E2F is a key regulator of p19INK4d: implicances in DNA repair and cell cycle control

ABEL L. CARCAGNO; MARIELA C. MARAZITA; MARIA E. SCASSA; EDUARDO T. CANEPA

San Carlos de Bariloche, Rio Negro

Simposio; Gene Expression and RNA Processing; 2007

International Centre for Genetic Engineering and Biotechnology - Agencia Nacional de Promoción Científica y Tecnológica

E2F is a key regulator of p19ink4d: implicances in DNA repair and cell cycle control Abel L. Carcagno, Mariela C. Marazita, María E. Scassa, and Eduardo T. Cánepa p19INK4 is a member of the INK4 family of proteins that regulates G1/S cell cycle transition by inhibiting pRb phosphorylation by CDK 4/6 and consequently diminishing the  transcriptional activity of E2F. p19 results periodically expressed during the cell cycle, showing  the highest level in G1/S phase. Besides, we have previously reported p19 expression is induced upon DNA damage and that it plays a role in DNA repair. In silico analysis of p19 promoter sequence revealed potential E2F binding sites. Adding to this, the overexpression of E2F in asyncronic fibroblast (BHK) led to an increase in p19 mRNA in all of the  cell cycle phases. However, physiologic transcription factors regulating p19 expression remain elusive. The aim of this work was to evaluate if E2F is involved in p19 induction under two conditions, the first one linked to DNA damage response and the second one related to the periodic expression in the cell cycle and its physiological relevance.To begin with, p19 RNAm levels were evaluated following  E2F overexpression and sequestration  by an E2F-decoy. This treatment significantly decreased p19 induction after UV damage and moreover, p19 expression in G1/S phase was markedly reduced. We performed EMSA assays to assess the ability of E2F sites present in p19 promoter sequence to bind this factor. We evaluate the affinity towards E2F sites as well. We found E2F bound to this elements and that the affinity of E2F by the elements presents in p19 promoter is lower than the affinity for E2F reported consensus element. Finally, we compared the kinetics of cyclin E to p19 induction and a delay in p19 expression was observed. This delay was abolished by E2F overexpression. We conclude that E2F mediates p19 induction following DNA damage and that at least in part participates in p19 expression during cell cycle. We hypothesize p19 could be involved in a negative feedback mechanism loop controlling E2F transcriptional activity.