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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.


Related in: MedlinePlus

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).
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f2: 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).

Mentions: Zbtb11 is maternally deposited and then widely expressed early in development (Fig. 2a). After 24 hours post fertilization (h.p.f.), its expression wanes at many sites but is retained in the nervous system. Consistent with its expression pattern, mne also has a multisystem embryonic lethal phenotype including impaired craniofacial development (Fig. 2b) and hydrocephalus (Figs 1a and 2c). Injection of rhodamine dye into the fourth ventricle clearly shows its enlargement in mne compared to WT (Fig. 2c). However, early in haemopoietic development when primitive haemopoiesis prevails, mne displays a highly specific, lineage-restricted, myeloid phenotype. Consistent specifically with the myeloid-failure phenotype of mne, Zbtb11 is expressed in the zebrafish haemopoietic intermediate cell mass (Fig. 2a). Several pointers indicate an ongoing requirement for Zbtb11 in sustaining definitive haemopoiesis. By 5 d.p.f., when there is strong local expression of rag1-expressing T-cells in the thymus in the WT, mne lacks rag1 expression in the thymus despite development of the thymic primordia as marked by foxn1 (Fig. 2d). Thrombocyte numbers are also reduced by 82 h.p.f. (Supplementary Fig. 2e). Despite normal specification of HSCs, as defined by cells expressing runx1 and myb along the ventral wall of the dorsal aorta (Supplementary Fig. 2b), myb expression is absent in mne caudal haemopoietic tissue at 72 and 96 h.p.f., suggesting that maintenance of the stem cell pool is also disrupted in mne (Supplementary Fig. 2c,d). This indicates a broader failure to sustain definitive haemopoiesis later in development and highlights the sensitivity of granulocytes as the first lineage affected in mne. PCR with reverse transcription (RT–PCR) of fluorescence-activated cell sorting (FACS)-sorted adult zebrafish kidney marrow confirms expression of Zbtb11 in adult haemopoietic cells, with highest levels in myeloid cells (Fig. 2e). Consistent with public domain RNA expression profiles1718, we have confirmed that ZBTB11 protein is highly expressed in human Jurkat (T cells), K562 cells (a BCR-ABL positive blast crisis erythroleukaemia) and HL60 (promyelocytic leukaemia) cells (Fig. 2f). Lower expression in HepG2 liver cancer cells correlates with hepatocellular carcinoma expression profiling showing very low ZBTB11 expression20. FACS-sorted embryonic neutrophils from mne and WT stained with May-Grünwald Giemsa exhibited an abnormally higher proportion of immature neutrophils in mne. Hence, there is both a quantitative and qualitative myeloid development defect as a result of Zbtb11 dysfunction (Fig. 2g,h).


The Pu.1 target gene Zbtb11 regulates neutrophil development through its integrase-like HHCC zinc finger
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).
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f2: 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).
Mentions: Zbtb11 is maternally deposited and then widely expressed early in development (Fig. 2a). After 24 hours post fertilization (h.p.f.), its expression wanes at many sites but is retained in the nervous system. Consistent with its expression pattern, mne also has a multisystem embryonic lethal phenotype including impaired craniofacial development (Fig. 2b) and hydrocephalus (Figs 1a and 2c). Injection of rhodamine dye into the fourth ventricle clearly shows its enlargement in mne compared to WT (Fig. 2c). However, early in haemopoietic development when primitive haemopoiesis prevails, mne displays a highly specific, lineage-restricted, myeloid phenotype. Consistent specifically with the myeloid-failure phenotype of mne, Zbtb11 is expressed in the zebrafish haemopoietic intermediate cell mass (Fig. 2a). Several pointers indicate an ongoing requirement for Zbtb11 in sustaining definitive haemopoiesis. By 5 d.p.f., when there is strong local expression of rag1-expressing T-cells in the thymus in the WT, mne lacks rag1 expression in the thymus despite development of the thymic primordia as marked by foxn1 (Fig. 2d). Thrombocyte numbers are also reduced by 82 h.p.f. (Supplementary Fig. 2e). Despite normal specification of HSCs, as defined by cells expressing runx1 and myb along the ventral wall of the dorsal aorta (Supplementary Fig. 2b), myb expression is absent in mne caudal haemopoietic tissue at 72 and 96 h.p.f., suggesting that maintenance of the stem cell pool is also disrupted in mne (Supplementary Fig. 2c,d). This indicates a broader failure to sustain definitive haemopoiesis later in development and highlights the sensitivity of granulocytes as the first lineage affected in mne. PCR with reverse transcription (RT–PCR) of fluorescence-activated cell sorting (FACS)-sorted adult zebrafish kidney marrow confirms expression of Zbtb11 in adult haemopoietic cells, with highest levels in myeloid cells (Fig. 2e). Consistent with public domain RNA expression profiles1718, we have confirmed that ZBTB11 protein is highly expressed in human Jurkat (T cells), K562 cells (a BCR-ABL positive blast crisis erythroleukaemia) and HL60 (promyelocytic leukaemia) cells (Fig. 2f). Lower expression in HepG2 liver cancer cells correlates with hepatocellular carcinoma expression profiling showing very low ZBTB11 expression20. FACS-sorted embryonic neutrophils from mne and WT stained with May-Grünwald Giemsa exhibited an abnormally higher proportion of immature neutrophils in mne. Hence, there is both a quantitative and qualitative myeloid development defect as a result of Zbtb11 dysfunction (Fig. 2g,h).

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.


Related in: MedlinePlus