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p53 is a central mediator of cellular stress responses, and its precise regulation is essential for the normal progression of hematopoiesis. MYSM1 is an epigenetic regulator essential for the maintenance of hematopoietic stem cell (HSC) function, hematopoietic progenitor survival, and lymphocyte development. We recently demonstrated that all developmental and hematopoietic phenotypes of Mysm1 deficiency are p53-mediated and rescued in the Mysm1−/−p53−/− mouse model. However, the mechanisms triggering p53 activation in Mysm1−/− HSPCs, and the pathways downstream of p53 driving different aspects of the Mysm1−/− phenotype remain unknown. Here we show the transcriptional activation of p53 stress responses in Mysm1−/− HSPCs. Mechanistically, we find that the MYSM1 protein associates with p53 and colocalizes to promoters of classical p53-target genes Bbc3/PUMA (p53 upregulated modulator of apoptosis) and Cdkn1a/p21. Furthermore, it antagonizes their p53-driven expression by modulating local histone modifications (H3K27ac and H3K4me3) and p53 recruitment. Using double-knockout mouse models, we establish that PUMA, but not p21, is an important mediator of p53-driven Mysm1−/− hematopoietic dysfunction. Specifically, Mysm1−/−Puma−/− mice show full rescue of multipotent progenitor (MPP) viability, partial rescue of HSC quiescence and function, but persistent lymphopenia. Through transcriptome analysis of Mysm1−/−Puma−/− MPPs, we demonstrate strong upregulation of other p53-induced mediators of apoptosis and cell-cycle arrest. The full viability of Mysm1−/−Puma−/− MPPs, despite strong upregulation of many other pro-apoptotic mediators, establishes PUMA as the essential non-redundant effector of p53-induced MPP apoptosis. Furthermore, we identify potential mediators of p53-dependent but PUMA-independent Mysm1−/−hematopoietic deficiency phenotypes. Overall, our study provides novel insight into the cell-type-specific roles of p53 and its downstream effectors in hematopoiesis using unique models of p53 hyperactivity induced by endogenous stress. We conclude that MYSM1 is a critical negative regulator of p53 transcriptional programs in hematopoiesis, and that its repression of Bbc3/PUMA expression is essential for MPP survival, and partly contributes to maintaining HSC function.
p53 is a central regulator of cell fate and cellular responses to physiological and exogenous stresses.1 It is a DNA-binding protein that primarily acts as a transcriptional regulator.2 Acute p53 activation can trigger cell-cycle arrest, cellular senescence, or apoptosis. Different combinations of p53-induced effectors can execute these distinct cell fate decisions. For example, cyclin-dependent kinase inhibitor Cdkn1a/p21 is a significant p53-induced mediator of cell-cycle arrest,3, 4 whereas BH3-domain protein Bbc3/PUMA is an important mediator of p53-dependent apoptosis.5, 6
Knowledge of cell-type-specific features of the p53 pathway is important for the understanding of physiological stress responses and the development of clinical strategies for targeted p53 activation.7, 8 Many studies in this area have focused on hematopoietic stem and progenitor cells (HSPCs).9, 10 p53 was shown to have specialized roles in homeostatic regulation of hematopoietic stem cell (HSC) quiescence, self-renewal, and differentiation.11, 12, 13 p53-target gene Cdkn1a/p21 was originally implicated in enforcing homeostatic HSC quiescence,14 although this role was questioned in recent studies.15 Other novel p53-target genes were identified as regulators of HSC quiescence, including Gfi1 and Necdin.11, 16, 17
In hematopoietic responses to stress, Bbc3/PUMA was identified as a major mediator of p53-dependent apoptosis,18, 19, 20, 21 with some redundancy with other BH3-only family members such as Pmaip1/NOXA.22, 23, 24, 25 Other studies highlighted specific differences in the stress responses of HSCs and more mature hematopoietic progenitors,10 with HSCs in mouse bone marrow being less prone to induction of pro-apoptotic genes and cell death than progenitor cells.26, 27, 28 Despite a growing understanding of the specialized features of p53 responses in HSCs and hematopoiesis, and their obvious biological and clinical importance, the underlying molecular mechanisms remain poorly understood.
