- Research article
- Open Access
Differential regulation of p53 function by the N-terminal ΔNp53 and Δ113p53 isoforms in zebrafish embryos
© Davidson et al; licensee BioMed Central Ltd. 2010
- Received: 4 May 2010
- Accepted: 7 October 2010
- Published: 7 October 2010
The p53 protein family coordinates stress responses of cells and organisms. Alternative promoter usage and/or splicing of p53 mRNA gives rise to at least nine mammalian p53 proteins with distinct N- and C-termini which are differentially expressed in normal and malignant cells. The human N-terminal p53 variants contain either the full-length (FL), or a truncated (ΔN/Δ40) or no transactivation domain (Δ133) altogether. The functional consequences of coexpression of the different p53 isoforms are poorly defined. Here we investigated functional aspects of the zebrafish ΔNp53 ortholog in the context of FLp53 and the zebrafish Δ133p53 ortholog (Δ113p53) coexpressed in the developing embryo.
We cloned the zebrafish ΔNp53 isoform and determined that ionizing radiation increased expression of steady-state ΔNp53 and Δ113p53 mRNA levels in zebrafish embryos. Ectopic ΔNp53 expression by mRNA injection caused hypoplasia and malformation of the head, eyes and somites, yet partially counteracted lethal effects caused by concomitant expression of FLp53. FLp53 expression was required for developmental aberrations caused by ΔNp53 and for ΔNp53-dependent expression of the cyclin-dependent kinase inhibitor 1A (CDKN1A, p21, Cip1, WAF1). Knockdown of p21 expression markedly reduced the severity of developmental malformations associated with ΔNp53 overexpression. By contrast, forced Δ113p53 expression had little effect on ΔNp53-dependent embryonal phenotypes. These functional attributes were shared between zebrafish and human ΔNp53 orthologs ectopically expressed in zebrafish embryos. All 3 zebrafish isoforms could be coimmunoprecipitated with each other after transfection into Saos2 cells.
Both alternative N-terminal p53 isoforms were expressed in developing zebrafish in response to cell stress and antagonized lethal effects of FLp53 to different degrees. However, in contrast to Δ113p53, forced ΔNp53 expression itself led to developmental defects which depended, in part, on p21 transactivation. In contrast to FLp53, the developmental abnormalities caused by ΔNp53 were not counteracted by concomitant expression of Δ113p53.
- Ionize Radiation
- Acridine Orange
- Zebrafish Embryo
- Morphological Aberration
- Abridge Universal Amplification Primer
The p53 tumor suppressor coordinates the response of cells to genotoxic insults and other forms of cell stress either by inducing cell cycle arrest and allowing time for DNA repair or by causing elimination of damaged cells through apoptosis. The central role of p53 in cell stress responses lends significance to recent reports that p53 exists as a family of at least 9 different isoforms with different N- and C-termini due to differential splicing and promoter usage . Three human N-terminal p53 isoforms are known containing either the full-length transactivation domain (FL), or a truncated transactivation domain (ΔN/p47) or no transactivation domain altogether (Δ133). The functional importance of some of these isoforms in vertebrate cells and organisms has only recently come into focus. Specifically, transgenic overexpression of ΔNp53 in mice leads to premature aging associated with increased expression of the CDK inhibitor CDKN1/WAF1/p21 and reduced proliferation . In addition, both alternative N-terminal p53 isoforms have been observed to be differentially expressed in human tumor cell lines [1, 3–5]. Finally, the zebrafish ortholog of the human Δ133p53 isoform (Δ113) is induced by genotoxic stress, counteracts FLp53 function and protects zebrafish embryos against deleterious consequences of genotoxic stress . Whereas ΔNp53 expression is induced in an MDM2-dependent fashion in human colon carcinoma cells , expression and functional attributes of ΔNp53 in stress responses at the level of the whole organism have yet to be reported.
The present study seeks to investigate functional attributes of ΔNp53 in developing zebrafish embryos. We cloned the zebrafish ΔNp53 ortholog and, like Δ113/133p53, found it to be induced at the mRNA level by ionizing radiation (IR). Ectopic expression of zebrafish ΔNp53(z) isoform modulated the functional consequences of FLp53 expression in concert with Δ133p53 in zebrafish embryos. Furthermore, human ΔNp53(h) phenocopied the effects of zebrafish ΔNp53(z) when overexpressed in zebrafish embryos.
