Runx1 regulates zebrafish neutrophil maturation via synergistic interaction with c-Myb

• DOI: 10.1016/j.jbc.2021.100272
• Publication Date: 2021-01-10T23:27:30Z
• Authors: Zhibin Huang; Kemin Chen; Yali Chi; Hao Jin; Li Li; Wenqing Zhang; Jin Xu; Yiyue Zhang
• Keywords: MYB; RUNX1

Neutrophils play an essential role in the innate immune defense system vertebrates. During hematopoiesis, full function of neutrophils involves maturation granules and related enzymes. Yet, transcription regulators that promote neutrophil remain largely undefined. Here, two hematopoiesis-defective zebrafish mutants, runx1w84x c-mybhkz3, were used to investigate vivo roles Runx1 cooperation with c-Myb regulating maturation. Loss runx1 impairs primitive development. Additional regulation c-myb+/− c-myb−/− induces a more severe phenotypes suggesting synergistic genetic interaction between c-myb Further studies revealed factors act cooperatively control processes via transactivating series maturation-related genes. These data reveal while also indicating novel molecular orchestration myeloid cell The study will provide new evidence on during hematopoiesis. are most abundant phagocytes for first line system. 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Wen Z. regulates embryonic through negative feedback loop inhibiting Pu.1 expression.Blood. 119: 5239-5249Crossref (61) still debatable. It could regulate Mpx Elane lines (15Nuchprayoon Meyers Scott L.M. Suzow Friedman A.D. PEBP2/CBF, murine homolog AML1 PEBP2 beta/CBF beta proto-oncoproteins, genes immature cells.Mol. Cell. 1994; 14: 5558-5568Crossref 16Austin G.E. Zhao W.G. Regmi Lu J.P. Braun Identification upstream enhancer containing site (MPO) gene.Leuk. Res. 1998; 22: 1037-1048Crossref (19) 17Brightman D.S. Grant Ruzycki P.A. Suzuki R. Hennig A.K. Chen MLL1 retinal neurogenesis horizontal inner neuron integrity.Sci. Rep. 2018; 8: 11902Crossref (6) Similarly, recent mouse demonstrated RUNX1-haploinsufficient progenitors impaired vitro repressing Cebpe expression (18Ng K.P. Hu Ebrahem Q. 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Furthermore, achieved verification. we hematopoietic-defective (22Jin Sood English Definitive manifest distinct output VDA PBI.Development. 136: 647-654Crossref (70) Scholar) c-mybhkz3 (23Zhang Jin Qin F.X. cMyb mobilization hematopoiesis.Blood. 118: 4093-4101Crossref (55) determine mutants elucidate epistasis biochemical analysis. was cooperates myelopoiesis. This elucidates networks orchestrate improving our pathogenesis diseases. characterized cytoplasm, which be specifically stained Sudan Black B (SB) 24Le G.D. Redd M.J. Colucciguyon E. Murayama Kissa Briolat V. Mordelet Zapata Shinomiya Herbomel Origins unconventional behavior developing 2008; 111: 132Crossref (245) Similar phenotype myelopoiesis Scholar), runx1−/− had reduced number SB+ 36 h postfertilization (hpf) (Fig. 1, A–C). 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To relation maturation, approach compare single composite derived crossing c-myb+/−;runx1+/− double heterozygotes. heterozygous indistinguishable wild-type hpf focused comparisons c-myb+/−;runx1−/− additional heterozygotes would yield phenotypes. When one allele introduced runx1−/−, further 2, A–E). Consistently, c-myb−/−;runx1−/− homozygotes nearly no left (data not shown). DIC F–I, F'–I' J–K) residual I I') elicited characteristics, scarce almost lacking mpx:GFP c-myb+/−;runx1+/+ G G') c-myb+/+;runx1−/− H H'). biological consequences function, examined rate bacterial killing each group. As expected, additive attenuated activity, prolonged clearance time 2L). above suggest genetically cooperate potentiate mammals, required typical digestive enzymes, Lyz, Mpx, nephrosin (Npsn), produced 28Cuartero Weiss F.D. Dharmalingam Guo Ing-Simmons Masella Robles-Rebollo Xiao X. Wang Y.F. Barozzi Djeghloul Amano M.T. Niskanen Petretto Dowell R.D. et al.Control inducible links cohesin progenitor self-renewal differentiation.Nat. 19: 932-941Crossref (81) directly targeted transcriptionally regulated coactivated unclear. test combined effects, whole mount situ hybridization (WISH) performed expressions crossed embryos. Our laboratory lyz+ S1, A'), c-myb+/+;runx1+/+ comparable 3, B). results indicate (c-myb+/− heterozygous) does affect signals mutated. 