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L. Wang et al.
protein-protein interaction.46 In the present study, we determined that in the balance between granulocyte and macrophage commitment, zebrafish irf2bp2b acts as a direct target of C/ebpα to repress pu.1 expression. Our data also suggest that during the stage of definitive myelopoiesis in zebrafish, it is the C/ebpα-Irf2bp2b-Pu.1 axis, not the C/ebpα-Gfi1-Pu.1 one, that regulates NMP cell fate. Thus zebrafish Irf2bp2b acts as a novel player in NMP cell fate decision and adds a new layer of complexity to this fine-tuning process.
It should be noted that the primitive macrophages and neutrophils developed normally in irf2bp2b-deficient embryos (Online Supplementary Figure S3C, D, G, H, K, L). Previously it was reported that a Pu.1-Runx1 negative feed- back loop determines the macrophage versus neutrophil fate of cells originating in the rostral blood island.13 Runx1 was shown to inhibit the pu.1 promoter directly in the study; however, injection of runx1 mRNA into our irf2bp2b- deficient embryos could not rescue the aberrant myelopoiesis (Online Supplementary Figure S8G, I). To fur- ther elucidate whether irf2bp2b regulates primitive myeloid differentiation, we first determined whether irf2bp2b is present in primitive versus definitive progenitor cells (Online Supplementary Figure S9A). We then injected irf2bp2b mRNA into one-cell stage wildtype embryos. The biased myelopoiesis could only be observed in the ventral wall of the dorsal aorta at 48 hpf (Figure 3F-H). By contrast, c/ebp1, lyz, and mfap4 were all normally expressed in the rostral blood island at 22 hpf (Online Supplementary Figure S9B-H). Based on these observations, we believe that even though irf2bp2b is expressed in both primitive and defini- tive myeloid progenitor cells, distinct regulatory mecha- nisms are implicated in cell fate determination of NMP derived from the ventral wall of the dorsal aorta/caudal hematopoietic tissue and the rostral blood island.
The DNA-binding properties of IRF2BP2 have never been studied. Although C4-type zinc fingers are found in Irf2bp2, GATA, RARα, and RXR, there are still some dif- ferences. While a single C-X2-C-X17-C-X2-C type zinc finger exists in Irf2bp2, two consecutive ones are con- tained in GATA. RARα and RXR have two C-X2-C-X13- C-X2-C type zinc fingers. GATA binds specifically to a consensus sequence.47 Physiologically the RAR-RXR het- erodimer binds to responsive elements that consist of two
AGGTCA core motifs.48 To determine the binding site of Irf2bp2b within the pu.1 promoter, we first investigated whether it was similar to that of GATA or RAR/RXR. Two putative GATA binding sites (GS1, GS2) were predicted within the 132 bp A region, whereas no RAR/RXR bind- ing sites could be found. However, both GATA site dele- tion constructs could still be inhibited by Irf2bp2b (Online Supplementary Figure S10). Therefore, Irf2bp2b presumably has its own binding site.
The majority of APL patients bear a PML-RARα fusion gene. However, in APL variants RARα is fused with genes other than PML. Recently, four APL cases with a novel fusion, IRF2BP2-RARα, were identified.20-23 All X-RARα fusion-related APL are characterized by blockage at the promyelocyte stage and inhibition of a large set of differ- entiation-related genes targeted by co-repressors recruited onto the RARα moiety.49 It should be noted that the zinc finger motif of IRF2BP2 was intact in all four patients car- rying the IRF2BP2-RARα oncoprotein,20-23 thus two poten- tial DNA-binding domains from each moiety are retained simultaneously in the fusion. Such a phenomenon is very rare in a chimeric fusion protein composed of two tran- scription factors. This raises a few questions about IRF2BP2-RARα-related APL. Since dimerization is one of the prerequisites for all X-RARα fusions,50 does the IRF2BP2 moiety serve merely as an interface for dimeriza- tion of IRF2BP2-RARα, or does IRF2BP2 make other con- tributions, such as DNA binding, to the pathogenesis of APL? Does IRF2BP2-RARα arise at the NMP level? If it is expressed in NMP, could IRF2BP2-RARα trigger the biased choice of NMP cell fate favoring granulopoiesis? Further studies are needed to answer these questions.
Acknowledgments
The authors are grateful to Y Chen and J Jin (both from Shanghai Jiao Tong University School of Medicine, Shanghai, China) for technical support. We thank Dr. X Jiao, Dr. Maria Mateyak, and Dr. Sunny Sharma (all from the Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ, USA) for their critical reading of this manuscript.
Funding
This work was supported by research funding from the National Natural Science Foundation of China (31871471).
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