Vol.

177,

May

31, 1991

No.

1, 1991

AND

BIOCHEMICAL

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

370-376

Pages

INTERACTION

OF

RNFI

POLYMERCISE

NUCLEOSOMAL Manuel

PiXeiro,

Pedro

Jose

and Centro

de

Received

Madrid,

April

16,

Hernandez

Palacian Consejo

Cientificas

de

ACETVLATED

Francisco

Molecular,

Investigaciones

WITH

PARTICLES

Gonzalez,

Enrique

Biologia

II

CORE

and

Cantoblanco,

Superior

de

Universidad

28049

Flut6noma

Madrid,

Spain

1991

SUMMCIRV. Chemical acetylation of nucleosomal cores is accompanied by an increase in their efficiency as in vitro transcription templates. Low amounts of acetic anhydride cause preferential modification of the amino-terminal tails of core histones. Modification of these domains, which causes moderate structural efis apparently correlated with the observed stimulation of fects, RNA synthesis. In contrast, extensive modification of the globular regions of core histones, which is accompanied by a large structural relaxation of the particle, causes little additional effect on transcription. Acetylation of the amino-terminal domains of histones might stimulate transcription by changing the interaction of the histone tails with components of the transcriptional machinery. 0 1991 Academic Press, Inc.

In

previous

work

vitro-transcription bution

of

ticle,

individually

a

different

the

played

fects

terminal ma1

cores

efficiency

core

of domains, in

of

might

work

and

the

the

nucleosomal

to

of

the

interactions

in

the

globular

the

efficiency

templates.

The

results

which

is

370

to

eluci-

possible

roles partithe

improves extensive

ef-

between and

amino-

nucleoso-

shows mainly

as

incor-

of

histones,

Conversely,

act interac-

determine

present

transcription.

1991 by Academic Press, inc. reproduction in any form reserved.

is

substantially

the

help

weakening

on

to

of

of

$1.50 0

capacity

par-

particles

transcription

in

contri-

nucleosomal

understanding

histones

transcription

amino-terminal

0006-291X’91 Copyright All rights

of

acetylation

their cle

residues

the its

simple

the

mononucleosomal

present

progressive lysine

as

of

alterations of

a

evaluate

to

better and

the

(l-31, to

modifications of

domains

moderate

a

mechanism

the the

used

combination,

polymerase

purpose

of and

in

physiological

The

laboratory

been

structural

basic by

cles.

DNA

RNA different

date

our

features

template.

between

porating

has

structural or

transcription

tion

from assay

that located the

a on

partiacetyla-

Vol.

777.

tion large

No.

of

1, 1991

the

BIOCHEMICAL

histone

globular

structural

tional

in

BIOPHYSICAL

domains,

which

does

relaxation,

changes

AND

not

seem

transcriptional

the

RESEARCH

accompanied

is to

COMMUNICATIONS

produce

properties

by any

of

a

addi-

the

parti-

cle.

MATERIALS

AND

METHODS

Preparation of Nucleosomal Particles Nucleosomal cores were -._---------: prepared from chicken erythrocyte nut lei , isolated after lysis cells in the presence of Nonidet Nuclei were of the P40 (4). micrococcal nuclease digested with and extracted with 0.25 mM EDTA 8.0)(5). Nut leosomal particles containing Hl and H5 (PH were precipitated from the extract with 100 mM KCl, and the nut leosomal cores were isolated from the supernatant (1). Acetylation took place at room temperature by treatment of the (0.1 mg of DNA/ml), nucleosomal particles in 200 mM N-(tris(hydroxymethyl)-methyllglycine (Tricine)(pH 8.21, with the required amount of acetic anhydride disolved in dioxane (200 mg of acetic anhydride/ml). Reaction took place absence of in the base addition (6). To separate the acetylated nucleosomal particles, the modified preparations were centrifuged in 5-20X sucrose gradients containing 10 mM Tris-HCl (pH 8.21, 5 mM EDTA mM phenylmethanesulphonyl fluoride, in a Beckman SW40 and 0.1 4PC. The sedimentation rotor at 34,000 rev./min for 22 h at of free DNA for any of the treatments patterns show no release particles were employed (not shown). The isolated nucleosomal dialyzed at 40 C against 10 mll Tris-HCl (pH 8.2) and 5 mll EDTA. G~12lex~ Fsxx&i with polynucleotide RNA polymerase beled nucleosomal cerol gradients, lated fractions

on

-

II

Free DNFI (146 bp), labeled at its CY-=“PlATP, was incubated ki nase and in the presence and absence of competing the mixture was centrifuged particles, and the radioactivity was measured in

5’

ends with unlain glythe iso-

(1).

