Tissue-specific expression of human terminal deoxynucleotidyl transferase is regulated at the transcriptional level

Vol. 164, No. 2, 1969 October

BIOCHEMICALANDBIOPHYSICALRESEARCH

COMMUNICATIONS

31. 1989

Pages

750-757

TISSUE-SPECIFIC EXPRESSION OF HUMAN TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE IS REGULATED AT THE TRANSCRIPTIONAL LEVEL Theoni

Trangas

and Mary

Sue Coleman

Department of Biochemistry and the Lucille P. Markey Cancer Center University of Kentucky Medical Center Lexington, Kentucky 40536-0084 Received

September

5,

1989

Summary: Transcriptional regulation of expression of the terminal deoxynucleotidyl transferase gene in normal thymus and in differentiation arrested cells was demonstrated by analyzing steady-state levels of TdT RNA as well as the relative transcription rate of the gene. Terminal transferase transcripts were detected only in those cells and tissues that contained antigen and enzyme activity. The relative rates of transcription correlated with levels of mRNA as well as with levels of the protein. These data suggest that expression of this gene in normal and leukemic cells is modulated at the level of transcription.

lerminal directed

deoxyribonucleotidyltransferase DNA polymerase

prelymphocytes

of

that

both

cells

counterparts

of

lymphoid

the

of

this

normal cells

of

and in all

the

"lymphoid

terminal

in those

has

mutations(4.5). been

positively

signal

joints

are

fused),

but

details of the are incorporated

not

at

expression

information insertions(5.b).

relating

Inc. reserved.

times

cells

also

exhibit

higher

than

in the

marker

the joints

to

of the

in the

activity

of

leukemic

of

would several potential

transferase

receptor

generation

of pre--8

nucleotide

corresponding

(recombined

750

terminal

in murine

incidence

$1.50

0 1989 by Academic Press, of reproduction in any form

in other The malignant

and T-cell

enzyme

on DNA where

molecular events which into DNA are unknown,

nucleotide

Copyright All rights

transferase

coding

undetectable

tissues(l,2). 1000

in

lineage(3).

this with

is

a non-template

levels

a well-established

implicated

preliminary

0006-291X039

is

imnunoglobulin

position

in high

but

10 to

undergoing

correlated (the

levels

progenitor"

Terminal

is

transferase-containing

at

and temporal

cells

diversification

origin,

transferase

The tissue-specific gene

present

non-hemopoietic

enzyme,

Terminal

cells.

normally

B and T cell

hemopoietic activity

is

(EC 2.7.7.31)

somatic cells

insertion

recognition if

noncoded

recent

studies

mechanisms

of

at signals

junctions)(5).

occur

has

While

the

nucleotides have noncoded

provided

Vol.

BIOCHEMICAL

164, No. 2, 1989

The mechanisms enzyme hampered

are

by lack

modulated. leukemic thymus

cells

We have cells

therefore

arrested

and normal transferase

indicate

that

at

peripheral activity

transcription

employ

to

interest.

