Proteolytic digestion of histones indicates that the nucleosomes of nuclei and of sheared chromatin are similar

Vol. 74, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PROTEOLYTIC DIGESTION

OF HISTONES INDICATES

THAT THE NUCLEOSOMES OF NUCLEI AND OF SHEARED CHROMATIN ARE SIMILAR* Dawn B. Marks Department

Received

and Barbara

of Biochemistry, Temple Philadelphia,

November

J. Keller

University Pa. 19140

School

of Medicine

8,1976

SUMMARY: Calf thymus nuclei were treated with trypsin, chymotrypsin or Pronase, and the rate of digestion of the various histone fractions was determined. The results differed from those obtained by digestion of DNAfree histones with the same set of enzymes but were identical to those obtained by digestion of calf thymus chromatin. Because these enzymes have such different specificities, the results of these digestions indicate that the histone fractions have similar locations in the chromosomal substructures of nuclei and chromatin, L.e. that the structure of the nucleosomes which exist within nuclei is not changed markedly when chromatin is isolated from nuclei by a method which involves shearing.

Electron initial

microscopy

evidence

which

According

to this

structures

each

a protein histones

core

model

might

model,

have

digestion

current

model

consists

approximately This

(5,6).

core

is

these

(11)

of histones

along

from

nuclei

intact

additional

generated

(3,4)

for

composed

provided

chromatin

of a series 200 base

evidence

concern

that

pairs

the

structure.

of repeating

sub-

of DnA wound

around

of two molecules

from DNA under have

recently

DNA fibers. which

This work was supported Support Grant.

0 1977 by Academic of reproduction in any

certain

shown

treated

in part

Press. Inc. form reserved.

support

of each of the

of this

that

with

conditions vigorous (12)

134

for

of chromatin some time

(8-10)

have found

causes that

nuclease

RR 05417,

that

and Doenecke

shearing

micrococcal

by NIH Grant

"nucleosomeW

of isolation

has been known

No11 --et al.

were

in

the method It

substructures.

dissociate

and McCarthy

Copyright All rights

to the

chromatin

containing

to obtain

disturb

histones

*

lead

and nuclease

H2A, H2B, H3 and H4.

Efforts (7)

(1,2)

movement

DNA isolated yielded

General

Research

Vol. 74, No. 1, 1977

a series

BIOCHEMICAL

of discrete

bands

chromatin

treated

ting

the shearing

that

with

method

the chromosomal

chromatin

prepared

indicated had been

On the other discrete

hand,

DNA fragments

chromatin

isolated that

by sonication

revealed

fibers

similar

microscope

had

with

sults

after

a smear,

indica-

this

chromatin

had

micrococcal which

digestion

of

and of chromatin

of the histone

pre-

fractions

procedure.

(13)

obtained

nuclease involved

the same series

treatment

shearing,

of chromatin

substructures

trypsin

of nuclei

shearing

and Johns

microscopy

(v bodies)

of nuclei

and Olins sheared

when intact

nuclei

were

reported

the results

of digesting

of or of

--et al.

by blending

throughout

observed

the chromosomal

intact

calf

we obtained

chromatin, conventional which

by the

retains

enzymes,

of the possibility

destroyed

utilizing

the susceptibility

sheared

only

Furthermore,

that

the proteolytic

Because

(15).

isolating

DNA from

the

(14) or

chromatin

lysed

directly

calf

thymus

onto

grids.

We have previously chromatin

for

treatment

Woodhead

while

produced

nuclease

altered

electron

to those

nuclease

employed

by a method

have reported

gels

substructures.

by mild

by shearing

to proteolysis

on polyacrylamide

micrococcal

destroyed

pared

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

supporting preparative

that

for

were

methods

the nucleosome

the digestion to those

of Woodhead involving

structure

shearing found

within

and Pronase

of isolating

we repeated

identical

the conclusion

chymotrypsin,

our method

substructures,

thymus nuclei nuclei

trypsin,

chromatin

our

experiments

procedure.

The re-

we reported

for

and Johns forces

that yield

certain chromatin

the nucleus.