Ir.rln_s.A._e_t_itme

Transcription was estimated -&~.~~. incorporated into the radioactivity from C”HlUTP RN& as previously described (2). The nucleosomal free DNf9 were preincubated at 3OeC for 5 min with mM Tris-HCl (pH 7.9), and C”HlUTP, in 10 10 mM MnCl,, 2 mM dithioerythrytol, 5% (w/v) glycerol bovine serum albumin/ml. Transcription was initiated of RNA polymerase II from calf thymus. The size cription products was determined by electrophoresis fied RNA synthesized in the presence of C=PlCTP, taining 7 rt urea (7).

by measuring acid-insoluble particles or ATP, CTP, GTP (NHG)&Dti, 2 mM and 0.2 mg of by addition of the transof the puriin gels con-

RESULTS flcetylation

of

chemically

nucleosomal --.-

extent

of

ments

with

fied

with

with

modification acetic tails low

amounts

maximal

The

acetic

of

nucleosomal

anhydride.

nucleosomal

anhydride.

amino-terminal

dride/ml,

earticie_s_.

acetylated

The

cores ami

of

histones

are

of

reagent.

With

modification

of 371

no

for

10

1

present

pm01

of

the treatin

preferentially

amino-terminal

were

shows

different

groups

those

the

cores

Fig.

the modi-

acetic

anhytails

is

Vol.

177,

No.

1, 1991

BIOCHEMICAL

0 1.

terminal The figure

groups

Extent domains

acetvlation

of in

shows histones

of

COMMUNICATIONS

20 lpmol

/ml1 and of their aminonucleosomal cores. of the total amino groups present in

Modification

is

expressed

The extent tained by

cleosomal ual particle. synthesis

cleosomal 3).

practically

obtained.

A

modification

of

RNA

using

synthesis

acetylated

correlation

appears

amino-terminal

the as

domains

(Fig.

cores

exist

and

nucleosomal

decrease DNA

in

the

a

crude

somes

and

time,

the

creases the that

with in

two

amount

preparation

of

of the

of

in

untreated histones

denaturation,

and

histone

the

midpoint

observed

melting

DNA

DNA

Hl

(8).

for in

this

nucleosomal appear

since

to

the

cores.

be the

mainly extent

372

a

dinucleo-

Acetylation

(a

the

by

of nucleosomal previous results

temperatures

in

except melting

accompanied

containing

transitions

acetylation,

proportion

domains served

decrease the

to

is

which denaturation agreement with

in

mononucleosomal

a progressive ponding

I),

trinucleosomes,

the

1).

anhydride

at

(Table

of

templates

Modification

acetic

temperature

place

takes

ueing

with

cores

between

stimulation

transcription

j_n.-.-y.j.t-x-

nucleosomal

to

s_tr_l-~~.t.~raL..e~-g~_e_r_~eg..-Pf._t_he_a_cety_la_ted_.ea_rt~le_. of

as the

modified groups relative to the total number of groups present in the nucleosomal core. The degree of nucleosomal cores was determined, before and digestion (2), by measuring the radioactivity from anhydride incorporated into nucleosomal particles. of the amino-terminal tails was obof modification substracting from the modification of the whole nucore that corresponding to the trypsin-digested residThe broken line shows the relative increase in RIM obtained the corresponding preparations of nuwith cores used as transcription templates (see --in vitro

C1'+Clacetic

Fig.

extent

to), and domains (0).

the amino-terminal percentage of histone amino of acetylation after trypsin

RESEARCH

of histones anhvdride-treated ___--of modification that of the amino

acetic the

BIOPHYSICAL

10 ANHYDRIDE

ACETIC

Fig.

AND

causes (T,)

and

b).

lower highest

transition

Changes

At

corresthe

same

transition

in-

treatment is in

responsible of acetylation

where

the

same

the

globular

for

the

of

as

obthese

Vol.

177,

No.

1, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

TABLE

Thermal

Denaturation Different

Treatment (jam01 of

and Circular Dichroism Acetylated Nucleosomal

AA/ml)

1.25 2.5 5.0 10 20

ture

directly

midpoint

properties

of of of

(Table

!.Wxaction

Ha (%)

T,b (OC)

Hb (%)

F

61 60 60 57 53 50

31 37 41 43 49 31

75 74 72 70 65 60

69 63 59 57 51 69

1.00 ----

were

evaluated

particles 2

shows

dichroism

are

indicative

of

for

more

with

templates

and

of

nucleosomal

but

no

change

(not

the

decrease

when

the

acetylated When

to

acetylated

detected

The

in DNA,

2.5

absence 373

as is

of

any

RNA

accompanied

is

levels

Fmol

relative

of

syn-

transcrip-

increased

nucleosomal 20

inthan

assay

is

relative

and

their

of

cores

untreated

with

RNA

nucleosomal

levels

the

synthesis the

is

for

templates

transcription

RNFI

cores

RNA polymerase

Acetylation

the

N&l,

M is

II.

mix-

affinity

addition

nucleosomal

polymerase

RNA

in corre-

incubation

with

the

ace-

nucleosomal

obtained In

the

their

I _n..~L.t_ro. Fig. 3 shows

with free modified shown).

of in

The

different

transcription

0.15

thesis obtained nucleosomal cores

in

were

RNA synthesis. with

p lemented

present

shown).

particles.

obtained

formed

RNA polymerase,

efficient

non-modified

increased

results

of

from

complex

increase

&r_~,Ji. (not

affinity

II

acetylation

that

Similar

g3-c_herichia are

were

a substantial

II.

polymerase

dride/ml

tempera-

circular

structure

cores

unlabeled

creased

the

R!%~ee!.~me_ra~e.

polymerase.CB"PIDNA

of

by

times,

tertiary

nucleosomal

Fig.

tion

asymmetric

in

cores

polymerase

(1).

thesis

nucleosomal

RNA

accompanied

the

The

for

sponding

cores

decrease

transitions.

acetylated the

the

affinities amount

from

two

0.79 ---0.58 ----

circular dichroism spectra as previously described the temperature of maximum of altered DNCI structure difference band (3).

..,_.. _, __,__.__ of the _- .-_.._.acetylated_p_art~.cles_--witl. ---_ ._. ..__

tylated

ture

to

of

I).

relative the

the

the

a relaxation DNA

by

proportional

Parameters Cores

T,a (CC)

The thermal denaturation profiles and were obtained in 0.25 mM EDTA (pH 8.0) transcription midpoint, is (3). T,, H, hyperchromicity. F, fraction dH/dt. contributing to the circular dichroism

is

COMMUNICATIONS

I

0

regions

RESEARCH

sup-

2-3 of

cores

acetic increase

synand anhy-

in

Vol.

177,

No.

1,

1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

r

10

5

[ADDED

15

PARTICLE]

[32P-

TIME lminl

DNA]

affinities of acetylated nucleosomal p articles II. Affinity was estimated from the effect of the unlabeled particles on the amount of complex formed between polymerase and free DNA. RNA polymerase II was incubated with C3=P3DNA (146 bp) (2.5 xg/mll , at a molar ratio of polymerase to CaePIDNA equal to 2, in the presence of the following unlabeled particles: free DNA (146 bp) (x1, intact nucleosomal cores (01, and nucleosomal cores modified with 2.5 (~1 and 10 (ml xmol of acetic anhydride/ml. In the absence of added unlabeled particles, 41% of CSeP3DNA was bound to polymerase. Fig. for

2. RNA

Fig.

3.

Relative

p olvmerase

RNA

svnthesized

by

RNA

polymerase The template

usinq different concentration was 4 pg of DNA/ml, and the molar ratio of RNA polymerase II to particle was 2. The nucleosomal cores were treated with the following amounts of acetic anhydride (xmol/mll: 0 (0)s 1.25 ( A 1, 2.5 (V), 5.0 (01, 10 (01, and 20 (01. A control template of free DNA (146 base pairs) (xl was also included for each series of particles. After SO min of incubation 7 UTP molecules were incorporated per molecule of free DNA (xl.

acetvlated

RNA

synthesis

cates

the

of

temolates.

the

different

raise

in

DNA

during

acetylated

Malysis

loss

of

of

one

partially somal

free

highly

min.

cores,

with

that

release the

nucleosomal

the

H2A.H2B

eliminates allowing

nucleosomal

salt

and

transcription

RNA polymerase

of

from

E.

4). coli

the

(not

promote even

in

that,

intact length

results

50

like

the cores

nucleosomal of

were

the with for

nucleosomal

of

whole

Similar

not assay,

shows

present

indi-

incubation

acetylation

block

(Fig.

after

products

(2),

transcription

DNA template

does

transcription

preparation

the

preparations

concentration the

dimer

II

the

nucleo-

obtained

with

acetic

anhy-

shown).

