Enzymatic deacetylation of histone

Vol. 36, No. 1, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ENZYMATICDEACETYLATION OF HISTONE

Akira Inoue and Daisaburo Fujimoto Department of Chemistry, Faculty of Science, Tohoku University, Sendai, Japan

Received May 27, 1969

SUMMARY An.enzyme activity which catalyzed the deacetylation of histone was found in the calf thymus extract. 14C-Acetyl-histone prepared by incubating calf thymus nuclei with sodium14C-acetate served as the substate and treatment of the histone with pronase destroyed most of the susceptibility to the enzymatic deacetylation. 14C-Acetyl-histone prepared by chemical acetylation with 14C-acetic anhydride served as the substrate to a less extent.

Acetyl either

groups are present in many histone fractions

and they are attached

to the amino-terminal nitrogen of the polypeptide

chain( Phillips,

1963 ; 1968 ) or to the s-amino groups of lysine residuesc Gershey et al., 1968 ; Vidali acetyl

et al.,1968

; DeLangeet al.,1969

).

groups in histones is observed in the mammaliancells and the rate

varies amongvarious tissues(

Pogo et al.,1968

; Byvoet ,1968 >.

been proposed that the structural

modifications

and deacetylation

the interactions

would influence

DNA and might play a role in regulation ( Allfrey

et al.,1964

which catalyzes

It has

of histones by acetylation between histones and

of information

transfer

from DNA

).

This paper reports that the calf thymus extract ivity

Turnover of these

deacetylation

contains an enzyme act-

of histone.

METHODS Calf thymus ( 2.1 g ) was minced and homogenized in 0.14M NaCl ( 5.0 ml ) 146

Vol. 36, No. 1, 1969

BIOCHEMICAL

in a Potter-Elvehjem

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

type homogenizer for 3 min.

The homogenatewas centri-

fuged at 1OOOOxgfor 15 min and the supernatant was used as the enzyme preparation. Two kinds of 14C-acetyl histones were prepared.

"Biologically

acetylated"

histone was prepared by incubating calf thymus nuclei with sodium l-14Cacetatet. 20 uC / ca. 0.3 g nuclei,

specific

ing to the method of Gershey et al.(

activity

1968 ).

Whole histone was extracted

from the nuclei with 0.25M HCl( MacGillivray ated" histone was prepared by acetylation Whole histone sulfate( with l-14C-acetic

anhydride{ 20 PC ,l.l After

the treatment,

4.0 mg protein

volume of 0.6 ml in 0.025M Tris-HCl buffer, the reaction mixture was acidified 5u mole of carrier

pH 7.3.

) at 37O in a final After

the incubation,

Acetic acid was extracted

by shaking

Two ml of the organic layer was taken, mixed with

solution and the radioactivity

scintillation

with ethanol.

by the addition of 0.1 ml Of O.lM HCl con-

acetic acid.

with 3 ml of ethylacetate.

efficiency

histone was recovered by pre-

14C-acetyl histone( 2.5 mg ) was

incubated with the enzyme preparationc

liquid

acetyl-

u mole ) in 2.5 ml of 2M sodium acetate

with ethanolc 5 ml ) and washed throughly

the scintillator

Yhemically

of histone with 14C-acetic anhydride.

For the assay of the enzyme activity,

taining

,1968 ).

Murray ,1964 ) from calf thymus( 60 mg ) was treated

pH 8.1 at O" for 4 hours. cipitation

40 mC/ m mole ) accord-

counter,

of the extaction

Model GSL-111. with ethylacetatec

was measured by a Kobe Kogyo Values were corrected for the 80 % ) and for the efficiency

of the countingc ca. 80 % ). RESULTSand DISCUSSION

Fig. 1 shows the time course of the deacetylation ally

acetylated"

After

reaction

when "biologic-

histone was incubated with the calf thymus extract.

60 min incubation,

about 55 % of the 14C-acetyl groups was released.

Without the enzyme, no significant

deacetylation

In order to confirm the liberation

occurred.

of 14C-acetate and to check the possible 147

Vol. 36, No. 1, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. Time course 1

( dpm

1

of the deacetylation

( B 1 70

t500

t

e-Q-------------------o---

0

I

I

I

I

I

IO

20

30

60

1080 (

Time

( Minutes

The "biologically acetylated" dpm ) was incubated with ( e calf thymus extract at 37O .

contamination acetate the

of 14C-acetyl-amino

extract,

acetic

ethylacetate

acid

for

As shown in Table

results

out,

from

or acyl-lysine Deacetylation

the

different

90 % of the

ethylacetate

cooperation

rate

known

that

of proteases

with

of the

radioactivity. with

pronase

deacetylation.

acetyl

groups

aminoacylases.

acetate

from

i.e.14C-acetic

extractable

the

the

E.C.3.5.1.17

distillation

distillatec

enzymatic

attacks

other

in the ethyl-

14C-acetyl-histone

to the

deacetylase from

deacylase(

by azeotropic

of the

)

14C-acetyl-peptides

in the

the possibility the

or

Radioactivity

1, treatment

that

and is

therefore,

resulted

acids

most of the susceptibility indicate

molecule

about

)

histone ( 2.5 mg, 2100 ) or without ( o---o

was seperated

extract.

