The loss of p53-AS isoforms affects Ackr4 expression in Eμ-Myc male mice.
(A) B-cell subpopulations in spleens of 6 weeks-old p53+/+ Eμ-Myc and p53ΔAS/ΔAS Eμ-Myc mice. Means ± SEM (n=6 per genotype). (B-C) RNAseq analysis of spleens from p53+/+ Eμ-Myc (n=3) and p53ΔAS/ΔAS Eμ-Myc (n=4) 4-6 weeks-old male mice. Volcano plot (B), with differentially expressed genes (DEGs) in red. Unsupervised clustering heat-map plot (C), with DEGs ranked according to mean fold changes, and protein-coding genes in bold. (D) RT-qPCR analysis of candidate DEGs from spleens of p53+/+ Eμ-Myc males and p53ΔAS/ΔAS Eμ-Myc males. Means ± SEM (n=3-4 per genotype). (E) RT-qPCR analysis of indicated DEGs from spleens of 4-6 weeks-old p53+/+ Eμ-Myc males, p53ΔAS/ΔAS Eμ-Myc males, p53+/+ Eμ-Myc females and p53ΔAS/ΔAS Eμ-Myc females. Means ± SEM (n=3-4 per sex and genotype). (F) Ackr4 is transactivated by p53 in response to stress. Ackr4 mRNAs in untreated or doxorubicin-treated WT and p53-/- MEFs. Data from 2-3 MEFs per genotype (Younger et al., 2015). (G) A putative p53 response element in Ackr4 intron 1. Top: Map of the Ackr4 gene. (boxes: exons (brown box: translated region), black line: intron 1); middle: p53 ChIP in doxorubicin-treated MEFs according to ChIP-Atlas (SRX270554) (Oki et al., 2018); bottom: p53 Response Element (p53RE) consensus sequence (R=G or A, W=A or T, Y=C or T), the putative p53RE and its mutated counterpart. (H) Luciferase assays of the candidate p53RE. A 1.5 kb fragment containing the WT or mutant p53RE was cloned upstream a luciferase reporter, then transfected into p53-/- MEFs together with an expression plasmid for full length p53 (FL), p53-AS or the DNA-binding mutant p53R270H (RH). Means ± SEM (n=4-6). (I) In MEFs, p53 activation leads to an increased Ackr4 expression attenuated by estradiol. Ackr4 and Cdkn1a mRNAs were quantified by RT-qPCR from p53+/+ and p53ΔAS/ΔAS MEFs, untreated or treated with 10 μM Nutlin and/or 5 μg/ml 17-β estradiol (E2). Means ± SEM from 4 independent experiments. In A, D, E, F, H, I ***P<0.001, **P<0.01, *P<0.05, °P≤0.057, ns: non-significant in Student’s t or Mann-Whitney tests.