Activation of p53 function by human transcriptional coactivator PC4: role of protein-protein interaction, DNA bending, and posttranslational modifications
Authors: Tapas K Kundu
Kiran Batta
Journal: Molecular and Cellular Biology (2007)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Tumor suppressor p53; cell cycle checkpoints; apoptosis; genes; proteins
Affiliations: Transcription and Disease Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, P.O. Bangalore-560064, India.

Abstract:  Tumor suppressor p53 controls cell cycle checkpoints and apoptosis via the transactivation of several genes that are involved in these processes. The functions of p53 are regulated by a wide variety of proteins, which interact with it either directly or indirectly. The multifunctional human transcriptional coactivator PC4 interacts with p53 in vivo and in vitro and regulates its function. Here we report the molecular mechanisms of the PC4-mediated activation of p53 function. PC4 interacts with the DNA binding and C-terminal domains of p53 through its DNA binding domain, which is essential for the stimulation of p53 DNA binding. Remarkably, ligation-mediated circularization assays reveal that PC4 induces significant bending in the DNA double helix. Deletion mutants defective in DNA bending are found to be impaired in activating p53-mediated DNA binding and apoptosis. Furthermore, acetylation of PC4 enhances, while phosphorylation abolishes, its ability to bend DNA, activate p53 DNA binding, and, thereby, regulate p53 functions. In conclusion, PC4 activates p53 recruitment to p53-responsive promoters (Bax and p21) in vivo through its interaction with p53 and by providing bent substrate for p53 recruitment. These results elucidate the general molecular mechanisms of activation of p53 function, mediated by its coactivators. 

