Fig 2: Setd1b-deficiency leads to disturbed HSPC homeostasis in BM and spleen.(A) FACS analysis of hematopoietic stem and progenitor cell (HSPC) populations in BM. The LSK (Lin- Sca-1+ c-Kit+) compartment (depicted in red) is subdivided into LT-HSCs, MPPs, and LMPPs based on differential expression of CD34 and Flt3. The LK (Lin- Sca-1- c-Kit+) population (depicted in blue) divides into CMPs, GMPs, and MEPs based on differential expression of CD34 and CD16/32. The given percentages are relative to total cells analyzed. (B) HSPC ratios 7–25 weeks after tamoxifen induction (Ctrl n = 9, cKO n = 11). The LSK ratio in cKO BM shifted toward a higher percentage of MPPs at the expense of LMPPs. (C) Quantification of FACS data with respect to total cellularity in both hind limb bones (x106). In cKO BM, the LSK compartment has roughly doubled (p*=0.011). (D) This increase resulted from an enriched pool of MPPs (p**=0.001), while LMPPs were significantly reduced (p**=0.006). Of note, none of the myeloid progenitor populations were majorly affected. Data in (B–D) are representative of six independent experiments. (E) BrdU proliferation assay 6 hr after injection (Ctrl n = 7, cKO n = 7). Only in case of LMPPs (p**=0.038) and MEPs (p**=0.007) minor changes were detected in cKO BM. The data were derived from five independent experiments. (F) qRT-PCR analysis of Setd1b expression. Similar levels of Setd1b mRNA were detected in all FACS-sorted HSPC populations from WT mice (n = 3) relative to Rpl19 expression. (G) FACS analysis of HSPCs in the spleen. Other than in controls, the c-Kit + compartment was highly enriched in cKO spleens, indicating fully active hematopoiesis. The given percentages are relative to total cells analyzed. (H) Quantification of FACS data normalized to spleen weight (mg) 7–25 weeks after induction (Ctrl n = 7, cKO n = 10). Both LSK (p***<0.001) and LK compartments (p**=0.001) were significantly increased in cKO spleens. The data are representative of six independent experiments. The graphs in (B) and (D)-(F) depict the mean ± SD; in (C) and (H) the mean ± SEM is provided.10.7554/eLife.27157.011Figure 4—source data 1.Numerical data used to generate Figure 4B,C and D (HSPCs BM RC).

Fig 3: Zbtb16-tdTom Reporter Reveals Fluctuating Expression throughout Hematopoiesis(A) Flow-cytometric gating strategy for HSC, CLP, CHILP, and ILC2P subsets in Zbtb16+/tdTom bone marrow.(B) Flow-cytometric analysis of Zbtb16-tdTom in bone-marrow-derived CHILP (Lin-CD45+IL-7Ra+a4ß7hiSca1int/loFlt3-CD25-).(C) Flow-cytometry analysis of Zbtb16-tdTom expression in the ILC subsets in the siLP of Zbtb16+/tdTom mice.(D) Flow-cytometry analysis of Zbtb16-tdTom expression in bone marrow-derived HSCs (Lin-CD45+Kit+Sca1+Flt3-).(E) Flow-cytometry analysis of Zbtb16-tdTom expression in bone-marrow-derived CLPs (Lin-CD45+ IL-7Ra+Flt3+).(F) Flow-cytometry analysis of Zbtb16-tdTom expression in bone-marrow-derived ILC2Ps (Lin-CD45+IL-7Ra+a4ß7+Sca1hiFlt3-CD25+).Data are representative of 2 independent experiments. Please also see Figure S5.

