Functional characterization of p53 pathway components in the ancient metazoan Trichoplax adhaerens

The identification of genes encoding a p53 family member and an Mdm2 ortholog in the ancient placozoan Trichoplax adhaerens advocates for the evolutionary conservation of a pivotal stress-response pathway observed in all higher eukaryotes. Here, we recapitulate several key functionalities ascribed to this known interacting protein pair by analysis of the placozoan proteins (Tap53 and TaMdm2) using both in vitro and cellular assays. In addition to interacting with each other, the Tap53 and TaMdm2 proteins are also able to respectively bind human Mdm2 and p53, providing strong evidence for functional conservation. The key p53-degrading function of Mdm2 is also conserved in TaMdm2. Tap53 retained DNA binding associated with p53 transcription activation function. However, it lacked transactivation function in reporter genes assays using a heterologous cell line, suggesting a cofactor incompatibility. Overall, the data supports functional roles for TaMdm2 and Tap53, and further defines the p53 pathway as an evolutionary conserved fulcrum mediating cellular response to stress.


Results
We first carried out pull-down assays using proteins expressed in vitro to measure interactions. Both full-length and C-terminally truncated p53 variants (p53Δ , Tap53Δ ) were used. p53 was truncated to remove the p53 C-terminal domain not critical for Mdm2 interaction and the corresponding truncation was made in Tap53 (Fig. 1). The results showed the expected strong interaction between human Mdm2 (referred to as MDM2 hereon) and both full length and C-terminally truncated p53. Similarly, TaMdm2 was seen to interact with Tap53 and Tap53Δ (Fig. 2a). Whilst the interaction with full-length Tap53 appears weaker, this is most likely due to its comparatively reduced expression levels. Notably, cross species interaction was observed, with MDM2 showing strong interaction with both full-length and truncated Tap53. TaMdm2 showed very weak interaction with p53 and p53Δ . Pull-down assays using proteins expressed in the p53-null H1299 cell line 19 essentially recapitulated the in vitro data. TaMdm2 interacted with Tap53 and p53, albeit much weaker than for corresponding interactions of MDM2 (Fig. 2b). However, the amount of Tap53 pulled down is likely to be under-represented due to presence of competing endogenous MDM2 in H1299 cells.
The major interaction site of p53 with MDM2 resides in its N-terminal domain. This is defined by a contiguous sequence (amino acids [19][20][21][22][23][24][25][26] that comprises three signature amino acids (F19, W23 and L26) whose side chains interact with discrete pockets in a hydrophobic cleft of the MDM2 N-terminal domain (Fig. 1) 20 . Stapled peptide antagonists derived from this sequence have been shown to be potent inhibitors of the p53-MDM2 interaction 21 . Two stapled peptides, PM2 and MO11 22 were both able to inhibit the interaction of MDM2 with Tap53Δ when compared to a negative control stapled peptide (P-CON) (Fig. 2c, left panel). In the case of the Tap53Δ -TaMdm2 interaction, only MO11 was able to inhibit the reaction (Fig. 2c, right panel). MO11 is a higher affinity derivative of PM2. As both these peptide antagonists compete with the N-terminal domain of p53 for binding to MDM2 23 , this indicates conservation of interaction of the N-terminal domain of Tap53 with TaMdm2. The prototypical small molecule MDM2 inhibitor Nutlin 24 did not inhibit the in vitro Tap53Δ -TaMdm2 interaction (Fig. S1). This is not surprising as Nutlin binding can be compromised by very small changes in MDM2 structure 25,26 . However, homology modeling further highlights overall structural conservation between the MDM2 and TaMdm2 N-terminal domains and interactions with p53/Tap53 N-terminal interacting region (Fig. S2).
Of the three signature residues in the MDM2-interacting region of p53, only W23 is conserved in Tap53, with F19 and L26 respectively being replaced by L and M (Fig. 1). We therefore mutated the methionine in Tap53 to leucine, as this larger side chain is readily accommodated by MDM2 20 . The results show increased interaction of Tap53-M26L with MDM2 (Fig. 2d), further defining the key role of this region for MDM2 engagement. Interestingly, the interaction of this mutant with TaMdm2 was similar to wild-type. This may indicate plasticity in the N-terminal domain of TaMdm2 that accommodates the larger side chain and possibly greater dependence on a secondary p53-MDM2 interaction site 27-29 that may be conserved in the Tap53-TaMdm2 interaction.