Myb-like SWIRM and MPN domains 1 (MYSM1) is a chromatin-binding protein29 previously implicated in transcriptional regulation through histone H2AK119 (histone H2A lysine 119) deubiquitination.30 Mysm1 loss in mice results in the arrest of lymphocyte development and severe defects in HSPC functions, including loss of HSC quiescence and impaired survival of multipotent progenitors (MPPs).31, 32, 33 Patients with rare MYSM1 mutations display lymphopenia and HSC exhaustion, confirming the essential role of MYSM1 in hematopoiesis and lymphocyte development in humans.34, 35
There are two perspectives on the role of MYSM1 in hematopoiesis. In a series of recent papers, Mysm1−/− hematopoietic defects were linked to impaired expression of various hematopoietic lineage differentiation genes, namely Ebf1, Id2, Gfi1, and Flt3.32, 33, 36, 37 In contrast, we and others demonstrated that Mysm1−/− phenotypes are rescued in the Mysm1−/−p53−/− mouse model,38, 39 indicating that p53 activation is the common mechanism mediating HSC dysfunction, MPP depletion, and lymphopenia in Mysm1 deficiency. However, the mechanisms leading to p53 activation and its effects in Mysm1-deficient HSPCs remain unclear. Although Gatzka M et al.39 indicated induction of Cdkn2a/p19ARF as the cause of p53 activation in the Mysm1−/− thymus, it is unknown whether these findings extend to other Mysm1−/− hematopoietic cells, particularly HSPCs. Furthermore, the specific p53-induced effectors that trigger dysfunction in Mysm1-deficient HSPCs remain unknown.
Here we show that MYSM1 is a novel regulator of p53 transcriptional programs that antagonizes the expression of p53 stress-response genes through direct effects on chromatin at their promoters. We further show that Bbc3/PUMA is the primary non-redundant mediator of MPP depletion in Mysm1 deficiency and contributes to HSC dysfunction, whereas depletion of lymphoid-lineage cells involves PUMA-independent p53 activities.
To understand the causes of p53 activation and downstream p53 effector functions leading to hematopoietic failure in Mysm1-deficiency, HSPC gene expression was analyzed. Inducible Mysm1 deletion in the Rosa26CreERT2Mysm1flox/flox model40 was used to focus on direct effects of Mysm1 loss rather than indirect effects of chronic Mysm1 deficiency. HSPCs (Lin−cKit+Sca1+) were sorted from tamoxifen-treated Rosa26CreERT2Mysm1flox/flox mice against control Rosa26CreERT2Mysm1+/flox mice and untreated Mysm1flox/flox animals. qRT-PCR validated loss of Mysm1 expression, without significant changes in p53 transcript (Figure 1a). There was a strong induction of p53 stress-response genes including Bbc3/PUMA, Pmaip1/NOXA, Bax, Cdkn1a/p21, and Ccng1 (Figure 1b).
We validated the reduced expression of HSC quiescence regulator Gfi1 previously reported in Mysm1−/− HSPCs,37 and demonstrated the downregulation of other HSC quiescence regulators, namely Necdin and Cdkn1c/p5716, 17, 41, 42, 43 (Figure 1c and d). Overall, this indicated severe dysregulation of both p53-mediated stress responses and cell-cycle target genes in HSPCs upon Mysm1 deletion. Importantly, Cdkn2a/p19ARF expression was undetectable in all samples and not elevated in Mysm1-deficient HSPCs (Supplementary Figure S1a). As induction of Cdkn2a/p19ARF was previously implicated in p53 activation in Mysm1−/− thymocytes,39 we conclude that the mechanisms of p53 activation in Mysm1-deficient HSPCs are distinct and p19ARF-independent.
As both p53 and MYSM1 are transcriptional regulators,2, 30 we hypothesized that the increased induction of p53-target genes in Mysm1-deficient HSPCs indicated a direct role of MYSM1 in their regulation. The recruitment of MYSM1 to p53-target gene promoters was tested using chromatin immunoprecipitation (ChIP) in a Ba/F3 hematopoietic progenitor cell line stably expressing FLAG-MYSM1 (Supplementary Figure S2a). MYSM1 binding was detected at known p53-binding sites within Bbc3/PUMA and Cdkn1a/p21 promoters,44 and enhanced with irradiation (Figures 2a and b).
Given the recruitment of MYSM1 to genomic p53-binding sites, we tested for interaction between MYSM1 and p53 proteins. In the Ba/F3 line expressing FLAG-MYSM1, endogenous p53 was co-immunoprecipitated with FLAG-MYSM1 (Figure 2c), and similar results were obtained in reciprocal co-immunoprecipitations in HEK293T cells (Supplementary Figure S2b). MYSM1 therefore interacts with p53, either directly or more likely as part of a larger protein complex. The interaction of MYSM1 with p53 and their colocalization to p53-target gene promoters, in addition to p53 transcriptional signature activation in Mysm1−/− HSPCs, suggest a role for MYSM1 in regulating p53 transcriptional activity.
