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Nucleic Acids Research 2004 32(21):6454-6467; doi:10.1093/nar/gkh981
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Published online 8 December 2004

Nucleic Acids Research, Vol. 32 No. 21 © Oxford University Press 2004; all rights reserved

A novel E2 box-GATA element modulates Cdc6 transcription during human cells polyploidization

Nuria Vilaboa, Rodrigo Bermejo, Pilar Martinez, Rafael Bornstein1 and Carmela Calés*

Department of Biochemistry, Instituto de Investigaciones Biomédicas ‘A. Sols’, Universidad Autónoma-CSIC, Arturo Duperier, 4, 28029 Madrid, Spain and 1 Madrid Cord Blood Bank, Hospital Universitario ‘Doce de Octubre’, Km. 5.4, 28041 Madrid, Spain

* To whom correspondence should be addressed. Tel: +34 91 5854469; Fax: +34 91 5854401; Email: ccales{at}iib.uam.es
Present address: Nuria Vilaboa, Department of Experimental Surgery, Bone Metabolism Laboratory, Hospital ‘La Paz’, Madrid, Spain
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors

Received August 21, 2004; Revised and Accepted November 16, 2004

Cdc6 is a key regulator of the strict alternation of S and M phases during the mitotic cell cycle. In mammalian and plant cells that physiologically become polyploid, cdc6 is transcriptionally and post-translationally regulated. We have recently reported that Cdc6 levels are maintained in megakaryoblastic HEL cells, but severely downregulated by ectopic expression of transcriptional repressor Drosophila melanogaster escargot. Here, we show that cdc6 promoter activity is upregulated during megakaryocytic differentiation of HEL endoreplicating cells, and that Escargot interferes with such activation. Transactivation experiments showed that a 1.7 kb region located at 2800 upstream cdc6 transcription initiation site behaved as a potent enhancer in endoreplicating cells only. This activity was mainly dependent on a novel cis-regulatory element composed by an E2 box overlapping a GATA motif. Ectopic Escargot could bind this regulatory element in vitro and endogenous GATA-1 and E2A formed specific complexes in megakaryoblastic cells as well as in primary megakaryocytes. Chromatin Immunoprecipitation analysis revealed that both transcription factors were occupying the E2 box/GATA site in vivo. Altogether, these data suggest that cdc6 expression could be actively maintained during megakaryocytic differentiation through transcriptional mechanisms involving specific cis- and trans-regulatory elements.


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