Nuclease-sensitivity of methylated DNA as a probe for chromatin reconstitution by genotoxicants

Vol. 107, No. 2, 1982 July 30, 19812

BIOCHEMICAL

AND BIOPHYSICAL

NUCLEASE-SENSITIVITY

OF METHYLATED DNA AS A PROBE

FOR CHROMATIN RECONSTITUTION Akira Department 39th Received

June

Nishio*

of Pharmacology, St. and Rainbow 11,

RESEARCH COMMUNICATIONS Pages 485-491

and

BY GENOTOXICANTS+

Edwin

M. Uyeki*

The University of Kansas Blvd., Kansas City, Kansas

Medical 66103,

Center, USA.

1982

DNA iln Chinese hamster ovary cells was labeled with [W]thymidine and [methylsH]-1-methionine in culture, and their nuclei were digested with nuclease. Not until 10 percent of bulk DNA was digested did micrococcal acid-soluble fraction. methylated DNA appear in the When these cells were exposed to UV-radiation, alkylating agents and intercalating agents in culture, the resistance of methylated DNA to digestion by the nuclease was largely or completely eliminated. The change in the sensitivity of methylated DNA to the induced by the nuclease indicates a conformational change in chromatin genotoxicants.

INTRODUCTION The bulk 200-base

pair

base pairs

of il

units,

DNA

(1,2).

replication.

inf'..uence repair however,

is

nucleosome. around

The

"phasing"

(

nucleosome

in genes

the

eukaryotes

There is

is

generally

the

be

consensus

that (3).

of

linker of

of

such

core

into

the

because

the

can also

are

been

subject

and

of nucleosomes significantly

by physical/chemical have

DNA

location

gene expression

structure

146

60 base

to the

specific

distribution

studies studies

of about relative

a means of regulating

Nucleosome

of about

each of histones

consisting

nucleosome

a non-random

approximately

consists

two molecules

region

of DNA damage induced

Nuclease-digestion results

A nucleosome

much interest

gene may

obtained

the distribution processes.

arrangement

) has received

in a

known to be packed

an octamer

H3 and H4, and a nucleosome

of

seqt..ence

DNA in

of DNA winding

H2a, H2b, pairs

of

agents

invaluable to

and

probes;

contradictory

reorganization of nucleosome structure interpretations because of the possible -~ * : supported by NIEHS Grant No. ES-02046, and to whom all correspondence should be addressed. **: present address: 2nd Department of Medicine, Kyoto Prefectural University of Medicine, Kajii-cho, Kawaramachi Hirokoji, Kamikyoku, Kyoto 602, Japan.

0006-291X/82/140485-07$01.00/0 485

Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 107, No. 2, 1982

BIOCHEMICAL

by damage

processes

the

and repair

detection

sequences

are

for

2 to

nucleosome

structure

9),

portions

(10,

differential in nucleosome

7

has (7);

times

up

to 75

11).

(MeCyt)

percent

MeCyt

several and

changes

5-methylcytosine

of

(8,

Further

RESEARCH COMMUNICATIONS

development

in

of

chromatin

with

the only

minor

the methods respect

for

to

DNA

required.

distribution

of DNA is

(4,5).

conformational

of

In mammals, accounts

AND BIOPHYSICAL

In

been the

higher

this

total

cytosine

reported

abundance than

percent

nuclease-sensitivity reconstitution

of

is

of study, of

induced

with

respect

of MeCyt

in highly

in moderately total

residues

MeCyt

to

DNA sequences

repetitive

is

in

the and bulk

possibility DNA to

and

sequences

or unique

located

in DNA and Non-random

(6).

repetitive

we investigated methylated

base

sequences

nucleosome

core

of using detect

a

changes

by genotoxicants.

