JBC Invitrogen Ultrasensitive Cytokine Assays

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Originally published In Press as doi:10.1074/jbc.M007008200 on October 18, 2000

J. Biol. Chem., Vol. 276, Issue 3, 1881-1888, January 19, 2001
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Transcription Factors Zic1 and Zic2 Bind and Transactivate the Apolipoprotein E Gene Promoter*

Enrique SaleroDagger , Raquel Pérez-SenDagger , Jun Aruga§, Cecilio GiménezDagger , and Francisco ZafraDagger

From the Dagger  Centro de Biología Molecular "Severo Ochoa," Facultad de Ciencias, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain and the § Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute, Wako-shi, Saitama 351-0198, Japan

We have used the yeast one-hybrid system to identify transcription factors that bind to specific sequences in proximal regions of the apolipoprotein E gene promoter. The sequence between -163 and -124, that has been previously defined as a functional promoter element, was used as a bait to screen a human brain cDNA library. Ten cDNA clones that encoded portions of the human Zic1 (five clones) and Zic2 (five clones) transcription factors were isolated. Electrophoretic mobility shift assays confirmed the presence of a binding site for Zic1 and Zic2 in the -136/-125 region. Displacement of binding with oligonucleotides derived from adjacent sequences within the APOE promoter revealed the existence of two additional Zic-binding sequences in this promoter. These sequences were identified by electrophoretic mobility shift assays and mutational analysis in regions -65/-54 and -185/-174. Cotransfection of Zic1 and Zic2 expression vector and different APOE promoter-luciferase reporter constructs in U87 glioblastoma cell line showed that the three binding sites partially contributed to the trans-stimulation of the luciferase reporter. Ectopic expression of Zic1 and Zic2 in U87 cells also trans-stimulated the expression of the endogenous gene, increasing the amount of apolipoprotein E produced by glial cells. These data indicate that Zic proteins might contribute to the transcriptional activity of the apolipoprotein E gene and suggest that apolipoprotein E could mediate some of the developmental processes in which Zic proteins are involved.

* This work was supported by Spanish Dirección General de Enseñanza Superior e Investigación Científica Grant PM98-0006 and an institutional grant from the Fundación Ramón Areces.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 34-913978720; Fax: 34-913974799; E-mail: fzafra@cbm.uam.es.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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