Fig 2: Deregulated Canonical NF-κB Activation in HSCs Leads to Pancytopenia and Progressive BM Failure(A) NF-κB target gene expressions in total BM of 14-day-old CA/CA and control mice. Representative data from two independent experiments.(B) NF-κB target gene expressions in HSCs (CD150+ LSK cells) of 14-day-old CA/CA and control mice. Representative data from two independent experiments.(C) Representative pictures of CA/CA and control mice at 2 weeks old (left) and 4 weeks old (right).(D) Kaplan-Meier survival curve analysis of CA/CA and control mice (n = 18). Significance (****p < 0.0001) was assessed using the log-rank test.(E) Complete blood count (CBC) analysis of CA/CA and control mice (n = 3–6 at each time point). Hb, hemoglobin; Plt, platelet.(F) Cell number of hematopoietic organs from CA/CA and control mice (n = 3–6 at each time point). Sp, spleen; Thy, thymus.(G) Normalized (cell number per gram body weight) cell counts of hematopoietic organs from CA/CA and control mice (n = 8 at each time point).All of the data represent means ± SEMs. Two-tailed Student’s t tests were used to assess statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig 3: Mof exhibits dynamic expression along erythropoiesis, and its reduction has a pronounced impact on erythroid lineage commitment.(A) Heatmap representing gene expression patterns of KATs and HDACs along the erythroid trajectory [data from (18)]. (B) Top: Schematic representation of fate-bias analysis, showing the cell fate-bias score (x axis) versus transcript expression (y axis). The fate-bias score indicates the probability of a given cell to belong to target cell types A, B, or C. A score of 1.0 indicates that the given cell has 100% probability of belonging to a target cell type. The example cell expressing Gene X has a 75 to 100% chance of belonging to cell type A but only a 0 to 20% chance of belonging to cell types B or C. In the analysis represented in the bottom panel, we selected erythroid, myeloid, and lymphoid cells as our target cell types. Bottom: Dot plot from 4763 cKit+ cells, showing the cell fate-bias score determined by FateID plotted against RaceID-normalized transcript expression in erythroid [Gata1+Klf1+Gypa+Hba-a1+; cluster 3; erythroid versus Mof expression r = +2 × 10-2; Pearson correlation, P = 4 × 10-2), myeloid (Mpo+Elane+; cluster 1; myeloid versus Mof expression r = -3.6 × 10-2; Pearson correlation, P = 6.3 × 10-3), and lymphoid (Ebf1+Rag1+; cluster 7; lymphoid versus Mof expression r = +6 × 10-3; Pearson correlation, P = 0.32) lineages; original data from (12)]. For t-distributed stochastic neighbor embedding (t-SNE), maps, and cluster annotation see fig. S1 (A and B). (C) Box plot showing the total number of RBC, hemoglobin (HB) quantity, and hematocrit (HCT) percentage (n = 4 per genotype) and scatter plot showing the HB concentration measured using an enzyme-linked immunosorbent assay (ELISA)–based assay (wild type, n = 7; Mof+/-, n = 13; P < 0.001). (D) Left: Box plot showing the total area of the colonies obtained from the single-cell colony-forming unit (sc-CFU) assay on fluorescence-activated cell sorting (FACS)–sorted HSCs (LSK+Flt3-CD34-CD48-CD150+). Right: Box plot showing the total cell number per colony obtained in the sc-CFU assay. (E) Pie charts representing the lineage potency from the sc-CFU assay. (F) Stacked bar plot showing the fraction of lineage output from each clone. Final populations were defined on the basis of cell surface markers: myeloid (Mye; cKit-CD11b+, number of cells: 208,961 wild type and 905,455 Mof+/-), erythroid (Ery; cKit-Ter119+, number of cells: 205,963 wild-type and 27,564 Mof+/-), lymphoid [Lym; cKit-IgD+, number of cells: 65,093 wild-type and 63,815 Mof+/-], and progenitor cells (Prog; cKit+, number of cells: 98,988 wild-type and 124,690 Mof+/-). Significant enrichment by Fisher’s exact test was set as P > 0.05. sc-CFU data from 72 wild-type and 147 Mof+/- colonies from three independent animals. (G) Bar plots showing the CFU capacity output of 100 FACS-sorted HSCs (wild-type, n = 6; Mof+/-, n = 3). (H) Same as (G), but using HSCs sorted from Cag-Cre:ERT2Tg/+(control) or Moffl/flCag-Cre:ERT2Tg/+transgene (Mof-iKO) animals. After sorting, cells were cultured with 4-hydroxytamoxifen (4-OHT) to induce Mof depletion in vitro. Bar plots show CFU capacity output of HSCs (control, n = 3; Mof-iKO, n = 3). (I) Serial CFU assay scheme and serial colony formation capacity from (G) or (H). (J) CFU assay scheme and CFU capacity output of FACS-sorted MEPs (Lin-cKithighSca-1-IL-7R-CD34-FcRgII/III-) from wild-type or Mof+/- (wild-type, n = 6; Mof+/-, n = 3) animals. (K) same as (J), but from control or Mof-iKO–sorted MEPs, wherein Mof depletion was induced in vitro after sorting and plating. Error bars represent means ± SEM, and biological replicates are represented as the overlaid dots. Experimental significance was determined by one-way analysis of variance (ANOVA), P < 0.05. Related to figs. S1 to S4.

