (1975) Ann. Rev. Biochem. 44, 725-774. Mayfield, J. E., and Bonner, J. (1972) Proc. Nat. Acad. Sci. USA 69, 7-10.

1223

Vol. 70, No. 4, 1976

21. 22. 23. 24.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Gurley, L. R., Walters, R. A., and Tobey, R. A. (1974) Arch. Biochem. Biophys. 164, 469-477 * Chen, C., Smith, D. L., Bruegger, B. B., Halpern, R. M., and Smith, R. A. (1974) Biochemistry 13, 3785-3789. Sung, M. T., Dixon, G. H., and Smithies, 0. (1971) J. Biol. Chem. 246, 1358-1364. Gutierrez-Cernosek, R. M., and Hnilica, L. S. (1971) Biochim. Biophys. Acta 247, 348-354.

1224