Deubiquitinase catalytic activity of MYSM1 is essential in vivo for hematopoiesis and immune cell development

Loss of catalytic activity in the MYSM1
^D660N mutant protein

To generate an allele encoding a catalytically inactive MYSM1 in mouse we chose to introduce the Mysm1^D660N point mutation at the highly conserved aspartic acid 660 residue within the JAMM motif of the MPN catalytic domain (Fig. 1A). This residue is considered essential to the catalytic mechanism and is predicted to interact with both Zn²⁺ and the substrate^3,4. To confirm that the catalytic activity of MYSM1^D660N is indeed impaired, we expressed and purified both wild-type MYSM1 and MYSM^D660N proteins from Sf9 insect cells, and performed an in vitro catalytic activity assay using Ubiquitin-Rhodamine 110 as substrate. The proteins were purified from Sf9 insect cells at a similar yield (0.78 mg/L of cells for wild type MYSM1, 0.83 mg/L of cells for MYSM1^D660N) and eluted in a retention volume slightly lower than 13 mL in size exclusion chromatography, demonstrating that they were not aggregated and likely not misfolded (Fig. S1A). We found that wild-type MYSM1 cleaved the substrate in a dose-dependent manner with a K_m of 8.5 μM for the substrate, whereas the activity of MYSM^D660N was completely abrogated (Fig. 1B). This confirms that the D660N mutation inactivates the catalytic activity of mouse MYSM1 protein.

Development and validation of the mouse model expressing a catalytically inactive MYSM1. (A) Domain structure of the mouse MYSM1 protein indicating the mutation in the MPN catalytic domain predicted to render the protein catalytically inactive. (B) Catalytic activity assay of recombinant mouse MYSM1 against ubiquitin-Rhodamine substrate demonstrates that the D660N mutation results in a full loss of the DUB catalytic activity. (C) Sanger sequencing of the Mysm1 locus in the genomic DNA of wild type control and Mysm1^DN/+ heterozygous mice, indicating the DNA sequences and the corresponding amino acid sequences of the wild type and mutant proteins. (D) High embryonic lethality of Mysm1^DN/DN mice: offspring genotypes obtained from the mating of two Mysm1^+/DN heterozygous parents show strong deviation from expected Mendelian ratios. (E) Length and weight of the age- and sex- matched mice of Mysm1^+/+, Mysm1^−/− and Mysm1^DN/DN genotypes; bars represent means ± SEM, statistical analysis with ANOVA comparing each group to the control, *p < 0.05, **p < 0.01, ***p < 0.001, NS—not significant. (F) Representative image of the age- and sex- matched mice of Mysm1^+/+, Mysm1^−/− and Mysm1^DN/DN genotypes, showing reduced body size and tail dysmorphology. (G) Western blot of mouse bone marrow cell lysates showing comparable MYSM1 protein levels in Mysm1^+/+ and Mysm1^DN/DN samples. (H) Western blot of splenocyte lysates from tamoxifen-treated Cre^ERT2 transgenic mice of Mysm1^fl/+, Mysm1^fl/fl, and Mysm1^fl/DN genotypes, showing strong depletion of MYSM1 protein in the Mysm1^Δ/Δ cells, and comparable MYSM1 protein levels between Mysm1^Δ/+ and Mysm1^Δ/DN samples. β-actin is used as a loading control in (G,H).

Generation of the Mysm1
^D660N mouse strain

We used CRISPR/Cas9-mediated genome editing in zygotes to introduce the point mutation into exon 16 of Mysm1. C57BL/6 zygotes were co-injected with Cas9 protein and a gRNA, along with two homology-dependent recombination (HDR) templates. Since conventional knockout of MYSM1 causes partial embryonic lethality, we used two HDR templates to increase the efficiency of the procedure: one HDR template for the introduction of the D660N mutation and a second template introducing silent mutations to disrupt the gRNA recognition site²⁴. The founder harboring both the D660N and silent mutations was backcrossed onto C57BL/6 mice to generate heterozygous Mysm1^D660N mice lacking the silent mutations on the other Mysm1 allele. Sanger sequencing of the genomic DNA demonstrated successful introduction of the point mutation that translates into the MYSM1^D660N amino acid substitution in the protein (Fig. 1C). The sequencing window covered 371 nucleotides (NCBI GRCm39 Mysm1, Gene ID: 320713, range from 94,840,309 to 94,840,679), and included the entire Mysm1 exon 16 and ≥ 80 nucleotides of the flanking introns at both ends. This demonstrated no other mutations apart from those shown in Fig. 1C and resulting in the D660N substitution in the protein. As the mutations are located > 20 nucleotides away from the 3′ splice site of Mysm1 exon-16 they are not expected to disrupt splicing²⁵; and further analysis of the Mysm1^D660N allele with the Spliceator online tool (www.lbgi.fr/spliceator/) predicted no changes in splicing. Furthermore, RT-qPCR analysis of mouse bone marrow cells with the primer pairs spanning Mysm1 exon junctions 15–16 and 16–17 demonstrated no changes in the levels of Mysm1 transcript successfully spliced across these exon junctions in Mysm1^DN/DN compared to Mysm1^+/+ control cells (Fig. S1B).

