Specific binding of alu sequences by HeLa nuclear extracts

Vol. 117, No. 2, 1983 December 16, 1983

BIOCHEMICAL

SPECIFIC

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 378-384

BINDING OF ALU SEQUENCES BY HELA NUCLEAR EXTRACTS

Barbara

Dunn,

Michael

Seidman

and Michael

Bustin

Laboratory of Molecular Carcinogenesis National Cancer Institute National Institutes of Health Bethesda, Maryland 20205 Received

October

24,

1983

SJiMARY: Plasmid Blur 8 which contains the 300bp human Alu consensus sequence and plasmid pBR322 were digested with restriction enzymes and the fragments obtained end labelled with 32P-y-ATP. The end labelled fragments were incubated with HeLa nuclear extracts and the incubation mixtures passed through a nitrocellulose filter. The 300bp alu consensus sequence was preferentially retained on the filter. The HeLa nuclear extract did not preferentially bind any fragments generated from pBR322 and histones which bind nonspecifically all DNA fragments did not preferentially bind the alu sequence. We conclude that the HeLa nuclear extract contains components which specifically bind the human alu sequence.

The Alu

family

of short,

predominant

species

of primates

and rodents

human genome

(2-4).

of sequences

ranges

in

humans,

and the less,

ural the

ally

from

in vivo

or functional,

interaction the major

bind

conformation. a protein

Copyrighr All rights

unknown.

of the

site,

as genetic

that

specifically

the

copy the

III

with

do not

sequences

Neverthe-

the

whether struct-

in terms

of

proteins. bind

specific-

proteins

or to DNA with

of human HeLa cells Alu

in (5,6)

sequence,

specific

does contain

recognizes

378

group

mobility,

(7-10).

abundant

histones,

nuclei

genomes

this

In many instances

nucleus

$1.50

0 1983 by Academic Press, Inc. of reproduction in any form reserved.

highly

in question the

the

to some 300 base pairs

genome can be understood

proteins,

to single

which

rat

in the

is

of 3 - 6% of the

restriction

features

remains

Here we report

a minimum

by RNA polymerase

of DNA sequence,

component

0006-291X/83

by such

DNA sequence

preferentially

in

of this

chromosomal

to any type

which

a common Alu

role(s)

aspects of the

for

130 base pairs

DNA sequences

DNA present

accounting

to be transcribed

and functional

While

(l-3),

interspersed

repetitive

characterized

the precise

structural

of moderately

Named for

and is

ability

repeated,

sequences.

altered contain

BIOCHEMICAL

Vol. 117, No. 2, 1983

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

MATERIALS AND METHODS Nuclear proteins were isolated from purified HeLa nuclei essentially The nuclei were extracted with as described by Nordheim et al (12). buffer A (20 mM Tris-HCl, pH 7.5, 0.4 M NaCl, 1 mM dithiothreitol, 0.1 mM PMSF, 0.1% Aprotinin) and sonicated. After bringing the NaCl concentration to 1.5 M, the mixture was centrifuged and the DNA precipitated by making the supernatant 0.4% in polyethyleneimine. After centrifugation the supernatant was made 70% in (NH4)2SO4 and centrifuged. The pellet was suspended in buffer B (5 mM Tris, pH 8.0, 0.15 M NaCl, 0.1 ti PMSF, 5% glycerol), mixed 0.1 4 EDTA, 0.1 NM dithiothreitol, with sonicated E. coli DNA which was covalently bound to Sepharose 48, The dialysate was centrifuged and the and the mixture was dialysed. supernatant, containing protein at a concentration of approximately 30 stored at -200C. ug/ml,

Our search employed (a gift sequence

for

RESULTS AND DISCUSSION binding proteins in the HeLa nuclear

Alu

a nitrocellulose from

Dr.

inserted

Carl

filter Schmid)

at the

and BamHI to generate

five

1 Fig.

I

binding which

assay

contains

(11-13). the

Plasmid

of pBR322

(14)

distinct

DNA bands

(Fig.

1, lane

5

6

3

4

Blur

300 bp human Alu

BamHI site

2

extract,

was digested

7

consensus

with

2).

8

BglI

Following

8

Specific retention of Alu fragments by HeLa nuclear extracts detected by autoradiographic analysis of 3*P-labeled DNA fragments. Lane 1: Ethidium bromide stained polyacrylamide gel revealing the restriction pattern of pBR322 digested with BglI, 8amHI and EcoRI. Lane 2: Same, except plasmid Blur 8 was digested with BglI and BarnHI. Lane 3: Autoradiograph of a gel containing 32P-end labeled pBR322 fragments. Lane 4: Autoradiograph of the 32P-end labeled fragments derived from Blur 8. Lane 5: Fragments eluted from nitrocellulose filters after pBR322 fragments were incubated with histones. Lane 6: Same as 5 except Blur 8 fragments were used. Lane 7: HeLa nuclear extract was used for binding pBR322 fragments to nitrocellulose. Lane 8: Same as 7 except using Blur 8 fragments. The two experiments shown demonstrate the reproducibility of the observation. Asterisk denotes the 300 bp Alu fragment. Numbers on left denote the size of the fragments in base pairs.