Fig 4: Mof expression is associated with global chromatin accessibility and H4K16ac levels.(A) Representative flow cytometry histograms showing ATAC-see staining intensities. The dark-gray shading shows the ATAC-see in wild-type animals, and orange shows the ATAC-see for Mof+/-animals. The black line marks ATAC-see oligo autofluorescence, and light gray shading shows the fluorescence minus one (FMO) control in which the ATAC-see oligo was omitted. (B) Summary box plot showing the quantification of the ATAC-see FACS median fluorescence intensities (MFIs) of HSCs (LSK+CD34-CD150+), MPPs (LSK+CD34+), CMPs (Lin-KithighSca1-CD34+CD16/32dim), GMPs (Lin-KithighSca1-CD34+CD16/32high), and MEPs (Lin-KithighSca1-CD34-CD16/32-) from wild-type (gray bars) and Mof+/- (orange bars) mice. Data represent n = 7 independent animals. P values were calculated by two-tailed Mann-Whitney test, with confidence level set as 95%. (C) Representative images of ATAC-see (green), MOF immunofluorescence (red), and 4',6-diamidino-2-phenylindole (DAPI; blue) in sorted MEPs from n = 3 animals and two independent experiments. Scale bars, 2 µm (see also fig. S8, A and B). (D) Density plot showing the mean number of ATAC-seq peaks found in human HSCs (dash line), MPPs (blue line), MEPs (red line), and erythroblasts (gray line). Data from GSE74912. (E) Box plots showing FACS MFI signal of H4K16ac in HSC (LSK+CD34-Flt3-CD48-CD150+), MPP1 (LSK+Flt3-CD34+CD150+CD48-), MPP2 (LSK+Flt3-CD34+CD150+CD48+), MPP3 (LSK+CD34+Flt3-CD150-CD48+), CMP, and MEP populations [as in (B)] normalized by their total H4 levels in wild-type and Mof+/- mice. Number of animals n = 4 from two independent experiments. For representative histogram, see fig. S8C. Mean value is shown as a line. P value was determined by two-tailed Mann-Whitney test, with confidence level set as 95%. A.U, arbitrary units. (F) Ex vivo erythropoiesis assay. Bar plot showing reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses of Mof levels in HSCs isolated from wild-type (gray) or Mof+/-(orange) animals and cultured in methylcellulose-based medium for the indicated number of days. Mof expression was determined by [CT-Mof - CT-Hprt, dCT] and then normalized by the smallest dCT (normalization was conducted considering each genotype and day of interest), ddCT. The fold change is 2ddCT. Each overlaid data point represents the number (n) of independent animals. Error bars represent means ± SEM. Significance was determined after normal distribution analysis by two-tailed Mann-Whitney test. (G) Scheme of CFU experiment. Wild-type, Cag-Cre:ERT2Tg/+(control), or Moffl/flCag-Cre:ERT2Tg/+transgene (Mof-iKO) HSCs were sorted and cultured with 4-OHT in methylcellulose medium for 10 days. After 10 days in culture, ATAC-see signal was evaluated. (H) Left: Representative immunofluorescence image of MOF (red) and ATAC-see (green) from wild-type, Cag-Cre:-ERT2Tg/+, or Moffl/fl Cag-Cre:ERT2Tg/+ (Mof-iKO) mice. Right: MFI of ATAC-see, MOF, and DAPI signal (number of animals, n = 4). After normality test, two-tailed Mann-Whitney test was applied for statistical significance. Related to fig. S7.

Fig 5: Hematopoietic stem cells are derived from the Hoxb8 lineage. (A-C) Representative flow cytometry plots of Lin− Sca-1+ Kithi (LSK+) cells gated for CD48 and CD150 to identify LT-fHSCs (CD150+ CD48−), ST-fHSCs (CD150− CD48−) and MPPs (CD150− CD48+) from fetal livers of (A) E12.5 and (B) E14.5 embryos, and (C) bone marrow of 2-month-old mice. These LSK+ subsets were further gated for tdTomato to identify Hoxb8 lineage (B8L) and non-Hoxb8 lineage (nB8L) cells. (D-F) Percentage of B8L and nB8L cells in the (D) ST-fHSC, (E) LT-fHSC and (F) MPP populations. FL, fetal liver; Sp, spleen; BM, bone marrow. n=6 biological replicates per group per time point; two-way ANOVA with post-hoc analysis comparing nB8L cells with B8L cells across all times points examined, P<0.0001. Gray circles are individual data points. Data are mean±s.e.m. ****P<0.0001. Data in all graphs are from two independent experiments per time point.