Gene, 118 (1992) 231-238 0 1992 Elsevier Science

GENE

Publishers

B.V. All rights reserved.

231

0378-l 119/92/SO5.00

06604

The carbamyl phosphate synthetase promoter for C/EBP-related proteins (Recombinant

DNA;

Monique Laga&“*, Gordon C. Shorea ‘I Department

contains multiple binding sites

rat liver; footprinting)

Ing Swie Goping”,

Christopher

R. Muellerb,

Maribeth

Lazzaro”

and

of Biochemistry. McGill University, Montreal, Quebec, Canada; and ’ Cancer Research Laboratory, Queen’s Uniwr.sity, Kingston. Ontario.

Canada. Tel. 1613)545-6751 Received

by R. Rachubinski:

24 January

1992; Revised/Accepted:

14 February/21

February

1992; Received

at publishers:

18 May 1992

SUMMARY

The promoter of the gene (CPS) encoding rat carbamyl phosphate synthetase I has been mapped 5’ to a segment of about 525 nucleotides upstream from the transcription start point and, when analyzed in liver nuclear extracts, contained six well-defined protein-recognition elements, designated CPS sites Z-VI. All six elements were recognized, with varying affinities, by CAAT and enhancer-binding protein (C/EBPa) produced in bacteria. Oligodeoxyribonucleotides corresponding to CPS site II or to the C/EBPa-recognition element of the ALB promoter, site D, competed with the six CPS-promoter elements in footprinting assays. However, mutagenesis of the C/EBPr-recognition element, 5’-GTTGCAAC, at the core of site II was sufficient to abolish transactivation of the CPS promoter by C/EBPc( in co-transfected HepG2 cells. These findings indicate that the CPS promoter contains multiple recognition elements for factors with DNA-binding specificities similar to C/EBP proteins. Activation by C/EBPa, however, requires promoter site II.

INTRODUCTION

The mammalian urea-cycle enzyme, carbamyl phosphate synthetase (CPS), is an essential detoxifying enzyme that is located in the mitochondrial matrix compartment, and is

Correspondence to: Dr. G.C. Shore, Department

of Biochemistry,

McGill

University, 3655 Drummond St., McIntyre Medical Science Bldg., Montreal, Quebec H3G lY6, Canada. Tel. (514)398-7282; Fax (514)398-7384. * Present address:

I’Institut du Cancer de Montreal,

Montreal,

H2L 4M1, Canada.

Quebec

Abbreviations: C/EBP,

ALE, albumin-encoding

CAAT and enhancer-binding

synthetase

(E.C.

6.3.4.16);

strand(ed);

kb, kilobase

Hopital Notre-Dame,

Tel. (514)876-7078. gene (promoter); protein;

CPS, gene encoding or 1000 bp; LUC,

bp, base pair(s);

CPS, carbamyl rat CPS;

luciferase;

phosphate ds, double

LUC, gene en-

coding LUC; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; polymerase chain reaction; tsp, transcription start point(s).

PCR,

produced at detectable levels in only two cell types: hepatocytes and epithelial cells of the small intestinal mucosa (Ryall et al., 1985; and references therein). In the rat, expression of the CPS gene begins, in both tissues, at about day 16 of fetal development and reaches maximal (adult) levels shortly after birth (Ryall et al., 1985; Adcock et al., 1984). The enzyme is one of a cohort of proteins that contributes to the phenotype of the terminally differentiated hepatocyte; manipulations leading to hepatic dedifferentiation are usually accompanied by loss of CPS expression (Rozen et al., 1983). Here, we have identified protein-recognition elements in the CPS promoter and characterized their ability to interact with protein(s) exhibiting properties similar to those of C/EBP, a transactivator of gene transcription in liver (Friedman et al., 1989) and in a limited number of other tissues (adipose and intestine, and at lower levels in lung and skin; Birkenmeier et al., 1989). C/EBP (now called

232

+ strand

- strand

12345678

12345678 -1q3

‘7 proximal probe

Fig. 1. DNase combined

I protection

analysis

with phosphorylation

erate three probes

covering

of the CPS promoter

with T4 polynucleotide

CPS promoter

regions

in liver nuclear

extracts.

kinase and [;>-3’P]ATP

from nt -163 to t 7, -547

Restriction followed

digests of the plasmids,

by polyacrylamide-gel

to - 140. and

-547 to -235,

pGEM161CPS

clectrophorcsis,

or pGEMCPS412,

were employed

to gen-

labelled on either the ( t ) or ( - ) strand.

Adult rat liver nuclear extracts were prepared according to Gorski ct al. (1986) with the modifications described in Maire et al. (1989); various amounts of nuclear extract, indicated above each lane (pg protein), were used in DNase I protection assays which were carried out according to Lichtsteiner et al. (1987), with minor modifications; 1 fmol of probe (5000 cpm) and 500 ng ds poly(dI)-poly(dC)/2 mM Na vanadate/l mM KF were included per reaction. A pool of the reagents was combined with nuclear reactions containing less than 10 pg of extract

extracts and the binding reactions conducted for 15 min at 4°C before DNase I was added. For protein, bovine serum albumin was added to augment the quantity of protein to

233 + strand

- strand

0 0.5 2 5 1020400

Extra