of a model

terminal tissue

which

of considerable

AND BIOPHYSICAL

system

in which

employed various

the

stages

and antigen. terminal

activity

mechanistic the

enzyme

an approach

lymphocytes, of the

regulate

However

RESEARCH COMMUNICATIONS

can

based

of this

studies

been

be experimentally

on the

of differentiation which

have use of

as well

vary

in their

The data

presented

transferase

gene

level

as of

in this is

human

paper

highly

specific. HATERIAL

AND METHODS

Cell lines were maintained in RPM1 1640 medium supplemented cell cultures. with 10% of either horse, fetal calf or Nu-serum V (Collaborative Research. lhe human cells represented various differentiation-arrested Bedford, MA). Normal mononuclear cells were obtained early I3 and early T cells (Table I). by leukapheresis. Tissues were obtained through surgical pathology services. Enzyme Assay and Antiqen Imunoassay. Terminal transferase activity was measured in a standard reaction mixture(7) containing dA5G as initiator and 3H dGTP as monomer. Cell extracts were prepared as previously described A Dynatech Imulon II 96 well microtiter plate (Fisher Scientific) was (7). coated overnight with a diluted (11500) goat anti-terminal transferase antiserum (PL Eiochemicals). The plate was overcoated with a 1% BSA solution, incubated at room temperature for 30 min and then washed with water. To each well, 50~1 of cell extract (2.5~105 cells) was added. The plate was incubated at room temperature for 90 min and washed with water. Diluted (1:2500) rabbit anti-terminal transferase antiserum (50~1) was added to each well(8) and incubated for 90 min at room temperature. The wells were then washed as before and 50~1 of a peroxidase labeled goat anti--rabbit antibody (diluted 1:2500) (Kirkegaard & Perry Laboratories, Inc.) was added. The plate was incubated for 60 min at room temperature and washed 5 times with water. o--Phenylene-diamine dihydrochloride solution (5mgIml) was added. The reaction was stopped with 1N sulfuric acid and the optical Antigen levels were calculated on the basis density was measured at 490nm. of a standard curve. RNA Isolation and Analvsis. Total RNA was isolated by homogenization of approximately 109 cells, or lgm of tissue, in 20 mls of extraction buffer (3mg/ml heparin. 1OmM vanadyl ribonucleoside complexes, 1OmM sodium acetate, pH 5.2, 6M urea, 3M LiC12, 0.1% SDS) and incubated overnight at 4'C. Precipitated RNA was extracted several times with phenol/chloroform. Polyadenylated RNA was isolated by oligo dT cellulose chromatography For dot blots, RNA was immobilized on nitrocellulose in 7.5X SSC(lX (9). SSC -0.15M sodium chloride, 0.15M sodium citrate, pH 7) containing 4.6H formaldehyde. For northern blots, RNA was heated in 50% formamide/2.2M formaldehyde in 1X MOPS buffer for 5 min. at 65°C. Samples were cooled to room temperature and electrophoresed in 2.2 M formaldehyde/l.2% agarose gels with 1X MOPS running buffer. RNA was transferred to nitrocellulose in 20X ssc . lhe membranes were baked at 80°C and prehybridized for 1 h at 42°C in 50% formamide, 5X Oenhardt's solution, 0.5% SOS, 5X SSC, and 100 ug/ml salmon sperm DNA. lhe cDNA probes used were a Barn HI/Hint II 1128 bp fragment of terminal transferase(l0) or actin cDNA(11) (kindly provided by riming. The specific L. Kedes. Stanford University) labeled by random The blot was activities of both probes were approximately 1X10 Ii cpm/pg. hybridized for 12-18 h at 42"C, washed twice in 2X SSC/O.l% SDS, twice in 0.1X SSC/O.l% SDS at room temperature, followed by a wash in 0.1X SSC/O.l% SDS for 1 h at 65°C. Kodak X-Omat XAR-5 film was exposed for various lengths of time at -80°C using an intensifying screen. 751

Vol. 164, No. 2, 1989

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

Run-on Transcription. Nuclei were isolated from 2 X 108 cells by the method of Oingam(l2). The run on transcription reaction mixture contained 5mM Tris hydrochloride, pH 8.0; 2.5mM MgC12; 150mM KCl; 0.25mM each of ATP, GTP, CTP and 500 rtCi 32P UTP and was incubated for 30 min at 30°C. The reaction was terminated by the addition of 10 units of RQl ONase (37°C for 10 min.). RNA was isolated using a standard procedure(l3). The RNA pellet was dissolved in hybridization buffer (O.OlM Tris hydrochloride, pH 7.4, O.OlM EDTA, 0.3m NaCl, 0.2mg/ml Ficoll, 0.2mg/ml polyvinylpyrrolidone. 0.2mg/ml bovine serum albumin, lOOunlts/ml RNasin, 0.25mg/ml E. coli tRNA). The labeled RNA was passed through a slot blot apparatus on to prehybridized nitrocellulose strips containing 5ug of fixed, denatured terminal transferase cDNA(lO), actin cDNA and pBR322. After 48 hours of hybridization at 65°C the filters were washed in 2X SSC for 10 min. at room temperature, 50 min. at 65°C. and treated with lOmg/ml RNAse for 30 min. at 37°C. Finally, the filters were washed with 2X SSC for 1 h at 37°C. dried and autoradiographed. RESULTS AND DISCUSSION Terminal

transferase

Thymus,

a tissue

thyroid,

spleen,

antigen,

were

terminal

transferase

in

normal

mRNA levels

colon,

of

no terminal

(Fig.

Since

RNA dot leukemic

blot

low

analysis

the

normal

serially

by dot

and steady-state

blot

in the which

I).

analysis other

were

of

tissues terminal

selected

with

activity.