MATERIALS AND METHODS Chemicals: Doxorubicin was supplied by The Drug Synthesis and Chemical Branch, National Cancer Institute (Bethesda, MD). Dichlorvos was supplied by Analytical Chemistry, U.S. Environmental Protection Agency (Research Triangle Louis, Park, NC). Ethyl methanesulfonate (EMS) was obtained from Sigma ( St. [methylsH]-1-methionine (specific activity: 12 Ci/mmole) was obtained MO). from New England Nuclear (Boston, MS). [2-l%] thymidine (54 mCi/mmole) and [methylsH]thymidine (48 Ci/mmole) were from Schwarz/Mann (Orangeburg, NY). Micrococcal nuclease was obtained from Worthington ( Freehold, NJ). Cell culture: Chinese hamster ovary (CHO) cells were grown as a monolayer in RPM1 1640 medium supplemented with 10 percent fetal calf serum, an additional 10 rig/ml insulin and penicillin/streptomycin at 37'C in a water2 gm/L glucose, Radio-labeling of DNA was done overnight by adding jacketed CO2 incubator. [methyl-sH]methionine, ['4C]thymidine or [aH]thymidine at concentrations of 5, 0.05 and 1 uCi/ml, respectively. For the treatment with genotoxicants the medium containing radioactivity was discarded, and cells were washed once with phosphate buffered saline (PBS), chased with fresh medium for 2 hr, and then exposed to chemicals for 3 hr or W-light (predominantly 254 nm) in PBS. of CHO cell nuclei followed Digestion by nuclease: Preparation the method Nuclei were suspended in a buffer consisting reported by Muramatsu et al (12). of 0.25 M sucrose, 0.1 mM CaC12, 1 mM Tris-HCl (pH 7.8) at a concentration of 108 nuclei/ml. Micrococcal nuclease was added at a concentration of 50 U/10* nuclei. Digestion was continued in a 37X water bath and terminated by adding of 10 percent perchloric acid (PCA) or 10 mM EDTA and chilling an equal volume in ice. Digestion of bulk DNA was determined by measuring the radioactivity of [ 14C]thymidine in the cold PCA-soluble and -insoluble fractions of nuclease-treated nuclei, after solubilization of DNA with 5 percent PCA by heating at 90 'C for 20 min.

rig/ml),

Extraction pronase

of DNA: The EDTA-added nuclei (0.5 mg/ml) and sodium dodecyl 486

were treated with (1 percent) sulfate

RNase A (0.5 in a buffer

Vol. 107, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

containing 0.2 M NaCl, 5 mM EDTA and 10 mM Tris-HCl (pH 7.8) at 37'C for 1, 4 and 2 hr, respectively. DNA was extracted twice with phenol: chloroform: isoamyl alcohol (24: 24: 1) and precipitated overnight with 3 volumes of ethanol with 5 pg calf thymus DNA as carrier at -20 'C. For measurement of radioactivity, DNA was solubilized in 5 percent PCA by heating at 90°C for 20 min and ad.ded to a xylene-based scintillation counting fluid. Radioactivity of [methyl-sH] methionine in the ethanol-precipitated DNA was rendered completely acid-soluble by DNase I, but not by RNase A or pronase. The possibility that radioactivity resides in the methyl group of thymine and purine rings can not be excluded (1.1, 13, 14), since the radioactivity among the DNA bases was not determined. RESULTS kinetics

of

methylated

shown in

Fig

1 A.

Digestion nuclease acid

are

soluble

using

did

the

mouse L cells

Met-)NA

is

digested

Met-DNA

initiated,

a

accord is

reports

compared

with

digested

in

parallel

with

of

total

of bulk

and this

bulk

DNA by micrococcal DNA was rendered

We obtained

of others

manner

( 40 percent

remained

occur.

observations), the

and bulk

10 percent

of Met-DNA

with

plateau

of Met-DNA

Not until

(unpublished

proceeded

DNA reached percent

in

digestion

DNA (Met-DNA)

nuclease-

(10,111.

DNA. bulk

similar

In

Digestion

DNA.

resistance Fig.

undigested

of

1 B, the

of Met-DNA,

When digestion

DNA remained

results,

once

of bulk 1, 50 - 60

undigested.

fl40 0

3 : 30 I

o$

a 2o iFI 1 10

,OOk

15

/ I" 9'

;c 0L 0

TIMECMIN)

A

/

d""

10 20 -1. DIGESTED

30 40 BULK DNA

50

of methylated DNA and bulk DNA in nuclei by micrococcal --Fig. 1. Digestion nuclease. A: kinetics of digestion. CHO cells were labeled overnight with [methyl-aH]-1-mzthionine and ['4C]thymidine, 5 and 0.05 pCi/ml, respectively. Isolated nuclei were digested with micrococcal nuclease ( 50 U enzyme/108 nuclei 1 at 37 'C for the indicated periods. Undigested DNA was purified, and the radioactivity was determined by scintillation counting. o: methylated DNA ([sH]). .: bulk DNA ([WI). B: Digestion of methylated DNA as a function of bulk DNA. Digestion of methylated DNA was plotted against bulk DNA. 0, a, 0 represent

three

different

experiments.