379

Vol. 117, No. 2, 1983

end labeling

of both

kinase,

the

in the

nuclear

nuclear

restriction

fragments

ments derived

used

prepared

were

incubated

from

over

were

extract

extracts

passed

BIOCHEMICAL

the

nitrocellulose

on the

presence

of HeLa extract.

of the

input

tration

of nuclear

At this

off. the

input

difference Alu

sequence

HeLa nuclear

existence

of Alu

clusively

by analyzing

In the

to

filter.

amount

to the

results

filter

superimposed

the

sequence.

The

autoradiogram

on some nonspecific Alu Alu

This

fragment. constitutes

neither possibility

the

filters

conclusion

largest its

nor

the

specific 380

filters.

proteinase

by ethanol from

pattern

an intermediate

that

with

resulting

binding

more con-

nitrocellulose

at 37 o, followed

from the

eliminated

Alu

the

by incubation

90 minutes

1 shows the

is

of DNA,

can be demonstrated

eluted

Alu

any type

activity

4) to that

the

bind

binding

filters

Since

in the

specific

each

precipitation

two different to the

8) clearly

is a selective reinforced sized

The

K in the

of DNA applied (lane

is

presence

presence

for

from

only

to the

which

on the

up

leveled

is the

affinity

DNA retained

filter,

and 55% of

the

specific

the

dilu-

the concen-

binding

Since

10%

20% of the

on the

8 fragments

of components

restriction

wherein

about

8 fragments

point

about

at a 1:lOO

the

filters.

8, our

of the

control

Blur

quantity

extract

between

point

was

of DNA binding

of nuclear

of DNA retained

input

Comparison

to the

of the

proteins)

at which

frag-

8, and the mixture

correlation

in Blur only

of the

By contrast,

a direct

adhered

dilutions

dependence

1.5 ng total

72% of the

components

of end-labeled

counting

absence

pBR322 and Blur

of 2% SDS for

binding

amount

a strong

experiments.

that

Various

binding

with

DNA was recovered

(lane

-ATP and polynucleotide

specific

Radioactive

ug proteins)

not

components

Lane 8 in Fig.

a constant

between

extract

but also

presence

with

on the

concentration

Alu

HeLa cells.

and the

pBR322 fragments

sequence of the

(0.375

3*P-y

from

We observed extract

to a 3.5 dilution

for

indicated

(equivalent

DNA was bound.

to assay

RESEARCH COMMUNICATIONS

of pBR322 or Blur

filter

DNA was retained

input

with

filters.

on each

of the extract

digests

restriction

of DNA retained

tion

AND BIOPHYSICAL

Rlur

indicates

enrichment by the

of

internal

8 fragment.

smallest

fragment,

retention

on the

we have filter

is

Vol. 117, No, 2, 1983

due to binding gains

as a function

additional

histones

BIOCHEMICAL

meaning

replaced

binding. exhibit

a strong

basis

of sequence.

binding

patterns

filters

and those

to the

offer

the

nuclear

of the

filters

following were

and integrating ities

between

(broken

line

fragment Fig.

end-labeled extract

pBR322 for

The results marized constituted

filters

are conservative material the

Alu

which

binding

after

failed

peak to the

area

under

the

scan did the

gel.

adjacent 381

the

HeLa

1, 3, 5 and 7).

digest

The ratio peak

(due

are

sum-

fragment

eluted

comprised

54% of the

include

peaks

the Alu

material

not

enrichment

of the

of the

extract.

filters

with

and in the mixture

for

line

performed

affinity

1, lanes

by HeLa nuclear

to penetrate

from

experiment

restriction

accounts

the

(solid

shows a marked

the Alu

similar-

to a filter

histones

densitometry

by histones

since

applied

eluted

(Fig.

binding

values

with

28)

close

gels

of the corresponding

DNA fragment

Alu

to and eluted

of the

material

material,

relative

autoradiographs

no specific

In contrast,

of Alu

bound

or HeLa

revealed

total

were

applied

fragment

A similar

quantitative

histones,

histones

proportion

In the original

14.9% of the

total.

from the

from

to nitrocellulose

The scan reveals

(Fig.

fragment.

fragments

1.

following

of the

Alu

obtained

the

scan of the

HeLa extract

any particular

in Table

filters

the

pair

either

of each

relative

electrophoretic

4 and 6).

fragment

peak.

they

on the

no enrichment

due to interaction

In contrast,

300 base

each

in that

the

applied

with

with

proportion

to a filter

with

of each

under

DNA filter

binding

were

by scanning

2A) and the

interaction

in the

area

wherein

for

made between

1, lanes

interaction

relative

Fig.

bound

2A).

after

the

Fig.

obtained

the

is

we observe

quantity

control

mediator

incubation

(compare

to Alu

does not discriminate

which

following

fragments

extract

DNA which

such filters,

binding

of nonspecific

8 fragments

which,

enhanced

protein

When a comparison

Measurements from

for

RESEARCH COMMUNICATIONS

to an external

as the

a paradigm

affinity

from

other

This

when compared

of Blur

to and eluted

of size.

