DNase I preferentially digests chromatin containing hyperacetylated histones

Vol. 82, No.4, June29,1978

BIOCHEMICAL

1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1346-1353

DNASE I PREFERENTIALLY

DIGESTS CHROMATIN

CONTAINING HYPERACETYLATED HISTONES

Daniel

A. Nelson,

Michael

Linda

Perry,

Sealy

and Roger

Chalkley

Department of Biochemistry College of Medicine Universtiy of Iowa

Iowa City, Received

Iowa 52242

May 8,1978 SUMMARY

Hepatoma Tissue Culture (HTC) cell cells and from cells treated with sodium acetylated histone. Nuclei from sodium dramatic increased rate of digestion with nuclei. Micrococcal nuclease showed no hyperacetylated histones.

nuclei were isolated from untreated butyrate to increase the levels of butyrate treated cells exhibited a DNase I as compared to control cell preference for chromatin containing

INTRODUCTION DNase I and micrococcal chromatin

structure

the way in which tected

against

(1).

nuclease micrococcal

somal

spacer

region

within

the

nucleosome

charged

residues

to a greater is

in the

thought

same time

by acetylation

(for

out of twenty-eight

as this

is

time

histone

acetylation

the

as described

location

above

not perhaps

of the

has been it

could

might

implicated

histones

might

can

positively

be surmized

within

the

histones.

to greater

stages

internucleo-

to four

in transcriptional

pro-

extensively

Since

it

acetylated

nuclease,

1346

to nick

inH4up

be generated

lead

are

At early

(2-4).

instance

conceivably

to micrococcal

0 1978 by Academic Press, Inc. of reproduction in any form reserved.

periods

which

more in the

the ability

of

to reflect

extent.

to attack

extensively

0006-291X/78/0824-1346$01.00/0 Copyright All rights

or lesser

as probes

thought

regions

can be neutralized)

DNase I sensitivity

core

is

DNA,producing

DNase I possesses

core

the additional

DNase I, but

susceptibility

with

nuclease

whereas

'that

Since,

interact attack

extensively

have been used extensively

The nuclease

histones

in digestion

be modified

nuclease

activity sensitivity

we have endeavored

nucleosome For some (5,6). to to test

Vol. 82, No. 4, 1978

whether

there

and little

BIOCHEMICAL

is a correlation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

between

or no correlation

bettieen

acetylation

and DNase I susceptibility

acetylation

and micrococcal

nuclease

attack. MATERIALS

AND METHODS

Growth of Hepatoma Tissue Culture (HTC) cells was as described by Oliver et al. (7). Labelling of log phase cells with [3H] thymidine (200 uCi/l) was carried out for 10 hr prior to isolation. Cells treated with sodium butyrate were exposed for 6 hr to 6 mM sodium butyrate prior to isolation. Nuclei were isolated by two washes in 0.25 M sucrose, 60 mM KCl, 15 mM NaCl, 10 mM MgC12, 1 mM CaCl 6 mM Na butyrate, 10 mM MES (E[N-morpholino] ethane sulfonic acid), pH 6.5, p?,us 0.5% Triton X-100. Subsequent nuclease digestion of nuclei was in the above buffer without Triton X-100. DNase I (1 unit/50 ug DNA) or micrococal nuclease (2 units/50 ug DNA) (both purchased from Worthington) were used, acid solubility being determined as described by Garel and Axe1 (8). RESULTS To determine

whether

hyperacetylated treated

histones,

wtih

(9,lO).

6 mM sodium

This

acetylated

causes

form.

control

nuclei

(figure

1A).

all

indicated

tion

times

(one

to three

are

shown

in the

figure.

the

two digestion

while tion

phase

levels

of histone

ratios,

to enhance

except

20% of histone

fold

min).