Mysm1
^DN/DN mouse model: partial embryonic lethality and developmental phenotypes

Mysm1^DN/DN mice were born in sub-Mendelian numbers, with only ~ 4% of offspring from an intercross of two heterozygous parents having the Mysm1^DN/DN genotype, indicating that the loss of MYSM1 catalytic activity causes increased embryonic lethality (Fig. 1D). At adulthood, Mysm1^DN/DN mice were significantly smaller in length and weight than their littermates (Fig. 1E,F), and had abnormally short tails (Fig. 1F), as previously seen in the Mysm1^−/− mice^8,9. Importantly, we demonstrated similar levels of MYSM1 protein in Mysm1^DN/DN and control Mysm1^+/+ bone marrow cells, and the expected loss of MYSM1 protein expression in Mysm1^−/− cells (Fig. 1G). Overall, we highlight the similarity in the gross developmental phenotypes of the Mysm1^DN/DN and Mysm1^−/− mouse strains, and establish the essential role of the MYSM1 DUB catalytic activity in vivo.

Mysm1
^fl/DNCre^ERT2 mouse model for an inducible loss of the MYSM1 catalytic activity

Given the partial embryonic lethality and low availability of the Mysm1^DN/DN mice, we crossed the mice to the Mysm1^fl/flCre^ERT2 mouse strain that allows highly efficient Mysm1^fl to Mysm1^Δ allele conversion with tamoxifen treatment, as demonstrated in our previous studies^17,26. Here we derived cohorts of Cre^ERT2-transgenic mice of Mysm1^fl/+, Mysm1^fl/fl, and Mysm1^fl/DN genotypes, which were born in normal Mendelian numbers, lacked any obvious developmental phenotypes, and bred normally (data not shown). Following tamoxifen treatment, we demonstrated a strong depletion of MYSM1 protein in Mysm1^Δ/Δ mouse splenocytes, but comparable retention of MYSM1 protein levels in Mysm1^Δ/+ and Mysm1^Δ/DN samples (Fig. 1H). The Cre^ERT2 Mysm1^fl/DN model will test the effects of the loss of MYSM1 DUB catalytic activity on the maintenance of hematopoiesis, leukocyte development, and other aspects of mammalian physiology, independently of its roles in mouse development and the significant developmental phenotypes seen in the Mysm1^DN/DN mouse strain.

Severe hematologic dysfunction in Mysm1
^DN/DN and Mysm1
^fl/DNCre^ERT2 mice

Hematology analyses of the blood of Mysm1^DN/DN and Mysm1^−/− mice relative to the Mysm1^+/+ controls, demonstrated severe hematopoietic dysfunction, characterized by macrocytic anemia, with reduction in blood erythrocyte counts, hematocrit, and hemoglobin concentration, as well as an increased in mean corpuscular volume (MCV, Fig. 2A). Severe depletion of leukocytes and lymphocytes in Mysm1^DN/DN relative to control Mysm1^+/+ mice was also observed (Fig. 2A). Overall, the reported anemia and leukopenia phenotypes of Mysm1^DN/DN mice are highly consistent with those observed in the Mysm1^−/− mouse model (Fig. 2A), and also clinically in the patients with MYSM1 loss-of-function mutations^1,5,6,7.

Hematologic dysfunction in the mouse models with the loss of MYSM1 DUB catalytic activity. Hematology analyses were conducted on the blood of (A) Mysm1^+/+, Mysm1^−/−, and Mysm1^DN/DN mice, and (B) Cre^ERT2-transgenic tamoxifen-treated mice of Mysm1^fl/+, Mysm1^fl/fl, and Mysm1^fl/DN genotypes. Data is from (A) 3–8 mice per genotype consolidated from two independent experiments, or (B) 5–6 mice per genotype consolidated from two independent experiments. Bars represent means ± SEM; statistical analysis with ANOVA comparing each group to the control, *p < 0.05, **p < 0.01, ***p < 0.001, NS—not significant; MCV—mean corpuscular volume.