HeLa extract

Histones

AND BIOPHYSICAL

from 11.0% of

material

eluted

Furthermore, the

undigested

of the to the

these

area

under

559 base

pair

Vol. 117, No. 2, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1.25 -

47

67

07

107

115

MIGRATION

Fig.

fragment;

2

bound

with

1.41

in the

fragments.

presence

following originally

RNAse,

followed retained

undigested

a dot

bound

and

to filters

fragments

nitrocellulose

the filters

fainter

of eluted via

of

reduced

Accordingly,

in

the

original

and 6.47

Blur

382

is

HeLa stable

in

proteases.

However,

digestion

radioactive

counts

in plasmid

to l/3

of those

radioactive

8 and PBR fragments

a predigested

unbound

in the

in HeLa extract

a variety

by trypsin,

extract.

1.55

component(s)

DNAse

by the

2) is

Fig.

histone

electrophoresis bound

in

The binding

of

pepsin,

fragments for

with

marked

fragments,

the

Densitometric scans of the autoradiograms obtained from gels in which 32P-end labeled restriction fraoments derived from Blur 8 were electrophoresed. A) Broken line; original end-labeled fragments (see lane 4, Fig. 1). Solid line, fragments eluted from the filter after binding by histones (Fig. 1, lane 6). 8) Fragments eluted from the nitrocellulose filter after binding by HeLa nuclear extract. The Alu fragment is marked with a solid triangle. The adjacent 559 base-pair fragment, with a solid circle.

HeLa extract

bands

observed appeared

which than

were

when

BIOCHEMICAL

Vol. 117, No. 2, 1983

The 300 filters

Table 1 cellulose

AND BIOPHYSICAL

bp Alu fragment by HeLa nuclear Area

RESEARCH COMMUNICATIONS

is preferentially extracts.

(% of

retained

Total) Bound

Plasmid Blur

Fragment 8

(bp)

on nitro-

Original

by: HeLa

Histones

Extract

2319

35.3

49.8

5.4

1248

21.0

23.6

13.3

650

14.6

6.4

11.9

559

9.5

8.3

12.9

300*

14.9

11.0

54.1

234

1.2

>0.6

>0.6

The autoradiograms of polyacrylamide gels containing separated 32P-end labeled fragments were analysed with a Beckman DU-8 gel scanner. The values given are the % that each peak constitutes of the total area of all peaks. "Original" refers to the total mixture applied to nitrocellulose "Bound by histones" or "HeLa extract" indicates fragments filters. recovered from the filter. For example, the 300 bp Alu fragment constitutes 14.9% of the total fragments. It constitutes 11% of the 32P fragments bound to nitrocellulose after addition of histones and 54% of the fragments bound to the nitrocellulose after addition of HeLa nuclear extract.

the

filter

binding

shown). the

We conclude binding

entire

on

to

any

preferentially DNA genes a distinct

a protein but

of

such

type

of

DNA

(15),

exclusively

Among

which

protein(s)

Alu

sequence

with

one

or

more

may can initiation

be

ranging

from

through

those

DNA. well

transcribed factors

least

that

those

cells

that

which

to

clustered,

Drosophila

Insight

into

from by

the RNA

(25-29).

383

contain

are

an

capable

of

(12,13,16,17) regions

be

localized

for

a highly

the

exact

sequences

add

to

that

III the

this

list

repetitive nature

knowledge

However

bound

repeated

We now

polymerase

or

selectively

highly

(16,24).

specificity

come

of

recognize

discrete shown

the

not

a portion

DNA conformations

displays of

revealed

regions

in

at

(data

protein.

have

are

DNA

segment

pair

the

extract

that

including

fraction

satellite

undigested

is

specific

(21-23).

component

Alu-binding

protein,

(13,18-20),

interspersed

extract proteins

segments

the

by

observations

HeLa

binding

protein

comprising

mediated these

in

or

specific within

from

DNA

spectrum

binding

been

factor(s)

Studies

by

had

of the

in

this 300

base-

conjunction

polymerase

III

Vol. 117, No. 2, 1983

BIOCHEMICAL

5s gene transcription

factor

the

range

with totally

1. 2. ii: 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. k 17. 18. 19. 20. 21. 22. 2 2 27. 28. 29. 30.

by itself

of 5s on nitrocellulose

does not bind

to DNA.

a component independent

of the

AND BIOPHYSICAL

filters

The possible polymerase

function

does not

remains

(30)

identity

III

complex

RESEARCH COMMUNICATIONS

cause

retention

and plymerase of the

protein(s)

or with

of DNA in

III

by itself in question

some protein

of

to be elucidated.

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