BIOCHEMICAL

Vol. 66, No. 2, 1975

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

FRACTIONATION OF CHICK OVIDUCT CHROMATIN:

IV.

ASSOCIATION

OF PROTEIN KINASE WITH TRANSCRIPTIONALLY

ACTIVE

CHROMATIN

R.K. J.F.

Keller,'

Krall,*

Department

of

S.H.

T. Chandra

Cell

Biology,

July

and B.W. O'Malley

Baylor

Houston,

Received

Socher,

Texas

College

of

Medicine

77025

18,1975 SUMMARY

Chromatin isolated from the oviducts of estrogen treated chicks was fractionated into template active and template inactive components by sucrose gradient centrifugation and by ECTHAM cellulose chromatography. The fractionated chromatin was assayed for protein kinase activity. Chromatin protein kinase activity is enriched in the transcriptionally active fraction of chromatin. INTRODUCTION Considerable nonhistone

demonstrated

bind

to the

More

the

the

nuclear

NHPP are

DNA or origin.

recently,

into

(NHPP)

that

in the

synthesis

has been accumulated

phosphoproteins

have

proteins

evidence

protein

with

the

this

these

kinases

of

if

or template

we have

used

oviduct.

nuclear

protein

inactive sucrose

1

Present College

address: Department of Medicine, Tampa,

of Biochemistry, Florida 33620.

2

Present tration

address: Hospital,

Endocrinology California

of phospho-

of chromatin

NHPP increase to gain we have

kinase

region gradient

and

order

In the

out

specific, level

phosphorylation,

carry

Molecular Sepulveda,

that

and in

chick

(2)

region

reported

findings

of nonhistone the

active

implicating

Teng --et al.

tissue

an increased

have

aspects

active study,

(4)

of

in determining

template

reported

years

(1).

heterogeneous,

or transcriptionally

In light

regulatory

interested

highly

Shea and Kleinsmith

--in vitro.

were

in gene regulation

Frenster

"diffuse"

in recent

present

of oviduct

Laboratory, 91343.

insight studied

is

we

associated

chromatin.

centrifugation

University

RNA

study,

activity

(3).

To

and ECTHAM

of South Veterans

Florida,

Adminis-

VoL66,No.

cellulose

BIOCHEMICAL

chromatography

inactive ably

2, 1975

regions.

enriched

to fractionate

We find

in the

AND BIOPHYSICAL

that

fraction

chromatin

chromatin

into

protein

of chromatin

which

RESEARCH COMMUNICATIONS

template

kinase

active

activity

is

is more active

and consider-

transcrip-

tionally. MATERIALS AND METHODS Preparation and Fractionation of Chromatin: Chromatin was prepared from purified nuclei isolated from oviducts of diethylstilbestrol (DES) treated chicks according to the method of Spelsberg and Hnilica (5). Chromatin was sheared in a French pressure cell and the active and inactive regions separated by centrifugation on linear sucrose gradients as described previously (6). Chromatin was also fractionated on ECTHAM-cellulose by a modification (7) of the method of Reeck --et al. (8). Chromatin Template Activity: E. coli RNA polymerase was prepared according to Bur ess (9). Each terngate capacity assay of 0.25 ml contained: Tris-HCl (pH 7.9 7 , 10 umoles; MgCl 2.5 umoles; B-mercaptoethanol, 0.5 pmoles; KCl, 37.5 pmoles; 0.075 nmoles eaci'of ATP, UTP, GTP, and CTP; 0.0015 $Zi of [3~]-~~~ (Schwarz-Mann, 14.5 Ci/mmole) or 0.07 uCi of [14c]-~~p (Schwarz-Mann, 47 mCi/mmole); 20 ng of polymerase and 5 ng of DNA as chromatin or purified chick DNA. Assays were incubated for 10 minutes at 37°C. Reactions were terminated with an excess of 5% CClsCOOH. After 30 minutes, the acid precipitates were filtered onto Whatman GF/C glass fiber filters and washed with 2% CCl,COOH. Filtered precipitates were digested with NCS tissue solubilizer (Amersham-Searle Corp.) at 50°C for 60 minutes and counted in a toluenespectrofluor (Amersham-Searle Corp.). Samples were counted in a Beckman LS-250 liquid scintillation counter with fixed window isoset modules for 3H and 14C. Counts registered in the 3H window were corrected for 14C spillover. Template activity of chromatin or DNA was calculated from 3H-GTP incorporation alone. Protein Kinase Assay: Protein kinase was assayed in a total volume of 0.1 ml which contained 5.0 pmoles sodium phosphate buffer, 1 mole MgC12, 1 nmole [Y-~~P]ATP (25 cpm per fmole) and 0.3 mg casein as exogenous substrate. Reactions were carried out for 6 minutes at 37"C, and stopped by the addition The acid precipitates were filtered onto 0.45 of 1.5 ml of 5% CClsCOOH. nitrocellulose filters (Millipore Corp.) and washed with 5% CClsCOOH. Filters were dried and counted in toluene-spectrofluor.