RNA samples the

between

terminal

The

expressed

Cells

total

quantitate

was found

that

studied.

transferase

diluted to

mRNA abundance

include

(Table

of terminal

or

of

indicated

tissues

tissue to

cells

was used

A correlation

activity

all

detected

analysis

levels of

and thymus mRNA.

enzymatic (Table

and

cells

transferase

only

leukemic

intermediate

for

and

activity

presence

transferase

cDNA probe

used

and antigen

no enzyme

whether

mRNA was detected

was the

mRNA, we expanded

with

determine

was easily

and tissues

activity

terminal

RNA were

transferase

thymus

differentiation-arrested high,

to

to an actin

total

lines

tissues

reflected

in thymus

RNA, while transferase

analysis

cell

transferase

and kidney,

activity

transcript 1).

for

leukemic

terminal

Hybridization

amounts

specific

high liver

selected

cells.

equivalent

with

in

level

of

relative

from terminal

differences

transferase

in

mRNA in cells

II).

Selected further

undifferentiated

analyzed

leukemic

by northern

cells

blotting.

as well

As shown

predominate approximately the specific

RNA species hybridizing to the terminal 16s indicating a transcript size of transcript was estimated by staining

used

analysis

in the

transcript region

length of

migrated

the

is

human

slightly

to

a transcript

the

NALM-6

with

cell

consistent terminal

faster of line

ethidium

3500

bromide

(Fig.

the

prediction

with transferase

28).

gene(lO).

28s was also

detected

bases.

When poly

A containing

in the

northern 152

RNAs were

2A,

the

transferase cDNA was The size of 2100 bases. the polyacrylamide gel

than

and used

as thymus

in Figure

analysis,

The estimated based

on the

A faint which

coding

band that would

correspond

RNA was isolated only

the

2100b

from RNA

BIOCHEMICALAND

Vol. 164, No. 2, 1989

BIOPHYSICAL

RESEARCH COMMUNICATIONS

KIDNEY LIVER COLON SPLEEN THYROID THYMUS

FIGURE 1: Dot blot analysis of total RNA isolated from normal tissues. Total RNA (long), isolated as described in Methods, was inmobilized on nitrocellulose filters using a blotting apparatus. The filters were hybridized to 32P terminal transferase cDNA (5XlU6cpm/ml hybridization buffer). The filter was stripped in boiling water and re-hybridized to actin cDNA (3XlO%pm/ml).

species

was detected

represents Terminal

the

exerted

measured

mature

transferase

In order is

(not

to at

the

gene

relative

assay allows presence of

transcripts 32P-UTP.

immobilized the

nitrocellulose

molecules

on the

of

is

this

in human

probably

cells transferase

in normal

gene transcription

mononuclear

species

transferase.

of terminal

transcription

and

expression

leukemic

by nuclear

cells

and

leukemic

cells, run-on

cells.

initiated in vivo to be elongated in vitro Labeled RNAs were isolated and hybridized to transferase,

nitrocellulose gene

that

terminal

control

thymus.

cDNAs (terminal to

of

levels of

for

transcription

whether

some stage

nuclei

indicating

mRNA coding

examine

in isolated

denatured

shown),

pBR322

filters.

an indication of

interest

The amount of

at

or actin)

the 753

the

of

activity time

of

of cell

that

we assays This

in the excess

were

radioactivity

bound

RNA polymerase lysis

(Figure

3).

to

Vol.

164,

No.

2, 1989

BIOCHEMICALAND

Table

Cell

Line

I.

Differentiation-Arrested

Characteristic

JH

HPCA-1 OKT 10, OKT 9, OKT 10, OKT 10, OKT 9, Leu OKT 10, OKT-1, l/12/13

NALH-6 697 CEM MOLT-4 KTl HPB-ALL KE-37 K562

BIOPHYSICAL

Leukemic

Surface

Antiqens

J5, 84, IaL243, CALLA J5, CALLA OKT 3. OKT 6 OKT 10. Leu 1, 5, Leu 9 OKT 3. OKT 6 Leu 1

order

to

compare

and to

insure

that

4,

of

newly

the

using

a

range

dilution

of

actin

concentrations

II.

