Degradation of chromosomal proteins during dissociation and reconstitution of chromatin

Vol. 57, No. 3, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DEGRADATION OF CHROMOSOMAL PFXXEINS DURING DISSOCIATION AND RECONSTITUTION OF CHROMATIN Chi-Born Department Received

Chae and Donald

of Biochemistry, Chapel Hill,

February

B. Carter

University North Carolina

of North 27514

Carolina,

22,1974

and nonhistone chromosomal proteins are degraded when chroSUMMARY. Histones matins exposed to 2 M NaCl-5 M urea (pH 6-8) which has been most often used for dissociation and reconstitution of chromatin. Histones and nonhistone proteins are also degraded in 5 M urea (pH 6-8). Isolated presence

chromatin

mediates

of RNA polymerase

the dissociation criterion

tion

of RNA's homologous

for

correct

results

was dissociated

lysis

against also

tionate

dissociated

nonhistone

proteins

sively

reconstitution

reports (5-111,

transcribed

were

pH 6-8.3

in 2 M NaCl-5

appeared

on

obtained

(5-9).

with

chromatin

and reconstituted

M urea

impor-

transcrip-

chromatin

of NaCl in the presence

chromatin

in the

has been the

from native

chromatin M urea,

have

RNA's

and the most

of chromatin

reconstituted

by dia-

of 5 M urea.

in order

to frac-

(7,9-13).

report

we present

in

2 M NaCl-5

degraded

several

chromatin

concentrations

have

In this

of

in 2 M NaCl-5

decreasing

of tissue-specific

Recently

to those

with

which

Others

(l-4).

and reconstitution

tant

Most of the

the synthesis

evidence M urea

that

chromosomal

at pH 6-8

and in

proteins 5 M urea

are extenat pH 6-8.

METHODS Preparation

Chromatin

of chromatin:

as described

before

(14)

and sheared

of chromatin:

Chromatin

was prepared at 20 volts

from pure for

rat

90 seconds

liver in

nuclei a VirTis

homogenizer. Dissociation NaCl-5 matin for

M urea solution

15-16

(deionized

Copyright All rights

or 5 M urea

hours. ultra

by stirring

was centrifuged The urea pure

slowly

in a Beckman used

in this

study

grade).

0 1971 by Acndemic Press, Inc. in nny form reserved.

of reproduction

was dissociated

740

at 4'C

in freshly

prepared,

3 hours,

and the

for

No. 40 rotor was

at 35,000

from Schwartz/Mann

2 M chro-

rpm at 4°C

Vol.

57, No. 3, 1974

Sodium

dodecyl

chromatin

BIOCHEMICAL

sulfate

dissociated

NaHSO3 , which and then

is

against

5 M urea

against

the

dissolved

in

2 M NaCl-5

1% sodium

was dialyzed

supernatant

0.1

vol.

with

1% sodium

dodecyl

against

obtained

mixture. sulfate

sulfate

were

50 mM

for

3 hours

(pH 7)-O-l%

from chromatin dodecyl

fi-

dissociated

sulfate

The pellet

dodecyl

from

cold

(15),

M phosphate

of 10% sodium

sulfate

1% sodium

COMMUNICATIONS

obtained

protease

sulfate-O.01

the

was mixed

1 ml of the

M urea

dodecyl

RESEARCH

The supernatant

of chromatin-bound

However,

in

BIOPHYSICAL

electrophoresis:

an inhibitor

mercaptoethanol. in

gel

AND

and dialyzed

containing

mixture

with

DNA was

vigorous

stirring. The samples (0.6

x 0.5

cm)

0.1% sodium phate,

in sodium (16)

stained

with

for

sulfate,

of histones:

.20 hours

obtained

after

dialyzed

against

to 2.5% stacking

separating

gel

5 n-&l EDTA, and 0.1

as described

For isolation

at 4'C,

but

with

centrifuqation cold

not cold

before

(0.6

gel

x 10 cm) in

M sodium

(14).

of histones

centrifuged,

phos-

The gels

0.4 N H2SO4 for

were

N acetic

separated

electrophoresis

the

chromatin

was dialyzed

of the chromatin

7 M urea-O.9

The histones

2.5 M urea)

M urea,

applied

were

Blue.

~21 NaHS03 and extracted

night.

2.5

and electrophoresed

Coomassie

Electrophoresis iated

on top of 7.5% acrylamide

dodecyl

pH 7 (17)

dodecyl

30 min.

acid-O.1

cold

50

The supernatant

at 10,000

by acid-urea

against

dissoc-

xq for

10 min was

% O-mercaptoethanol

polyacrylamide

gel

over(15% in

(18).

