Nuclear interacting SET domain protein 1 inactivation impairs GATA1-regulated erythroid differentiation and causes erythroleukemia

Abstract

The nuclear receptor binding SET domain protein 1 (NSD1) is recurrently mutated in human cancers including acute leukemia. We show that NSD1 knockdown alters erythroid clonogenic growth of human CD34⁺ hematopoietic cells. Ablation of Nsd1 in the hematopoietic system of mice induces a transplantable erythroleukemia. In vitro differentiation of Nsd1^-/- erythroblasts is majorly impaired despite abundant expression of GATA1, the transcriptional master regulator of erythropoiesis, and associated with an impaired activation of GATA1-induced targets. Retroviral expression of wildtype NSD1, but not a catalytically-inactive NSD1^N1918Q SET-domain mutant induces terminal maturation of Nsd1^-/- erythroblasts. Despite similar GATA1 protein levels, exogenous NSD1 but not NSD^N1918Q significantly increases the occupancy of GATA1 at target genes and their expression. Notably, exogenous NSD1 reduces the association of GATA1 with the co-repressor SKI, and knockdown of SKI induces differentiation of Nsd1^-/- erythroblasts. Collectively, we identify the NSD1 methyltransferase as a regulator of GATA1-controlled erythroid differentiation and leukemogenesis.

Fig. 1. NSD1 knockdown alters clonogenic erythroid differentiation of human CD34⁺ hematopoietic cells.

a Relative NSD1 mRNA expression (1/dCt) in peripheral blood CD34⁺ cells transduced with pLKO.1 expressing control shRNA (Ctrl) or NSD1 shRNA (#372) harvested from the first and second plating in growth-factor-containing MC (H4434). Bars represent average relative expression normalized to (n = 5 per group). b Numbers of colonies formed by 4 × 10⁴ peripheral CD34⁺ cells transduced with pLKO.1 expressing control shRNA (Ctrl) or NSD1 shRNA in the first plate (n = 5) and upon replating (n = 2) in growth-factor-containing MC (H4434). c Representative images of colonies formed in MC (H4434) by 4 × 10⁴ peripheral CD34⁺ cells transduced with pLKO.1 expressing control or NSD1 shRNA. d Flow cytometric analysis of cells harvested from the first and second plating in MC (H4434) revealed accumulation of CD71^high and glycoprotein A (GPA)⁻ cells upon replating. The plots represent one out of three independent experiments. e Representative images of Wright Giemsa-stained cytospin preparations from control shRNA (Ctrl) or NSD1 shRNA-expressing CD34⁺ cells harvested from the MC (H4434) cultures after the first and second plating, illustrating the overall predominance of cells with erythroblast morphology upon replating (one out of three experiments) (×1000, the size bar = 10 μM). Values are presented as individual points, bar graphs represent the mean value of biological replicates, error bars as standard error of the mean. Statistical significances in a, b was tested with paired two-tailed t-test.

Fig. 2. Hematopoietic ablation of Nsd1 leads to a fully penetrant and transplantable leukemia-like disease in mice.

a Kaplan Meier plot of disease-free survival of Nsd1^fl/fl (n = 12, black line) and Nsd1^−/− (n = 24; red line) mice. Median survival of Nsd1^−/− mice was 91 days; Nsd1^fl/fl mice did not develop any disease. b Spleen and liver weight of Nsd1^fl/fl and symptomatic Nsd1^−/− mice in grams (n = 12 per group) (* indicates a p value smaller than 1 × 10⁻¹⁵). c Representative image of spleens of Nsd1^fl/fl (left) and diseased Nsd1^−/− (right) mice. d Representative images (from 1 out of 24 mice) of HE-stained sections (×200, size bars = 50 μm) of (i) spleen, (ii) lung, and (iii) liver of diseased Nsd1^−/− mice showing significant cell infiltrations in all organs. e Peripheral red blood cell counts (RBC, ×10¹² cells/l), f reticulocyte counts (RTC, ×10¹² cells/l), g hemoglobin levels (HGB, g/l), and h platelet counts (PLT, ×10¹² cells/l) in diseased Nsd1^−/− compared to Nsd1^fl/fl littermate controls (n = 12 per group). i Representative image of a Wright Giemsa-stained peripheral blood smear of a symptomatic Nsd1^−/− mouse with the presence of an erythroblast (from 1 out of 24 mice, ×600, the size bar = 10 μM). j Spleen and liver weight in grams of WT mice transplanted with whole BM from diseased Nsd1^−/− mice (red bars) (n = 4) or Nsd1^fl/fl littermate controls (black bars) (n = 2) or in a 1:1 mixture of Nsd1^−/− (CD45.2) and B6.SJL (CD45.1) cells (orange bars) (n = 6). Values are presented as individual points, bar graphs represent the mean value of biological replicates, error bars as standard error of the mean. Statistical significances in a was tested with log-rank Mantel Cox test, and in b, e–h, j with an unpaired two-tailed t-test.