DISCUSSION When dride

under

nucleosomal the

conditions

cores used

are in

374

the

treated present

with work,

the

amino

Vol.

177,

No.

BIOCHEMICAL

1, 1991

FREE DNA (146 base

AND

BIOPHYSICAL

INTACT

pairs1

RESEARCH

CORES

I

ACETYLATED

III

ACETYLATEO

CORES 12.5 Rmol AA/mL)

RNA LENGTH [number

I

located

erentially

in

the

modified

globular

domains,

of nucleotides)

thermal

denaturation

tone

as

amino-terminal

thermal lar

stability regions

cleosomal sition

of accompanied

particle

with

midpoints

of

(T,a

nucleosomal

cores

the

histone

amino-terminal

tion

of

effect

as

nied

by

the

causes

a and

appears

the

transcription.

be

histone

1). only

in

the

point

amino-terminal

globuthe

two

nutran-

structural be

particle

produces

accompa-

efficiency

as

acetylation a

large

additional a

of acetyla-

small might

little to

acetylation

Therefore, a

of

destabiliincrease

specific

domains.

315

the

modification

extensive

which causes

the

the

by to

(Fig.

contrast,

regions,

results

in

denaturation,

In

of

induced

causes

thermal

on

of

correlated

1s

which

particle, These

of

to

improvement

globular of

that

his-

I).

synthesis

tai

effect

destabilization

(Table RNA

The

of

small

decrease

T,b) of

a

the

(91.

acetylation

whereas

substantial

prefto

results

that

large

are

corresponding

only

a

histones

previous

DNCI, by

template.

histone

acetylation

domains

synthesized bv nut 1 eosoma 1 electrophoresis template partiand the molar Incubation took

of

those

wi th indicate

by

substantial

transcription

zation

with

studies

domains, determined

a

compared agreement

stimulation

these

tails

nucleosomal

is

The

a

amino-terminal

in

1

CORES (10 Rmol AA/mLl

of the RNA molecules Fiq. 4. Size distribution using different acetvlated RNA polvmerase II Densitometric tracings obtained after templates. RNA molecules synthesized with different of the of DNQ/ml, cles. Template concentration was 4 gg ratio of polymerase to particle was equal to 2. place for SO min.

groups

COMMUNICATIONS

effect Since

in of

elimination

the

Vol.

177,

of

the

does the 1Ylr

in

No.

1, 1991

BIOCHEMICAL

amino-terminal

tails

significantly stimulation

not observed

deblocking interacting by the

panies

loss

BIOPHYSICAL

the

appears

these

by

even

is

more

properties

(21, Apparent-

by a specific amino-terminal

the

of

digestion,

remarkable.

produced of

COMMUNICATIONS

trypsin

transcription

suppression

of

RESEARCH

histones,

change

non-specific

the

of

transcription capabi li ties

of

the

not

AND

interactions

change

domains,

which

accom-

regions.

Prof. Francisco Montero (Universidad &&r!s.wl.eds~n.tsWe thank Complutense, Madrid) for making available to us the Jobin-Yvon Mark III dicrograph. Chicken blood was kindly donated by hvesur, Madrid. This work was supported in part by the Direcci6n General de InvestigaciAn Cientifica y Tecnica (PBO384), Spain, and by an institutional grant from the Fundaci6n Areces.

REFERENCES

1. 2.

Gonzalez, Biol. Chem. Gonzalez,

P-J.,

Martinez, 11280-11283.

2b2, P.J.,

and

and

Palacian,

(1989)

J.

C.,

E.

Palacian,

E.

Biol.

(1987)

J.

Chem.

264,

Chem.

265,

18457-18462. 3.

Gonzalez, 8225-8229.

P.J.,

and

4.

Weintraub, 85-110.

l-l.,

Palter,

5.

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Flxel,

72,

E.,

Nieto, 623.

7.

Maniatis, Molecular

M.CI.,

Wallace, (1977) Tack,

R.

and Palacian,

T., Cloning, Cold

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K.,

E. and

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Van Lente, Proc.

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8.

Palacian,

R.B., Proc. L.O.,

Natl.

E.

Fri

tsch, E-F., Laboratory Spring Harbor, A

Sargent, F\cad.

and Simpson,

T.D., Sci.

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(1987) and Guide, NY.

Biochem. Sambrook,

Co Id

Murphy, LlSFI 74,

R.F., 3244-3248.

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Biochemistry

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621(1982) Harbor

Bonner,

18,

J. 3110-