Identification and expression of zebrafish ΔNp53
Oligonucleotide sequences used for RT-PCR, RT-qPCR analyses and morpholino design
Primers for ΔNp53 PCR amplification (see Fig. 1A)
Primers for quantitative PCR (see Fig. 5)
Primers for semiquantitative PCR (see Figs. 1C/2A)
ΔNp53 mismatch control
Effects of forced ΔNp53 expression on zebrafish development
Combined effects of ectopic ΔNp53 and Δ113p53 isoform expression on FLp53-induced embryonal lethality
Previous work assessed the effects of transgenic overexpression of either ΔNp53 in mice  or Δ113p53 expression in zebrafish . However, it is not known how simultaneous expression of the two alternative N-terminal p53 variants affects p53 responses of the whole organism. This is a relevant question because we observed coincident induction of ΔNp53 and Δ113p53 expression upon IR exposure of zebrafish embryos (Fig. 1C). To address this question directly we examined the consequences of ectopic expression of both alternative isoforms together with FLp53. To this end, we injected triple mRNA combinations encoding the different isoforms at different ratios ranging between 0.1 - 2 ng/embryo (Fig. 4A). These experiments confirmed that Δ113p53 was more efficient than ΔNp53 in rescuing FLp53-induced lethality. They further revealed that a combination of both alternative N-terminal isoforms does not add to the extent of rescue achievable with ΔNp53 alone (1 ng/ml). It is possible that morphological aberrations induced by ectopic ΔNp53 expression alone restrain the extent of rescue achievable by Δ113p53 expression. In contrast to ΔNp53 and as described previously , ectopic Δ113p53 expression, even at very high levels (2 ng/embryo) had no apparent effect on zebrafish morphology or development (not shown). Finally, ectopic expression of Δ113p53 (1 ng/embryo) did not affect the incidence of ΔNp53-dependent hypoplasia although the two isoforms could be coimmunoprecipitated (Fig. 4B and Additional file 2). At present, it is unclear why forced expression of Δ113p53 clearly counteracts the effects of FLp53 on zebrafish survival but not those of ΔNp53. It seems possible that the two alternative N-terminal isoforms in combination affect transcription of a subset of target genes that are deleterious to the developing embryo.
The ΔNp53 isoform regulates p53 target gene expression in concert with FLp53
This study demonstrates that IR exposure of zebrafish embryos increased steady-state transcript levels of the two known alternative N-terminal p53 isoforms, Δ113p53 and ΔNp53. These isoforms shared the ability to counteract lethal effects of FLp53 expression in the developing fish. However, in contrast to Δ113p53, forced expression of ΔNp53 induced morphological aberrations itself, notably hypoplasia of the head and somites associated with a moderate degree of lethality. These effects of ΔNp53 were contingent on the presence of FLp53 and due, in part, to p21 induction. Furthermore, the developmental aberrations caused by forced expression of ΔNp53 limited the extent of rescue of FLp53-induced lethality by this isoform either alone or in combination with Δ113p53. Future work will address the relevance of ΔNp53 to the genotoxic stress response of specific tissues as induction of p53 targets occurs in a tissue-specific manner in mice  and ΔNp53 is expressed differentially in normal human tissues and malignancies .
Zebrafish Husbandry and Radiation Protocol
Zebrafish husbandry and embryo maintenance were performed according to standard procedures as published previously [17, 18] and with approval by the IACUC at Thomas Jefferson University. Zebrafish were maintained at 28.5°C on a 14-h light/10-h dark cycle. Embryos were irradiated (20 Gy) at 24 hours post fertilization (hpf) using a 250 kVp X-ray machine (PanTak) at 50 cm source-to-skin distance with a 2-mm aluminum filter. Dosimetric calibration was performed before each experiment using a thimble ionization chamber with daily temperature and pressure correction.
G-capped mRNA production
Generation of G-capped mRNA was performed using the mMessage-mMachine-sp6 kit (Ambion). Specifically, cDNA sequences were cloned into the pCS2+ vector containing an upstream sp6 promoter and a downstream SV40 polyA sequence. The vector was linearized using Sac II restriction enzyme (Promega) and 1 μg of linear plasmid was used as template for in vitro transcription according to the manufacturer's protocol. The mRNA was precipitated and diluted to 1 μg/μl in water prior to injection.
