Two Initiator-like Elements Are Required for the Combined Activation of the Human Apolipoprotein C-III Promoter by Upstream Stimulatory Factor and Hepatic Nuclear Factor-4

Danièle Pastier,Jean-Marc Lacorte,Jean Chambaz,Philippe Cardot,Agnès Ribeiro

doi:10.1074/jbc.m200227200

Danièle Pastier, Jean-Marc Lacorte + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m200227200

Copy DOI

Abstract

Human apoC-III (-890/+24) promoter activity is strongly activated by hepatic nuclear factor (HNF)-4 through its binding to the proximal (-87/-72) element B. This site overlaps the binding site for an activity that we identified as the ubiquitously expressed upstream stimulatory factor (USF) (Ribeiro, A., Pastier, D., Kardassis, D., Chambaz, J., and Cardot, P. (1999) J. Biol. Chem. 274, 1216-1225). In the present study, we characterized the relationship between USF and HNF-4 in the activation of human apoC-III transcription. Although USF and HNF-4 binding to element B is mutually exclusive, co-transfection experiments in HepG2 cells surprisingly showed a combined effect of USF and HNF-4 in the transactivation of the (-890/+24) apoC-III promoter. This effect only requires the proximal region (-99/+24) of the apoC-III promoter and depends neither on USF binding to its cognate site in element B nor on a USF-dependent facilitation of HNF-4 binding to its site. By contrast, we found by electrophoretic mobility shift assay and footprinting analysis two USF low affinity binding sites, located within the proximal promoter at positions -58/-31 (element II) and -19/-4 (element I), which are homologous to initiator-like element sequence. Co-transfection experiments in HepG2 cells show that a mutation in element II reduces 2-fold the USF transactivation effect on the proximal promoter of apoC-III and that a mutation in element I inhibits the combined effect of USF and HNF-4. In conclusion, these initiator-like elements are directly involved in the transactivation of the apoC-III promoter by USF and are necessary to the combined effect between USF and HNF-4 for the apoC-III transcription.

Highlights

Introduction in theϪ99/ϩ24 C-IIIBM1 reporter of the mutation IIm (Table II), which avoids USF2a binding to element II, results in a 2-fold reduction in the transactivation capacity of USF2a but without altering its capacity to potentialize the effect of hepatic nuclear factor (HNF)-4 (Fig. 4, B versus A)
We found by electrophoretic mobility shift assay and footprinting analysis two upstream stimulatory factor (USF) low affinity binding sites, located within the proximal promoter at positions ؊58/؊31 and ؊19/؊4, which are homologous to initiator-like element sequence
Cotransfection experiments in HepG2 cells show that a mutation in element II reduces 2-fold the USF transactivation effect on the proximal promoter of apoC-III and that a mutation in element I inhibits the combined effect of USF and HNF-4

Summary

Introduction

Introduction in theϪ99/ϩ24 C-IIIBM1 reporter of the mutation IIm (Table II), which avoids USF2a binding to element II, results in a 2-fold reduction in the transactivation capacity of USF2a but without altering its capacity to potentialize the effect of HNF-4 (Fig. 4, B versus A). USF and HNF-4 binding to element B is mutually exclusive, co-transfection experiments in HepG2 cells surprisingly showed a combined effect of USF and HNF-4 in the transactivation of the (؊890/؉24) apoC-III promoter.

Results

Conclusion