Supplementary Materials Wang et al. function of Irf2bp2b. Hence, Irf2bp2b is definitely a novel determinant in the choice of fate of neutrophil-macrophage progenitor cells. Intro Hematopoiesis is the process by which uncommitted hematopoietic stem cells proliferate and differentiate into all adult blood cell types.1 The stepwise development of multipotent hematopoietic stem cells undergoes sequential lineage potential limitations toward oligopotent and unipotent progenitor cells, eventually restricting their output.2 The molecular network governing every stage of hematopoiesis involves an interplay between multiple lineage-specific transcription factors/cofactors and epigenetic modifiers.3 Any tiny disturbance of these factors could bias the lineage-restricted cell fate toward an alternate fate.4 Neutrophil-macrophage progenitors (NMP) generate neutrophil-macrophage lineage cells, mainly neutrophils, monocytes, and macrophages. The gene regulatory network governing NMP cell fate is composed of main determinants, CCAAT enhancer binding protein alpha (C/EBP) and spleen focus forming disease proviral integration oncogene (PU.1), along with secondary determinants Gfi and Egr/Nab.5,6 Neutrophil cell fate specification requires C/EBP, whereas macrophage cell fate specification depends on PU.1.7,8 The relative levels of C/EBP and PU.1 determine the choice of NMP cell fate. A low C/EBP:PU.1 percentage shifts the balance toward macrophage differentiation, whereas a high ratio directs granulocyte differentiation.6 To keep myeloid lineage fidelity, the interplay among 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 downstream transcription factors of C/EBP and PU.1, function as mutually antagonistic repressors to inhibit lineage-specific genes in mice.5,9 It has also been reported that the suppression of knockout mice even develop a chronic myeloid leukemia-like disease.11,12 Mechanistically, interferon regulatory factor 8 (IRF8) impedes the ability of C/EBP to stimulate neutrophil differentiation by preventing its binding to chromatin.12 In addition to the transcription factors involved in the C/EBP and PU.1 network, Runx1 was shown to repress in a Pu.1-Runx1 negative feedback loop and determine macrophage neutrophil fate.13 Interferon regulatory factor 2 binding protein (IRF2BP)2 is a member of the IRF2BP family that was initially identified 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 KLRK1 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 Ievidence demonstrating that a deficiency of 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/ebp, inhibits expression. We further reveal that SUMOylation is indispensable for the transcriptional Isoorientin 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 knockout zebrafish, guide RNA targeting exon 1 of was designed using an online tool, ZiFiT Targeter software. Plasmid construction The zebrafish gene and its serial mutants were cloned into PCS2+ vector. The upstream sequences of zebrafish and genes were Isoorientin cloned into PGL3 promoter vector (Promega). Whole-mount in Isoorientin situ hybridization Digoxigenin-labeled RNA probes were transcribed with T7, T3 or SP6 polymerase (Ambion, Life Technologies, USA). Whole-mount 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 at 30C for 90 min) in a retroviral supernatant supplemented with cytokines and 4 g/mL polybrene (Sigma). Transduced cells were selected by G418 treatment (800 mg/mL, Sigma). Statistical analysis The statistical significance of a difference between two means was evaluated by the unpaired Student causes a reduction of the neutrophil population and a simultaneous expansion of the macrophage population during definitive myelopoiesis The gene family includes three members, and named and in zebrafish, whereas a unique gene exists in the human genome, which generates two isoforms.