We conducted further hematology analyses on tamoxifen-treated Cre^ERT2 transgenic mice of Mysm1^+/fl, Mysm1^fl/fl, and Mysm1^DN/fl genotypes, and observed highly similar hematopoietic phenotypes in the Mysm1^Δ/DN mice, including macrocytic anemia, leukopenia, and lymphocyte depletion (Fig. 2B). We further observed an increase in platelets in Mysm1^DN/Δ mice (Fig. 2B), while platelets were not quantified in the Mysm1^DN/DN model due to increased clotting of the blood samples. Elevated platelet counts were previously reported for the Mysm1^−/− mouse strain^1,8, and although the mechanisms remain poorly understood they may be linked to elevated inflammatory response in Mysm1^−/− mice^14,15,16, as thrombocytosis is a common feature of systemic inflammation²⁷. Overall, we demonstrate that the loss of MYSM1 DUB catalytic activity in either constitutive or inducible mouse models results in a severe hematologic dysfunction with highly similar phenotypes to the previously characterized Mysm1^−/− and Mysm1^Δ/Δ mouse strains.

Depletion of lymphoid and myeloid immune cells in the Mysm1
^DN/DN mice

Severe reduction in lymphocyte numbers, including B cells, CD4 T cells, CD8 T cells, and NK cells, was also observed in the spleen of the Mysm1^DN/DN and Mysm1^Δ/DN mice, with the overall phenotype being highly similar to that of the Mysm1^−/− and Mysm1^Δ/Δ mouse models (Fig. 3A–D). An increase in the proportion of dead cells was also observed, particularly for splenic B cells and NK cells in Mysm1^DN/DN relative to control Mysm1^+/+ mice (Fig. S2A). Further analyses confirmed the depletion of splenic transitional (T1–3) and follicular B cells in the Mysm1^DN/DN and Mysm1^Δ/DN mice, with a somewhat milder depletion of the marginal zone B cell population (Fig. S3A–B). The numbers of myeloid cells, including monocytes, macrophages, and neutrophils, were somewhat more variable across the experimental groups, but also showed a depletion in the Mysm1^DN/DN and Mysm1^Δ/DN mice (Fig. 3E,F, Fig. S2B).

Depletion of splenic lymphoid and myeloid immune cells with the loss of MYSM1 DUB catalytic activity. Flow cytometry analyses were performed on (A,C–E) Mysm1^+/+, Mysm1^−/−, and Mysm1^DN/DN mice, and (B,F) Cre^ERT2 transgenic mice of Mysm1^fl/+, Mysm1^fl/fl, and Mysm1^fl/DN genotypes at > 20 weeks after tamoxifen treatment, to quantify (A,B) B cells (CD19⁺CD3⁻), CD4 T cells (CD3⁺CD4⁺CD8⁻), CD8 T cells (CD3⁺CD4⁻CD8⁺), NK cells (CD3⁻NK1.1⁺), and (E,F) monocytes (CD45⁺CD3⁻NK1.1⁻CD11b⁺Ly6C⁺Ly6G⁻), macrophages (CD45⁺CD3⁻NK1.1⁻CD11b⁺Ly6G⁻Ly6C⁻F4/80⁺CD64⁺), and neutrophils (CD45⁺CD3⁻NK1.1⁻CD11b⁺Ly6G⁺Ly6C⁻). The data is from (A,E) 3–10 mice per genotype consolidated from two independent experiments, or (B,F) 8–11 mice per genotype consolidated from three independent experiments. Bars represent means ± SEM; statistical analysis with ANOVA and Dunnett’s post-hoc test, comparing each group to the control, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS—not significant. (C,D) Representative flow cytometry plots of the spleen of Mysm1^+/+, Mysm1^−/−, and Mysm1^DN/DN mice gated on live cells and showing the depletion of (C) CD19⁺ B cells and (D) CD4⁺ and CD8⁺ T cells in Mysm1^−/− and Mysm1^DN/DN mice; the average frequencies of cells in the gates are presented as mean ± st. dev.