Retracted: No

PubPeer comments:
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 Thank you for pointing this out. We are currently trying to find old raw data used in the paper. Since the work was performed over 15 years ago, we are having difficulties in finding the raw data. If there was an image duplication it was not done intentionally and it does not change any conclusions made from this study. , 
 Raw data relating Figure 9C that show the same conclusion as Figure 9E. ![file] , 
 In Figure 9, we were trying to understand how PC4 and itu2019s different truncation mutants affect p53 binding to its cognate sites and thereby induce Bax expression and apoptosis. We found all mutants except PC4 1-67 were able to induce p53 dependent apoptosis. PC4 62-127, C-terminal domain of PC4, was able to induce p53 binding to its cognate sites (Figure 9A), induce Bax expression (Figure 9C) and also induce apoptosis (Figure 9F). All these data indeed confirm that PC4 62-127 could induce p53 dependent apoptosis. We presented representative images in Figure 9E showing the effect of PC4 62-127 on p53 dependent apoptosis. We apologize for the unintentional error in image processing. Though we have the raw data for Fig 9F, which quantitatively show the effect of PC4 and its mutant on the induction of Apoptosis, we are unable to retrieve the old image data. We will continue our efforts to retrieve old data and correct the image by submitting erratum. However, we would like to draw the kind attention of the readers that the conclusions based on the experiments in Figure 9 do not change at all due to this image processing mistake. , 
 https: mcb.asm.org content 40 19 e00364-20 AUTHOR CORRECTION Volume 27, no 21, p. 7603u20137614, 2007, https: doi.org 10.1128 MCB.01064-07. Page 7612, Fig. 9E: Due to an inadvertent error in assembling images, the bottom panels are duplicates. The image duplicated was a representative image; quantification of the same data is shown in panel F and is further supported by the data from panels A and C. Therefore, the conclusions of Fig. 9 and that of the paper have not changed due to the unintentional mistake. Since the work was performed almost 15 years ago, we do not have the original image files used in this paper, and as ASM policy is not to allow new data in corrections, the figure could not be replaced. We sincerely apologize for this error and any confusion it may have caused. , 
 Previous work from our lab showed that transcriptional coactivator PC4 activates tumor suppressor p53 function . In this manuscript, we investigated molecular mechanisms of activation of p53 function by PC4. We showed a role for PC4-p53 interaction, PC4 posttranslational modifications, and PC4 DNA bending ability in the activation of p53 function. Several research articles both from our group as well as the others corroborate these conclusions. Briefly, we present here some of the research articles in support of our conclusions. A role for PC4-p53 interaction: https: www.ncbi.nlm.nih.gov pmc articles PMC3137081 : This article showed inhibiting PC4-p53 interaction by a recombinant peptide reduced PC4 mediated activation of p53 dependent gene transcription. https: pubmed.ncbi.nlm.nih.gov 31236588 : This article showed the importance of PC4-p53 interaction in p53 dependent gene expression and apoptosis. https: pubmed.ncbi.nlm.nih.gov 21193408 : This article showed 22-91 aa of PC4 is important for its interaction with transcription factor SP-1 similar to what has been shown for PC4u2013p53 interaction in our study. A role for DNA bending: https: pubmed.ncbi.nlm.nih.gov 18459978 : This article discussed the DNA bending ability of PC4 in activating gene transcription. https: pubmed.ncbi.nlm.nih.gov 19525231 : This article showed the importance of DNA bending single-strand DNA binding domain (62-127 aa) of PC4 in activating p53 function. https: www.ncbi.nlm.nih.gov pmc articles PMC3227220 : This article also discussed the role of single strand DNA binding DNA bending domain of PC4 in transcriptional activation. A role for post-translational modifications: https: pubmed.ncbi.nlm.nih.gov 21193408 : This article showed PC4 phosphorylation inhibits its interaction with transcription factor SP-1 similar to what has been shown for PC4 and p53. This article also discussed multiple ways in which PC4 stimulates transcriptional activation similar to what has been proposed for PC4 mediated activation of p53 function. https: pubmed.ncbi.nlm.nih.gov 20305379 This review discussed the role of PC4 posttranslational modification in its transcriptional activation repression function. https: www.ncbi.nlm.nih.gov pmc articles PMC3137081 https: pubmed.ncbi.nlm.nih.gov 14759370 https: www.ncbi.nlm.nih.gov pmc articles PMC5834259 These three articles together showed that acetylation of p53 that recruits HAT CBP, is important for PC4-p53 interaction suggesting that PC4 interaction with p53 leads to acetylation of both of these proteins Additional support: https: pubmed.ncbi.nlm.nih.gov 20453859 : Showed addition of recombinant PC4 stimulated p53 dependent in vitro transcription. https: www.ncbi.nlm.nih.gov pmc articles PMC5062974 : Interaction between coactivator Sam68 and p53 is important for p53 dependent Bax and p21 gene expression. In Figure 9 of the manuscript, following extensive biochemical characterization, we explored the mechanisms of PC4 mediated activation of p53 function in cells using different PC4 mutant constructs. We used multiple approaches to derive our conclusions. We looked at the recruitment of p53 to Bax promoter, p53 dependent Bax p21 gene expression, and p53 mediated apoptosis in cells overexpressing both p53 and different PC4 constructs. In Figure 9A, we showed both PC4 22-127 and PC4 62-127 could induce p53 recruitment to Bax promoter as determined by ChIP-QRT-PCR. In Figure 9C, we showed both PC4 22-127 and PC4 62-127 could induce p53-dependent Bax gene expression as determined by RT-PCR. The raw data presented in comment 3 above should read relative p21 levels instead of Bax levels, we apologize for the confusion. In Figure 9E and 9F, we quantified the number of apoptotic cells manually by visualizing apoptotic nuclei under a fluorescent microscope. In Figure 9F, we showed both PC4 22-127 and PC4 62-127 could induce p53 dependent apoptosis. In Figure 9E, we presented representative images showing levels of apoptosis. As noted representative images when overexpressing PC4 22-127 and PC4 62-127 are the same. None of the conclusions made in this manuscript were based on Figure 9E alone. Therefore, the unintentional, rather accidental duplication in one panel of Figure 9E, does not alter the conclusion of Figure 9, as well as the paper. We sincerely apologize for this error and any confusion it may have caused. ,