Fig. 3. Cellular and molecular characterization of the erythroleukemia-like disease of Nsd1^−/− mice.

a CD71⁺ and Kit⁺ cell populations (given in %) in single-cell suspensions of spleen and BM of healthy Nsd1^fl/fl (spleen, n = 8, BM, n = 9, black bars) and diseased Nsd1^−/− mice (spleen, n = 8; BM, n = 8, red bars). b Comparative flow cytometric analysis of erythroid maturation (R1–R4) of single-cell suspensions of total BM of healthy Nsd1^fl/fl (n = 9, black bars) and diseased Nsd1^−/− mice (n = 9, red bars). c Colony types formed by 4 × 10⁴ BM cells of Nsd1^fl/fl (n = 9, black bars) and Nsd1^−/− mice (n = 9, red bars) in growth-factor-containing MC (M3434). * indicating a p value smaller than 1 × 10⁻¹⁵. d Representative images (illustrating one out of four experiments) of MC (M3434) cultures of Nsd1^−/− BM cells demonstrating (i) abnormal large red colonies, (ii) partially benzidine-staining-positive colonies, (iii) a large dense isolated colony, and (iv) Wright Giemsa-stained cytospin of an isolated colony (×4 and ×1000, size bar = 10 μM). e Number of colonies in four consecutive rounds of plating in MC (M3434) formed by 4 × 10⁴ BM cells of Nsd1^fl/fl (black dots; 1^st plating: n = 9, 2^nd plating: n = 4, 3^rd plating: n = 3, 4^th plating: n = 2) and Nsd1^−/− mice (red squares; 1^st plating: n = 9, 2^nd plating: n = 7, 3^rd plating: n = 6, 4^th plating: n = 3). f Number of LT-HSC, ST-HSC, and MPP (×10⁴) in lineage-marker-depleted single-cell BM suspensions of Nsd1^fl/fl (n = 3, black bars) and Nsd1^−/− mice (n = 4, red bars) relative to the total number of lineage-depleted cells obtained during each procedure. g Representative HE-stained biopsies of E19.5 fetal livers from a Nsd1^fl/fl (left panel, illustrating one out of two experiments) and Nsd1^−/− (right panel, illustrating one out of four experiments) mouse (×400, size bar = 10 μM). h CD71⁺ cells (%) in E19.5 fetal livers of Nsd1^fl/fl (n = 3, black bar) and Nsd1^−/− (n = 3, red bar) mice. i Representative images of colonies in MC cultures (M3434) and Wright Giemsa-stained cytospin preparations from 4 × 10⁴ E17.5 fetal liver-derived hematopoietic cells of Nsd1^fl/fl (left panels, illustrating one out of three experiments) and Nsd1^−/− (right panels, illustrating one out of three experiments) mice (×2 and ×1000, size bars = 10 μM). j Gene set enrichment analysis (GSEA) (weighted Kolmogorov–Smirnov-like statistics, two-sided, with adjustment for multiple comparisons) of selected signatures of differentially expressed gene between Nsd1^−/− mice (n = 5) and littermate controls (n = 3). Values are presented as individual points, bar graphs represent the mean value of biological replicates, error bars as standard error of the mean. Statistical significances in a–c, e, f, h was tested with unpaired two-tailed t-test.

Fig. 4. Impaired erythroid maturation of Nsd1^−/− erythroblasts.

a Experimental setup: lineage-depleted E17.5 fetal liver cells of Nsd1^fl/fl and Nsd1^−/− mice were grown in maintenance medium (>6 days) before induction of maturation in differentiation cultures. b Representative images (illustrating one out of two maturation experiments) of Wright Giemsa-stained cytospin preparations of E17.5 Nsd1^fl/fl and Nsd1^−/− fetal liver-derived erythroblasts expanded in maintenance medium (day 0) and induced to maturate in differentiation medium, shown at days 2, 3, and 6 (×1000, the size bar = 10 μM). c Growth of fetal liver-derived Nsd1^fl/fl (n = 6, black line) and Nsd1^−/− (n = 4, red line) erythroblasts in differentiation medium. Living cells were counted using trypan blue exclusion. d Forward scatter-negative (FSC⁻) Nsd1^fl/fl (black bars) and Nsd1^−/− (red bars) living cells (%) before (day 0) and after 3 days in differentiation medium (n = 3). Values are presented as individual points, bar graphs represent the mean value of biological replicates, error bars as standard error of the mean. Statistical significances in c, d was tested with unpaired two-tailed t-test.