(data

not

Quantitation In

RNAb (IN) 10 5 2.5 1.25 0.63 0.31

Activitye Antigenf

the

shown)

two

In

each

remained that

transferase lines and

(1986),

were

carried

sample

constant

sufficient

mRNA. tissuesa

transferase of hybridizing

assays

cONAs.

indicating

of terminal selected cell

F.,

A.H.,

and terminal

run-on

transferase)

9,

& Ragab,

cDNA was capable

RNA, nuclear

a a a

Res.

h Hecht,

of actin

actin

of

(actinkerminal

series

Table

inmnobllized

synthesized

of transcription

rates

C

T cell

Leukemia C.

a a b a a

T cell T cell

Intermediate Late T cell Hyeloid

transcription the

Lines

Reference

Stem cell Early B cell Early B cell Early T cell Intermediate Uncommitted

CALLA

Leu

Cell

COMMUNICATIONS

TYDe

aDrexler, H.G., 6. Gaedicke, J. Minowada. (1985). 209-229. bFindley, H.W., Cooper, N.D., Kim, T.H., Alvarado, (1982). Blood, 60. 13051309. cSmith, S.D.. Morgan, R., Link, H.P., McFall. P. Blood 67, 650-656.

In

RESEARCH

activity

the

across

all out ratio

the

cDNA was

and

antigen

CEH

K562

HPB-ALL

KE-37

JM

MOLT-4

697

NALM-6

KTl

CHL Spleenc

Thymus

n.dd n.d n.d n.d n.d n.d

n.d n.d n.d n.d n.d n.d

n.d n.d n.d n.d n.d n.d

n.d n.d n.d n.d n.d n.d

5 2.5 n.d n.d n.d n.d

17

:: 12 B 4 n.d

15 B 4 n.d n.d n.d

20 10 5 2.5 n.d n.d

50

: n.d n.d n.d

38 20 9 5 n.d n.d





:

108 18

107 65

1;:

95 62

80 50

1;:

aThe quantitation of mRNA levels is expressed as the peak area of each autoradiogram scan. bAmounts of total cellular RNA indicated were applied to nitrocellulose membranes and subsequently hybridized with 32P-labeled terminal transferase cDNA probe (5X106cpm/ml). Hybridization to an actin cDNA (lX105cpm/ml) indicated that equivalent amounts of RNA were present in each of the matched sets (data not shown). The filters were washed and exposed radiographic film. The autoradiogram was scanned using a laser densitometer. CSpleen of a patient with chronic myelogenous leukemia, infiltrated with terminal transferase positive leukocytes. dn.d. is not detectable. cone unit is defined as lnmol dGTP polymerized per h. fAntigen was measured in a microtiter plate assay, and is reported as ng/lO* cells.

754

:: 9 4 n.d

to

Vol. 164, No. 2, 1989

BIOCHEMICALAND

BIOPHYSICALRESEARCH

COMMUNICATIONS

B FIGURE 2: Northern blot analysis of total RNA isolated from thymus, spleen infiltrated with leukemic cells or leukemic cell lines. Total RNA samples were fractionated by formaldehyde agarose gel electrophoresis and transferred to nitrocellulose membranes. A. The membranes were hybridized to 32P labeled terminal transferase cDNA, washed and autoradiographed as described in Methods. 8. Migration and quantitation of ribosornal RNA was monitored by ethidium bromide staining of the gel before transfer.

present.

In addition

terminal thymus) there

the

transferase the

rate

of

was a direct transferase

activity

and antigen

activity

(data

of

radioactive

not

not

and the

but currently

label

l/10

shown).

terminal

about

2/3

cell

lines

examined

transcription

transferase

mononuclear

cells

(P8L)

transferase to

number

(in of

was observed

of terminal

exhibited

no enzyme

transcripts.

identify

the

relative

contribution

of RNA polymerase II initiation and elongation(14) to transcriptional control of the terminal transferase gene and to characterize cis-acting sequences, gene

and trans-acting

in pre-lymphocytes.

transcriptionally

regulated

proteins

that

The terminal lymphoid

regulate

transferase specific 755

the

mRNA,

The same correlation a large

underway

of

that

4) to

Of leukemic of

II).

indicated

Molt

rate

contained

or terminal

(in

relative

levels

to Table

thymus

shown)

are

from

between

(refer where

transcripts,

Experiments

(data

genes

tissues

transferase

actin correlation

terminal in normal

ratios

was transcribed

genes.

transcription gene

is

In the

of similar 5'

DNA this

to other region

Vol. 164, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

KT-ALL

-

-

,

KE-3'7

4

-

K562

-

HPB-ALL

JM

+/-

MOLT-4

+

K-T1

+

697

++

NAL.M-6

+++

PBL

-

THYMUS

+++

FIGURE 3: Competence of human cell nuclei for gene transcription. Nitrocellulose filters bound with 5pg of actin cDNA. terminal transferase run on transcripts from cDNA or pBR322 DNA were hybridized to 32P labeled nuclei isolated from the leukemic lymphoblasts, thymus, or peripheral blood leukocytes indicated on the figure.

upstream are element

from

present

the as

has

been

transcription

well

as found

the to

start octamer be

site, element

associated

TATA-

and

5'ATGCAAAl-3'(10). with

756

several

promoters

CAAATThis

of

immunoglobulin

boxes octamer

Vol.

