Fig. 4

Fig. 4

ZBTB11 is regulated by myeloid transcription factors and directly represses TP53.

(a) Transient co-transfection of human ZBTB11 2.9 kb promoter luciferase reporter and transcription factors into 293T cells shows ZBTB11 is regulated by PU.1 (positively) and GFI1a/b (negatively). Triangles represent increasing concentration of transcription factors (n=3 experiments; mean ±s.e.m.; two-way ANOVA). (b) A zebrafish zbtb11 2.3 kb promoter reporter is positively regulated by Pu.1 and C/ebpα, and negatively regulated by all three Gfi1 paralogs. Triangles represent increasing concentration of transcription factors (n=3 experiments; mean ±s.e.m.; two-way ANOVA). (c) ChIPseq shows PU.1 occupies the Zbtb11 locus in mouse granulocytes at the promoter, 5′ untranslated region of exon 1 and within intron 1. (d) Whole-mount in situ hybridization shows overexpression of Δ113p53 in the brain at 48 h.p.f. in mne but not phenotypically WT sibling embryos. (e) Transient co-transfection of ZBTB11 and a human TP53 luciferase reporter into 293T cells shows direct repression of TP53 by ZBTB11. Triangle represents increasing concentration of ZBTB11 (n=3 experiments; mean ±s.e.m.; two-way ANOVA). (f) ZBTB11 is enriched at the TP53 locus by ChIP–qPCR in human K562 (endogenous ZBTB11) and 293T HEK cells (overexpressed mouse ZBTB11). Using four primer sets tiled across the TP53 promoter, primer set 1 yields little enrichment over normal rabbit serum control, while primer sets 2–4 show 12–25-fold enrichment (K562: n=5 experiments, mean ±s.e.m.; 293T: n=2 experiments, mean ±s.e.m.). (g) Antisense morpholino oligonucleotide knockdown of tp53 suppresses excessive apoptosis and increases neutrophil number in mne embryos. (h) Quantification of mne neutrophils in control and tp53 morphants (n=3 experiments; mean ±s.e.m.; two-tailed paired t-test). (i) Quantification of mne neutrophils in tp53 WT and tp53^M214K/M214K at 2 and 5 d.p.f.; (n=3 experiments; two-tailed paired t-test). (j) Cell death marked by Annexin secA5-mVenus is prominent in mne CNS on tp53 WT background and rescued on mne/ tp53^M214K/M214K. (k) 2 × 2 Contingency table χ² analysis shows rescue of CNS cell death in mne on the tp53^M214K/M214K mutant background. Data for three independent experiments; Exp1, n=13, 14; Exp2, n=9, 20; Exp3, n=11, 17; exact P values are shown. Where indicated: *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001; scale bars, 300 μm (d), 200 μm (g,j).