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First published online 28 September 2004
doi: 10.1242/jcs.01384

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p73 competes with co-activators and recruits histone deacetylase to NF-Y in the repression of PDGF ß-receptor

Hidetaka Uramoto^*, Daniel Wetterskog, Anders Hackzell, Yoshiki Matsumoto and Keiko Funa

Department of Cell Biology, Institute of Anatomy and Cell Biology, Göteborg University, Box 420, SE-405 30 Gothenburg, Sweden

View larger version (19K): [in a new window]   Fig. 1. Trichostatin A (TSA) increases platelet-derived growth factor ß-receptor (PDGFRB) promoter activity through the CCAAT motif and attenuates its repression by p73. (A) The HindIII/SacI promoter construct containing the CCAAT motif without the GC box and the SacI/SacI constructs with a mutated or wild-type CCAAT motif were co-transfected into NIH3T3 cells with or without TSA treatment. Values represent mean luciferase activity and error bars indicate standard deviation of triplicate samples. (B) The SacI/SacI construct was co-transfected with p73 or vector alone (mock) and treated with or without TSA. (C) The SacI/SacI construct containing the mutated or wild-type CCAAT motif was co-transfected with CBP, p300, or vector alone.

View larger version (26K): [in a new window]   Fig. 2. NF-YB and NF-YC bind P/CAF and NF-YB also binds p300. COS-1 cells were co-transfected with various Flag-NF-Y subunits and HA-P/CAF, p300, CBP plasmids or HA-vector, as indicated. Cells were lysed and immunoprecipitated with HA antibody and immunoblotted with Flag antibody. Cell lysate was divided and used for immunoprecipitation (IP) or immunoblotting (IB). The transferred membrane was immunoblotted with Flag-M2 antibody (upper panel). The expression of HA-tagged P/CAF, p300 or CBP was confirmed using HA antibody (lower panel).

View larger version (40K): [in a new window]   Fig. 3. p300 and p73 reciprocally regulate the PDGFRB promoter through the CCAAT motif. (A) The SacI/SacI promoter construct was co-transfected to NIH3T3 cells with vector alone (mock), p73, p300 or various amounts of p300 and p73, as indicated. (B) The SacI/SacI construct was co-transfected with mock vector, p73, P/CAF or various amounts of P/CAF and p73. Values represent mean luciferase activity and error bars indicate standard deviation of triplicate samples.

View larger version (41K): [in a new window]   Fig. 4. Effects of p73 on p300-induced acetylation of NF-Y. (A) NIH3T3 cells were co-transfected with various NF-Y-subunits, HA-NFYA, Flag-NFYB and Flag-NFYC in combination with HA-vector, HA-p73, His-Np73 and HA-p300 plasmids, as indicated. Cell lysates were immunoprecipitated (IP) with Flag antibody and immunoblotted (IB) with indicated antibodies. (B-D) Increasing amounts of HA-p73 with HA-p300 or P/CAF (1 µg) and Flag-NF-YB or NF-YC (1 µg) were transfected into NIH3T3 cells, as indicated. Cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-HA antibodies.

View larger version (23K): [in a new window]   Fig. 5. HDAC1 binds p73 in vivo. (A) COS-1 cells were co-transfected with Flag-tagged p73, or Flag-vector together with HA-tagged HDAC1 or HA vector, respectively. Cell lysates were reciprocally immunoprecipitated (IP) and immunoblotted (IB) with Flag antibody or HA antibody, as described in legend to Fig. 4. Cell lysates were immunoblotted with HA antibody (upper panel) or Flag-M2 antibody (lower panel). (B) GST-HDAC1 and HA-p73 fusion proteins were incubated in vitro and immunoprecipitated with GST antibody. Binding was detected using antibodies against HA and GST. (C) Flag-tagged p73 or its C-terminal deletion mutants were co-transfected with HA-tagged HDAC1 or vector, and the interaction was examined as described above. (D) Interaction between Np73 and HA-tagged HDAC1 was examined as described in (A). In addition, anti-Np73 was used for immunoprecipitation and immunoblotting.

View larger version (30K): [in a new window]   Fig. 6. p73 and acetylated proteins were found to be bound in vivo to the promoter. (A) Chromatin extracted from 3T3 cells treated with or without Trichostatin A (TSA) was immunoprecipitated with p73 antibody, acetyl-lysine antibody, or pre-immune serum. DNA was purified and analysed by PCR together with whole cell lysate (input), using primers specific for the proximal and distal areas of the PDGFRB promoter. (B) Chromatin was extracted from cells harvested at indicated time points following serum stimulation after being cultured in serum-free media for 48 hours. Samples were immunoprecipitated with antibodies against p73, Np73, NF-YB, p300 and HDAC1, respectively and DNA was detected as described above. Primers for the proximal promoter sequence were used as controls.

View larger version (60K): [in a new window]   Fig. 7. Nuclear colocalisation of TAp73 and Np73. (A-C) Double immunofluorescence labelling of 3T3 fibroblasts 6 hours after serum stimulation with anti-TAp73 antibody (A, red) and anti-HDAC1 antibody (B, green), and the merged image (C). The majority of cells in this field express both molecules in the nucleus. Bar, 5 µm.