Page 325 - 2022_01-Haematologica-web

Letters to the Editor
viously described, and correspond to phenotypic stages like those of B2 fractions.7 These results suggested that Ikaros deficiency blocks B1-cell differentiation at the fraction C' stage, similar to its effect on B2-cell develop- ment, using the same Cre transgene.4
In order to evaluate the molecular pathways regulated by Ikaros, we analyzed the gene expression changes
A
B
between WT and cKO BM B1 progenitors by RNA sequencing (RNA-seq) (Online Supplementary Figure S2). Pathway enrichment and gene set enrichment analyses (GSEA) associated Ikaros with cell migration, adhesion, proliferation, and B-cell differentiation and maturation (Figure 2C and D). As Ikaros antagonizes STAT5 binding to DNA in pre-B2 cells, we asked if Ikaros has a similar
C
D
Figure 2. Ikaros deficiency in B1 progenitors induces a differentiation block at fraction C' and deregulation of genes involved in B-cell differentiation. (A) VDJ rearrangement as indicated, following published protocols.4 Spleen and kidney cells were used as positive or negative controls, respectively. Germ: germline configuration. Numbers correspond to kilobases. (B) Bone marrow (BM) cells were analyzed for different B-cell stages by flow cytometry: B1 progenitors (IgM- CD93+CD19+B220-/lo) were further gated on CD24 and BP-1 to give fraction (Fr.) A (CD24-BP-1-), Fr. B (CD24+BP-1-), Fr. C (CD24+BP-1+), Fr. C' (CD24+BP-1++). Graphs show frequencies (left) and absolute numbers (right) of B progenitor fractions as indicated. Data from 3 independent experiments with 5 mice for wild- type (WT) and 7 for conditional knockout (cKO). ns: not significant; **P<0.01; ****P<0.0001 (unpaired Student's t-test). (C) RNA sequencing (RNA-Seq) data of BM B1 progenitors from 3 WT and 3 cKO mice (4 weeks old) were evaluated by pathway enrichment analysis (http://metascape.org)15 for genes upregulated in cKO (left) or WT B1 progenitors (right). (D) Gene set enrichment analysis (GSEA) of 3 gene sets as indicated, compared with genes upregulated (red) or down- regulated (blue) in cKO vs. WT B1 progenitors. The STAT5 gene set corresponds to STAT5 genes antagonized by Ikaros.8 NES: normalized enrichment score; P: P-value; q: false discovery rate (FDR) q-value.
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