Materials and Methods: Trypsin and a-chymotrypsin were purchased from Worthington Biochemical Corporation and Pronase from Calbiochem. Nuclei were isolated from frozen calf thymus tissue by the method of Allfrey -et --al suspended in 0.3 M sucrose, 1 mm CaC12 (to prevent lysis (16). Nuclei and aggregation) and 10 mM sodium borate, 2 mM sodium citrate (pH 7.3) at a concentration of 300 ug DNA/ml were treated at room temperature with trypsin (5 pg/ml),chymotrypsin (30 pg/ml), or Pronase (7.5 ug/ml). Two ml aliquots were taken at the times indicated in Fig. 1 and processed as previously described (15). The acid extracted histones were subjected to electrophoresis according to the procedure of Panyim and Chalkley (17), stained, scanned, and the amounts of the individual histone fractions were determined (15). At each

135

Vol. 74, No. 1, 1977

BIOCHEMICAL

z e

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

4

8

4

8 TIME

12

I6

20

12 I6 (M1N-I

20

60 20

Fig. 1. Proteolytic digestion of nuclei. Calf thymus nuclei (300 ug DNA/ml) were suspended in 0.3 M sucrose, 1 mM CaC12, 10 mM sodium borate, and 2 mM sodium citrate (pH 7.3) and treated at room temperature with (A) trypsin (5 ug/ml), (B) chymotrypsin (30 pg/ml), and (C) Pronase (7.5 ug/ml). Aliquots were taken at the indicated times and histones were extracted as previously described (15). The acid-soluble proteins were subjected to electrophoresis on polyacrylamide gels in 0.9 N acetic acid. The amount of each histone fraction was determined at each time point and is presented as the amount of protein relative to its amount at zero time. Controls indicated that within this time period there was no change in histone fractions of nuclei in the absence of these enzymes. (A), Histone Hl; (m >, HZA;( a), H2B; (0), H3; (0) H4.

time point the ratio present at zero time bated in the absence of these experiments.

of the amount of a given histone fraction to the amount The histone fractions of nuclei incuwas calculated. of these enzymes did not change within the time span

Results:

of protein

times Fig. sults

The amount during

1.

digestion

Although

in

terms

these of the

thymus

nuclei

amount

of histone

with

digestion

H3 also

in

each histone

each of the proteolytic

enzymes

are similar.

remaining

have very

Histone decreases

Hl is most quite

136

enzymes

different

of the various

rapidly.

fraction

at various

is presented

specificities,

the re-

histone

fractions

in calf

rapidly

digested,

and the

H2B is

digested

more

in

BIOCHEMICAL

Vol. 74, No. 1, 1977

slowly,

and H2A is most

enzymes,

being

almost

as susceptible These chymotrypsin been

will

H4 differs

to trypsin

are virtually

in a Waring

RESEARCH COMMUNICATIONS

for

the various

and chymotrypsin

as H2A and

as H3. identical

digestion

from disrupted

of chromatin

calf

Blendor

to those

thymus

(15).

nuclei

at 35 V, the

we obtained This

by a method

lowest

speed

by trypsin,

chromatin which

at which

had

involved the blender

operate. Our results

Discussion: from

calf

thymus fractions

enzymes

can be used

chromosomal residues

molecules.

break

bonds

leucine

in the

a mixture

of proteolytic

pancreas,

is

assumed

that

enzymes

are

probably

other

a positon

appears

portions

of the molecule

involve

are

is are

secreted

residues enzymes,

by all

the nucleosomes, Since

possible

that

buried,

perhaps

137

by all

of the nucleosome

of the nucleosomes.

within

H4 is

affected

the NH2-terminal in

the major

and

Pronase,

by the mammalian

attacked

digested

to

to be

molecules.

