TATA binding protein associated factor 3 (TAF3) interacts with p53 and inhibits its function

Abstract

Background: The tumour suppressor protein p53 is a sequence specific DNA-binding transcription regulator, which exerts its versatile roles in genome protection and apoptosis by affecting the expression of a large number of genes. In an attempt to obtain a better understanding of the mechanisms by which p53 transcription function is regulated, we studied p53 interactions.

Results: We identified BIP2 (Bric-à-brac interacting protein 2), the fly homolog of TAF3, a histone fold and a plant homeodomain containing subunit of TFIID, as an interacting partner of Drosophila melanogaster p53 (Dmp53). We detected physical interaction between the C terminus of Dmp53 and the central region of TAF3 both in yeast two hybrid assays and in vitro. Interestingly, DmTAF3 can also interact with human p53, and mammalian TAF3 can bind to both Dmp53 and human p53. This evolutionarily conserved interaction is functionally significant, since elevated TAF3 expression severely and selectively inhibits transcription activation by p53 in human cell lines, and it decreases the level of the p53 protein as well.

Conclusion: We identified TAF3 as an evolutionarily conserved negative regulator of p53 transcription activation function.

Figure 1

Dmp53 interacts with DmTAF3. (A) Full length DmTAF3 and the clones identified from Y2H screen as p53 interacting proteins (clone 5 and clone 11) are depicted. HFD, histone fold domain; PHD, plant homeodomain. (B) The various portions of Dmp53 and human p53 that were fused to lexA-DBD and used in Y2H experiments are depicted. TA, transactivation domain; DBD, DNA binding domain; O, oligomerization domain; B, basic regulatory domain. (C) The indicated lexA-DBD fusion constructs were co-introduced into yeast with DmTAF3 amino acids 514 to 924 fused to Gal4 activation domain (AD). Interacting proteins result in complementation of His-auxotrophy and induction of β-galactosidase activity, which was detected by filter assay. Negative control was DBD-ADA2A with the same DmTAF3 clone. Positive control was DBD-rpb4 with AD-rpb7. The p53ΔN-DmTAF3 interaction was examined on a separate filter with the same positive and negative controls, and the image was fitted into the figure. (D) Direct binding of DmTAF3 to Dmp53C in vitro. Coomassie stained gel of bacterially expressed GST (lane 1) and GST-Dmp53C (lane 2). DmTAF3 (aa 514–924) was expressed in vitro in the presence of ³H-labelled leucine and incubated with GST (negative control) or GST-Dmp53C. Glutathione-agarose bead bound proteins were eluted, electrophoretically separated and detected by autoradiography (lane3 and 4).

Figure 2

In vitro and in vivo binding of mTAF3 to Dmp53 and p53. (A) GST and GST-p53 was expressed in bacteria as described in Methods, SDS-PAG was stained with Coomassie Brilliant Blue. (B) Mammalian TAF3 binds human p53 and Dmp53 as well. Cell extract from Sf9 cells overexpressing full length murine TAF3 was incubated with GST, GST-Dmp53C or GST-p53 bound to glutathione-agarose beads. Beads were washed with various concentrations of KCl as indicated above the lanes, then bound proteins were eluted and analysed with Western blotting using anti-TAF3 antibody. (C) TAF3 binds p53 in vitro. Drosophila ADA2A, human p53 and firefly luciferase were in vitro translated and transcribed using ³⁵S-methionine. The proteins were then mixed with cell extract overexpressing mTAF3, an aliquot was taken for input control that was analysed by autoradiography (upper panel). Binding proteins were immunoprecipitated using anti-TAF3 antibody, and precipitated radioactive proteins were detected as above (lower panel). (D) p53 binds TAF3 in vivo. Plasmids overexpressing human p53 and mTAF3 (+) or empty vector (-) were co-transfected into 293T cells as indicated above the lanes. Complexes were immunoprecipitated using anti-TAF3 antibody and Western blot was done using anti-p53 and anti-TAF3 antibodies.

Figure 3

Overexpression of mTAF3 strongly inhibits the transcriptional activity of p53. (A) U2OS cells were transfected with a p53-responsive luciferase reporter construct and the indicated amounts of plasmid overexpressing mTAF3 (0, 0.5, 1, 2 μg) supplemented with empty vector to a total of 3 μg DNA. (B) HeLa cells were co-transfected with 0.5 μg plasmid overexpressing p53 where indicated by a +, and the same luciferase reporter as in A, and plasmid overexpressing mTAF3 (0, 0.5, 1, 2 μg as indicated) supplemented with empty vector to a total of 3 μg DNA. Luciferase activity, measured 24 h post transfection, is given in arbitrary units, error bars indicate SD, **P < 0.01, *** P < 0.005 in t-test.

Figure 4

Inhibitory activity of TAF3 is specific for p53 and it does not require the PHD finger motif. (A) TAF3 has no effect on CMV enhancer and promoter activity. HeLa cells were co-transfected with pCDNA-luciferase reporter construct, plasmid overexpressing p53 (+), and the indicated amounts of plasmid overexpressing mTAF3 (0, 0.5, 1, 2 μg) supplemented to a total of 3 μg with vector DNA. Luciferase activity was measured 24 h post transfection and it is given relative to vector-transfected control. (B) Lack of the C terminal PHD finger motif of TAF3 has no effect on its p53-inhibitory activity. HeLa cells were co-transfected with a plasmid overexpressing p53, the p53-responsive luciferase reporter as in figure 3, plasmid overexpressing hTAF3ΔC (0, 1, 3, 5 μg) and empty vector up to a total amount of 6 μg DNA. Luciferase activity measured 24 h post transfection is given in arbitrary units, error bars indicate SD, * P < 0.05 in t-test.

Figure 5

Overexpression of TAF3 decreases the level of p53 protein but not p53 mRNA. (A) U2OS cells were untransfected (U) or co-transfected with various amounts of plasmid expressing mTAF3 (0, 1, 2, 4 μg as indicated above the panel), and empty vector to a total of 4 μg DNA. p53 protein level in the cells was examined by Western blotting and the same blot was immunoblotted with anti-actin antibody for internal control. (B) HeLa cells were co-transfected with p53 expression construct, GFP-expression construct for control, and empty vector (0) or 4 μg of plasmid expressing mTAF3 (4). Cell lysates were prepared 24 h after transfection and analysed by Western blotting using anti-TAF3, anti-p53, or anti-GFP antibodies as indicated. (C) U2OS cells were co-transfected with various amounts of plasmid expressing mTAF3 (0, 1, 2, 4 μg) and empty vector to a total of 4 μg DNA. p53 and for internal control, GAPDH mRNA level was examined by QPCR, p53 mRNA level was normalized to GAPDH mRNA level in each sample, and expressed relative to normalized p53 mRNA level in vector-transfected control.