Severe depletion of lymphocyte precursors in the Mysm1
^DN/DN mice

We further analyzed for the B and T cell precursor subsets in the bone marrow and thymus of Mysm1^DN/DN and Mysm1^−/− mice, relative to the Mysm1^+/+ controls. We observed a strong depletion of most B cell precursor subsets, including pre-pro-B and pro-B cells (Fractions A-B), pre-B cells, and the immature and mature bone marrow B cell populations in the Mysm1^DN/DN and Mysm1^−/− mice (Fig. 4A,C). Similarly, we observed a strong depletion of most thymocyte populations in the Mysm1^DN/DN and Mysm1^−/− mice (Fig. 4B,D). Overall, this indicates that the loss of MYSM1 DUB catalytic activity or the loss of MYSM1 protein expression both result in a severe defect in B and T lymphocyte development.

Depletion of B and T lymphocyte precursors in the bone marrow and thymus in mice with the loss of MYSM1 DUB catalytic activity. Flow cytometry analyses were performed on Mysm1^+/+, Mysm1^−/−, and Mysm1^DN/DN mice, analyzing (A) the bone marrow for the following B cell populations: Fraction A (FrA, B220⁺IgM⁻IgD⁻CD43⁺CD24^loBP1^lo), Fraction B (FrB, B220⁺IgM⁻IgD⁻CD43⁺CD24⁺BP1^lo), Fraction C (FrC, B220⁺IgM⁻IgD⁻CD43⁺CD24⁺BP1⁺), large pre-B cells (B220⁺CD19⁺IgM⁻IgD⁻CD43⁻IL7Rα^hiFSChi), small pre-B cells (B220⁺CD19⁺IgM⁻IgD⁻CD43⁻IL7Rα^loFSC^lo), immature B cells (B220⁺IgM⁺IgD⁻), mature B cells (B220⁺IgM⁺IgD⁺); and (B) the thymus for double negative thymocytes DN1 (CD45⁺CD4⁻CD8⁻CD44⁺CD25⁻), DN2 (CD45⁺CD4⁻CD8⁻CD44⁺CD25⁺), DN3 (CD45⁺CD4⁻CD8⁻CD44⁻CD25⁺), and DN4 (CD45⁺CD4⁻CD8⁻CD44⁻CD25⁻), double positive thymocytes (DP, CD45⁺CD4⁺CD8⁺), and single positive thymocytes (CD45⁺CD4⁺CD8⁻ and CD45⁺CD4⁻CD8⁺). The data is from 3 to 10 mice per genotype consolidated from two independent experiments. Bars represent means ± SEM; statistical analysis with ANOVA and Dunnett’s post-hoc test, comparing each group to the control, *p < 0.05, **p < 0.01, ***p < 0.001; bone marrow cell counts are presented per two tibias and femurs. (C) Representative flow cytometry plots of the mouse bone marrow stained for B220 and CD19 B cell markers and (D) of the mouse thymus stained for CD4 and CD8; the average frequencies of cells in the gates are presented as mean ± st. dev.

Hematopoietic progenitor depletion and hematopoietic dysfunction in the Mysm1
^DN/DN mice

To further characterize the dysfunction in hematopoiesis resulting from the in vivo loss of MYSM1 DUB catalytic activity, Mysm1^DN/DN, Mysm1^−/−, and control Mysm1^+/+ mice were analyzed for the numbers of hematopoietic progenitor cells across the different lineages, as well as for the multipotent progenitors (MPPs) and hematopoietic stem cells (HSCs). We observed a significant depletion of common lymphoid (CLP), common myeloid (CMP), and granulocyte monocyte (GMP) progenitors in Mysm1^DN/DN mice (Fig. 5A), while changes in megakaryocyte erythroid (MEP) and megakaryocyte (MkP) progenitors did not reach statistical significance (Fig. 5A). The numbers of HSC and MPP1-3 cells were highly variable between the Mysm1^DN/DN mice, and showed trends for expansion, which however did not reach statistical significance (Fig. 5B,C), and this likely reflects the competing effects of the loss of HSC quiescence and increased cell apoptosis, as previously reported in the Mysm1^−/− mouse models^10,19. Importantly, there was a severe depletion of the lymphoid primed MPP4 cells in both Mysm1^DN/DN and Mysm1^−/− relative to control mice (Fig. 5B,C), further supporting the essential role of MYSM1 DUB catalytic activity for lymphopoiesis. Furthermore, an increase in the proportion of dead cells was observed particularly for lymphoid biased MPP4 and CLP cells in Mysm1^DN/DN relative to control Mysm1^+/+ mice (Fig. S2C-D).

