FIG. 2.

hSirT1 modulates E2F1 recruitment and transcriptional activity on the P1p73 promoter. (a) U2Os cells were transfected with the P1p73 reporter and the indicated expression vectors and exposed for 24 h to NAM (10 to 25 mM), TSA (100 nm), and VPA (10 mM). (Top) Representative anti-HA (α-HA) and anti-FLAG immunoblot (IB) of exogenously expressed E2F1 and hSirt1/mthSirt1. (b) U2Os cells were transfected with the P1p73 reporter and the indicated expression vectors or exposed for 24 h to NAM (10 mM). (c, top left) Chromatin from untreated and NAM (10 mM)-treated U2Os cells was immunoprecipitated with either the relevant control IgG or anti-E2F1 antibodies and analyzed with P1p73 primers by PCR. Control reactions using distant primers (C) did not amplify anti-E2F1 ChIP products from either untreated or NAM-treated cells (data not shown). (Top right) Chromatin from untreated and NAM (10 mM)-treated U2Os cells was immunoprecipitated with either the relevant control IgG or anti-E2F1 antibodies and analyzed with the Bim promoter primers by PCR. Control reactions using distant primers (C) did not amplify anti-E2F1 ChIP products from either untreated or NAM-treated cells (data not shown). (Bottom left) TAp73 transcripts from untreated and NAM-treated U2Os cells were quantitated by qRT-PCR as described in the legend to Fig. 1B. (Bottom right) TAp73 and E2F1 protein levels detected by immunoblotting using specific antibodies. (d, left) Modulation of E2F1 binding to the P1p73 promoter by the hSirT1 deacetylase activity. HA-E2F1, wild-type hSirT1, and deacetylase-defective hSirT1-H355Y were exogenously expressed in U2Os cells and immunoprecipitated in a ChIP assay using a polyclonal anti-HA antibody. (Right) Densitometric quantification with Image J software.