We next investigated the DNA binding function of Tap53 using a real-time PCR based assay 30 . In addition to probing with a consensus p53 response element (ConA) 31 , we used three sequences identified upstream of the TaMdm2 coding region (TXRe320, TXRe350 and TXRe540, respectively 320, 350 and 540bp upstream of coding region) as potential Tap53 binding sites (Fig. 3). As part of a negative feedback loop, MDM2 is a direct transcriptional target of p53. Hence these binding sites may represent evidence for the feedback loop in Trichoplax. The results show increased binding of both Tap53 and Tap53Δ to all response elements tested when compared to a control DNA sequence, with binding to TXRe540 being favoured (~30-fold increased binding over control DNA for Tap53) (Fig. 3). p53 also bound this sequence, albeit relatively weaker than the consensus sequence (respectively ~10 versus 120-fold binding over control DNA).
Tap53 was then assayed for transactivation function using p53 reporter gene assays in H1299 cells. In addition to using a reporter gene driven by tandem copies of the consensus p53 response element (2ConA), we also looked at the TXRe540 response element as Tap53 bound this preferentially (Fig. 3). Tap53 was unable to transactivate either reporter gene construct. In contrast, both p53 and p53Δ showed activity with both reporters. This was reduced ~4-fold for the TXRe540 reporter, in agreement with the comparatively weaker binding of p53 to the TXRe540 response element (Fig. 4a,b).
MDM2 and TaMdm2 were next co-transfected with p53 into H1299 cells and p53 transactivation measured using the 2ConA reporter gene. MDM2 co-transfection led to the expected reduction of p53 function (Fig. 4c). TaMdm2 co-expression also reduced the activity of p53, albeit not as efficiently as MDM2 considering its very high expression level in this cell line (Fig. 4c). This is likely due in part to the weak interaction between p53 and TaMdm2 observed both in this cell line and in vitro (Fig. 2). To further address TaMdm2 cellular function, plasmids encoding TaMdm2 and Tap53 were co-transfected into the p53/Mdm2-null DKO cell line and the p53/ Mdm2/Mdm4-null cell line TKO 13 . The TaMdm2 was tagged with the HA epitope at either the N-or C-terminus. The results showed that only N-terminally tagged TaMdm2 was able to reduce levels of co-expressed Tap53 (Fig. 5, compare lanes 3 and 5 or lanes 11 and 13). The ability of the proteasome inhibitor MG132 to block this degradation (compare lanes 3 and 4 or lanes 11 and 12) implies that, as with vertebrate Mdm2-p53, TaMdm2 is targeting Tap53 for proteasomal degradation. The inactivity of the C-terminally tagged TaMdm2 agrees with studies showing that MDM2 ubiquitination function can be inhibited by addition of tags/fusion partners adjacent to the C-terminal RING domain important for E3 ligase activity 32 . Likewise, mutation of a critical cysteine residue in the RING domain (equivalent to MDM2 C464) prevents the degradation of Tap53 (compare lanes 7 and 2 or lanes 15 and 11). These results indicate that ancient TaMdm2 interacts with mammalian E2 proteins and other components in the proteasomal pathway and emphasize the co-evolution of TaMdm2 and Tap53 in one of the earliest animal species.
The ribosomal protein component L11 has been shown to interact with MDM2 and regulate its function 33,34 . As with MDM2, TaMdm2 showed clear interaction with human L11 when measured by pull-down assay using in vitro expressed proteins (Fig. 6a). Similarly, both proteins were able to pull down human L11 in H1299 cells when expressed ectopically (Fig. 6b). The zinc finger domain of MDM2 has been shown to mediate interaction with L11 35,36 . This domain is the most highly conserved between the human and Trichoplax proteins ( Fig. 1), again demonstrating strong evolutionary conservation of TaMdm2 function.
As a transcription factor, human p53 is predominantly located in the nucleus. Sequence alignment of Tap53 shows conservation of positively charged residues in the region of the known C-terminal bipartite human p53 nuclear localization sequence (NLS) 4,37 . Also, there is strong conservation of a hydrophobic motif in the tetramerization domain comprising the nuclear export signal (NES) 38 . Immunocytochemistry analysis shows both nuclear and cytoplasmic distribution of Tap53 in H1299 cells, suggesting the presence of functional NLS and NES motifs (Fig. 7). Additional analysis of 4 other divergent species (frog, zebrafish, elephant shark, lamprey) also indicated similar p53 staining patterns.

Discussion
The identification of p53-pathway components in the genome of Trichoplax adhaerans suggests exceptional evolutionary conservation in this early metazoan. We have recapitulated several of the core functions attributed to two of the key players in this pathway, a p53 family member and Mdm2. The p53 family member was shown to bind both a high affinity consensus p53 response element and a sequence identified in the promoter region of TaMdm2 with similarity to the canonical p53 response element. Human p53 was able to drive transactivation of a reporter gene using the Trichoplax response element. Mdm2 transcription in higher eukaryotes is regulated directly by p53 binding to response elements in its promoter. The functional sequences identified in Trichoplax therefore provide evidence towards conservation of the p53-Mdm2 feedback loop. Confirmation will require further studies in the native organism.