RESULTS AND DISCUSSION Table

1 summarizes

chromatin tin

(mixtures

(5 M urea).

histones

of salt There

when thymus

(8,19),

but

strates

that

the

exposure

is

dissociated

the

as far

composition and urea)

have been chromatin

is

and for

several

nonhistone

of chromatin

in

preparation

proteins

of nonhistone

741

for

dissociation

of reconstituted

on the partial

to 5 M urea-2

there

the solutions

used

washing

reports

exposed

as we can determine

chromosomal

for

of solutions

M NaCl-10

has been no report are or are not listed

in Table

proteins

(9-13)

1.

of chroma-

degradation

of

mM Tris,

pH8

which degraded

demonduring

When chromatin or reconstitution

Vol. 57, No. 3, 1974

BIOCHEMICAL

AND BlOPHYSlCAl

RESEARCH COMMUNKATIONS

\

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-

2

1

4

“3

9

8

IO

Figure 1. Sodium dodecyl sulfate gel electrophoresis of chromatin dissociated in 2 M NaCl-5 M urea. Control chromatin (undissociated) (1); 2 M NaCl-5 M urea-50 mM acetate, pH 6, supernatant (2) and pellet (3); 2 M NaCl-5 M urea50 mM phosphate, pH 7, supernatant (4) and pellet (5); 2 M NaCl-5 M urea-10 mM Tris, pH 8, supematant (6) and pellet (7); 2 M guanidine'HCl-5 M urea, pH 6.5, supernatant (8) and pellet (9); control chromatin (10). Experimental details are described in Methods.

of chromatin

(5-ll),

chromatin

is

HCl and urea

between

16 hours

and

such condition degraded.

considerable

at pH 6, I,

Methods,

it

1).

In the

sodium

molecular

weight

proteins

Figure

obtained 1 but

and we found

liver

nonhistone

than more

not

proteins

chromatin

the

supernatant which

are not

chromatin, F2al

proteins of salt

histone

F2al

extensive

at

the

chromatin

centrifuged

(not

seen

which

M urea

is

a considerable in native

appear pH 8 than

while

in

in

protein

that amount

mainly in

polypeptides

the electrophoretic at lower

dissociated shown here). 742

chromatin

the smallest

are decreased

in the

pHs. same

2 M NaCl-5 in

amount

dissolved

patterns

and urea

in

under are

as described

was directly

histone

that

and histones

is dissociated

contains

from the electrophoretic

with

at 4"C,

of nonhistone

appears

smaller

is

days

mixture,

in the mixture

degradation

several

sulfate

weight

weights

were

case of control

It

dissociated

of NaCl or guanidine.

and 8 and in 2 M guanidine*HCl-5 that

dodecyl

chromatin.

when rat

was found

low molecular

to a mixture

amounts

For example,

M urea

exposed

the

(Figure in the

1%

in liver high chromatin

of molecular patterns.

The same results solutions

of

used in

The

Vol. 57, No. 3, 1974

BIOCHEMICAL

CJ 1

-

AND BIOPHYSICAL

X-J‘ 5

I

2

RESEARCH COMMUNICATIONS

7

6

8

Figure 2. Sodium dodecyl sulfate gel electrophoresis of chromatin dissociated in 5 M urea. Control chromatin (1); 5 M urea-50 mM acetate, pH 6, supernatant (2) and pellet (3); 5 M urea-50 mM phosphate , pH 7, supernatant (4) and pellet (5); 5 M urea-10 mM Tris , pH 8, supernatant (6) and pellet (7); control chroExperimental details are described in Methods. matin (8). In this experiment chromatin was exposed to 6-7'C during 3 hour stirring before centrifugation.

High molecular in the chromatin 2.

However,

weight dissociated

As also

shown

tones,

are dissociated from

proteks

in 5 M urea

the degradation

pH 8.

proteins

nonhistone

are also

from

less

2 most of nonhistone

chromatin

DNA by 4 M urea-O.1

in quantity

at pH 6, 7, and 8 as shown

seems to be somewhat

in Figure

decreased

M phosphate

at pH 6 and 7 than

proteins,

by 5 M urea.

in Figure

but

Dissociation

at

not hisof nonhistone

(pH 7) has also

been

reported

(20). Since

histones

trophoresis,

we extracted

sociated tones

M urea

separated

by acid-urea

3 histones

from the

of degradation

considerably siderable 6 and 7 but

degraded amounts not

products

gel

at different

electrophoresis dissociated

M urea migrate

are before

M urea

pHs,

are

M urea

but

On the other

Histones

dis-

and the his-

at pH 6

although

histone,

degraded

elec-

As shown in

intact

F2al

gel

chromatin

in 2 M NaCl-5

at pH 8.

at pH 8.