increase

for

butyrate

The average of the

values

inherent

of endogenous might

nucleases

affect

histone

more sensitive

removing

possible

of treated

rate

rate

of digestion

of

of three

at early

separate in

comparing

reproducing

nuclei/ variable

we decided

to attempt

in diqestion variations

diges-

digestions

and the possible binding,

and

digestion

was observed

to differences

most of the

to be in the

ini,tial

initial

accurately

were

acetylation

kinetics

difficulties

such as the need for

containing

HTC cells

example,

in the

in the

sodium

Because

system

at the same time

H4,

difference

with

RNA which

an assay

the

chromatin

of the DNase I digestion

treated

the problem with

of a culture

butyrate

curves,

rates

between

diges-

runs. Our approach

of both fit

of log

A two to three cells

to develop

a portion

a substantial

from

contamination

digests

A comparison

the nuclei

enzyme

DNase I preferentially

untreated

activity

involves

prelabelling

and sodium

butyrate

of the DNA released

the treated

when nuclei

1347

DNA with cells, from

[3H]

thymidine

and then untreated

in a portion

analyzing

the spec

and hyperacetylated

Vol. 82, No. 4, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Time (min)

Figure 1. DNase I digestion kinetics as assayed by the release of acid soluble oligonucleotides. Digestion of (0) untreated and (0) sodium butyrate treated cells as described in the Materials and Methods.

cells

are

butyrate during

mixed

and digested.

as diagrammed the

time

Nuclei

in figure

course

2.

treated

cells,

leased

into

the acid

soluble

fraction

amount

of nuclei by nuclei

be discerned

by a plot

The results demonstrated, soluble

as shown

the

cern

if

that

the

synthesized

either

untreated

figure

3A would

early

digestion

ratio

fl

thymidine

or butyrate

#3.

specific

the

only

deviate

from

the

times.

This

could

cells,

other

Fig.

lead

1348

3.

digested

an equal DNA

can easily

It

separately,

for

set

was was to

to our

example, in

a higher

to an incorrect

This

was related

one of the

was first

of the acid-

of digestion.

associated,

by showing

DNA re-

of the

activity

This

then

with

difference

DNA somehow more accessible

treated

DNA in nuclei

activities.

#2 if

artifact. were

This

DNase I,

of the

activity

specific

the course

the

are mixed

shown in

and nuclei

of one other

DNA, and this

nuclei

are

that

throughout

labelled

specific

sodium

If

activity

#l

the

of the

3A,

from nuclei

possiblity

newly

with

in figure

the same or constant

eliminate

than

with

as follows: digests

when nuclei

equally

of the

is

the specific

of such a DNase I digest

DNA released

either

then

be greater

#2 mixed

and treated

preferentially

butyrate

#4, will

labelled

The strategy

of digestion,

from sodium

released

were

nuclei

with from

of points

specific

interpretation

con-

in

activity of the

at

Vol. 82, No. 4, 1978

BIOCHEMICAL

Log

Label with

(200

$0' [ H] dill)

I I

be seen,

#1

Nuclei

#2

in figure

circles

(butyrate

(control)

nuclei

is

the

Nuclei

acid

soluble

that

obtained

fraction

activity

the

cells)

and is

nuclei

#4,

solubilzed

associated positive

presented

deviation

This

are

from

mixed

with

hyperacetylated linearity

#4

fall

butyrate

cells

of the

is

divided

1349

acti-

cells).

When

divided

When nuclei

digestion

by

#3,

the preferred

(l-5

ratio specific

from

untreated

#l are

DNA compared

times

After

1.111 (the

by that

#2 and nuclei

obtained.

open

of the DNA in the

at about

released

is

(that

#4, we see a constant

purposes.

histones

treated

activity

As can

same curve

of DNase I digestion

treated

At early

on the

3B.

DNA specific

nuclei

nuclei

in figure

of

in the overall

line

is

I

above the

horizontal

mixing

obtained.

from

course

activity

compared

specific

time

comparative

Nuclei

are each slightly

and sodium

butyrate for

I

63

of points

t3 and the the

specific

3B are

with

cells)

38).

DNA obtained

in figure

treated

#2 are

DNA from

and the

sets

during

figure

for

3A, both

nuclei

when nuclei

circles,

upon mixing

untreated

with

"0 treatment

cells, and the designation in figures 3 and 4.

due to a 10% difference

DNA from

#l are mixed

(open

activites

though,

between

treat for 6 hr with 6 mM Nabutyrate I I I I

Nuclei

the specific

solid

circles

cles

Isolate

2. Method of labelling and treating batch numbers, for the experiments

when

vity

No l.lhel

I I

Nucle:

the

HTC cells

no treatment

I

data

phase

4 hr thymidine

Treat for 6 hr with 6 mM Nabutyrate I I

Figure nuclear

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mixed

to that

the closed min),

substrate, one mintuteof

with of

cir-

the

DNA

since digestion,

a

BIOCHEMKAL

Vol. 82, No. 4, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

:: D 2 2-Y

20-

lo-

20

I 120

60 Time IminI

3

1.00

%K-CJ-

0

0

I

0

c

t

0

3

10

20 Time

I

,

40

60

0 4

(min)

o.oo-

, 0

1 10

I 20

Time

fmin)