To overcome the limitations of the severe depletion of primary HSPCs in Mysm1−/− mice, Mysm1 shRNA knockdown in Ba/F3 hematopoietic progenitor cells was employed as a model for further analysis of MYSM1 functions in p53-target gene regulation. The shMysm1-knockdown lines showed a 10-fold reduction in the MYSM1 protein as compared with firefly luciferase shRNA (shFF) control lines (Figure 3a). To validate the model's relevance, shMysm1 lines were tested for the phenotypes observed in primary Mysm1−/− HSPCs, including activation of the p53 stress response and apoptosis. Following irradiation, shMysm1 cells displayed increased susceptibility to cell death, and slightly increased levels of p53 protein. Furthermore, shMysm1 cells showed elevated induction of p53 stress-response genes such as Bbc3/PUMA and Cdkn1a/p21 at both transcript and protein levels (Figures 3b–e; Supplementary Figure S3a). The knockdown Ba/F3 cells therefore reproduced all the major phenotypic and transcriptional features of Mysm1−/− HSPCs, despite having only partial reduction in MYSM1 protein. This confirms that shMysm1-Ba/F3 cells are an appropriate model for assessing the role of MYSM1 in regulating p53-target gene expression.
We analyzed p53 recruitment and histone modification at the Bbc3/PUMA and Cdkn1a/p21 promoters in untreated and irradiated Mysm1-knockdown cells. At the Bbc3/PUMA promoter, Mysm1 knockdown resulted in increased p53 recruitment and striking increases in histone H3 lysine 27 acetylation (H3K27ac) and histone H3 lysine 4 trimethylation (H3K4me3) histone modifications (Figures 3f and g; Supplementary Figure S3b). At the Cdkn1a/p21 locus, increased H3K27ac and H3K4me3 levels were also observed, but without increased p53 binding (Supplementary Figure S3b), which indicates that MYSM1 can have an impact on H3K27ac levels independently of effects on p53 recruitment. Increased levels of H3K27ac and H3K4me3, hallmarks of promoter activity, are known to correlate with increased transcription. Therefore, even partial Mysm1 knockdown results in significant changes in chromatin dynamics at its binding sites within p53 target gene promoters. As the chromatin state represents a propensity for transcription, we conclude that MYSM1 acts as a transcriptional cofactor that restricts p53 stress-response transcriptional activity by modulating the epigenetic state of activation at p53-target gene regulatory elements (Figures 3f and g; Supplementary Figure S3b).
Our data implicate MYSM1 as a transcriptional regulator of p53-target genes Bbc3/PUMA and Cdkn1a/p21, with MYSM1 loss resulting in increased p53 transcriptional activity in vitro and in vivo (Figures 1–3). Considering our recent demonstration that Mysm1-deficiency phenotypes are p53-mediated,38 we tested the roles of PUMA and p21 as specific mediators of Mysm1−/− phenotypes by generating Mysm1−/−Puma−/− and Mysm1−/−Cdkn1a−/− mouse lines.
Mysm1−/−Puma−/− mice showed rescue of Mysm1−/− coat pigmentation defects, hindlimb and tail dysmorphology, and partial rescue of growth retardation (Figures 4a and b). There was a significant increase in bone marrow cellularity in Mysm1−/−Puma−/− relative to Mysm1−/− controls, suggesting some rescue of hematopoiesis; however, thymus and spleen cellularity remained low (Figure 4c). In contrast, Mysm1−/−Cdkn1a−/− mice showed high levels of embryonic and neonatal mortality, and the few surviving animals had no observable rescue of Mysm1-deficiency phenotypes (Supplementary Figures S5 and 6). Overall, this implicated PUMA, but not p21, in mediating developmental and hematopoietic abnormalities in Mysm1 deficiency.
B cells, CD4 T cells, and CD8 T cells remained strongly diminished in Mysm1−/−Puma−/− spleens (Figures 4d and e). There was also no significant rescue of common lymphoid progenitor cells (CLPs), bone marrow B-cell precursors, or thymic T-cell precursors in Mysm1−/−Puma−/− mice relative to Mysm1−/− controls (Supplementary Figure S4). We conclude that the lymphoid-lineage differentiation arrest in Mysm1−/− mice is independent of Bbc3/PUMA, and is mediated through other p53 effector functions, as it was rescued in Mysm1−/−p53−/− mice.38, 39
Surprisingly, the severe loss of lymphoid-primed MPPs (MPP4s; cKit+Lin−Sca1+CD150−CD48+Flt3+CD34+) characteristic of Mysm1 deficiency was fully rescued in Mysm1−/−Puma−/− mice.31, 33, 37 This implicates PUMA-mediated apoptosis in the depletion of this cell population (Figure 5). Restricted myeloid progenitor cell numbers were also rescued, further demonstrating that the p53/PUMA axis mediates Mysm1−/− hematopoietic dysfunction (Figure 4f). Moreover, we observed an expansion of HSC numbers in Mysm1−/−Puma−/− mice not only relative to the Mysm1−/− group but also significantly above wild-type and Puma−/− levels (Figure 5; Supplementary Figure S7). This suggested that the loss of HSC quiescence previously reported in the Mysm1−/− line33 persists in Mysm1−/−Puma−/− mice. We conclude that PUMA is the essential non-redundant mediator of p53-driven depletion of MPP4s and myeloid lineage cells in Mysm1 deficiency.