3C), homozygous one-third those 3A). Importantly, inadequate background introduced, average per embryo 30 3C) 10 D E). lyz. proper sequential it hypothesized effects likely lyz, well other Zebrafish Npsn both granzymes microbe (28Cuartero 29Wang Fang Ma Lin Myeloperoxidase-deficient show augmented challenge Candida albicans.Fish Shellfish 44: 109-116Crossref mpx+ npsn+ alleles F–O, Fig. B, C, B' C'). Moreover, granule-formation-related glycoprotein-srgn greatly downregulated srgn c-Myb. Expectedly, srgn+ greater inhibition P–T D'). least amount there downstream Taken together, Runx1, c-Myb, positively (lyz, mpx, npsn, srgn) regulatory regions analyzed. putative binding sites 4A). Chromatin immunoprecipitation polymerase chain reaction (ChIP-PCR) assays ascertain bind vivo. address this question, injected Myc-tagged plasmids overexpress ChIP-PCR analysis coprecipitated using anti-MYC antibody 4B). result demonstrates respectively. support hypothesis, reporter assay expressing constructs together overexpression reported, increased activity present report, 4C). Compared overexpressing only, enhanced coexpression suggests targeting Next, asked physically interact functions. Coimmunoprecipitation (Co-IP) experiments 293T cotransfected runx1-Flag Myc-c-myb constructs. lysates immunoprecipitated anti-FLAG antibody, followed western blot antibodies. 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Top. Microbiol. 211: 149-157PubMed 39Friedman C/EBPalpha myelopoiesis.Int. Hematol. 101: 330-341Crossref (68) 40Zhang D.E. Hohaus Voso H.M. Smith L.T. Hetherington Tenen Function PU.1 (Spi-1), C/EBP, myelopoiesis: CSF receptor promoters.Curr. 137-147PubMed myeloid-lineage-specific great interest future studies. why pairs determined. possible sequentially subpopulations and/or same factor. derive list Runx1. find interacting factors, manner. improved disease pathogenesis. (Gene ID: 58126) 30519) study. following strains utilized: AB, Tg(mpx:GFP)i114 mutant harbors nucleotide substitution resulting premature truncation protein. removes residues CBFb binding, nuclear signal. splice synthesis transactivation domain. maintained accordance Guidelines Animal Care Use Committee South China University Technology. All experimental protocols approved Division Cell, Developmental Integrative Biology, School Medicine, Fish anesthetized 90% PBS + 10% FBS 0.02% tricaine. After tail clipping syringe needle, collected pipetting cytospun onto slides centrifugation 450 rpm 3 min Cytospin 4 (Thermo Scientific). then airdried subjected (Merk) according standard protocol. group ∼150 WISH onward report 41Westerfield Book. Guide Laboratory (Danio rerio).4th Ed. Oregon Press, Eugene2000Google evident detection. Relative ratio calculated Image software. Heat-shock-inducible (hsp70:Myc-c-myb hsp70: Myc-runx1) generated inserting Myc-runx1 pTol vectors heat shock 70 (hsp70) promoter. Embryos incubated 39.5 °C 1 24 hpf. grown DMEM supplemented bovine calf serum. transfection, extracts preparation, immunoprecipitation, Blot (42Huang Qian Ang Chromatin-remodelling BRG1 selectively activates subset interferon-alpha-inducible genes.Nat. 4: 774-781Crossref (120) Constructs Flag-tagged transfected cells. immunoprecipitants Input represents total immunoprecipitation. Anti-MYC antibodies obtained Santa Cruz (sc-40) Sigma (ab6658), VE done 60×/1.00 NA water-immersion objective mounted Nikon 90i microscope embryos, easy detected since accessible lens. Putative identified JASPAR online one-cell stage. Six hundred harvested dpf brief fixation. Cross-linked chromatin anti-Myc anti-IgG (negative control) procedure Hart al. (43Hart D.O. Raha Lawson N.D. Green Initiation haematopoiesis TATA-box-binding protein-related Trf3.Nature. 450: 1082-1085Crossref semiquantitative PCR. ChIP primers Table S1. anaesthetized tricaine (MS-222; Sigma-Aldrich) muscle approximately 200 c. f. u. dsRed-labled coli. 1% (w/v) low-melting-point agarose time-lapse Bacterial counted. Data analyzed GraphPad Prism6 software Student t-test groups one-way variance (ANOVA; Tukey posttest adjustment) among groups. Significance accepted p < 0.05. mean ±standard error (SEM). An asterisk indicates statistical difference (∗p ≤ 0.05, ∗∗p 0.01, ∗∗∗p 0.001, ∗∗∗∗p 0.0001). included published article [and supplementary information files]. authors declare competing financial interests. thank Koichi providing vector, Tingxi sharing constructs, Paul mutants. Contribution: experiments; helped perform assay; characterization; wrote article; discussed commented article. work supported National Key R&D Program ( 2018YFA0800200 2018YFA0801000 ), Natural Science Foundation 31922023 Guangdong Province Universities Colleges Pearl River Scholar Funded Scheme (2019), Fundamental Research Funds Central 2019ZD54 ).