1 40

Figure 3. DNase I digestion kinetics as assayed by the specific actiyity ([3~] thymidine cpm in the acid soluble fraction divided by the absorbance at 260 nm in the acid soluble fra tion) of the DNA released. (A) Nuclei from [ 5 H] thymidine labelled cells were digested without mixing with unlabeled nuclei. (of Digestion of nuclei #7. (0) Digestion of nuclei #2. (6) (0) Nuclei #l were mixed with an equal amount (as determined by absorbance at 260 nm) of nuclei #3 and the specific activity of the acid soluble DNA released during digestion divided by that released by the digestion of nuclei #2 mixed with nuclei #4. (0) Nuclei #l were mixed with an equal amount of nuclei #4 and the specific activity of the acid soluble DNA released during digestion divided by that released by the digestion of nuclei #2 mixed with nuclei #3.

Figure

4. Micrococcal nuclease digestion: (A) Micrococcal nuclease digestion kinetics as indicated by the release of acid soluble oligonucleotides. Nuclei from (0) untreated and (m) sodium butyrate treated cells. Ratios of the specific activities of the acid soluble DNA released during digestion: (B) Nuclei #l we\e mixed with an equal amount of nuclei #3 and compared to nuclei #2 mixed with an equal amount of nuclei #4. (C) Nuclei #1 were mixed with an equal amount of nuclei #4 and compared to nuclei #2 mixed with an equal amount of nuclei #3.

1350

I 60

Vol. 82, No. 4, 1978

this

ratio

is about

performed (ll),

BIOCHEMICAL

in the

this

ratio

after

4C).

In this

apparent times

when the

the

(data

DNA in the

compared tained

after

amount

and the

nuclei

from

tion

from

mixing

is

same rate

fraction

from

nuclei

are

in

butyrate

is figure

of digestion

untreatedand

kinetics

ratio

illustrated

could

labelled

cells,

for

DNase

I,

in

nuclear

figure

are

thus

in

without

activities treated

(the

There

(figure

digested

of butyrate

a constant

4A (2

digestion

of the specific

cells,

(figure

RNA, resulting

at early

cells

4B).

there

histones

of

cells,

similar

ob-

the

nuclease

results

as

result

is apparently

of the RNA by micrococcal treated

genes

nuclease,

not be detected

supernatant

the

untreated

micrococcal

as illustrated

digests

Fortunately

acid-soluble

to nuclei

in the

active

hyperacetylated

also

C3H]-thymidine

shown).

with

kinetics

nuclease

DNase I are

3 to 1.

above

in rate

activities

from

is

with

with

to solubilize

reported

digestion

micrococcal

nuclei

of digestions

performed

DNA associated

specific

not

of digestion was also

nuclease

RESEARCH COMMUNICATIONS

used by Weintraub

ug DNA) a difference

case,

reduced

mixing

the

even slower

of enzyme/50

same series

to the observations for

units

buffer

one minute

in contrast

With

If the

of digestions

was no preference 4).

to 1.

digestion

The same set however,

6.5

AND BIOPHYSICAL

of the

same

in the

DNA diges-

valid. DISCUSSION

That sensitive

of chromatin

containing

to DNase I whereas

micrococcal

nuclease.

Since

genes are selectively

active there

part

to DNase I.

micrococcal

nuclease

digestion.