RESULTS Centrifugation previously

described

dient

was divided

ratio

of

Table

I were

lighter pellet

of sheared (6) yielded into

incorporation

fraction

four

the

chromatin profile

fractions

Template fractions,

(Table

I).

activity while

the

RNA polymerase

454

on linear

shown in

and assayed

of ATP to UTP into

obtained.

sedimenting

oviduct

for

Figure

was consistently

assays

1.

template

RNA calculated,

lowest

sucrose

activity carried

the

gradients When the

activity data

associated was found out

using

as gra-

and

shown in with in the [14C]-ATP

the

BIOCHEMICAL

Vol. 66, No. 2, 1975

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

25 -

-2

II TOP

Figure

1.

base

I

IO

I5

20

FRACTION

that,

composition

RNA transcribed that

,

5

BOTTOM

Distribution of protein kinase in sucrose gradient fractionated oviduct chromatin. Chromatin was fractionated and protein kinase activity was determined as described in Methods and Table 1. (----) absorbance at 260 nm, (---) protein kinase activity.

and C3HJ-GTP revealed the

1

of

from

transcribed

the

the

from

in addition RNA products

pellet

both

to differences varied

was markedly

the

template

in template

across enriched

active

the

capacity,

gradient.

The

in G-C content

fraction

over

and unfractionated

chromatin. In a separate described Figure

above 1 shows

edge of kinase

the

faster

were

the

the

bound

stimulated

were

main

of

peak

absorbance

run

peak. enzyme

containing

The protein than

remained

that

chromatin

fractions

represented

were

chromatin.

and the

major

activity

gradients

experiment,

free

kinase nuclear

to chromatin. by the

addition

was sheared assayed

kinase To rule

released

salt-extracted activity protein

for

and centrifuged protein

activity out from

occurred

the the

nuclear in chromatin

kinase,

Interestingly, of low6

455

kinase

none of M CAMP in the

that

chromatin,

that the

this

and sheared considerably the

enzyme

fractions

protein

trailing

duplicate

sedimented

indicating

activity.

on the

possibility

kinase

as

kinase

tested assay.

VoL66,No.

2, 1975

BIOCHEMICAL

TABLE 1.

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Template activity of sucrose gradient fractionated chick oviduct chromatin.

CHROMATIN

TEMPLATE ACTIVITY (UNFRACTIONATED

FRACTION

CHROMATIN= 1.0)

Sheared Chromatin

1 .oo

1.68

Purified Chick

4.00

1.72

DNA

Gradient Fractions

2-4

1.53

1.69

Gradient Fractions

5-7

1.33

1.69

Gradient Fractions

9-10

0.86

1.59

Gradient Fractions

11-15

0.57

1.35

0.34

1.18

Pellet Fraction

Chromatin (0.7 mg DNA/ml) was sheared in French pressure cell at 7000 psi. Insoluble material was removed by centrifugation at 10,000 x g for 20 minutes. The solubilized chromatin was layered onto a 13 ml gradient of 5 to 30% sucrose and spun at 154,000 x g for 6 hours. After centrifugation the gradients were fractionated and monitored at 260 nm. The fractions were then assayed for template capacity as described in Materials and Methods.

Chromatin Simpson

and co-workers

chromatin properties Using

was also

into

early

expected

a modification

chromatin

fractionated (8)

(I)

have

shown that

and late

of active of

by ECTHAM-cellulose

this

on ECTHAM-cellulose

(II)

eluting

and inactive method

we have

and assayed

456

this

chromatography.

technique fractions

chromatin, fractionated Fractions

separated which

exhibited

respectively. chick

oviduct

I and II for protein

Vol. 66, No. 2, 1975

kinase

activity.

scriptionally more the

BIOCHEMICAL

protein

portion

kinase

possibility

the

that

material

IIa,

(Fraction little

kinase

of

activity

have been extracted used,

II shows that

Table active

per

between

Fractions

activity,

fraction

indicating

that

during

chromatography.

from

both

sucrose

gradient

centrifugation

the

that

transcriptionally

active

Table

2.

protein

bound

II.

To eliminate

salt

kinase the

gradient

relatively

was not results

extracted obtained

and ECTHAM chromatography

of

activity

is

associated

chromatin.

Protein kinase activity chromatin and Fractions ECTHAM-cellulose.

in unfractionated

I and 11 from

Protein Kinase Activity pmole 3zP1 + Casein

- Casein

Unfractionated Chromatin

6.15

2 0.01

0.95

+ 0.01

Fraction

I

8.21

2 0.01

1.25

+ 0.01

Fraction

IIa 2

0.67 ~0.01

Fraction

II

0.85 + 0.01

0.11

pg chromatin

- DNA

per

II might

and assayed

to contain

Hence,

tranlo-fold

to Fraction

due to the

protein

kinase

region

the

approximately

Fraction

was found

chromatin

conclusion

DNA than

I and II

the

the

representing

possessed

originally

from

support

I,

RESEARCH COMMUNICATIONS

I and II was collected

between This

genome,

ug of

kinase

2).

