Commonly used cat reporter vectors contain a cAMP-inducible, cryptic enhancer that co-operates with NF-KB-sites

Gene, 151 (1994) 331-332 0 1994 Elsevier Science B.V. All rights

reserved.

331

0378-l 119/94/$07.00

GENE 08316

Commonly used cat reporter vectors contain a CAMP-inducible, cryptic enhancer that co-operates with NF-KB-sites (Context;

E-selectin;

ELAM; forskolin

inducible;

HUVEC;

Paola Ghersa, James Whelan, Rosanna Rob Hooft van Huijsduijnen

IL-l;

PKA; tetracycline;

Pescini, John F. DeLamarter

transcription)

and

GLAXO Institutejtir Molecular Biology, CH-1228 Geneva, Switzerland Received by P.F.G. Sims: 18 April 1994; Revised/Accepted:

21 June/l

July 1994; Received at publishers:

1 August

1994

SUMMARY

Commercially element capable is presented

available and widely used cat expression vectors of co-operation with NF-kB-sites in test promoters.

that allows investigation

An often-overlooked, promoters

inherent

in expression

plasmids

of the effects of Fs and other agents

problem

in the study of

is the influence

of the

surrounding vector sequences on the expression system. Current dogma holds that promoter activity relies on the interplay of multiple DNA-binding and non-binding (‘tethered’) factors. DNA-binding sites can be quite short or degenerate in sequence, requiring a particular promoter context for activity. For these reasons isolated promoter elements the surrounding

are prone to artefactual influences from vector sequences (reviewed by Kushner

et al., 1994). We have investigated

the IL-l

Correspondence to: Dr. R. Hooft

induction

van Huijsduijnen,

of an endo-

GLAXO

Chemin des Aulx, Case Postale 674, 1228 Plan-les-Ouates, Switzerland. Tel. (41-22) 706-9701; Fax (41-22) 794-6965;

IMB, 14, Geneva,

e-mail: [email protected] Abbreviations: aa, amino acid(s); ATF-a; activating transcription factor a; bp, base pair(s); CAMP, cyclic AMP; CRE(B), CAMP-responsive element (binding); C/EBP, CAAT/enhancer binding protein; ELAM, endothelial leukocyte adhesion molecule (renamed E-selectin); Fs, forskolin; hCMV, human cytomegalovirus; HSV, herpes simplex virus; HUVEC, human umbilical vein endothelial cord (cells); IL-l, interleukin-1; kb, kilobase or 1000 bp; NF-rB, nuclear factor KB; nt, nucleotide(s); PKA, protein kinase A; Tc, tetracycline; tet, Tc-resistanceencoding gene; tk, gene encoding HSV thymidine kinase; VP16, virion protein 16.

SSDI

0378-l

119(94)00527-3

were found to contain a forskolin (Fs)-inducible An alternative NF-kB-dependent reporter system that augment

intracellular

cyclic AMP.

thelial adhesion molecule, E-selectin (Hooft van Huijsduijnen et al., 1993, and references therein). Fs, an inducer of adenylate cyclase activity, augments intracellular CAMP and PKA-activity, resulting in phosphorylation and activation of CREB and probably other transcription factors. We have found that in HUVEC (human umbilical endothelial cord) cells Fs strongly inhibits E-selectin induction by IL-l. As we found this inhibition to be at the transcriptional level (P.G., R.H.v.H., J.W., Y. Cambet, R.P. and J.F.DeL., data not shown), we assayed 2.5kb and 383-bp E-selectin promoter fragments in a cat expression vector (pCATbasic@). To our surprise, Fs increased, rather than inhibited IL-l induction of the E-selectin promoter (Fig. lA,B). A control construct containing three NF-ELAMl/ATF elements is not induced by IL-l or Fs (Fig. 1D). Two possible explanations are that: (i) The E-selectin promoter has multiple Fs-responsive DNA elements: one (or more) proximal site(s) mediate increased IL-l inducibility; one (or more) distal sites act negatively and these overcome the effect of the proximal, positive site. (ii) The cut vector contains Fs-responsive DNA-elements that only reveal themselves in cells induced with IL-l. We considered the first possibility unlikely, given that both very long E-selectin promoter constructs (up to 2.5-kb promoter sequence; Fig. IA) and a construct