Thus since

and not at all

is

reflecting

We surmize

that

systems

this

other

hyperacetylation,

the extent

is

spacer

regions

have shown

of histone-DNA

1351

that

within binding

is

gene

that

clear

that

regions

and

the nucleosome

and

at early

in

at early

we conclude

interactions

particularly towards

it

active within

shown only

nuclease

histone-DNA

workers

by DNase I (8,11,12)

towards

effect

is

susceptibility

DNase I seems to cleave directed

histones

no differential

in several

between

by micrococcal

phenomenon

is

solubilized

is a good correlation

sensitivity

there

hyperacetylated

times we are

the

times

by DNase I detecting

nucleosome

is decreased

core.

as a result

a

Vol. 82, No.4,

BIOCHEMICAL

1978

of hyperacetylation

I thus This

leading last

point

lated

histones

direct

kinetic

matin

revealed

formed tions

is

that

cleavage

illustrated if

are

most

nuclei

are

analysis

(in

strength

buffer).

consistent

DNA varies

or unacetylated

if

the

and if

the variation

DNA does not change

the subsequent

rate

enzyme

with

of acetylated

is

presented

chromatin these lar

it

will

preferentially

chromatins depending

Evidently

are

at low ionic

somewhat consideration

to test

tically

active

this

idea

of the the

notion

that

types

advantage

indicate

as modified

shown

is

less

chromatin

as a result that

it

must change this

report

nucleosomes

type

to perceive

in this

of hypermodification

hypermodified

at higher

analysis

does not

if

control

kinetic

held

of

be simi-

both

Again

experiments

tightly

though

for

1.

The observations

the

will

availability

a more detailed

acety.

However,

not seen:

in fig.

of mixing

further.

of the

are

with

and environment.

similar

is more compact,

the nucleosome

specifically

experiments

conformation

chromatin

material.

by histones

in rate

affin-

regions

of hydrolysis

is

the

case if

and unacetylated

availability

Certainly,

regions

to unfold

the

if

of enzyme and

In this

rates

observa-

associated

of affinity

chrowas per-

these

in excess,

acetylated

relative

and differences

to results

in availability.

the

inary

the

emphasizes

support

tin

strength

where

leading

ferences

then

is

that

the DNA is

of DNA governed

chromatin

strengths

needed

not mixed

the

a

and hypermodifed

of cleavage.

digest

upon availability

and hypermodified ionic

a mixture

hyper-acety-

1 the experiment

substrate

whether

(13).

However,

We have concluded

depending

histone

massively

of normal to figure

experiments.

observation

the supernatant.

digestion

by DNase

in the mixing

DNase I,

into

contrast

bound

by a recent

with

released

no differences

of enzyme for

particularly dramatically

of nuclease

can be internally

can be more easily

digested

selectively

in a low ionic

lated

the substrate

to a selective

It was noted

ity

so that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of dif-

will would

in the gene-

together require

the

chroma-

and in fact

prelim-

have the

same overall

material. ACKNOWLEDGMENTS

We wishtothank

Mary Ellen

Eichner

for

1352

assistance

with

be

the

figures

and

Vol. 82, No. 4, 1978

Tom Slattery from

for

BIOCHEMICAL

help

the NIH Cancer

in cell Institute

AND BIOPHYSICAL

culturing.

This

#CA-10871

work

RESEARCH COMMUNICATIONS

was supported

by grants

and CA-20509.

References (1) (2) (3) (4) (5) (6)

(7) (8) (9) (10) (11) (12) (13)

Felsenfeld, G.(1968) Nature 271, 115-121. Sollner-Webb, B., and Felsenfeld, G. (1975) Biochem. 14, 2915-2920. Axel, R. (1975) Biochem. 14, 2921-2925. Whitlock, J. P., Jr., and Simpson, R. T. (1976) Biochem. 15, 3337-3314. Delange, R. J., and Smith, E. L. (1971) Ann. Rev. Biochem. 40, 279-314. Allfrey, V. G. (1971) in Histones and Nucleoproteins (Phillips, D. M. ed.) pp. 241-294, Plenum Press, London. :i;ver D., Granner, D.,and Chalkley, R. (1974) Biochem. 13, 746-749. Garel,'A., and Axel, R. (1976) Proc. Natl. Acad. Sci USA 73, 3966-3970. Riggs, M. G., Whittaker, R. G., Neumann, J. R., and Ingram, V. M. (1977) Nature 268, 462-464. Hagopian, H. K., Riggs, M. G., Swartz, L. A., and Ingram, V. M. (1977) Cell 12, 855-860. Weintraub, H., and Groudine, M (1976) Science 193, 848-856. Reeves, R. (1977) Eur. J. Biocuem. 75, 544-560. Sealy, L., and Chalkley, R. (1978) Nucleic Acids Res., submitted.

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