) accounted

destroyed

acid

18hr

1

The in a large

The results

liberation

ruled

from histone

aminoacylase(

E.C.3.5.1.14

)

).

"biologically 148

acetylated"

histone

and that

of

Vol. 36, No. 1, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Comparison

of the

Substrate

deacetylation

Activity substrate

in

( min

preparations

14C-Acetate

released Degree of deacetylation

Activity )

(%I

( dpm )

2100

0 10 30

97 912 1017

5 44 49

2100

0 10 30

36 133 157

2 6 7

10000

0 10 30

349 524 888

3 5 9

Pronase-digested "biologically acetylated"histone* "Chemically acetylated"histone

of the histone

Incubation time

( dpm 1 "Biologically acetylated"histone

1

14C-acetyl-histone( 2.5 mg ) was incubated with the calf thymus extract at 37O. * --The histone( 2.5 mg ) was digested with pronase( 0.014 mg > in 0.3 ml of 0.05M Tris-HCl buffer ,pH 7.3, at 37O for 60 min. After the digestion, the solution was heated at 100° for 15 min and served for the deacetylation reaction.

the

"chemically

acetylated"

After

10 min incubation,

ated"

histone

of the

"chemically

specific

lysine

"chemically

terminal

The deacetylase

acids

the

only

comparedc

et al.,1968

a high

groups

function

are some indications

groups degree

in the

of acetyl that

acetylation

149

groups

to s-amino

into

acetyl

in the

groups

of the

of lysine

The

).

but

a-amino

case

acetylated"

et al.,1969

characterized,

of specificity

groups

"biologically

"biologically

of a number

histone

1 1.

in the

; DeLange

been

non-specifically

and s-amino

may have

to attach

has not

introduced

groups

In the

are known Vidali

Table

5 % of 14C-acetyl

histone.

" histone

acetyl-amino

Although there

residues(

probably

amino

specific

groups

acetylated

are

while

acetylated"

14C- acetyl

were

44 % of 14C-acetyl

was released

histone,

groups

histone

14C-acetyl

groups

of N-

residues.

and distinguish

some

molecule in histone

is

not yet

and deacetylation

clear are

,

involved

in

Vol. 36, No. 1, 1969

the

activation

1968

).

BIOCHEMICAL

and repression

The deacetylase

Nohara

et a1.,1968;

control

mechanism

of the

demonstrated

Gallwitz,1968 in higher

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

gene

( Allfrey

et al.,

as well

as histone

here ) may play

an important

1964;

role

Pogo et al.,

acetylases

(

in genetic

organisms.

ACKNOWLEDGEMENT The authors wish to thank Professor ment and Professor S.Seto for his kind counter .

N.Tamiya permission

for

his interest to use liquid

and encouragescintillation

REFERENCES

Allfrev. _ - V.G.,

Faulkner, R., and Mirsky, A.E., Proc. Nat. Acad. Sci. U.S., 51, 786 ( 1964 1 B,yvoet, P., BiochiK Biophys. Acta, 160, 217 (1968 ) J. Biol. Chem., DeLange, R.J., Fambrough, D.M., Smith, E.L., and Bonner, J., 244, 319 ( 1969 > Gallwitz, D., BiocG; Biophys. Res. Commun., 32, 117 ( 1968 ) Gershey, E.L., Vidali, G., and Allfrey, V.G., J.xol. Chem., 243, 5018 ( 1968 ) Biochem. J., 2, MacGillivray, A.J., 181 ( 1968 ) Murray, K., Biochemistry, 3, 10 ( 1964 ) Nohara, H., Takahashi, T., and Ogata, K., Biochim. Biophys. Acta, z, 529 ( 1968 ) Biochem. J., 87, 258 ( 1963 ) ; ibid.,%, 135 ( 1968 ) Phillips, D.M.P., Pogo, B.G.T., Pogo, A.O., Allfrey, V.G., 'and Mirsky, A.E., Proc. Nat. Acad. Sci. U.S., 59, 1337 ( 1968 ) J. Biol. Chem., x, 6361 Vidali, G., Gershey, E.L., and Allfrey, V.G., ( 1968 )

150