Bone marrow chimeras were employed to further assess the role of Bbc3/PUMA in Mysm1-deficient HSPC dysfunction. B6.SJL-CD45.1 bone marrow was mixed with CD45.2 bone marrow from wild-type, Mysm1−/−, Puma−/−, or Mysm1−/−Puma−/− mice, and were transplanted into four groups of lethally irradiated recipients. As previously reported, Mysm1−/− donors were strongly impaired in hematopoietic reconstitution capacity.31, 33 However, the performance of Mysm1−/−Puma−/− donors was significantly enhanced in comparison (Figure 6). Specifically, Mysm1−/−Puma−/− bone marrow was rescued in HSPC compartment reconstitution (Figures 6a and b), indicating partial rescue of HSC functions. There was a strongly improved contribution to the myeloid cell lineage, including progenitors and total CD11b+ cells of the bone marrow, spleen, and blood (Figure 6c, Supplementary Figure S9). Lymphoid-lineage reconstitution, however, remained severely impaired, with no rescue of B- and T-cell reconstitution, and only a minor increase in the reconstitution of CLP and NK cells (Figures 6d and e,Supplementary Figures S8 and 9). The results show a significant yet incomplete rescue of Mysm1−/−Puma−/− HSC function and further establish that Bbc3/PUMA is an important mediator of the hematopoietic failure in Mysm1 deficiency.
Our data indicate that PUMA is an important mediator of MPP4 depletion and HSC dysfunction in Mysm1 deficiency. To further explore the p53/PUMA axis as the mediator of Mysm1−/− HSPC stress phenotypes,31, 33 we analyzed Mysm1−/−Puma−/− and Mysm1−/−p53−/− HSPCs for oxidative stress, DNA damage, and apoptosis. Increased levels of apoptosis in Mysm1−/− MPP4 cells were fully rescued upon deletion of either p53 or Bbc3/PUMA (Figure 7a), indicating that Mysm1−/− MPP4s are depleted via p53-dependent apoptosis, with PUMA as its primary non-redundant mediator. Increases in reactive oxygen species (ROS) and DNA damage (Phospho-Serine 139 Histone H2A Variant X, γH2AX) in Mysm1−/− HSPCs were also restored to normal levels in Mysm1−/−p53−/− and Mysm1−/−Puma−/− mice (Figure 7b and c). This indicates that they are neither direct products of MYSM1 loss nor primary inducers of p53, but rather downstream results of p53-PUMA activation and apoptosis.45, 46
Previous studies reported reduced quiescence and increased proliferation of Mysm1−/− HSCs.33 Using Ki67 as a marker of non-quiescent cells, we show that Mysm1−/− HSC loss of quiescence is rescued in Mysm1−/−p53−/− HSCs (Figure 7d, right). This is consistent with the full rescue of Mysm1−/−p53−/− HSC functions reported in our recent work.38 Cell-cycle analysis was also performed in Mysm1−/−Puma−/− HSCs by staining for Ki67 and DNA content to resolve G0, G1, and S–G2–M cell-cycle phases. This revealed a partial rescue, with normalized percentages of proliferating Mysm1−/−Puma−/− HSCs in the S–G2–M phases of the cell cycle, but persistent loss of quiescence with a significant reduction in G0-phase cells and increase in G1-phase cells (Figure 7d, left). The significant but incomplete rescue of HSC quiescence in the Mysm1−/−Puma−/− model is consistent with the aforementioned partial rescue of Mysm1−/−Puma−/− HSC functions (Figure 6).
Finally, we assessed p53 protein levels in Mysm1−/−Puma−/− mice and found that they were still significantly elevated in both HSC and MPP populations, although partially reduced in the latter (Figure 7e). Overall, this suggests that continued dysregulation of p53 effector functions beyond the upregulation of Bbc3/PUMA contributes to persistent Mysm1−/− HSC loss of quiescence, and also to the lymphoid differentiation arrest of Mysm1−/−Puma−/− MPP4s.
We establish PUMA as the primary effector of p53-mediated depletion of MPP4 and myeloid cells, but not CLPs and lymphoid cells in Mysm1 deficiency. To gain insight into the functional defects in Mysm1−/−Puma−/− MPP4s contributing to cell death and downstream lymphopoiesis failures, we performed RNA-sequencing (RNA-Seq) of MPPs (Lin−cKit+Sca1+CD150−) from Mysm1−/−Puma−/−, Mysm1−/−Puma+/−, Puma−/−, and wild-type mice (Mysm1−/−Puma+/− phenocopies Mysm1−/−). Unbiased clustering analysis of differentially regulated genes yielded four main groups (Figure 8a). Puma−/− cells displayed no significant gene expression differences as compared with wild-type controls.
