Fig 2: H2afy is a target gene of U2AF1(S34F)-induced alternative splicing(A) Expression of U2AF1(S34)-induced alternatively spliced isoforms in eight genes in common myeloid progenitors (CMPs), monocytes (MONO) and B cells in mice were examined using a Nanostring assay. Log2-fold change of U2AF1(S34F) samples compared to U2AF1(WT) samples are shown (n = 2–9 biologic replicates per tissue).(B) Diagram showing two H2AFY splice isoforms, H2AFY1.1 and H2AFY1.2.(C–G) Whole bone marrow cells from H2afy+/+, H2afy+/- and H2afy-/- littermates were transplanted into lethally irradiated WT congenic recipient mice. (C) Overview of the non-competitive bone marrow transplant assay. (D) Peripheral blood WBCs at 4 months post-transplant (n = 13–17). (E) Peripheral blood lineage cell counts at 4 months post-transplant (n = 13–17). (F) Bone marrow lineage cell frequencies at 12 months post-transplant (n = 5–15). (G) Pre-B cell colonies in the CFU-pre-B assay (n = 10–16).Data are shown as fold change of colonies of H2afy+/- or H2afy-/- pre-B cells relative to H2afy+/+ colonies. Statistical analysis by two-tailed Student’s t test. Error bars represent mean ± SD. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: Expression of H2AFY1.1 in H2afy-/- bone marrow cells rescues reduced B cells ex vivo and in vivoH2afy-/- bone marrow cells were transduced with control lentivirus (GFP alone), H2AFY1.1-, or H2AFY1.2-expressing lentivirus that co-expressed GFP and transplanted into lethally irradiated congenic CD45.1+ recipients.(A) Overview of the experimental design.(B) Limiting dilution assay of functional CLPs (GFP+). Top panel: percent negative wells are plotted against the number of cells seeded per well. Bottom table: quantification of frequencies of functional CLPs with 95% confidence intervals. Data were pooled from four independent experiments, each consisting of 12–24 replicates.(C) Peripheral blood GFP+ lineage cells at 4 months post-transplant (n = 8–10).(D) Bone marrow GFP+ B cells at 4 months post-transplant (n = 5–7).Statistical analysis by two-tailed Student’s t test, except for (B). Error bars represent mean ± SD. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: H2afy-/- mice have defective B cell developmentA competitive repopulation assay was performed by transplanting bone marrow cells from H2afy+/+ or H2afy-/- littermates (CD45.2+) with the same number of WT congenic competitor marrow cells (CD45.1+/CD45.2+) into lethally irradiated congenic recipients (CD45.1+).(A) Overview of the competitive repopulation assay.(B) Peripheral blood chimerism of CD45.2+ cells at 1, 2, and 4 month(s) post-transplant (n = 16). Differences (two-way ANOVA) are indicated at each time point.(C) CD45.2+ bone marrow lineage cell frequencies at 4 months post-transplant (n = 7).(D) Donor-derived CD45.2+ B cell progenitors (ALP and BLP) and Hardy fractions (n = 7).Statistical analysis by two-tailed Student’s t test, except for (B). Error bars represent mean ± SD. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: Abnormal chromatin environment at stalled forks in LSH-deficient cells.a Representative images (left panel) and quantification of PLA signals (mH2A1: EdU, right panel) from U2OS cells transfected with the indicated siRNA after exposure to 4 mM HU for 5 h. The scale bar represents 10 µm. Quantification of PLA signals (mH2A1: EdU) is based on EdU-positive cells. Representative of n = 3 independent experiments. Data are represented as median ± 95% CI (n = 200 cells). ****p < 0.0001 by two-tailed Mann–Whitney test. b Quantification of PLA signals (mH2A2: EdU) from U2OS cells transfected with the indicated siRNA after exposure to 4 mM HU for 5 h. Quantification of PLA signals (mH2A2: EdU) is based on EdU-positive cells. Representative of n = 3 independent experiments. Data are represented as median ± 95% CI (n = 200 cells). ****p < 0.0001 by two-tailed Mann–Whitney test. c Quantification of PLA signals (H3: EdU) from U2OS cells transfected with the indicated siRNA (siCtrl or siLSH) after exposure to 4 mM HU for 5 h. Quantification of PLA signals (H3: EdU) is based on EdU-positive cells. Representative of n = 3 independent experiments. Data are represented as median ± 95% CI (n = 200 cells). ns = not significant by two-tailed Mann–Whitney test. d Quantification of PLA signals (H3K4me3: EdU) from U2OS cells transfected with the indicated siRNA (siCtrl or siLSH) after exposure to 4 mM HU for 5 h. Quantification of PLA signals (H3K4me3: EdU) is based on EdU-positive cells. Representative of n = 3 independent experiments. Data are represented as median ± 95% CI (n = 200 cells). ****p < 0.0001 by two-tailed Mann–Whitney test. e ChIP-qPCR analysis for detection of H3K4me3 enrichment at CFSs in mock-treated (DMSO) or aphidicolin-treated (APH) U2OS cells transfected with shLSH or shCtrl. Data are represented as mean ± SD (n = 3 independent experiments). *p < 0.05, ***p < 0.001 by Student’s two-tailed t test. f Quantification of PLA signals (H3K4me3: EdU) from U2OS cells transfected with the indicated siRNA (siCtrl or simH2A) after exposure to 4 mM HU for 5 h. Quantification of PLA signals (H3K4me3: EdU) is based on EdU-positive cells. Representative of n = 3 independent experiments. Data are represented as median ± 95% CI (n = 200 cells). ****p < 0.0001 by two-tailed Mann–Whitney test.