Page 97 - 2020_02-Haematologica-web

Irf2bp2b regulates zebrafish NMP cell fate choice
matically decreased.13 In pu.1G242D/G242D homozygous embryos, biased myelopoiesis toward neutrophils occurred, as expected. It should be noted that no obvious rescue effect was observed in the irf2bp2b-/-pu.1G242D/G242D double-mutant embryos compared to pu.1G242D/G242D embryos, indicating that pu.1 is indeed downstream of Irf2bp2b in determining NMP cell fate (Figure 4K-O).
Irf2bp2b represses pu.1 gene transcription by binding directly to its promoter
IRF2BP2 has frequently been described as a co- repressor.14,16,17 We therefore set out to investigate how
Irf2bp2b represses pu.1 expression. The C-terminal C3HC4-type ring finger motif of IRF2BP2 is responsible for mediating its binding with interacting partners.14,16,17 The N-terminal C4-type zinc finger motif was believed to enable homo- and hetero-dimerization/multimerization between different IRF2BP2 family members.15 However, C4 zinc fingers are typically found in DNA-binding domains of transcription factors including GATA1-6 as well as nuclear receptors RAR and RXR.36,37 The possibility that IRF2BP2 functions as a transcription repressor by directly binding DNA should not, therefore, be excluded.
To characterize how Irf2bp2b represses transcription in
AEF
B
GH
IJ KL
MN
O
C
D
Figure 4. Irf2bp2b dictates neutrophil-macrophage progenitor cell fate through inhibition of pu.1 expression. (A) Quantitative reverse transcriptase polymerase chain reaction analysis of neutrophil and macrophage development-related genes in 32Dcl3 cells constitutively expressing human IRF2BP2b. Error bars represent the mean ± standard deviation (SD) of at least three replicates. ns: not statistically significant; **P<0.01; ***P<0.001 (Student t test). (B) Schematic diagram of the -8.5kb zebrafish pu.1 promoter dual luciferase report vector (top panel). Dual luciferase vectors each with a fragment of the zebrafish pu.1 promoter, as indicated, were co-transfected into HEK293T cells with an irf2bp2b-expressing vector or empty vector pCS2+. Luciferase activity with irf2bp2b expression was detected and nor- malized to empty vector pCS2+ which was set to 1.0 (bottom panel). Error bars represent the mean ± SD of at least three replicates. ***P<0.001; ****P<0.0001 (Student t test). (C, D) Representative fluorescent images of transient mCherry expression at 48 hours post-fertilization (hpf) of wildtype (WT) and irf2bp2b- overex- pressing embryos injected with a -8.5 kb pu.1:mCherry construct. (E-N) Whole-mount in situ hybridization (WISH) assay of mpx and mfap4 in WT embryos, (E, F), irf2bp2b-/- mutant embryos (G, H), irf2bp2b-/- mutant embryos injected with pu.1 morpholino (I, J), pu.1G242D/G242D mutants (K, L), and irf2bp2b-/-pu.1 G242D/G242D double- mutant embryos (M, N). (O) Statistic result for E-N. Error bars represent the mean ± standard error of mean of 15-30 embryos. **P<0.01; ***P<0.001 (analysis of variance followed by the least significant difference post-hoc test for multiple comparisons).
haematologica | 2020; 105(2)
331