in an inaccessible

readily

of the

tyrosine tend

of these

these

in

has been shown

at numerous

position

surface

regions

acids

to those

to H3 and H2A. it

amino

with

and arginine

phenylalanine,

are readily

more slowly

to be buried

enzymes,

hand,

of their

fractions

lysine

of the histone

which

Hl and H3 are

intermediate

each of these

the NH2-terminal

in a superficial

on the

for

specificities

fractions

located

hand,

in

histones

isolated

Digestion

of histone

specific

similar

of attacking

attacked

histones

enzymes.

is

regions

enzymes

and chromatin

of the susceptibility

location

which

of the different

are

terms

These hydrophobic

COOH-terminal

located

nuclei

on the other

molecules

histone

which Since

to be clustered

capable

Because

the

Trypsin

(18-22).

clustered

chain.

in

Chymotrypsin,

residues

tions

to determine

in histone

both

by proteolytic

substructures. tend

that

are similar

to digestion

which

histone

indicate

tissue

histone

are

Histone

as resistant

and pronase

isolated

shearing

resistant.

to Pronase results

AND BIOPHYSICAL

along

the

we have three while

frac-

position. three Histone while

enzymes,

they

H2A, on the H2B may be in

differently

by

and COOH-terminal groove

of the DNA

Vol. 74, No. 1, 1977

helix,

while

BIOCHEMICAL

portions

AND BIOPHYSICAL

of the molecule

that

RESEARCH COMMUNICATIONS

are

susceptible

indicate

that

to Pronase

are

more exposed. The results

presented

in a configuration chromatin. that

in nuclei

Our results

certain

involve

(12)

they

utilized

full

speed

for

in a Waring

whether

or not

ditions,

such disruption,

isolating

it

could

chromatin. Omnimixer

Blendor

(our

the nucleosome

such as the ionic

is

structure

structure.

they

Although

the extent

the major

will

environment

even though

No11

versus

factor

be destroyed,

and pH, might

play

a different

order

method

of blending

by No11 et al.)

method),

(13)

be due to the particular

Perhaps

(utilized

isolated

and Johns

chromatin,

nucleosome

are arranged

in

of Woodhead

of isolating disrupt

histones

configuration

the conclusion

do not

in a Sorvall

speed

to their

methods

forces,

observed

paper

similar

support

conventional

shearing

--et al.

in this

--i.e.

the lowest

which

determines

although

other

an equally

con-

important

role. It

should

of digestion

be noted

that

by trypsin

H2A = H4) than

that

we observed

of the histones

observed

of calf

by No11 --et al.

thymus

(12)

for

of the rate

nuclei rat

(Hl > H3 > H2B >

liver

(H3 > H2A >

H2B = H4). Both (23)

these

sets

and of Sollner-Webb

data,

from visual

clude

that

H5 are

in

cleaved

and H2B, also their

of results

gels

chromatin

then

as a pair.

histones

that Hl,

in trypsin

at similar

that

H4 is degraded our results

far

differ

of Chatterjee

of Weintraub they

Weintraub

cleaved

--et al.

but

those

(23)

con-

Hl and

as a pair

followed

by H2A

from visual treated

inspection

of

duck erythrocyte rates

more slowly

than

and H2A, H2B Hl,

H5 and H3.

in nuclei.

mentioned

and Walker

no quantitative

and Van Lente

at similar

more slowly from

present

histones

or chymotrypsin

rates

and Van Lente

chromatin

(24)

H5 and H3 are digested

They state

those

those

erythroblast

Sollner-Webb

digested

with

gels

H3 and H4 are

also

ever,agree

Although

chick

and H4 are

Although

(24).

of their

trypsin-treated

conclude

from

--et al.

inspection

first,

differ

(25)

above, who also

they

do,

digested

howcalf

BIOCHEMICAL

Vol. 74, No. 1, 1977

thymus

chromatin

of digestion there

is

in different

with

trypsin

of the histone a difference organisms,

AND BIOPHYSICAL

and chymotrypsin. fractions

in these

in the way in which or perhaps,

A comparison five

histones

in different

RESEARCH COMMUNICATIONS

studies are

of the rates suggests

associated

with

that DNA

tissues.

References 1. 2. 3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16. 17.

18. 19. 20. 21.

22. 23. 24. 25.

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139