In cluster 1, the strong p53 stress-response signature of Mysm1−/−Puma+/− MPPs, and Mysm1−/− HSPCs (Figure 1), was fully retained in Mysm1−/−Puma−/− MPPs (Figures 8a–c; Supplementary Figures S11a–c). Gene ontology analysis revealed that genes implicated in ‘positive regulation of apoptosis' and ‘cell-cycle arrest' were strongly enriched (Figure 8b), including p53-target genes such as Pmaip1/NOXA, Bax, and Cdkn1a/p21 (Figure 8c, Supplementary Figures S11a–c), and downstream apoptotic mediators including Casp12 and Bcl2l11/BIM (Supplementary Figure S11f). This indicates a persistent activation of p53-mediated stress-response pathways in Mysm1−/−Puma−/− MPPs.
In cluster 4, Mysm1−/−Puma−/− MPPs showed a striking downregulation of genes required for cell-cycle progression, such as Ccne1, Ccna1, Cdca5, histone genes,47 and many others, compared with control and Mysm1−/−Puma+/− levels (Figure 8d). This was associated with a reduced proportion of cells in the S–G2–M cell-cycle phases (Supplementary Figure S10). However, HSC-specific quiescence regulators were either unchanged or undetectable in expression in MPPs across all genotypes (Supplementary Figure S11i; Figure 1). The downregulation of cell-cycle progression genes, together with the upregulation of p53 effectors in Mysm1−/−Puma−/− MPPs further supports persistent stress-response activation in these cells. Importantly, as with HSPC qPCR analysis (Supplementary Figure S1), Cdkn2a was not expressed in Mysm1-deficient MPPs (Supplementary Figure S11d), further distinguishing MYSM1 functions in HSPCs and thymocytes.39
In cluster 3, the downregulation of many lymphoid-lineage genes observed in Mysm1−/−Puma+/− cells was rescued in Mysm1−/−Puma−/− cells (Figure 8e; Supplementary Figure S11h). Interestingly, Flt3, Satb1, Dntt, Il7r, and most other genes in this cluster were recently shown to be upregulated in the differentiation of lymphoid-primed MPP4 cells.48 This indicates that their downregulation in Mysm1 deficiency represents PUMA-dependent death of MPP4s, rather than an intrinsic requirement for MYSM1 to induce their expression. Notably, all the genes implicated as direct MYSM1 targets in previous studies, including Gfi1, Flt3, Id2, and Ebf1,32, 33, 36, 37 did not differ in expression between Mysm1−/−Puma−/− and control MPPs (Supplementary Figure S11g). This indicates that, at least in MPPs, Mysm1-deficiency phenotypes are independent of these genes, and instead are p53 stress-response-mediated, as supported by all our other analyses.
We conclude that the rescued Mysm1−/−Puma−/− MPP4 viability, despite a strong upregulation of other apoptotic mediators such as Pmaip1/NOXA, Bax, and others, demonstrates the key non-redundant role of PUMA as the mediator of p53-dependent apoptosis within the MPP4 population. The other upregulated p53 stress-response genes identified in the transcriptome analysis likely contribute to the p53-dependent but PUMA-independent phenotypes in Mysm1 deficiency.
Our work demonstrates that MYSM1 represses p53 stress-response gene expression by localizing to p53-binding sites within Bbc3/PUMA and Cdkn1a/p21 promoters and antagonizing local histone modification (H3K27ac and H3K4me3) and p53 recruitment (Figure 3g). We further establish that PUMA, but not p21, is an important mediator of the p53-driven hematopoietic dysfunction in Mysm1 deficiency, with Mysm1−/−Puma−/− mice showing full rescue of MPP4 cell viability, partial rescue of HSC functions, but persistent depletion of CLPs and lymphopenia. We demonstrate that PUMA is an essential non-redundant effector of p53-induced MPP4 apoptosis, and through global transcriptome analysis of Mysm1−/−Puma−/− MPPs identify other p53 effectors that remain induced and may contribute to the other p53-dependent but PUMA-independent Mysm1−/− phenotypes. Whereas recent studies established p53 activation as the common mediator of all Mysm1−/− hematopoietic phenotypes,38, 39 our current work provides novel mechanistic insight into the distinct outcomes of p53 activation in different hematopoietic populations. This highlights the Mysm1−/− and Mysm1−/−Puma−/− lines as unique models for the study of p53 hyperactivity within different hematopoietic populations without exogenous stress. We conclude that MYSM1 is an essential regulator of p53 transcriptional programs in hematopoiesis, acting to maintain hematopoietic progenitor viability by antagonizing p53-driven Bbc3/PUMA expression.
