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p65-NFB synergizes with Notch to activate transcription by triggering cytoplasmic translocation of the nuclear receptor corepressor N-CoR

Centre Oncologia Molecular, Institut de Recerca Oncologica, Hospitalet, Barcelona 08907, Spain

View larger version (16K): [in a new window]   Fig. 1. Synergistic effect of p65-NFB and Notch-IC in the transactivation of the Hes1 promoter. (A) N1-IC (0.5 µg) was cotransfected along with the Hes1-luc reporter (0.75 µg) in NIH-3T3 cells and increasing amounts of p65 wt. Lower panels show N1-IC and p65 protein levels. Protein loading for each lane was normalized according to ß-galactosidase expression. (B,C) p65-NFB can synergize with different Notch-IC homologs. 0.5 µg of N2-IC (B) or N3-IC (C) were cotransfected with p65 wt (0.5 µg) along with the Hes1-luc reporter plasmid (1µg) in NIH-3T3 cells. (D) Synergistic effect of p65-NFB and Notch-IC is not modified by ectopic expression of p50-NFB. NIH-3T3 cells were cotransfected with different combinations of equivalent amounts (0.5 µg) of p50 subunit, N1-IC and p65 wt expression vectors. Luciferase activity is represented as the fold induction relative to the basal level measured in cells transfected with empty vector. The average and standard deviation of duplicates from a representative experiment are presented.

View larger version (24K): [in a new window]   Fig. 2. Transcriptional activation of NFB-dependent genes is not required for p65-mediated upregulation of the Hes1 promoter. NIH-3T3 cells were transfected with increasing amounts of IB32-36 in the presence of N1-IC (0.5 µg) and p65 wt (0.5 µg). The reporter constructs were (A) 2xB-IL2-luc (1 µg) or (B) Hes 1-luc (1 µg). (C) Immunofluorescence assays to determine the subcellular localization of N1-IC and p65wt when coexpressed with IB32-36 in NIH-3T3 cells. Cells were cotransfected with N1-IC, p65 wt and IB32-36. N1-IC was detected with the 9E10 antibody and a FITC-labeled secondary antibody. p65 was detected with -p65 and a Cy3-conjugated secondary antibody. (D) Luciferase fold activation obtained by cotransfecting the indicated amounts p65TA with N1-IC (0.5 µg) and the Hes1-luc reporter (1 µg). Luciferase activity is presented as the fold induction relative to the basal level measured in cells transfected with empty vector. The average and standard deviation of duplicates from a representative experiment are presented. Lower panels show N1-IC and p65TA protein levels. Protein loading for each lane was normalized according to ß-galactosidase expression.

View larger version (12K): [in a new window]   Fig. 3. The Hes1 promoter is repressed by N-CoR, and this effect is reversed by p65-NFB overexpression. (A) N-CoR represses N1-IC activation of the Hes1 promoter. Indicated amounts of N-CoR were cotransfected with N1-IC expression vector (0.5 µg) into NIH-3T3 cells. (B) p65TA can revert inhibition mediated by N-CoR. Increasing amounts of p65TA were cotransfected with N1-IC (0.5 µg) and N-CoR (2 µg). Luciferase activity is presented as the fold induction relative to the basal level measured in cells transfected with empty vector. The average and standard deviation of duplicates from a representative experiment are presented. (C) Hes1 expression in 293T cells transfected with N2-IC and N-CoR expression vectors or coexpressed with p65wt is shown in the upper panel. Expression of N2-IC plasmid to compare transfection efficiency is shown in the lower panel.

View larger version (36K): [in a new window]   Fig. 4. N-CoR is translocated to the cytoplasm in the presence of p65TA. (A) Immunofluorescence staining of 293T cells transfected with N-CoR in the presence (lower panels) or absence (upper panels) of p65TA. N-CoR-flag was detected by incubating with -flag antibody and a FITC-conjugated secondary antibody (left panels), -p65 staining is represented in the middle panels corresponding to Cy3 signal and nuclei were visualized by DAPI staining (right panels). Ectopic expression of p65TA does not affect N-CoR protein levels. (B) Immunoblotting analysis of whole cell extracts isolated from 239T cells transfected with N1-IC and N-CoR-flag (lane 1) and N1-IC, N-CoR-flag and p65TA (lane 2). Cell lysates were separated on a 6% acrylamide gel, and immunoblots were performed with the following antibodies: -flag (N-CoR), -N1(N1-IC) and -p65. Protein loading for each lane was normalized according to ß-galactosidase expression and confirmed with -N1 immunoblot. The western blot for p65 shows endogenous p65 (upper band) and transfected p65TA (lower band).

View larger version (32K): [in a new window]   Fig. 5. p65-NFB nuclear export is required for cytoplasmic retention of N-CoR. (A) Immunofluorescence staining of 293T cells transfected with N-CoR-flag and p65TA in the absence (upper panel) or presence (lower panel) of LMB (10 ng/ml). (B) Immunofluorescence staining of 293T cells transfected with N-CoR-flag and p65NES. N-CoR was detected with an -flag antibody and a FITC-conjugated secondary antibody (left panels), -p65 staining is represented in the middle panels corresponding to Cy3 signal, and nuclei were visualized by DAPI staining (right panels). (C) Hes1-luc transcriptional activity of NIH-3T3 cells cotransfected with N1-IC, N-CoR or/and p65TA plasmids as indicated. Luciferase activity is represented as the fold induction relative to the basal level measured in cells transfected with empty vector. The average and standard deviation of duplicates from a representative experiment are presented. Lower panels show p65 protein levels. Protein loading for each lane was normalized according to ß-galactosidase expression.

View larger version (13K): [in a new window]   Fig. 6. p65TA increases the activity of promoters repressed by N-CoR. NIH-3T3 cells were contransfected with the above indicated luciferase reporters (A). N1-IC (0.5 µg) and N-CoR (1 µg) were contransfected in the presence or absence of p65TA (0.5 µg). Fold activation was calculated relative to cells transfected with N1-IC alone. (B) Cotransfection of p65TA (0.5 µg) with or without N-CoR (1 µg) expression vector. Cells were incubated for 12 hours in the presence of 20% FBS, and fold activation was calculated relative to cells transfected with the reporter alone. (C) Cells were transfected with a c-fos expression vector (0.5 µg), p65TA (0.5 µg), and/or N-CoR (1 µg). Fold activation was calculated relative to cells transfected with c-fos alone.