p21, ccng1, foxo3b and fbxw7 contribute to p53-dependent cell-cycle arrest [MPNP timecross]

p53 is a transcription factor that plays a critical role in cancer prevention. However, the mechanisms by which p53 exerts its tumor-suppressive function is still unclear. While PUMA/BBC3 and NOXA/PMAIP1 are known to be important in p53-dependent apoptosis, and p21/CDKN1A is crucial for p53-dependent cell-cycle arrest, we demonstrate that zebrafish lacking puma, noxa, and p21 do not show a predisposition to cancer. This suggests that additional p53 transcriptional targets are sufficient for its tumor suppressive function. Contrary to the prevailing belief that p21 is the key regulator of p53-dependent cell-cycle arrest, we provide evidence that p53 can still induce cell-cycle arrest in the absence of p21, following DNA damage or loss of mdm2 (p53 activation in the absence of stress). This implies the involvement of other p53 transcriptional targets in mediating p53-dependent cell-cycle arrest. Since p53 tumor suppression is conserved across multiple vertebrate species, we conducted a cross-species comparative analysis of p53-dependent transcriptional profiles to identify a conserved set of 136 p53-upregulated transcripts. Our analysis stresses the importance of ortholog to paralog analysis across species, since in many cases the paralog but not ortholog in differing species is p53 dependent. Additionally, we performed a CRISPR/Cas9 G0 “crispant” screen in a genetic background lacking mdm2, puma, noxa, and p21 to identify key components involved in p53-dependent cell-cycle arrest. Our findings revealed that ccng1, fbxw7, and foxo3b play an important role in this process. Overall design: RNA-sequencing was performed on zebrafish of un-genotyped progeny from mdm2+/-; pnp-/-intercrosses at various developmental stages (8, 10, 12, 14, 16 hpf) as the mutant group (N=~50 embryos for each group, referred to as MPNP). While this approach generated a dilution of RNA transcripts, since only approxmiately ¼ of the embryos were mpnp-/-, it nonetheless allowed us to glimpse at the dynamics of p53 target gene induction in mpnp-/- animals. Same-stage progeny from pnp-/- intercrosses were harvested to extract RNA for comparison, referred to as PNP.