Although PUMA is a major mediator of p53-dependent apoptosis, its functions overlap significantly with other BH3-only proteins, for example, with NOXA in lymphocyte responses to irradiation, in B-cell lymphomagenesis, and in non-hematopoietic cells such as fibroblasts and oocytes.22, 23, 49 Similarly, BIM contributes to apoptosis during lymphocyte development50 and exposure to mutagens.24, 25 In contrast, we demonstrate an essential non-redundant role of PUMA in p53-mediated apoptosis of MPP4s but not CLPs in the context of Mysm1 deficiency. Transcriptional profiling of Mysm1−/−Puma−/− MPPs provides valuable insights into the p53 transcriptional network of this hematopoietic population, and identifies upregulation of many p53-target genes, including pro-apoptotic Pmaip1/NOXA, Bax, Trp53inp1, and others. The full rescue of Mysm1−/−Puma−/− MPP4 viability, despite strong upregulation of other apoptotic mediators, further demonstrates that PUMA is the key effector of MPP4 apoptosis. Considering that Mysm1−/−p53−/− mice displayed complete rescue of hematopoietic dysfunction,38 the other p53 effectors identified in our Mysm1−/−Puma−/− transcriptome analysis may contribute to the PUMA-independent but p53-dependent Mysm1−/− phenotypes.
Lymphoid cell depletion in Mysm1 deficiency was previously attributed to several mechanisms, involving either MYSM1-mediated Cdkn2a/p19ARF repression to restrict p53 activation in thymocytes,39 or MYSM1-mediated induction of lymphoid-lineage genes, such as Ebf1 and Id2.32, 36 Importantly, Cdkn2a expression was undetectable in Mysm1-deficient HSPCs or MPPs, demonstrating the distinct mechanism of p53 activation in Mysm1−/− HSPCs, as compared with thymocytes.39 Furthermore, the full-transcriptome analysis of Mysm1−/−Puma−/− MPPs showed no global changes in lymphoid-lineage priming, and normal expression of all genes previously implicated as MYSM1 targets.32, 36, 33, 37 Although lymphoid genes were downregulated in Mysm1−/− MPPs, this reflected PUMA-mediated death of lymphoid-primed MPP4 cells, as the gene expression was restored in Mysm1−/−Puma−/− MPPs together with restoration of MPP4 viability. This shows that MYSM1 has no direct role in transcriptional regulation of MPP lymphoid priming, but instead maintains MPP4 survival by regulating p53 transcriptional programs. Lymphoid cell depletion via p53-dependent38, 39 but PUMA-independent mechanisms suggests the involvement of other p53-induced effectors. The upregulation of p53-regulated pro-apoptotic mediators, together with downregulation of many genes essential for normal cell-cycle progression in Mysm1−/−Puma−/− MPPs, may contribute to the p53-mediated depletion of downstream CLPs and lymphopenia in Mysm1 deficiency.
Our study also provides important insights into the unique effects of p53 stress responses in HSCs, where p53 activation in Mysm1 deficiency does not result in apoptosis, but promotes loss of quiescence. Our findings support the conclusions of several studies, which indicated that HSCs are more resistant to apoptosis compared with downstream progenitors, and have lower induction of pro-apoptotic genes in response to exogenous stress such as irradiation.10, 26, 27, 28, 51 We also demonstrate that Mysm1−/− HSC hyperproliferation is in part a compensatory effect driven by PUMA-dependent depletion of downstream progenitors, similar to the compensatory mechanisms operating following irradiation-induced MPP depletion.27, 51
Although the rescue of HSC quiescence and function in Mysm1−/−Puma−/− mice was significant, it was incomplete. This represents an important distinction from the complete rescue in Mysm1−/−p53−/− mice and indicates a cell-intrinsic role of MYSM1 in HSC homeostasis in crosstalk with p53. p53 is known to promote homeostatic HSC quiescence by regulating its HSC-specific targets Gfi1 and Necdin.11, 16, 17 We confirm downregulation of Gfi1 in Mysm1−/− HSPCs,33 and also show downregulation of Necdin. This suggests that MYSM1 may have distinct effects at different p53-target genes, repressing classical stress-response genes such as Bbc3/PUMA to promote survival while supporting expression of HSC-specific targets such as Gfi1 and Necdin to maintain quiescence (Supplementary Figure S12). Although MYSM1 was initially characterized as a transcriptional activator acting via histone H2AK119 deubiquitination,30, 52 recent studies linked H2AK119 deubiquitination by other proteins to transcriptional repression.53 Our work shows that MYSM1 represses p53 stress-response genes by antagonizing other histone modifications (H3K27ac and H3K4me3). Further understanding of the mechanisms through which MYSM1 regulates p53 transcriptional activity in HSCs to maintain HSC quiescence and function is an important focus for future work.