Optimal degradation of p53 by Mdm2 requires heterodimer formation via RING domain interaction with its paralog, Mdm4, in higher eukaryotes [39][40][41][42][43] . Both Mdm2 and Mdm4 likely arose from a gene duplication event more than 440 million years ago, prior to the emergence of vertebrates 44,45 . In common with other invertebrates including the sea squirt (C. intestinalis), acorn worm (S. kowalevskii), Florida lancelet (B. floridae), bay mussel (M. trossulus), and owl limpet (L. gigantean), only a single Mdm gene is evident in Trichoplax 44,46 . The product of this gene was able to degrade Tap53 in Mdm2/Mdm4-null TKO cells (Fig. 5), suggesting it can function independently. Homology modeling provides support for a TaMdm2 RING domain homodimer. This structure shows high similarity to a model of the TaMdm2-mouse Mdm4 RING domain heterodimer and the solved MDM2-MDM4 RING domain heterodimer structure (Fig. 8). The TaMdm2 homodimer is therefore likely to play similar functional roles in the degradation of p53, in agreement with the experimental results.
In TKO cells, the levels of Tap53 slightly decreased even in the absence of an Mdm2 protein and increased when co-expressed with catalytically inactive TaMdm2 variants (Fig. 5). This suggests the presence of another protein in TKO cells capable of targeting Tap53 for degradation. It is possible that binding of the TaMdm2 mutant to Tap53 shields it from degradation. Potential candidates include HUWE1 and COP1, ubiquitin ligases shown to target p53 independently of Mdm2/Mdm4 47,48 with homologues present in the Trichoplax genome.   The p53 family member identified in Trichoplax was not capable of significant reporter gene activation in a human cell line, despite translocation to the nucleus. Compared to other domains, the N-terminal transactivation domain of this family member shows the least homology with human p53. This would suggest an inability to interact with basal transcription factors that are recruited by p53. Also, there may be subtle contextual variation between human and Trichoplax p53 response elements that is not reflected in the human p53 reporter gene assay employed. As in Trichoplax, p53 family members identified in other divergent species such as the sea lamprey and mussel do not show transactivation in heterologous cells 45,46 .
Our results provide increasing evidence for the presence of a single functional p53 family member and an independently competent Mdm2 in Trichoplax adhaerens. The minimised p53 pathway set in this simple animal further validates its use as a reductionist model organism to study development and cancer.

Methods
Unless otherwise specified, all oligonucleotides and codon-optimised genes (derived from UniProt sequences B3RT05 and B3RZS6) were from Integrated DNA Technologies (Singapore), restriction enzymes from NEB and chemical reagents from Sigma. Amplification of pET22b with either jwConA-petF2, TXRe-540-F2, TXRe-320-F2 and TXRe-350-F2 with petR was carried out to create short DNA fragments with response elements ConA, TXRe530, TXRe320 and TXRe350 appended at the N-terminus. Control DNA (minus response element) was created by using petF2 and petR to amplify pET22b. Purified DNA was used for DNA binding assay (described below).

Immunoprecipitation of in vitro-expressed proteins and western blot analysis. Protein G beads
were incubated with anti-HA antibody (1 μ g per 10 μ L beads) for 1 hour in PBST-3%BSA and subsequently washed twice in PBST-0.1%BSA. IVT-expressed MDM2/TaMdm2 was incubated with the beads on a rotator for 30 mins and removed. Stapled peptides were added at indicated concentrations and incubation carried out for 45 mins before removal. IVT-expressed p53/Tap53/L11 was then added to the mixture and incubation allowed for 45 mins. Beads were finally washed thrice in PBST-0.1% BSA and thrice with PBS, and bound proteins eluted by resuspension in 20 μ L SDS-PAGE loading buffer and incubation at 95 °C for 5 minutes. Both the eluates and inputs were subjected to electrophoresis, transferred to nitrocellulose membranes and probed for p53 with horseradish peroxidase conjugated DO1 antibody (Santa Cruz), for Tap53/L11 with horseradish peroxidase conjugated anti-FLAG, or for MDM2/TaMdm2 with anti-HA antibody or followed by rabbit anti-mouse (Dakocytomation).
The western blots were identified by Clarity ™ ECL western blotting substrate (Bio-Rad).