487

BIOCHEMICAL

Vol. 107, No. 2, 1982 The resistance were

with

treated

hr.

of

2

Fig.

A shows

digestion

of

the

digestion

as

is

a

investigated;

a

known

and

protein similar

J/m')

curve of

on did

in the

the

the nuclease not

abolish

abolition

chemicals

0

5 10 TIMECMIN)

DNA

on

in

cells

bulk

protein

(16).

preferentially

treated

% DIGESTED

BULK

with

EMS is

methylates

and

genotoxicants although

completely,

a

(Fig.

3).

DNA was

also

the

drugs

15 DNA

Fig. 2. A: Digestion kinetics of methylated and bulk DNA in nuclei treated with dichlorvos. The radiolabeled cells were treated for 3 hr with 10 mM dichlorvos in culture, and isolated nuclei +re subjected to digestion by micrococcal nuclease under similar conditions to Fig. 1. o: methylated DNA. .: bulk DNA. B. Effects of dichlorvos, EMS and doxorubicin on the nuclease sensitivity of methylated DNA. This figure is represented in a similar way to Fig. 1 B. 0: control. ., A, . : cells were treated with 10 mM dichlorvos ( . ), 20 mM EMS ( A ) and 20 uM doxorubicin ( u ) for 3 hr in culture.

488

a an

was observed bulk

and

Dichlorvos,

of Met-DNA

were

the

Met-DNA.

(15).

chemical

of

with

and

of chromatin;

kinetics

B shows

disappeared,

agent

resistance

3

DNA in

2

treated

both

The three

bulk

Fig.

to digestion

the resistance

digestion

EMS.

in nuclei

sensitivity

of this

cells

by the nuclease.

anticancer

pesticide,

[sH]thymidine-labeled

50’

and

and

digested

fashion

DNA (17).

Met-DNA

and

of Met-DNA

DNA

than

rather

of

equally

intercalating

when

(10 mM) and EMS (20 mM) for

of bulk

parallel

alkylates

effect

(100

dose response Effect

in

were

was abolished

doxorubicin

organophosphate

phosphorylates

W-radiation

with

resistance

well

anti-cholinesterase

a

DNAs

RESEARCH COMMUNICATIONS

digestion

kinetics

a function

initial

mutagen

showed

both

proceeded

Doxorubicin strong

digestion

obtained

Met-DNA

genotoxicants; the

the

were

nuclease

(20 JIM), dichlorvos

nuclei;

results

to

doxorubicin

dichlorvos-treated Similar

Met-DNA

AND BIOPHYSICAL

in

BIOCHEMICAL

Vol. 107, No. 2, 1982

60’.

03

' ' 3 5 TIME(MIN)

0 1

Fig. DNA. -&n an additional bulk DNA. (0 ).

3.

AND BIOPHYSICAL

04 50. 0

4 10

' 7

RESEARCH COMMUNICATIONS

of W-radiation Effects were exposed to W-radiation 3 hr. The condition 0, A, q : methylated DNA,

5 TIME

10 (MINI

15

on

the digestion kinetics of methylated at 8, 20 and 100 J/msand cultured for .: for digestion was similar to Fig. 1. irradiated 8 ( a ), 20 ( o ) and 100 J/m2

Fig’ 4 Digestion kinetics of bulk DNA in nuclei treated with Llchlorvos. -c ('HO cellls were labeled with overnight L3Hl- or [ ‘4C] thymidine conditlonH ['HI-labeled cells were treated with dichlorvoa under similar to Fig. 1. Isolated [sH]- and ['4C]-labeled nuclei were mixed and subjected to digestion by micrococcal nuclease. o: dichlorvos-treated DNA. .: control DNA.

culture,

and

controls.

digestion

Fig. 4 shows

betws?en with

the

was compared that

there

dichlorvos-treated

doxorubicin

and

and EMS ( data

are

that

of

no differences

control not

with

cells.

shown

['4C]thymidine-labeled in nuclease-sensitivity

Similar

results

were

obtained

1.