Fig. 5. Aberrant regulation of GATA1 expression in Nsd1^−/− erythroblasts.

a Relative Gata1 mRNA expression levels (1/dCt) in BM-derived erythroblasts from Nsd1^fl/fl (n = 4, black bars) and Nsd1^−/− mice (n = 4, red bars) in maintenance medium (day 0) and after 2 days in differentiation medium. Ct values were normalized to Gapdh expression. b GATA1 protein levels in Nsd1^fl/fl (left panels) and Nsd1^−/− (right panels) BM-derived erythroblasts expanded in maintenance medium (0 h) and in differentiation medium (2.5, 5, and 24 h). LAMIN-A/C was used as immunoblot loading control for nuclear proteins (one out of two experiments). c Number of colonies formed by 5 × 10³ lineage-marker-depleted BM-derived erythroblasts in MC (M3434) from Nsd1^fl/fl (black bars) and Nsd1^−/− mice (red bars) transduced with pMSCV-puro (Ctrl) or pMSCV-mGata1-puro (Gata1) (n = 6 per group). d Ter119 expression (Ter119⁺, in %) in maintenance medium (0 h) and after 2 and 4 days in differentiation medium of Nsd1^fl/fl (black and gray bars) and Nsd1^−/− (red and pink bars) BM-derived erythroblasts transduced with control virus (Ctrl, black and red bars) or Gata1-expressing virus (Gata1, gray or pink bars) (n = 3 per group). e Representative images (one out of two experiments) of Wright Giemsa-stained cytospin preparations and cell pellets (small insets) of Nsd1^fl/fl and Nsd1^−/− BM-derived erythroblasts transduced with control virus (Ctrl) or Gata1-expressing virus (Gata1) after 5 days in differentiation medium (×600, size bars = 10 μm). f HbbA and g Spi1 mRNA levels in BM-derived Nsd1^−/− BM-derived erythroblasts transduced with control virus (Ctrl, black dots) or Gata1-expressing virus (Gata1, red dots) measured 0, 5, 24, 48, and 72 h in differentiation medium. Values are residual ΔCT relative to Gapdh, after adjustment for effect of individual mouse (Tukey test for difference in expression between transductions at 24 h in linear model with interaction between time a and transduction, adjusting for effect of mouse, two-sided, adjusted for multiple comparisons). Values are presented as individual points, bar graphs represent the mean value of biological replicates, error bars as standard error of the mean. Statistical significances in a, c was tested with unpaired two-tailed t-test. Statistical significance in d was tested with either unpaired (Nsd1^fl/fl.Gata1 vs. Nsd1^−/−.Gata1) or paired (Nsd1^−/−.Ctrl vs. Nsd1^−/−.Gata1) two-tailed t-test. Statistical significance in f, g was tested using Tukey test for difference in expression between transductions at 24 h in linear model with interaction between time a and transduction, adjusting for effect of mouse.

Fig. 6. NSD1-SET is essential for in vitro erythroblast maturation.

