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In vivo analysis of the state of the human uPA enhancer following stimulation by TPA

Oncogene volume 18, pages 2836–2845 (1999)Cite this article

Abstract

We have analysed in vivo the −2.0 kb enhancer of the human urokinase-type plasminogen activator (uPA) gene in HepG2 cells, in which gene expression can be induced by phorbol esters. The results reveal that, within the regulatory region, the enhancer, the silencer and the minimal promoter become hypersensitive to deoxyribonuclease I (DNase I) upon induction of transcription. The hypersensitivity of the enhancer can be reversed after removal of the inducer. In vivo footprinting analysis indicates that all the cis-acting elements of the enhancer, previously identified in vitro, are occupied in vivo upon 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulation of HepG2 cells. Micrococcal nuclease (MNase) cleavage of this region fails to reveal discrete nucleosomal boundaries in vivo in close proximity of the enhancer, either before or after stimulation. Furthermore, this region does not lose its nucleosomal configuration after TPA induction of transcription. An approximately 600 bp long region around the enhancer becomes more, but not fully, accessible to restriction endonucleases upon stimulation. A time-course experiment shows that this accessibility reaches a plateau after a 1 h TPA treatment suggesting the persistent presence of nucleosomes. These results indicate that TPA induces the binding of transcription factors to the uPA enhancer without chromatin remodelling of this region.

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Abbreviations

uPA:

urokinase-type plasminogen activator

DNase I:

deoxyribonuclease I

TPA:

12-O-tetradecanoyl-phorbol-12-acetate

MNase:

micrococcal nuclease

COM:

cooperation mediator

uCOM:

upstream COM

dCOM:

downstream COM

DMEM:

Dulbecco's modified Eagle's medium

SDS:

sodium dodecyl sulphate

PBS:

phosphate buffered saline

DTT:

dithiotreitol

EDTA:

ethylene diamino tetra acetic acid

BSA:

bovine serum albumin

PMSF:

phenyl methyl sulphonyl fluoride

UV:

ultraviolet

LTS LM-PCR:

linker tag-selection ligation-mediated polymerase chain reaction

OP-Cu:

1, 10-phenantroline-copper

GAPDH:

glyceraldehyde 3-phosphate dehydrogenase

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Acknowledgements

We thank Peter Becker and Jean-Pierre Quivy for many helpful suggestions on the LTS LM – PCR procedure. We also thank Drs Michael Bustin, Valerio Orlando and Anna Mondino for critically reading the manuscript. This work was supported by grants from Associazione Italiana Richerche sul Cancro (AIRC) and the Italian Ministry of Education (MURST). II-T was supported by a fellowship of the `Comitato Promotore Telethon' (105/BS). This paper is dedicated to the memory of Odette Tallon and Gianni Crippa.

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  1. Inés Ibañez-Tallon

    Present address: Laboratory of Molecular Biology, The Rockefeller University, 1230 York Avenue, 10021-6399, New York, USA

Authors and Affiliations

  1. Laboratory of Molecular Genetics, DIBIT - Ospedale. S. Raffaele, Via Olgettina 58, Milano, 20132, Italy

    Inés Ibañez-Tallon, Giuseppina Caretti, Francesco Blasi & Massimo P Crippa

  2. Department of Genetics and Microbial Biology, University of Milano, Via Celoria 20, Milano, 20133, Italy

    Giuseppina Caretti & Francesco Blasi

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  1. Inés Ibañez-Tallon
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  4. Massimo P Crippa
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Ibañez-Tallon, I., Caretti, G., Blasi, F. et al. In vivo analysis of the state of the human uPA enhancer following stimulation by TPA. Oncogene 18, 2836–2845 (1999). https://doi.org/10.1038/sj.onc.1202644

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