Fig 2: (A) Schematic drawing of ZBTB11 (O95625) with functional domains and position of the pathogenic variants identified in the current study and in literature. Families A and B were described by Fattahi et al.5; individual UPN-0706 was described by Monies et al.6 aa = amino acid. (B) Homology model of Znf domain 11 and 12 (in grey) in the human ZTBT11 in complex with DNA. Thr890 and Arg912 (in red) are located in the β-hairpin of Znf domain 11 and in the linker region between the domains, respectively.

Fig 3: Cys116 is central in the HHCC domain and required for TP53 repression.(a) A pair of conserved His and Cys residues (in red) in Zbtb11 align with those in the HHCC domain of integrase genes, and two human genes, GIN1 and NYNRIN. Blue, conserved amino acids. (b) Mutation of each His or Cys residue (H79A, H86A, C116S, C119S) or all four (HHCC), but not mutation of a non-conserved gln (Q98A) abrogates Zbtb11 bioactivity in an in vivo mne rescue bioassay. (n≥3 experiments; mean ±s.e.m.; χ2 analysis; ****P≤0.0001). (c) Deletion of N terminus (HBTBZF, BTBZF) or zinc fingers (NBTB, NBTB+) does not abrogate rescue by Zbtb11. (n≥3 experiments; mean ±s.e.m.; χ2 analysis; ****P≤0.0001). Yellow box, HHCC domain; blue box, BTB domain; red box, zinc finger domain. (d) Modelling of amino acids 77–123 of zebrafish Zbtb11 using the integrase HHCC structure predicts a new domain in Zbtb11 that can form a zinc finger with each of the paired His and Cys residues, including Cys116 (cerise), coordinating a central metal ion (blue sphere). Green, homology model; grey, template. Conserved His and Cys residues are shown as thick sticks in the homology model. (e) Transient co-transfection of ZBTB11–C116S and a human TP53 or ZBTB11 luciferase reporter into 293T cells shows Cys116 is indispensible for the TP53 and autoregulatory repressor function of ZBTB11 (n=3 experiments; mean ±s.e.m.; two-way ANOVA; *P≤0.05; **P≤0.01). The WT ZBTB11 data are identical to Fig. 4e because it was the contemporaneous control for this C116S data.

Fig 4: Zbtb11 expression and mne phenotype including delayed neutrophil maturation.(a) Whole-mount in situ hybridization (WISH) showing widespread expression of Zbtb11 in the developing embryo up until 19 h.p.f., which becomes progressively restricted up until 80 h.p.f. Arrows indicate Zbtb11 expression in the intermediate cell mass (ICM). (b) At 96 h.p.f., mne exhibits ocular, craniofacial and cardiovascular defects. e, eye; h, heart; m, mandibular cartilage. (c) Injection of rhodamine at 48 h.p.f. shows enlarged dye volume in fourth ventricle in mne compared to WT. (d) Loss of rag1 expression in mne at 82 h.p.f. compared to WT. Foxn1 marking the thymic primordium is expressed in mne and WT. (e) RT–qPCR of Zbtb11 expression in FACS sorted adult zebrafish blood cell populations (mean±s.d.; *P≤0.05; n=1 experiment; triplicate replicates on cDNA isolated from purified haemopoietic populations derived from pooled kidney marrows). Ery, erythroid; Lym, lymphoid; Mye, myeloid; Pre, precursors; WKM, whole kidney marrow; Mann–Whitney test. (f) Immunoblot showing ZBTB11 is expressed in human myeloid and lymphoid cell lines and with lower expression in HepG2 hepatocytes (50 μg protein per lane); M, protein ladder with molecular weight in kDa as indicated. (g) Examples of FACS-sorted neutrophils from mne and WT following May–Grünwald Giemsa staining. (h) Quantification of neutrophil sub-populations in mne and WT according to maturity shown as percentage of total cells counted. Schema below graph defines how sub-populations were scored. Gran., granulocytes. n=3 biologically independent experiments (mean±s.e.m.); (a) whole embryo scale70; scale bars, 200 μm (b–d,g).

Fig 5: GO enrichment analysis of biological process terms among the genes targeted by ZBTB11 in ChIP-seq experiment in control fibroblasts. ChIP-seq data from controls showed that ZBTB11 targets genes encoding proteins involved in mitochondrial functions and in RNA processes. Gene concept network plot (cnet plot) of the 238 genes associated to the ChIP-seq consensus peaks in controls depicts the linkages between genes and biological processes. Two main clusters of biological processes were identified: mitochondrial gene expression and RNA metabolic processes. The node sizes are dependent on the number of genes associated with the biological process as indicated. We used ClusterProfiler (R Bioconductor package) enrichment tool to perform analysis of over-representation of GO terms linked with our list of 238 significant genes identified in association with the differentially bound peaks. The GO enrichment analysis of biological process terms was subsequently visualized using the gene concept network plot (cnet plot).