Fig 4: B cell development is negatively affected whenTOP2B is mutated. a Spleen to body weight ratio for wild-type (n = 3), Top2b−/−-mb1 cre (n = 3), and Top2b+/EE587E mice (n = 4) reveal smaller ratios in knockin and knockout mice compared to controls (shown as mean ± SEM). *p < 0.05 by Kruskal–Wallis test. b Total serum IgG is reduced in Top2b−/− mb1 cre mice (n = 6) and Top2b+/EE587E (n = 4) mice compared to controls (n = 7, shown as mean ± SEM). *p < 0.05 by Kruskal–Wallis test. c, d Heterozygous mutant mice fail to produce specific IgG antibody in response to vaccination with ovalbumin (OVA, n = 4, (c) or pneumovax-23 (d) (n = 3 mice per group, shown as mean ± SEM, *p < 0.05 by the Kruskal–Wallis test). Bone marrow (e) and spleen (f) cells were isolated from Top2b+/EE587E mice and littermate controls. Phenotypic analysis by flow cytometry demonstrates that the bone marrow of Top2b+/EE587E mice (n = 3) have fewer PrePro B cells (Fr. A, B220+, CD43−, BP1−, CD24−), Pre B cells (Fr. D, B220+, CD43−, IgD−, IgM−), and immature B cells (Fr. E, CD19+, B220+, CD43−, IgM+, IgD−) compared to control mice (e). In the spleen (f), there are overall fewer B cells with significant reductions in transitional (CD19+, B220+, IgDlo, IgM+, CD21/35int, CD23 variable), follicular (CD19+, B220+, IgMlo, IgDhi, CD38+) and activated (CD19+, B220+, IgDhi, IgMhi, Flt3+) B cells compared to control mice (n = 4, shown as mean ± SEM, absolute numbers calculated from whole tissue numbers). *p < 0.05; **p < 0.01, two-tailed Student’s t test. Expression of mutant Top2b results does not affect E2A (g, n = 6)) or Rag1 (h, n = 8) expression in common lymphoid progenitor cells (compared to WT n = 6), whereas B cell-specific transcription factors Pax5 (i) and Foxo1 (j) show reduced expression of compared to wild-type mice. (n = 4 mice per group, shown as mean ± SEM, *p < 0.05, two-tailed Student’s t test)

Fig 5: B-cell development is negatively affected in the absence of TOP2B. a Absence of TOP2B in B cells results in reduced CD19+ B cells in peripheral blood compared to littermates (n = 6 WT and 7 Top2b-/- mb1 cre mice per group, mean ± SEM, ****p < 0.0001). b Splenic white pulp subcompartments are reduced in size in the absence of Top2b. Area of three sub-compartments were evaluated in at least ten follicles across three sections (500 µM apart) with marker indicating average per mouse. (Mean SEM, *p < 0.05). Representative follicles from wildtype (c–f) and Top2b-/- mb1-cre spleens (g–j) show altered splenic architecture. More B220-expressing (red) cells are seen in the wildtype (c, f) than Top2b-/- mb1-cre mice (g, j). The metallophilic macrophages (blue) indicating the border between marginal zone and follicular region. The periarteriolar lymphoid sheath (PALS) subcompartment contains T cells (green). Scale bar indicates 200 µm. Bone marrow (k) and spleen (l) cells were isolated from wildtype and Top2b-/- mb1-cre mice. Flow cytometry demonstrates that the bone marrow of Top2b-/- mb1-cre mice have fewer PrePro B cells (Fr. A, B220+, CD43-, BP1-, CD24-), Pre B cells (Fr. D, B220+, CD43-, IgD-, IgM-), and immature B cells (Fr. E, CD19+, B220+, CD43-, IgM+, IgD-) compared to controls (k). In the spleen (l), there are overall fewer B cells with reductions in marginal zone precursors (CD19+, B220+, IgMhi, IgDhi, CD21/35+), follicular (CD19+, B220+, IgMlo, IgDhi, CD38+), and activated (CD19+, B220+, IgDhi, IgMhi, Flt3+) B cells compared to controls. (n = 4 mice per group, mean ± SEM, absolute numbers calculated from whole tissue numbers). *p < 0.05; **p < 0.01. m qPCR analysis of kappa-deleting recombination excision circles demonstrate that B cells isolated from Top2b-/- mb1-cre mice have reduced replication in the spleen (n = 6 WT or 9 Top2b-/- mb1 cre mice, shown as mean ± SEM). *p < 0.05. n Pax5 is significantly decreased in B cells isolated from Top2b-/- mb1-cre mice (n = 9 WT or 6 Top2b-/- mb1- cre mice), whereas Rag1 expression (o) is unchanged (n = 5 WT and 4 Top2b-/- mb1- cre mice per group shown as mean ± SEM, with technical triplicates), **p < 0.005). All by two-tailed Student’s t test