BIOCHEMICALAND

164, No. 2, 1989

heavy

and

with

light

several

either

positive

(18). in the

5' the

other

putative core

sequence

correspond

promoter

to

the

region

conserved

chain

are

any of sequences

are

also

human terminal

(pd

element)(20).

the

cis-acting

described

tissues

been

identified

transferase

AMP promoters(19).

in

modulated

in different

have

as

can function

apparently

present

sequences

in cyclic

enhancer

DNA sequence which

of the

enhancer

whether

as well

that

enhancer

found

heavy

roles

proteins

potential

COMMUNICATIONS

This

regulatory

regulatory

to determine

the

genes(15-18).

or negative

irmnunoglobulin

interest

and with

octamer-binding

Several

(10);

genes

housekeeping

by distinct

the

chain

BIOPHYSICALRESEARCH

gene

and an analog It

will

of

be of

DNA sequences

identified

above.

ACKNOWLEDGMENTS This

work

and fellowships IFOS-TWO assistance

was supported from

3994-011T.T.).

the

by the

National

UICC and the We are

and to Ms. Danna

Kent

Fogarty

grateful for

Cancer to

secretarial

Institute

International Mr.

John

May for

CA 19492 Center

(MSC)

-

technical

help.

REFERENCES 1. Chang, L.M.S. (1971) Biochem. Biophys. Res. Commun. 44, 124-131. 2. Coleman, M.S., Hutton, J.J., DeSimone, P. and Bollum, F.J. (1974) Proc. Natl. Acad. Sci. USA 71, 4404-4408. 3. Coleman, M.S. and Hutton, J.J. (1981) in Methods in Hematology, D. Catovsky (ed.) 2, 203-219. 4. DeSiderio, S-V., Yancopoulos, G.D., Paskind, M., Thomas, E., Boss, M-A., Landau, N., Alt. F.W. and Baltimore, D. (1984) Nature 311, 752-755. 5. Leiber, M-R., Hesse, J.E., Mizuuchi, K., and Gelbert, M. (1988) Proc. Natl. Acad. Sci. USA 85, 8588-8592. 6. Robbins. D.J. and Coleman, M.S. (1988) Nucleic Acids Res. 16, 2943-2957. 7. Coleman, M.S. (1977) Arch. Biochem. Biophys. 182. 525-532. 8. Deibel, M.R., Jr., Coleman, M.S., Acree, K. and Hutton, J.J. (1981) J. Clin. Invest. 67. 725.-734. 9. Kraus. J.P. and Rosenberg, L.E. (1982) Proc. Natl. Acad. Sci. USA 79, 4015.-4019. 10. Riley, L.K., Morrow, J.K., Danton, M.J. and Coleman, M.S. (1988) Proc. Natl. Acad. Sci. USA 85, 2489-2493. 11. Engle, J.N., Gunning, P.W. and Kedes, L. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4614-4618. 12. Dignam, J.D., Lebovitz. R.M. and Roeder, R.G. (1983) Nucleic Acids Res. 11, 1475-1489. 13. Groudine, Ft., Peretz, M. and Weintraub, H. (1981) Mol. Cell. Biol. 1, 281-288. 14. Proudfoot, N.J. (1989) Trends Biochem. Sci. 14, 705-110. 15. Staudt, L.M., Singh. H., Sen, R., Wirth, T.. Sharp, P.A. and Baltimore, D. (1986) Nature 323, 640.-643. 16. Sive, H.L. and Roeder. R.G. (1986) Proc. Natl. Acad. Sci. USA 83, 6382-6386. 17. Fletcher, C., Heintz, N. and Roeder. R.G. (1987) Cell 51, 773-781. 18. Lenardo. M.J.. Staudt, L., Robbins, P.. Kuang, A., Mulligan, R.C. and Baltimore, D. (1989) Science 243, 544-546. 19. Short, J.M., Wynshar-Boris, Short, H.P. and Hanson, R.W. (1986) J. Biol. Chem. 261, 9721-9726. 20. Falkner, F.G. and Zachau. H.G. (1984) Nature 310, 71-74. 757