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The Pu.1 target gene Zbtb11 regulates neutrophil development through its integrase-like HHCC zinc finger

View Article: PubMed Central - PubMed

ABSTRACT

In response to infection and injury, the neutrophil population rapidly expands and then quickly re-establishes the basal state when inflammation resolves. The exact pathways governing neutrophil/macrophage lineage outputs from a common granulocyte-macrophage progenitor are still not completely understood. From a forward genetic screen in zebrafish, we identify the transcriptional repressor, ZBTB11, as critical for basal and emergency granulopoiesis. ZBTB11 sits in a pathway directly downstream of master myeloid regulators including PU.1, and TP53 is one direct ZBTB11 transcriptional target. TP53 repression is dependent on ZBTB11 cys116, which is a functionally critical, metal ion-coordinating residue within a novel viral integrase-like zinc finger domain. To our knowledge, this is the first description of a function for this domain in a cellular protein. We demonstrate that the PU.1–ZBTB11–TP53 pathway is conserved from fish to mammals. Finally, Zbtb11 mutant rescue experiments point to a ZBTB11-regulated TP53 requirement in development of other organs.

No MeSH data available.


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 tp53M214K/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/ tp53M214K/M214K. (k) 2 × 2 Contingency table χ2 analysis shows rescue of CNS cell death in mne on the tp53M214K/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).
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f4: 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 tp53M214K/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/ tp53M214K/M214K. (k) 2 × 2 Contingency table χ2 analysis shows rescue of CNS cell death in mne on the tp53M214K/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).

Mentions: Zbtb11-dependent transcriptional networks and its upstream genetic regulators have not been defined. To determine where Zbtb11 is placed with regard to the known haemopoietic transcriptional hierarchy, 2.9 kb of the human ZBTB11 promoter and 2.3 kb of the zebrafish zbtb11 promoter were cloned and assayed for activity in the presence and absence of increasing concentrations of different human or zebrafish haemopoietic transcription factors in 293 human embryonic kidney (HEK) cells. The myeloid specification determinant Pu.1 (ref. 21) positively regulated both zebrafish and human ZBTB11 promoter reporters, whereas the erythroid transcription factor Gata1 did not (Fig. 4a,b), further supporting a myeloid-specific role for ZBTB11. Likewise, GFI1 and C/EBPα transcription factors, also implicated in myeloid specification22, respectively repressed and activated both zebrafish and human ZBTB11 promoter reporters in a dose-dependent manner (Fig. 4a,b). These findings are consistent with published chromatin immunoprecipitation (ChIP) sequencing data examining genome-wide loci occupancy for a series of haemopoietic transcription factors including PU.1 and GFI1 in mouse HPC7 haemopoietic progenitor cells23, and functionally demonstrate regulation of the ZBTB11 promoter specifically by these myeloid regulators. In addition, ChIP sequencing of mouse granulocyte chromatin demonstrated PU.1 occupancy at the Zbtb11 locus (Fig. 4c) in granulocytes. In mne neutrophils, canonical Pu.1 (Spi1b) expression is slightly elevated compared to WT at 48 h.p.f. (logFC=0.57; FDR=0.043), which could indicate Pu.1 modulation by a Zbtb11-mediated potential negative feedback loop, though this remains to be explored. Collectively, these data identify a new myeloid transcription factor-ZBTB11 axis that is evolutionarily conserved in fish and mammals.


The Pu.1 target gene Zbtb11 regulates neutrophil development through its integrase-like HHCC zinc finger
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 tp53M214K/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/ tp53M214K/M214K. (k) 2 × 2 Contingency table χ2 analysis shows rescue of CNS cell death in mne on the tp53M214K/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).
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f4: 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 tp53M214K/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/ tp53M214K/M214K. (k) 2 × 2 Contingency table χ2 analysis shows rescue of CNS cell death in mne on the tp53M214K/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).
Mentions: Zbtb11-dependent transcriptional networks and its upstream genetic regulators have not been defined. To determine where Zbtb11 is placed with regard to the known haemopoietic transcriptional hierarchy, 2.9 kb of the human ZBTB11 promoter and 2.3 kb of the zebrafish zbtb11 promoter were cloned and assayed for activity in the presence and absence of increasing concentrations of different human or zebrafish haemopoietic transcription factors in 293 human embryonic kidney (HEK) cells. The myeloid specification determinant Pu.1 (ref. 21) positively regulated both zebrafish and human ZBTB11 promoter reporters, whereas the erythroid transcription factor Gata1 did not (Fig. 4a,b), further supporting a myeloid-specific role for ZBTB11. Likewise, GFI1 and C/EBPα transcription factors, also implicated in myeloid specification22, respectively repressed and activated both zebrafish and human ZBTB11 promoter reporters in a dose-dependent manner (Fig. 4a,b). These findings are consistent with published chromatin immunoprecipitation (ChIP) sequencing data examining genome-wide loci occupancy for a series of haemopoietic transcription factors including PU.1 and GFI1 in mouse HPC7 haemopoietic progenitor cells23, and functionally demonstrate regulation of the ZBTB11 promoter specifically by these myeloid regulators. In addition, ChIP sequencing of mouse granulocyte chromatin demonstrated PU.1 occupancy at the Zbtb11 locus (Fig. 4c) in granulocytes. In mne neutrophils, canonical Pu.1 (Spi1b) expression is slightly elevated compared to WT at 48 h.p.f. (logFC=0.57; FDR=0.043), which could indicate Pu.1 modulation by a Zbtb11-mediated potential negative feedback loop, though this remains to be explored. Collectively, these data identify a new myeloid transcription factor-ZBTB11 axis that is evolutionarily conserved in fish and mammals.

View Article: PubMed Central - PubMed

ABSTRACT

In response to infection and injury, the neutrophil population rapidly expands and then quickly re-establishes the basal state when inflammation resolves. The exact pathways governing neutrophil/macrophage lineage outputs from a common granulocyte-macrophage progenitor are still not completely understood. From a forward genetic screen in zebrafish, we identify the transcriptional repressor, ZBTB11, as critical for basal and emergency granulopoiesis. ZBTB11 sits in a pathway directly downstream of master myeloid regulators including PU.1, and TP53 is one direct ZBTB11 transcriptional target. TP53 repression is dependent on ZBTB11 cys116, which is a functionally critical, metal ion-coordinating residue within a novel viral integrase-like zinc finger domain. To our knowledge, this is the first description of a function for this domain in a cellular protein. We demonstrate that the PU.1–ZBTB11–TP53 pathway is conserved from fish to mammals. Finally, Zbtb11 mutant rescue experiments point to a ZBTB11-regulated TP53 requirement in development of other organs.

No MeSH data available.