DISCUSSION An explanation earl,?

of

course

distributed reports

in on

the

preferentially whether

for

(Fig. cores

distribution

of

distributed

or not nuclease-sensitivity

nucleosome

core

requires

Many studies re

digestion

nucleosome

(19)

lationship

between

further

are

nuclease-resistance

the micrococcal

currently

methylated

1)

is

11).

(IO, MeCyt in the

that

Met-DNA

There

are,

in chromatin: linker

of Met-DNA

of Met-DNA

portion will

is

preferentially

however, it

is

in the

conflicting

randomly

(18)

of nucleosome.

be a useful

"marker"

or

Hence, for

the

investigation. under DNA and gene 489

investigation activity

dealing (20).

There

with

the

i s agreement

Vol. 107, No. 2, 1982

BIOCHEMICAL

that

of a

hypomethylation

active

transcription different

hypersensitive

to pancreatic

possibility,

higher

rendering

their

order

(doxorubicin), abolished

change

previously

was

show

a more observed

not

in the

abrupt

and steeper

higher

order

is

the

and

Genotoxicant

effects

conformational

presence

on these

changes

genotoxicants

nuclease-hypersensitivity

and,

these

occurred as of

either

DNA >,

The

effects

completely

is

that

nuclease

that

2 B),

is

that

a DNA

are now

in the

early

Met-DNA

curves

steeper

slope

this

and that

of an all-or-none

We

there

action,

of Met-DNA

or

3).

such a manner

fact

(Fig.

agent

2 and

DV-radiation

response,

is a toxicant-induced

Fig.

is

mobility is

3 )

between

presently proteins

our

study,

the hypomethylated,

in progress. 490

in the

loss

normally

of nuclease-

dependent

on histone

(HMG) of proteins

these in

change

chromatin,

somewhat

group not

topological

"active" resulting

proteins

observed

as

possibility.

there

high

in bulk

(Fig.

to

of transcriptionally

of

genes.

as well

toxicant-treated

Nuclease-hypersensitivity

acetylation

(25).

an intercalating

Met-DNA

further, slope.

first

is

"active"

occurring

resistance

and nuclease-hypersensitive,

hypersensitivity.

those

opaque

( instead

that

structure

and,

chromatin

configurations

and DNA in

Met-DNA

of this

possibility

hypomethylated

of

be lost

curve

the feasibility

one for

hence,

should

response

lessens

of

then

is

nuclease

with

(EMS and dichlorvos),

the case,

drug-treated

a dose

A second

were

chromatin

hydrolysis.

the nucleosome

in

showed

with

with

nuclease-hypersensitive,

UV-radiation,

the first

and,

to micrococcal

nucleosome

to acid

agents

between

this

of digestion

would

different

correlated

"active"

may correlate

( compared

"covered"

If

susceptible. course

studies

genotoxicants,

two possibilities;

(24);

micrococcal

nuclease-resistance

the

conformational sites,

with

alkylating

and

considered

our

in

the

is

"active"

and possibly

that

structures

the

with

sequences

chromatin

DNA more susceptible

Treatment

almost

bulk

exists

Such genes may be associated different

neighboring

DNase I,

DNA observed

RESEARCH COMMUNICATIONS

Transcriptionally

from

thus,

hypomethylated

gene and

(21-23).

topologically

The

AND BIOPHYSICAL

known.

(25,

26).

We believe

that

and DNA with resulted

"active"

DNA.

in

the

addition

abolition

Further

work

of is

BIOCHEMICAL

Vol. 107, No. 2, 1982 Using digestion

radio-labeled kinetics

genocoxicants.

Met-DNA of

association

with

rearrangement

digestion

showed

no

differences;

conformational

changes

be

detected

chromatin general

by DNA.

utility

by

of bulk we

nuclease

increase

We believe

that

study

the change the

by Met-DNA in the

It

of genotoxic

is

to

mean are

in

dependent a

on its

structural

noteworthy cells that

not

nuclease-digestible DNA and Met-DNA

the

large

that

assays

a

(Fig.

4)

putative

enough

fraction

the

several

by

indicates

and control

labeling

the use of bulk

a change

DNA is

probably

toxicants.

this

that

was induced

of nuclear

interpreted

suggested

RESEARCH COMMUNICATIONS showed

DNA in drug-treated

a gross

in the

we

micrococcal

proteins,

chromatin

comparative

probe,

nuclease-sensitivity

chromatin of

as a

DNA by

Because

AND BIOPHYSICAL

as to

of whole will

have

agents.

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