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L. Wang et al.
the determinants is important not only in initiating the differentiation toward one lineage, but also in inhibiting that of the other lineage. Gfi1 and Egr/Nab, the down- stream transcription factors of C/EBPα and PU.1, function as mutually antagonistic repressors to inhibit lineage-spe- cific genes in mice.5,9 It has also been reported that the suppression of irf8, a downstream gene of Pu.1, leads to a depletion of macrophages and an expansion of neu- trophils during zebrafish primitive myelopoiesis.10 Irf8 knockout mice even develop a chronic myeloid leukemia- like disease.11,12 Mechanistically, interferon regulatory fac- tor 8 (IRF8) impedes the ability of C/EBPα to stimulate neutrophil differentiation by preventing its binding to chromatin.12 In addition to the transcription factors involved in the C/EBPα and PU.1 network, Runx1 was shown to repress pu.1 in a Pu.1-Runx1 negative feedback loop and determine macrophage versus neutrophil fate.13
Interferon regulatory factor 2 binding protein (IRF2BP)2 is a member of the IRF2BP family that was initially identi- fied as an interferon regulatory factor 2 (IRF2)-dependent corepressor in inhibiting the expression of interferon- responsive genes.14 The IRF2BP family is highly conserved during evolution, and is structurally characterized by an N-terminal zinc finger motif which mediates homo- or hetero-dimerization/multimerization between different IRF2BP2 family members, and a C-terminal ring finger motif that interacts with its partners.15 IRF2BP2 is described as a corepressor in most published works.14,16,17 The significance of IRF2BP2 in hematopoiesis was first revealed by genetic studies in Irf2bp2-deficient mice. IRF2BP2, with its binding partner ETO2, and the NCOR1/SMRT corepressor complex, participates in ery- throid differentiation.16 As a ubiquitously distributed nuclear protein, IRF2BP2 plays multiple roles in various types of hematopoietic cells. For example, IRF2BP2 exerts a repressive effect on target genes of nuclear factor of acti- vated T cells (NFAT), which is another partner of IRF2BP2.17 IRF2BP2 has also been shown to restrain naïve CD4 T-cell activation by inhibiting proliferation and CD25 expression.18 Moreover, Irf2bp2-deficient macrophages were inflammatory in mice.19 In recent years, four patients with acute promyelocytic leukemia carrying a novel fusion IRF2BP2-RARα have been report- ed. Nevertheless, the potential role of IRF2BP2 in leuke- mogenesis is still unclear.20-23
In this study, we provide in vivo evidence demonstrating that a deficiency of irf2bp2b triggers biased NMP cell fate choice, favoring macrophage development during zebrafish definitive myelopoiesis, which adds Irf2bp2b to the repertoire of factors regulating NMP cell fate decision. Mechanistic studies indicate that Irf2bp2b, which is under the control of C/ebpα, inhibits pu.1 expression. We fur- ther reveal that SUMOylation is indispensable for the transcriptional repression of Irf2bp2b.
Methods
Maintenance and generation of mutant zebrafish
Zebrafish were raised, bred, and staged according to standard protocols.24 For the generation of crisp9-mediated irf2bp2b knock- out zebrafish, guide RNA targeting exon 1 of irf2bp2b was designed using an online tool, ZiFiT Targeter software.
Plasmid construction
The zebrafish irf2bp2b gene and its serial mutants were cloned into PCS2+ vector. The upstream sequences of zebrafish pu.1 and irf2bp2b genes were cloned into PGL3 promoter vector (Promega).
Whole-mount in situ hybridization
Digoxigenin-labeled RNA probes were transcribed with T7, T3 or SP6 polymerase (Ambion, Life Technologies, USA). Whole- mount in situ hybridization (WISH) was performed as described previously.25
Semi-quantitative reverse transcriptase polymerase chain reaction
The RNA preparation, cDNA synthesis, and quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) were performed as described in the Online Supplementary Methods.
Retroviral transduction
The IRF2BP2 cDNA was inserted into a pMSCV-neo vector. For retroviral transduction, plat-E cells were transiently transfected with retroviral vectors. 32Dcl3 cells were transduced by spinocu- lation (1,300 g at 30°C for 90 min) in a retroviral supernatant sup- plemented with cytokines and 4 μg/mL polybrene (Sigma). Transduced cells were selected by G418 treatment (800 mg/mL, Sigma).
Statisticalanalysis
The statistical significance of a difference between two means was evaluated by the unpaired Student t-test. For multiple com- parisons, one-way analysis of variance was performed, followed by a least significant difference post-hoc test for multiple compar- isons. Differences were considered statistically significant at P<0.05.
Ethics
The animal protocol described above was reviewed and approved by the Animal Ethical and Welfare Committee, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine (Shanghai,China).
Results
Deficiency of zebrafish irf2bp2b causes a reduction of the neutrophil population and a simultaneous expan- sion of the macrophage population during definitive myelopoiesis
The IRF2BP gene family includes three members, IRF2BP1, IRF2BP2 and IRF2BPL, which are highly con- served throughout evolution.15 All the family members bear a nearly identical N-terminal C4-type zinc finger motif and a C-terminal C3HC4-type ring finger motif, whereas the intermediate domain between the zinc finger and the ring finger motifs shows relatively low similarity at the protein level.15 There are two paralog genes of irf2bp2 named irf2bp2a and irf2bp2b in zebrafish, whereas a unique IRF2BP2 gene exists in the human genome, which generates two isoforms also named IRF2BP2a and IRF2BP2b due to alternative splicing. Human IRF2BP2a has a 16 amino acid-long additional sequence in its intermedi- ate domain compared with IRF2BP2b. This additional sequence in human IRF2BP2a is not conserved in zebrafish Irf2bp2a/2b (Online Supplementary Figure S1). Phylogenetic analysis showed that the two paralogs and human IRF2BP2 arose from a common ancestor, suggesting that
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haematologica | 2020; 105(2)