Fraction

the

protein

eluting Table

AND BIOPHYSICAL

---

3

f. 0.01

1

incorporated

2

pooled to the

3

calculated using the chromatin DNA content in fraction II and assuming that all kinase activity in Fraction IIa is eluted from Fraction II

column fractions eluted beginning of chromatin

457

from 100 mN NaCl Fraction II

with

Vol. 66, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

DISCUSSION In the cellulose

present

chromatography

and inactive

regions

procedure, in the

of "active"

variation

in base 1) is

accessible active

sucrose

were

employed

previously

more

expected

both

of oviduct

we have lighter,

(Table

work

the

to --E. coli

identical

to those

transcribed

transcribed

mouse,

guinea

a minor pig

demonstrated

logical

properties

In the casein

was employed occurred

that

endogenous nature

suggest were

if

that

with

nonhistones. nuclear

acidic

proteins,

assay

was omitted were

endogenous

protein

of the

Second,

after

kinase,

such as casein

the

substrate

assay

bound

458

of in

elsewhere

physical

and bio-

respectively. chromatin

fractions,

phosphorylation

(Table

2),

indicating

It was of interest

to ascertain

Several

of evidence

First,

lines if

nuclear

radioactivity

and partial specificity

and phosvitin,

Satellites

significant

of AT32P and the

extraction

satellite

various

substrates.

protein

G-C

be a heavy

both

nonhistones.

presence

90-95%

protein

from

relatively

chromatin,

on the

phosphorylated.

predominantly

in the

(12),

oviduct

kinase

were

phosphorylated

fractionated

and inactive

However,

that

necessarily

to be reported

of

performed

and in-

shown to be contained

exepcted

as a substrate.

these

genome.

ECTHAM fractionation

active

active

the

could

been

DNA sequences

not

of

I and II possess

casein

they

chick

the

the

however,

are

Fractions

proteins

of

of

properties

fractions

the

in the

The origin fraction

sediments

of

these

that

--in vitro

pellet

former

which

to realize,

have previously of

that

protein

also

the

component

from

identical

polymerase

in the

Studies

(7,ll)

not

to the

significance

synthesized

in vivo.

and crabs

heterochromatin.

the

active

has additional

do indicate

were

template

respect

peak

Although

differences

coli

the

and ECTHAM-

chromatin

active

It is important

to r.

is

sheared

polymerase

accessible

DNA in which

shown that

(6).

fractions.

sequences

#ith

of RNA's

DNA sites

rich

chromatin.

chromatin

uncertain,

centrifugation

to separate

transcriptionally

composition

chromatin

gradient

were

oviduct

nuclei

proteins

were

was associated purification

studies considerably

of

revealed better

the that sub-

BIOCHEMICAL

Vol. 66, No. 2, 1975

strates

than

protein

kinases

cytosol;

this

kinase

basic

which

(14).

We therefore

association

interest

phorylation

in

light

as histone

phosphorylate

to be the

for of of

that the

this the

protein

activity

the

kinase

role

the of

Third,

present

oviduct

of

with

(13).

generally

phosphorylation

proposed

in gene regulation

were

case with the

RESEARCH COMMUNICATIONS

and protamine

histones

feel

responsible

preferential of

such

has been shown

was primarily

is

proteins,

AND BIOPHYSICAL

histone

activity

we observed

nonhistones.

template

nonhistone

in the

The

active protein

fraction phos-

of eukaryotes.

ACKNOWLEDGEMENTS This the

National

work

was supported Institutesof

of an NIH Postdoctoral

by grants Health.

Fellowship

HD 8188,

One of

HO 7857,

us (R.K.K.)

and HD 7495 from

was the

recipient

HD 02118-01. REFERENCES

1. :: 4. 5. 6. 7. 8.

Allfrey, V.G., Johnson, E.M., Karn, J., and Vidali, G. (1973) Miami Winter Symposia, Vol. 5, p. 217. Teng, C.S., Teng, C.T., and Allfrey, V.G. (1971) J. Biol. Chem. -246:3597. Frenster, J.H. (1956) Nature 206:680. L.J.-(1973) Biochem. Biophys. Res. Commun. Shea, M., and Kleinsmith, 50:473. Gelsberg, T.C., and Hnilica, L.S. (1971) Biochim. Biophys. Acta 228:202. Socher, S.H., Krall, F.J., Jaffe, R., and O'Mal'ley, B.W. (1975) submitted. B.W. (1975) submitted. Stratling, W., Van, N.T., and O'Malley, Reeck, G.R., Simpson, R.T., and Sober, H.A. (1972) Proc. Nat. Acad. Sci. U.S.A. 69:2317. Burgess, R.RF(1969) J. Biol. Chem. 244:6160. Krall, J.F., Socher, S.H., Van, N.T.,nd O'Malley, B.W. (1975) Biochem. J. (in press). Stratling, W., and O'Malley, B.W. (1975) submitted. Parker, M., Sheehan, D., and O'Malley, B.W. (1975) in preparation. Keller, R-K., Chandra, T., and O'Malley, B.W. (1975) in preparation. Keller, R.K., Schrader, W.T., and O'Malley, B.W. (1975) J. Biol. Chem. (in press).

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