DNA binding assay and real-time PCR. Protein G beads (5 μ L) (Invitrogen) were incubated with anti-FLAG or DO1 antibody (1 μ g per 10μ L beads) for 1 hour in PBST-3% BSA and subsequently washed twice in PBST-0.1% BSA. A 10 μ L mixture comprising 10-20 nM IVT-expressed p53/Tap53 (3 μ L in vitro expression mix added), 60 nM DNA fragments (containing ConA, TXRe320, TXRe350, TXRe540 response elements or control DNA) and water was prepared prior to incubation with the beads on a rotator for 1 hour at RT. Beads were finally washed thrice in PBST-0.1% BSA and thrice with PBS, and bound DNA eluted by resuspension in 20μ L nuclease-free water and incubation at 95 °C for 5 mins. Real-time PCR quantifications of the eluates were performed using 250nM each of primers petF3 and WpetR1 using iQTM SYBR ® Green Supermix (Bio-Rad Laboratories) and quantified via CFX96 Real-Time System CCD camera (Bio-Rad Laboratories). Data was interpreted as fold differences (calculated based on cycle threshold differences) over non-specific DNA binding control (control DNA).
Cell culture. H1299 p53 −/− cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% (v/v) foetal calf serum (FCS) and 1% (v/v) penicillin/streptomycin. The cells were seeded at 1.4 × 10 5 cells/ well in 6-well plates, 24 hours prior to transfection. Cells were co-transfected with different combinations of MDM2/TaMdm2/p53/Tap53 plasmid using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For β -galactosidase assay, 2ConA/TXRe540 LacZ reporter plasmid and luciferase transfection efficiency plasmid were also included during transfection. In all cases, the total amount of plasmid DNA transfected per well was equilibrated by addition of the parental vector pcDNA3.1a(+ ). Mouse embryonic fibroblast p53/Mdm2 double-knockout (DKO) and p53/Mdm2/Mdm4 triple-knockout (TKO) cells (kind gifts from Guillermina Lozano) were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% (v/v) foetal calf serum (FCS) and 1% (v/v) penicillin/streptomycin. To ensure a high transfection efficiency for reproducible amounts of p53 protein, DKO and TKO cells were co-transfected with Tap53Δ , TaMdm2 and vector plasmids using Nucleofector ® II (Lonza, Singapore) according to the manufacturer's instructions for mouse embryonic fibroblasts with MEF2 Nucleofector solution and program A-023. In all cases, the total amount of plasmid DNA transfected per well was equalized by addition of parental vector. Where required, four hours prior to harvesting, cells were treated with proteasome inhibitor MG132 (Calbiochem/Merck/Millipore, Singapore).  supplemented with both protease and phosphatase inhibitors. 10 μ L of anti-HA antibody-coated protein G Dynabeads was used per reaction. Beads were washed twice with PBST-0.1% BSA and incubated with 200 μ g of cell lysate on a rotator at 4 °C overnight before washing thrice in PBST-0.1% BSA and thrice with PBS, and bound proteins eluted by resuspension in 20 μ L SDS-PAGE loading buffer and incubation at 95 °C for 5 minutes.

Immunoprecipitation of cell lysates
Immunoblotting was carried out with the relevant antibodies and identified by Clarity ™ ECL Western Blotting Substrate). 5 μ g of H1299 p53 −/− cell lysate per reaction was also used to check expression levels of relevant proteins via western blot. DKO/TKO cells were harvested 24 hours after transfection by lysis with 95 °C SDS buffer (20mM Tris pH 8, 2% SDS and 10% glycerol) and sonicated. Following protein concentration determination by BCA Protein Assay (Thermo Scientific Pierce, Singapore), equal protein amounts of sample were added to LDS-PAGE loading buffer and incubated at 95 °C for 5 minutes. The proteins were then separated by SDS-PAGE and transferred to nitrocellulose. Immunoblotting was carried out with the relevant antibodies (anti-FLAG for Tap53Δ , anti-HA for TaMdm2 and anti-β -actin for loading control). β-Galactosidase assay. H1299 p53 −/− cells were harvested 24hours after transfection and β -galactosidase activities were assessed using the Dual-light System (Applied Biosystems) according to the manufacturer's protocol. The β -galactosidase activity was normalized with luciferase activity for each sample.
Images were acquired on an inverted microscope (Olympus IX83) using bright field with an LUCPLFLN-40 × PH, NA 0.60 objective and a camera (Hamamatsu ORCA -Flash 4.0). Images were acquired at a resolution of 2048 × 2048 pixels, 16-bit grey scale and exported as TIFF using cellSens Dimension software (version 1.15). Images were cropped to 1200 × 1200 pixels and scale bar was added in Fiji ImageJ2 and then reduced in size for figure assembly using Illustrator (CS5; Adobe).