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New regulatory region in WAS intron 1 involved in megakaryocytic differentiation

A Hinckley Boned(1,2) S Sánchez Hernández(3) A Aguilar González(1) M Tristán Manzano(2) F Martín Molina(1,2)

1:UGR; 2:Lentistem; 3:Fundación Progreso y Salud/GENyO

Transcriptional regulatory sequences inside introns are often involved in cell-specific expression. Since the Wiskott-Aldrich syndrome (WAS) gene expression is restricted to hematopoietic cells, we performed an in silico analysis searching for regulatory regions in the first intron, followed by an in vitro analysis of these regions. We found and studied several transcription factor binding sites (TFBS) and proved the binding of Ets1, Stat1 and Hmg1, all involved in hematopoietic regulation. Interestingly, two of the Ets1 binding sites are placed in a palindromic fashion, a configuration previously shown to be associated with transcriptional silencing in tissue-specific genes. In order to investigate the role of this region in the transcriptional regulation of the WAS gene we: 1) analyzed the effect of disrupting the TFBSs on WAS gene expression levels, 2) investigated the effect of this intronic region on regulating the transcriptional activity of a strong, unrelated promoter and 3) analyzed the effect of this regulator on megakaryocytic differentiation. Our results showed that the intron1 of the WAS gene acts as a transcriptional repressor, that this repression is important to control megakaryocytic differentiation and that Stat1, Hmg1 and especially Ets1 play a major role in this regulation. This findings should help to refine gene therapy strategies for WAS based on achieving physiological expression of the transgene.

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