743

sulfate

(18).

relatively

F2b and F2a2 histones

as much in 5 M urea

dodecyl

0.4 N H 2SO 4 from the

and in 5 M urea

in 2 M NaCl-5

of Fl,

in sodium

by cold

chromatin

and 7 and in 2 M guanidine*HCl-5 amounts

resolved

histones

in 2 M NaCl-5 were

Figure

are not well

histones hand,

in 5 M urea

of the

chromatin

small are conat pH dis-

Vol. 57, No. 3, 1974

BIOCHEMICAL

4

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5

6

Figure 3. Acid-urea gel electrophoresis of histones extracted from chromatin dissociated in 2 M NaCl-5 M urea and in 5 M urea. (1) 2 M NaCl-5 M urea-50 mM acetate, pH 6; (2) 2 M NaCl-5 M urea-50 mM phosphate, pH 7; (3) 2 M NaCl-5 M urea-10 mJ4 Tris, pH 8; (4) 2 M guanidine.HCl-5 M urea, pH 6.5; (5) 5 M urea-50 mM acetate, pH 6; (6) 5 M urea-50 mM phosphate, pH 7, (7) 5 M urea-10 mM Tris, pH 8; {8) 2 M NaCl; (9) 2 M NaCl-10 mM Tris, pH 8; (10) control chromatin.

sociated

in 2 M NaCl are

degraded

in 2 M NaCl-10

The results degradation tution

contain rat

liver

from other protease.

tissues

However,

followed

could

during

it cell

the

are

depleted

In some instances

urea-10

mM Tris,

pH 8.3

the (10,ll)

case of liver

chromatin

of liver

as one expects

for

in the

final

the

listed

which

chromatin

removes

chromatin

a histone-DNA

is usually complex

744

reconsti-

such as liver that

chromatin

in Table

1 may also

procedure

molecular chromatin was washed

2).

We found

that

precipitated having

histone/DNA

with

weight

non-

(not

shown

with

most of the nonhistone

(Figure

and thymus

high

is

extensive

reported

reconstitution

reconstituted

reconstituted

histone

have been

possible

lines

reported

of F2al

activity

is quite

at pH 6 and 8, and as expected

here).

from DNA in the

there

to have protease

or cultured

We have

chromatin

that

amount

and histones

are known

(15,21).

proteins

buffer

suggest

proteins

histone

extracted

here

which

chromatin

a significant

pH 8.

of nonhistone

and thymus

but

mM Tris,

described

of chromatins

isolated

intact

5 M proteins

the urea-

in dilute ratio

of

Vol. 57, No. 3, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table

1

Reported Dissociation Conditions Chromatin and Tissues Studied

Dissociation

Solution

Tissues

2 14 NaCl - 5 14 urea-50 acetate, pH 6

mM

2 I? NaCl - 5 M urea-l phosphate, pH 7

mM

2 M NaCl - 5 !4 urea-10 Tris, pH 8 - 8.3 2 I4 guanidine.HCl 5 M urea

l-l.2

tin

- 5 M urea

amounts

isolated

of

chromatin

in transcription

cribed

from native

the

integrity

and thus transcription permit

the

erythrocytes

preparations

(20).

of nonhistone

proteins

are also

tissues

and cell

previously reported

to yield

, yielded

RNA's

validity

of the done under

of RNA transcripts

likely

dissociated

that

from chroma-

for

reconstitution

which,

when used

similar

to those

reconstituted

about

were

very

raise

in these

which

is

experiments

proteins

experiments,

It

conditions

chromosomal the

10,ll

HeLa cells

chromatin

the present

5,7, P-11,13

lines.

reported

experiments

doubts

12

6

chromatin

chromatin,

detection

thymus,

fibroblasts,

of the

create

liver,

Wl-38

been

plates

8

pH 8.3

although

have

thymus

embryo

from other

In summary,

liver,

chick

as in most

substantial

Reference

pea codyledon, liver, thymus, kidney, HeLa cells, Wl-38 fibroblasts

mM

- 10 mM Trls,

(22)

for

the

from repetitive

trans-

questions

interpretations conditions

as tem-

about

chromatins of the which

sequences

only

of DNA (23).

ACKNOWLEDGEMENT This work was supported by grants from the University of North (VF 336) and the American Cancer Society (IN15-0) and USPHS General Support Award (5 Sol-FR-05406).

Carolina Research

REFERENCES 1. 2. 3.

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BIOCHEMICAL

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