a Experimental setup: BM-derived Nsd1^fl/fl and Nsd1^−/− BM-derived erythroblasts were transduced with either pMSCV-GFP-Puro (Ctrl), pMSCV-Nsd1-GFP-Puro (Nsd1), or pMSCV-Nsd1^N1918Q-GFP-Puro (Nsd1^N1918Q) in maintenance medium, GFP⁺ cells were expanded in the presence of Puromycin before induced differentiation and analysis. b Representative pictures of Wright Giemsa-stained cytospin preparations transduced with Nsd1, Nsd1^N1918Q, or control virus in maintenance medium (day 0, top panels) and after 4 days in differentiation medium (middle panels). The lower panels show flow cytometric analysis of CD71 and Ter119 expression of the cells after 4 days in differentiation medium. These data represent one of four independent experiments (×1000, size bars = 10 μM). c Western blot analysis showing NSD1 protein expression in 1 × 10⁶ Nsd1^fl/fl and Nsd1^−/− (untransduced), and Nsd1^−/− transduced erythroblasts either expressing Nsd1 or Nsd1^N1918Q in maintenance medium. LAMIN-A/C was used as a loading control. These data represent one out of two experiments. d Growth of Nsd1 (red line), Nsd1^N1918Q (gray line), or the control (Ctrl, black line) virus transduced Nsd1^−/− BM-derived erythroblasts in differentiation medium (1–4 days). Nucleated living cells were counted by the Trypan blue exclusion (n = 3 per group). e Number of colonies formed by 1 × 10⁴ Nsd1 (red bar), Nsd1^N1918Q (gray bar) or the control virus (Ctrl, black bar) transduced Nsd1^−/− BM-derived erythroblast in MC (M3434) after 11 days (n = 3 per group). f Ter119⁺ stained Nsd1^−/− BM-derived erythroblasts transduced with Nsd1 (red bars) and Nsd1^N1918Q (gray bars) in maintenance medium (day 0) and after 1 and 2 days in differentiation medium (n = 4). Values are presented as individual points, bar graphs represent the mean value of biological replicates, error bars as standard error of the mean. Statistical significances in d–f tested with a paired two-tailed t-test.

Fig. 7. Nsd1 expression induces an erythroid gene and protein signature.

a Experimental setup: Nsd1^−/− BM-derived erythroblasts expressing either Nsd1 or Nsd1^N1918Q were analyzed during expansion in maintenance medium (0 h) and after 24 h in differentiation medium by RNA-seq and global proteome analysis. b Heatmap of the top 100 differentially expressed genes (corresponding to FDR < 1.06 × 10⁹) of Nsd1^−/− BM-derived erythroblasts expressing Nsd1 (brown squares) and Nsd1^N1918Q (black squares) in maintenance medium, and after 24 h in differentiation medium (Nsd1, red squares; Nsd1^N1918Q, gray square). Columns clustering was done by Wards linkage on correlations. c Gene set enrichment analysis (GSEA) (weighted Kolmogorov–Smirnov-like statistics, two-sided, with adjustment for multiple comparisons) of differential expression between Nsd1^−/− BM-derived erythroblast expressing Nsd1 before and after 24 h in differentiation medium. d GSEA (weighted Kolmogorov–Smirnov-like statistics, two-sided, with adjustment for multiple comparisons) of differential expression between Nsd1^−/− BM-derived erythroblasts expressing either Nsd1 or Nsd1^N1918Q kept for 24 h in differentiation medium. e Differential protein expression of Nsd1^−/− erythroblasts expressing Nsd1 or Nsd1^N1918Q kept for 24 h in differentiation medium (n = 3 per group, FDR < 0.05, p value <0.05). Labels are shown for proteins with both logFC > 3 and FDR < 0.05.

Fig. 8. Nsd1 expression increases GATA1 chromatin binding and changes GATA1 protein interaction partners during induced differentiation of Nsd1^−/− cells.