3 Prime Race, RT-PCR and RT-qPCR
The ΔNp53 transcript was cloned using the 3' RACE System (Invitrogen). First strand cDNA synthesis was performed using the Abridged Universal Amplification Primer (AUAP) and I2. A 1.7 kb amplification product consistent with ΔNp53 was isolated and purified for nested PCR using the primer pairs I2/E5 and I2/E11 yielding the expected 490 bp and 1.2 kb amplicons. The full-length 1.7 kb ΔNp53 cDNA was validated by direct sequencing.
For RT-PCR, RNA was prepared from 30-60 embryos using standard procedures. First strand cDNA synthesis for RT-PCR was done using Random Hexamers and AMV Reverse Transcriptase (Promega Madison). PCR was performed using Taq DNA Polymerase (Promega) and the GeneAMP PCR System 9700 (Applied Biosystems) with the following conditions: denaturation 95°C, 30 s; annealing 58°C, 30 s; extension 72°C, 1 min for 35 cycles. PCR products were analyzed in 1-2% agarose gels containing ethidium bromide. For primer sequences please refer to Table 1.
First strand cDNA synthesis for qRT-PCR was done using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). QRT-PCR was performed using the Power Syber Green PCR Master Mix and the ABI 7900 HT Sequence Detection System (Applied Biosystems) using the following conditions: denaturation 95°C, 30 s; annealing 55°C, 30 s; extension 72°C, 1 min for 40 cycles. For primer sequences please refer to Table 1. Relative mRNA expression levels were calculated using microinjection of control mRNA encoding green fluorescent protein (GFP).
Reporter Gene Assay
The zebrafish ΔNp53 5'UTR sequence containing exons 1, 2 and intron 2 including the putative start site M2 and followed by an XhoI site was synthesized (DNA2.0 Menlo Park, CA) and inserted into the pCS2+ vector using BamHI-XhoI. The firefly luciferase gene was inserted into the XhoI/SnaBI site of pCS2+. ARCA-capped ΔNp535'UTR-Luc mRNA was generated using mMessage-mMachine-sp6 kit (Ambion) and injected (1 ng/embryo) into 1-2 cell stage embryos. As a negative control the putative alternative start site (M2) was substituted with GGG encoding glycine by site-directed mutagenesis (Stratagene). Embryos (8 hpf) were assayed for luciferase activity using the Dual Luciferase Assay kit (Promega) and a luminometer (Turner Biosystems).
Acridine orange staining of whole embryos
Embryos were stained with acridine orange as previously described with minor modifications . To quantify apoptosis we used IMAGEJ software (NIH) to measure AO fluorescence intensity. Quantification of AO fluorescence intensity was determined by first converting the original images to greyscale. Using the Green stack (converted into greyscale) the embryo body excluding the yolk sac was selected. The image threshold was calibrated to control embryos to the point where controls exhibited the least pixel intensity within the outlined area. The ratio of white pixel area (representing green fluorescence intensity) to total area outlined was determined.
Coimmunoprecipitation of zebrafish p53 isoforms was carried out after cotransfection into Saos-2 cells. Cotransfections were performed using pCS2+ expression vectors containing the following N-terminally tagged p53 isoform sequences in combination: 1) HA-FLp53 and Myc-ΔNp53, 2) HA-FLp53 and Myc-Δ113p53, 3) HA-Δ113p53 and Myc-ΔNp53. All transfections were done using the Fugene 6 Transfection Reagent (Roche, Germany) at a ratio of 3:1 (Fugene 6: DNA). Protein extracts were prepared 48 h post transfection and incubated with agarose beads conjugated with Anti-HA tag antibody. Immunoprecipitations were performed using the HA Tag IP/Co-IP kit (Pierce, Rockford IL). Interacting p53 isoforms were detected by immunoblot with Anti-Myc tag antibody. Coimmunoprecipitation of protein lysates from Saos-2 cells cotransfected with pCS2+ expression vectors encoding HA-Akt  and Myc-ΔNp53 was used as a negative control. Coimmunoprecipitation experiments were performed in triplicate and representative results are shown.
UR and APD acknowledge support from the National Institutes of Health. WRD received a RuthL.Kirschstein fellowship (CA119951). Additional support was from the State of Pennsylvania, the Radiation Therapy Oncology Group and the Christine Baxter Foundation. The Zebrafish facility at the Department of Biochemistry at Thomas Jefferson University is gratefully acknowledged. The pCS2+ vector was a gift from Dr. Dave Turner, University of Michigan.
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