In conclusion, we establish MYSM1 as an essential regulator of p53 transcriptional activity and stress-response programs in HSPCs. We demonstrate that PUMA is an essential non-redundant mediator of p53-induced apoptosis in MPPs but not lymphoid cells in the context of Mysm1 deficiency. Together with the characterization of p53 stress-response transcriptional signatures in MPPs, this provides valuable novel insights into the cell-type-specific roles of p53 in hematopoiesis. Our work advances the understanding of human bone marrow failures resulting from MYSM1 mutations,34, 35 and highlights MYSM1 as a potential drug target for the activation of p53 and PUMA in hematopoietic progenitors for myeloablation or leukemia treatment.7, 8, 54
Mysm1tm1a(KOMP)WTSI mice derived from EPD0019_1_A05 ESC clone were previously described31, 55 and have over 100-fold reduction in the Mysm1 transcript in a homozygous state (referred here as Mysm1−/−). Mysm1flox/flox line was generated by crossing Mysm1tm1a mice to the Gt(ROSA)26 Sortm1(FLP1)Dym/Wtsi line with ubiquitous expression of Flp recombinase,56 and subsequently bred to Gt(ROSA)26Sortm1(cre/ERT2) mice for tamoxifen-induced Mysm1 inactivation.40 The p53 knockout and Bbc3/PUMA knockout lines were obtained from The Jackson Laboratory (JAX002101 and JAX011067). All lines were on C57BL/6 background. The mice were maintained under specific pathogen-free conditions; all experiments were in accordance with the guidelines of the Canadian Council on Animal Care, and protocols approved by the McGill Animal Care Committee.
For competitive bone marrow transplantation, recipient B6.SJL-PtprcaPepcb/Boy mice (JAX002014, congenic for CD45.1) were irradiated with two doses of 4.5Gy, delivered 3h apart, in the RS2000 irradiator (Rad Source, Brentwood, TN, USA). The mice were intravenously injected with 2.5 × 106 bone marrow cells from the following donor mice in a 1:1 ratio: B6.SJL cells mixed with C57BL/6 cells from either wild-type, Mysm1−/−, Puma−/−, or Mysm1−/−Puma−/− mice. The mice were kept on neomycin in drinking water (2g/l, BioShop, Burlington, ON, Canada) for 3 weeks, and were killed at 20 weeks for immunophenotypic analysis. Bleeds were performed at 12.5 weeks post transplantation.
For tamoxifen-induced Mysm1 gene inactivation in the Mysm1flox/flox Gt(ROSA)26Sortm1(cre/ERT2) mouse line, the mice were injected intraperitoneally with tamoxifen (T5648, Sigma, St. Louis, MO, USA) in sterilized corn oil (Sigma C8267) at 0.15mg/g per injection, with eight doses in total administered over 16 days. The mice were analyzed 16 weeks after the treatment. Successful deletion of Mysm1 exon 3 was validated by genotyping on genomic DNA extracted from hematopoietic and lymphoid organs with primers Mysm1_wt_F5 5′-CCACAGTGATTCCTGGCTG-3′, Mysm1_wt_Rv6 5′-CTAGGCTTCAGGCATTTTGC-3′, Mysm1_wt_R7 5′-CCACATGTTCTTGACCTTGC-3′,40 and the loss of Mysm1 transcript ENSMUST00000075872 was further confirmed using qRT-PCR analysis of HSPCs FACS-sorted from the mouse bone marrow. Mysm1flox/+ heterozygous mice expressing the same Gt(ROSA)26Sortm1(cre/ERT2) transgene and undergoing identical tamoxifen treatment, as well as sham-mice treated with corn-oil only, were used as control groups.