332

giving an enhanced signal as compared to the direct cat system (compare Fig. 1E and B). Fig. 1E shows that in this binary

system, Fs treatment

tion of the E-selectin by

over-expression

E-selectin

promoter

of

383bp-cat E

D

KB3;cat F

80

for HUVEC

2.0

1 1.5{

60

which

activates

the

co-operativity

is not specific

cells; we saw very much the same in HeLa

is more

studying

induc-

of this system

et al., 1994), is not affected

(data not shown). We conclude, system

the IL-l

Induction

ATF-a,

(Pescini

by Fs (Fig. 1F). The Fs-IL-1

2.5kb-cat

reduced

promoter.

suitable

therefore,

than

that the binary

‘standard’

Fs effects in promoter

cat vectors

for

activity.

Our data do not allow us to locate the DNA element(s) in the pCAT@ observed i

EL13pt

383bp-tet

@z m

promoters

383bp-tet

responsible

for the CREs can

Different

that

are co-induced

by IL-1

factors and sites may be involved

is not

clear.

in a combina-

m

tIL1tFs

B

tATFa

torial fashion to give to observed response. Our data are another illustration that promoter studies in artificial

tFs

m

tATFatFs

contexts

for various

promoter

(383 bp or 2.5 kb) or enhancers

gene

are

several imperfect

CONTROL tIL-1

Fig. 1. CAT assays fragments

NF-KB-binding

that

be found in these vectors, why these would only enhance

-1 rL7

plasmids

Fs effects. Although

plasmid

constructs

site or NF-ELAMl/ATF),

followed

carrying

should

be interpreted

with great care.

E-selectin

(three copies of the either

by the cat

ACKNOWLEDGEMENTS

(A, B, C, D) or the tet-VP16 fusion (E, F). For E and F, cells

were co-transfected by a minimal

with a reporter

tk promoter

IL-l, Fs, or co-transformed

containing

only

construct

plus tet-operators.

with the cat gene driven Cells were treated

with ATF-a expression

three

with

vector (ATFa).

E-selectin

NF-KB sites in a different, minimal promoter (pCATpromoter@, Fig. 1C) displayed the Fs superinduction. As we suspected that the cat-gene itself might contain Fs-responsive sequence(s), we adapted a binary induction system (Gossen and Bujard, 1992) to separate test promoter and cat gene. In this system the test promoter is cloned into a vector that drives a fusion protein of tet-repressor and the C-terminal 97 aa of HSV VP16, a strong transactivator. The second construct contains seven tet-repressor binding sites in a minimal hCMV promoter, followed by cat cDNA. The ter-repressor merely serves to deliver the VP16 transactivator by binding to the operator sites in the second construct, and makes the system repressible by Tc, but does not in itself inhibit transcription (Gossen and BujBrd, 1992). The CAT activity resulting from this system reliably reflects test promoter activity, while

We thank

Dr. H. Bujard

for providing

us with the

vectors pUHGlO-3 and pUHG151, Dr. C. Kedinger for the ATF-a expression vector and Dr. Jonathan Knowles for critically

reading

the manuscript.

REFERENCES Gossen, M. and Bujard, H.: Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89 (1992) 5547-5551. Hooft

van Huijsduijnen,

distinct

E-selectin promoter 3711-3717. Kushner,

R., Pescini,

NF-KB complexes

differing

kB element.

P.J., Baxter, J.D., Duncan,

R. and

DeLamarter,

Nucleic KG.,

Acids

DNA: the activator 8 (1994) 405-407.

bind the

Res. 21 (1993)

Lopez. G.N.. Schaufele.

Uht, R.M., Webb, P. and West, B.L.: Eukaryotic in plasmid Endocrinol.

J.F.: Two

in their larger subunit

protein-l

site

elements in pUC.

F.,

lurking Mol.

Pescini, R., Kaszubska, W., Whelan, J., DeLamarter, J.F. and Hooft van Huijsduijnen, R.: ATF-a0, a novel variant of the ATFjCREB transcription factor family, forms a dominant transcription inhibitor in ATF-a heterodimers. J. Biol. Chem. 269 ( 1994) 1159- I 165.