Hematopoietic dysfunction and altered hematopoietic progenitor cell numbers in Mysm1^DN/DN mice. Flow cytometry analyses were performed on the bone marrow of Mysm1^+/+, Mysm1^−/−, and Mysm1^DN/DN mice to quantify (A) common lymphoid progenitors (CLP, Lin⁻IL7Rα⁺cKit^loSca1^lo), common myeloid progenitors (CMP, Lin⁻cKit⁺Sca1⁻CD34⁺CD16/32⁻), granulocyte monocyte progenitors (GMP, Lin⁻cKit⁺Sca1⁻CD34⁺CD16/32⁺), megakaryocyte erythroid progenitors (MEP, Lin⁻cKit⁺Sca1⁻CD34⁻CD16/32⁻), and megakaryocyte progenitors (MkP, Lin⁻cKit⁺Sca1⁻CD150⁺CD41⁺); (B) hematopoietic stem cells (HSCs) and multipotent progenitors (MPP1-4), gated as LSK (Lin⁻cKit⁺Sca1⁺), followed by CD150⁺CD48⁻CD34⁻Flt3⁻ for HSCs, CD150⁺CD48⁻CD34⁺Flt3⁻ for MPP1, CD150⁺CD48⁺CD34⁺Flt3⁻ for MPP2, CD150⁻CD48⁺CD34⁺Flt3⁻ for MPP3, and CD150⁻CD48⁺CD34⁺Flt3⁺ for MPP4. The data is from 3–10 mice per genotype consolidated from two independent experiments. Bars represent means ± SEM; statistical analysis with ANOVA and Dunnett’s post-hoc test, comparing each group to the control, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS—not significant; bone marrow cell counts are presented per two tibias and femurs. (C) Representative flow cytometry density plots of the bone marrow of Mysm1^+/+, Mysm1^−/−, and Mysm1^DN/DN mice, gated on live Lin- cells and showing the LSK cell population (top), or gated on the LSK C150⁻CD48⁺ cells and showing the Flt3^lo MPP3 and Flt3^hi MPP4 cells; the average frequency of cells in the gates is presented as mean ± st. dev. While the LSK cell numbers are highly variable (top), a strong depletion of the lymphoid-primed MPP4 cells is consistently observed in all the Mysm1^−/− and Mysm1^DN/DN mice (bottom). (D) Colony forming units (CFU) assays showing depletion of pre-B and erythroid BFU-E progenitors in Mysm1^DN/DN and Mysm1^−/− mouse bone marrow; MMNC – marrow mononuclear cells.

The dysfunction in lymphopoiesis and erythropoiesis in the Mysm1^DN/DN mice was further demonstrated with colony-forming units (CFU) assays, showing a severe depletion of B-cell lineage and erythroid lineage CFUs in the Mysm1^DN/DN and Mysm1^−/− relative to the control mice (Fig. 5D). No analysis of myeloid CFUs was conducted, as in previous studies myeloid CFU numbers in Mysm1^−/− mice were not significantly impaired⁸.

Cell intrinsic role of MYSM1 DUB catalytic activity in hematopoiesis

To directly test the cell-intrinsic requirement for the MYSM1 DUB catalytic activity in hematopoiesis, competitive bone marrow chimeras were set up. CD45.1⁺ wild type bone marrow was mixed in a 1:1 ratio with the bone marrow of CD45.2⁺ Cre^ERT2 mice of Mysm1^+/fl, Mysm1^fl/fl, or Mysm1^DN/fl genotypes, and transplanted into three independent groups of lethally irradiated recipients (Fig. 6A). The recipient mice were bled at 12-weeks to confirm the normal reconstitution with donor bone marrow across the genotypes (data not shown), and subsequently all the mice were administered with tamoxifen to induce the Mysm1^fl to Mysm1^Δ allele conversion. The mice were analyzed for the relative contributions of the CD45.2⁺ bone marrow to the different hematopoietic lineage, across the three Mysm1 genotypes.

