Global re-wiring of p53 transcription regulation by the hepatitis B virus X protein

Abstract

Background: The tumour suppressor p53 is a central player in transcription regulation and cell fate determination. By interacting with p53 and altering its sequence-specific binding to the response elements, the hepatitis B virus X protein (HBx) was reported to re-direct p53 regulation of some genes.

Results: Coupling massively parallel deep sequencing with p53 chromatin immunoprecipitation, we demonstrate that HBx modulates global p53 site selection and that this was strongly influenced by altered interaction with transcription co-factors/co-regulators as well as post-translational modifications. Specifically, HBx attenuated p53-TBP-RB1 transcription complex recruitment and interaction and this was associated with hyper-phosphorylation of p53 at serine 315 by HBx. Concurrently, HBx enhanced p53 DNA occupancy to other response elements either alone by displacing specific transcription factors such as CEBPB and NFkB1, or in complex with distinct interacting co-factors Sp1, JUN and E2F1. Importantly, re-wiring of p53 transcription regulation by HBx was linked to the deregulation of genes involved in cell proliferation and death, suggesting a role of HBx in errant cell fate determination mediated by altered p53 site selection of target genes.

Conclusions: Our study thus presents first evidence of global modes of p53 transcription alteration by HBx and provides new insights to understand and potentially curtail the viral oncoprotein.

Keywords: ChIP-Seq; HBx; P53 transcription; Phosphorylation; p53 serine 315.

Figure 1

HBx confers p53 a preference for less conserved response elements. (A) Control and HBx‐expressing HepG2 cells employed for ChIP‐Seq and expression profiling (upper panel). UV‐treated HepG2 cells successfully transduced with either control or HBx‐expressing recombinant adenovirus harbouring the GFP gene. Comparable high levels of HBx and control adenovirus transduction efficiencies were achieved as seen from the green fluorescing cells. Number of ChIP‐Seq control‐ and HBx‐specific peaks containing a p53MH‐predicted p53 motif is tabulated (lower panel). (B) Genomic distribution of control‐ and HBx‐specific p53 peaks using criteria detailed in Experimental procedures. (C) Illustration of the p53 consensus sequence comprising two half‐sites, where R denotes purine, Y denotes pyrimidine and W denotes A/T (top). Distribution of p53 peaks based on their similarity to the consensus sequence, in 20% intervals (bottom). (D and E) Distribution of p53 peaks according to the p53 motif position‐specific bases (D) and spacer lengths (E). Statistically significant findings are denoted as *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 2

HBx disrupts p53‐TBP and p53‐TFAP2A transcription complex‐DNA binding and subsequent recruitment of RB1. (A) Schematic diagram showing the in silico strategies employed to shortlist high confidence p53 co‐localizing transcription factors and regulators. Bold: predicted by Centdist, GeneMania and IPA; Blue: predicted by GeneMania and IPA. (B) Shortlist of high confidence p53 co‐localizing transcription factors identified and their frequency of occurrence in control‐ and HBx‐specific p53 peaks are tabulated. (C) p53‐TBP‐RB1 (top panel) and p53‐TFAP2A‐RB1 (bottom left and right panels) DNA co‐occupancy in control cells is abolished in the presence of HBx. The strength of TF‐DNA binding is indicated by the gradient heat map of ChIP‐qPCR fold enrichment of the respective transcription factor. ChIP‐qPCR validation was performed using three independent ChIP experiments of the respective transcription factors/regulators. (D) Interaction of indicated proteins determined by PLA. Red PLA signals indicate sites of protein–protein interaction observed by fluorescence microscopy (left panel), blue DAPI signals indicate the cell nucleus (middle panel), merged PLA and DAPI images (right panel).

Figure 3

HBx enhances p53 recruitment to DNA. (A) p53‐Sp1 (top), JUN (middle) and E2F1 (bottom) DNA co‐occupancy is enhanced in the presence of HBx. The strength of TF‐DNA binding is indicated by the gradient heat map of ChIP‐qPCR fold enrichment of the respective transcription factor. (B) Interaction of indicated proteins determined by PLA. Red PLA signals indicate sites of protein–protein interaction observed by fluorescence microscopy (left), blue DAPI signals indicate the cell nucleus (middle), merged PLA and DAPI images (right). (C) p53‐DNA occupancy excludes NFkB1 and CEBPβ DNA occupancy in the presence of HBx. (D) Interaction of indicated proteins determined by PLA.

Figure 3

(continued).

Figure 4

P53 serine 315 phosphorylation is associated with HBx‐disrupted p53‐TBP‐RB1 transcription complex. (A) Gene Ontology terms (GO) enriched (Benjamini <0.1) for significantly (p < 0.01) deregulated genes linked to altered p53‐associated transcription complexes, as assessed by DAVID. (B) Gene expression levels of the shortlisted p53 transcription co‐factors and co‐regulators in control and HBx‐expressing HepG2 cells, determined using microarray expression profiling is tabulated. Corresponding protein levels of the respective p53 transcription co‐factors and co‐regulators in control and HBx‐expressing HepG2 cells determined by western blotting is shown (right). (C) Subcellular localization of p53 and the specific p53 transcription co‐factors and co‐regulators in control and HBx‐expressing HepG2 cells, determined using fractionation and western blotting using specific antibodies. (D) Serine 315‐ and 392‐phosphorylated p53 peptides identified using mass spectrometry of p53 proteins immunoprecipitated from control and HBx‐expressing cells (table, top). Mass spectrum of p53 peptide containing phosphorylated serine 315 from HBx‐expressing cells (bottom). (E) Western blot validation of elevated p‐p53(S315) levels in HBx‐expressing cells compared to control cells using phospho‐specific p53 antibodies. Total p53 protein levels are shown and GAPDH protein expression was used as loading control for western blotting. (F) p‐p53(S315) ChIP‐qPCR occupancy on control and HBx‐expressing cells exhibiting identical recruitment patterns to that of p53‐TBP‐RB1 transcription complex. The strength of TF‐DNA binding is indicated by the gradient heat map of ChIP‐qPCR fold enrichment of the respective transcription factor.

Figure 5

Model illustrating modes of p53‐associated transcription complex modulation by HBx. HBx attenuates p53‐TBP/TFAP2A‐RB1 DNA‐binding (left panel). HBx conversely enhances other p53‐TF binding to DNA including Sp1, JUN and E2F1 (middle panel). In other cases, HBx favours p53‐DNA binding while concurrently excluding the binding of TFs including CEBPB and NFkB1 to their binding sites (right panel). Together, altered p53 site selection results in the deregulation of genes chiefly involved in cell proliferation and death.