a Relative Gata1 mRNA expression levels (1/dCt) in Nsd1^−/− BM-derived erythroblasts virally expressing Nsd1 (red bars) or Nsd1^N1918Q (gray bars) expanded in maintenance medium (day 0) or after 1 and 2 days in differentiation medium. Values were normalized to Gapdh (n = 4 per group). b Western blot showing GATA1 protein expression in 1 × 10⁶ Nsd1^−/− BM-derived erythroblasts expressing Nsd1 or Nsd1^N1918Q upon expansion in maintenance medium (day 0), and after 1 and 2 days in differentiation medium. Actin was used as a loading control. This data represents one of two experiments. c Experimental setup of the ChIP-seq and IP-MS experiment. d Heatmaps of genome-wide ChIP-seq signals in Nsd1^−/− BM-derived erythroblasts expressing Nsd1 (left column) or Nsd1^N1918Q (right column) after 24 h in differentiation medium for GATA1, H3K27^ac, and H3K36^me3. All heatmaps are sorted decreasingly according to read coverage around transcriptional start sites (TSS) of GATA1 (leftmost). Input denotes sheared non-immunoprecipitated DNA (rightmost), serving as visual control. Density plots above each heatmap depicts corresponding averaged binding around TSS. e One-dimensional heatmap of logFC between gene expression of Nsd1^−/− BM-derived erythroblasts expressing Nsd1 or Nsd1^N1918Q after 24 in differentiation medium (as presented in Fig. 5h, j) sorted according to read coverage around TSS for H3K27^ac ChIP (data as shown in panel c, sorted independently. Only overlapping genes are displayed). f Integrated genome viewer (IGV) representation of GATA1, H3K27^ac, and H3K36^me3 ChIP peaks in the Pklr (top panel) and Art4 gene locus (lower panel) from Nsd1^−/− BM-derived erythroblasts either expressing Nsd1 or Nsd1^N1918Q after 24h in differentiation medium. Right panels show Pklr and Art4 mRNA relative expression levels (1/dCt) in Nsd1^−/− BM-derived erythroblasts expressing Nsd1 or Nsd1^N1918Q in maintenance medium (day 0) and after 24 h differentiation medium. Values are shown as relative expression normalized to Gapdh (n = 4). g Volcano plot of differential protein enrichments by GATA1 immunoprecipitation (GATA1-IP) in Nsd1^−/− BM-derived erythroblasts either expressing Nsd1 or Nsd1^N1918Q kept for 24h in differentiation medium, each group is normalized to IgG control (n = 2). Significantly reduced GATA1-SKI association (indicated by a black arrow) was observed upon expression of Nsd1 compared to Nsd1^N1918Q (FDR < 0.05). h Western blot analysis showing SKI protein expression in 1 × 10⁶ BM-derived Nsd1^−/− erythroblasts either expressing Nsd1 or Nsd1^N1918Q during expansion in maintenance medium (day 0), and after 1 and 2 days in differentiation medium. Actin was used as a loading control. This data represent one of two experiments. Values are presented as individual points, bar graphs represent the mean value of biological replicates, error bars as standard error of the mean. Statistical significances in a, f tested with a paired two-tailed t-test.

Fig. 9. SKI knockdown results in terminal differentiation of Nsd1^−/− erythroblasts.

a Experimental setup: BM-derived Nsd1^−/− erythroblasts were transduced with either pLMP-empty-shRNA-GFP-Puro (Ctrl-shRNA) or pLMP-Ski-shRNA-GFP-Puro (Ski-shRNA) in maintenance medium, sorted for GFP, and selected with Puromycin for 2 days before induced differentiation and analysis. b Representative images of Wright Giemsa-stained cytospin preparations of control (Ctrl-shRNA, left panel) and Ski shRNA (Ski-shRNA), right panel) transduced Nsd1^−/− BM-derived erythroblasts after 2 days in differentiation medium. The small insets show the cell pellets before analysis. These data illustrate one of three experiments (×1000, size bar = 10 μM). c Growth of Nsd1^−/− BM-derived erythroblasts transduced with Ski- (Ski-shRNA, blue line) or control shRNA (Ctrl-shRNA, black line) grown for 4 days in differentiation medium. Nucleated living cells were counted by Trypan blue exclusion (n = 3 per group). P value > 0.05 for all time points. d Fraction of Ter119⁺ cells (%) of Nsd1^−/− BM-derived erythroblasts transduced with Ski- (Ski-shRNA, blue bars) or control (Ctrl-shRNA, black bars) virus grown for 2 days in differentiation medium (n = 3 per group). e Western blot showing SKI and GATA1 protein expression in Nsd1^−/− BM-derived erythroblasts transduced with Ski (Ski-shRNA) or control (Ctrl-shRNA) virus during expansion in maintenance medium (day 0) and following 1–2 days in differentiation medium. Actin was used as a loading control. These data represent one of three experiments. f Total number of colonies counted at day 11 after plating of 1 × 10⁴ Nsd1^−/− BM-derived erythroblasts expressing either Ski shRNA (Ski-shRNA, blue bar) or control (Ctrl-shRNA, black bar) in MC (M3434) (n = 3 per group). g Total number of cells obtained from 1 × 10⁴ Nsd1^−/− BM-derived erythroblasts expressing either Ski shRNA (Ski-shRNA, blue bar) or control (Ctrl-shRNA, black bar) after 11 days in MC (M3434) (n = 3 per group). h Percentage of Kit⁺ living cells obtained from 1 × 10⁴ Nsd1^−/− BM-derived erythroblasts expressing either Ski shRNA (Ski-shRNA, blue bar) or control (Ctrl-shRNA, black bar) after 11 days in MC (M3434) (n = 3 per group). P value > 0.05 for all time points. Values are presented as individual points, bar graphs represent the mean value of biological replicates, error bars as standard error of the mean. Statistical significances in c, d, f, g, h tested with a paired two-tailed t-test.