Assessing the cell-intrinsic role of MYSM1 DUB catalytic activity in hematopoiesis and leukocyte development with competitive bone marrow transplantation. (A) Schematic representation of the mouse-to-mouse competitive bone marrow transplantation study. Wild type CD45.1⁺ bone marrow cells were mixed in a 1:1 ratio with Cre^ERT2 transgenic bone marrow cells of Mysm1^fl/+, Mysm1^fl/fl, or Mysm1^fl/DN genotypes, and the mixes were transplanted into three independent cohorts of lethally irradiated wild type CD45.1⁺ recipient mice. Following full hematopoietic reconstitution, the chimeric mice were administered with tamoxifen to induce the Mysm1^fl to Mysm1^Δ allele conversion. Clipart images were used toward the preparation of the Figure (http://clipart-library.com). (B,C) The relative contribution of Mysm1^Δ/DN, Mysm1^Δ/Δ, and control Mysm1^Δ/+ cells to the different hematopoietic and immune cell populations was evaluated by flow cytometry, quantifying the proportion of CD45.2⁺CD45.1⁻ cells within each cell population. Data is from 3–5 mice per group; bars represent means ± SEM; statistical analysis uses ANOVA and Dunnett’s post-hoc test comparing each group to the Mysm1^Δ/+ control; *p < 0.05, **p < 0.01, ***p < 0.001, or NS—not significant. Data is presented for the following cell populations: (B) splenic B cells (CD19⁺CD3⁻), CD4 T cells (CD3⁺CD4⁺CD8⁻), CD8 T cells (CD3⁺CD4⁻CD8⁺), and NK cells (CD3⁻NK1.1⁺); bone marrow monocytes (CD11b⁺Ly6C^hiLy6G^lo), neutrophils (CD11b⁺Ly6C^loLy6G^hi), and erythroid cells (CD71⁺); (C) bone marrow stem and multipotent progenitors (LKS, Lin⁻cKit⁺Sca1⁺), common myeloid progenitors (CMP, Lin⁻cKit⁺Sca1⁻CD34⁺CD16/32⁻), granulocyte monocyte progenitors (GMP, Lin⁻cKit⁺Sca1⁻CD34⁺CD16/32⁺), common lymphoid progenitors (CLP, Lin⁻cKit^loSca1^loIL7Ra⁺CD16/32⁻), megakaryocyte erythroid progenitors (MEP, Lin⁻cKit⁺Sca1⁻CD34⁻CD16/32⁻), and megakaryocyte progenitors (MkP, Lin⁻cKit⁺Sca1⁻CD16/32⁻CD150⁺CD41⁺). (D) Representative flow cytometry plots showing the analyses of splenic B cells, splenic T cells, and bone marrow neutrophils for CD45.1 versus CD45.2 marker expression; percentage of cells in the gates is shown as mean ± st. dev. of all the mice in each group. Gates for CD45.1⁺ and CD45.2⁺ cells were set independently for each cell population using control non-chimeric WT-B6 (CD45.2) and WT-SJL (CD45.1) mice, as shown in Fig. S4E.

We observed a significant reduction in the contribution of the Mysm1^DN/Δ donor hematopoiesis to the B cell, CD4 T cell, CD8 T cell, and NK cell populations in the mouse spleen (Fig. 6B), to monocyte and neutrophil populations in both spleen and bone marrow (Fig. 6B and not shown), and to all the leukocyte populations in the mouse blood (Fig. S4A). Similar defects in the reconstitution were observed for the Mysm1^DN/Δ hematopoietic progenitor cells, including the lineage committed progenitors (CMPs, GMPs, CLPs, MEPs, and MkPs, Fig. 6C), all the developing B cell subsets (Fractions A-C, pre-B, and immature B cells, Fig. S4B), and the majority of T cell precursor subsets within the thymus (Fig. S4C). Among the multipotent HSC and MPP hematopoietic cells there was no defect in Mysm1^DN/Δ reconstitution of the early HSC and MPP1-2 cells, likely reflecting the balancing effects of the loss of quiescence and increase in apoptosis among these cells, as in the Mysm1^−/− mouse models^10,19, however impaired reconstitution was seen for the latter myeloid-biased MPP3 and lymphoid-biased MPP4 subsets (Fig. S4D). Importantly, throughout the datasets presented above the Mysm1^DN/Δ phenotypes aligned very well with the Mysm1^Δ/Δ group, both showing strong impairment of hematopoietic function relative to the Mysm1^+/Δ control (Fig. 6, S4). Overall, this demonstrates the essential and cell-intrinsic role of the MYSM1 DUB catalytic activity in the regulation of hematopoiesis, and suggests lack of significant MYSM1 mechanisms of action that are independent of its catalytic function.