Cell suspensions of mouse tissues were prepared in RPMI-1640 (Wisent, Jean-Baptiste, QC, Canada) with 2% (v/v) fetal calf serum (FCS), 100μg/ml streptomycin, and 100U/ml penicillin (Wisent). Cells were stained for surface markers in PBS with 2% FCS and 0.2% (w/v) sodium azide (VWR, Amresco, Solon, OH, USA) for 20min on ice, with the following fluorophore-conjugated antibodies: FITC-conjugated antibody against CD34 (clone RAM34), CD48 (HM48-1, both from eBioscience, San Diego, CA, USA) or CD127 (A7R34, BioLegend, San Diego, CA, USA); PE-conjugated antibodies against CD11b (M1/70, BD Biosciences, Franklin Lakes, NJ, USA), CD150 (mShad150, eBioscience), IgM (II/41, eBioscience), or CD135/FLT3 (A2F10, BioLegend); PerCP/Cy5.5 antibodies against: CD45R/B220 (RA3–6B2), CD11b (M1/70), TER119 (TER119), CD4 (RM4–5), CD8a (53-6.7, all from BioLegend); APC antibodies against CD27 (LG3A10), CD49b (DX5), IgD (11–26c.2a), Sca-1 (E13–161.7, all from BioLegend), and Sca-1 (D7, eBioscience); Alexa Fluor 647 anti-CD150 (TC15-12F12.2, BioLegend); Pacific Blue antibodies against CD8a (53-6.7), CD11b (M1/70), cKit/CD117 (2B8, all from BioLegend); eFluor450 antibodies against CD48 (HM48-1) or CD122 (TM-b1, both from eBioscience); BV421 anti-CD34 (RAM34, BD Biosciences); PE-Cy7 antibodies against CD4 (RM4–5), CD19 (6D5), CD45.2 (104), CD71 (RI7217, all from BioLegend), and cKit/CD117 (2B8, BD Biosciences); APC-Cy7 antibodies against: CD45 (30-F11), CD45.1 (A20), or Sca1 (D7, all from BioLegend). Fixable Viability Dye eFluor 506 (eBioscience) was used to discriminate live and dead cells. Annexin V PeCy7 (eBioscience) was used for detection of apoptotic cells. Compensation was performed with BD CompBeads (BD Biosciences). The data were acquired on FACS Canto II flow cytometer (BD Biosciences) and analyzed with FACS Diva (BD Biosciences) or FlowJo (Tree Star, Ashland, OR, USA) software.
For intracellular staining, samples were pre-stained with Fixable Viability Dye eFluor506 (eBioscience), APC anti-Sca1 (D7, eBiosciences), Pacific Blue anti-cKit/CD117 (2B8, BioLegend), and PE anti-CD135/Flt3 (A2F10, BioLegend). The cells were fixed in 2% paraformaldehyde in PBS with 2% FCS at 37 °C for 10min, and permeabilized in 90% methanol for 30min on ice. The cells were then stained with PerCP/Cy5.5-conjugated antibodies against lineage markers and either with FITC anti-H2AX phospho-Ser139 (clone 20E3, Cell Signaling), or Alexa Fluor 488 anti-p53 (clone 1C12, Cell Signaling), or FITC anti-Ki67 (clone SolA15, eBioscience), or FITC Ki67 Set (clone B56, BD Pharmingen), or appropriate isotype controls. For ROS measurements the cells were loaded with carboxy-H2DCFDA (Life Technologies, Carlsbad, CA, USA) at 5μM in PBS for 30min at room temperature, and were washed twice before analyses.
Mouse bone marrow was flushed from femurs, tibiae, humeri, and ilia of mice with a 26-G needle in PBS supplemented with 0.1% BSA and 2mM EDTA, filtered through 40-μm cell strainers, and subjected to red blood cell lysis in ACK buffer (0.15M NH4Cl, 10mM KHCO3, and 0.1mM EDTA). The cells were stained with Biotin anti-mouse Lineage Panel (BioLegend) and lineage-positive cells depleted using biotin-binding Dynabeads (Life Technologies). The lineage-negative fraction was stained with PeCy7-cKit/CD117 (clone 2B8, BD Biosciences), APC-Sca1 (E13–161.7, BioLegend), FITC-CD48 (HM48-1, eBioscience), PE-CD150 (mShad150, eBioscience), and streptavidin-PECy5 (BioLegend). Propidium iodide or DAPI was added immediately before sorting for dead cell exclusion. Cell sorting was performed on the FACSAria instrument and was analyzed with FACS Diva software (BD Biosciences).
RNA was isolated from Ba/F3 cells using the EZ-10 DNAaway RNA mini-prep kit (Biobasic, Markham, ON, Canada) according to the manufacturer's protocol. Quality was assessed with nanodrop spectrophotometry and gel electrophoresis. cDNA was prepared using the M-MLV reverse-transcription kit (Invitrogen, Life Technologies).
RNA was isolated from FACS-sorted hematopoietic cells using the MagMAX total RNA kit (Ambion, Life Technologies) according to the manufacturer's protocol. Quality was assessed on Bioanalyzer Pico chip (Agilent, Santa Clara, CA, USA). KLS cDNA was prepared using the qScript cDNA Supermix (Quanta, Gaithersburg, MD, USA) with 5-ng input RNA.
All qPCR analyses were performed on a StepOnePlus instrument with Power SYBR master mix (Applied Biosystems, Life Technologies). Primers were purchased from IDT Technologies (Coralville, IA, USA), and primer sequences are provided in Supplementary Tables S1 and S2.
RNA integrity was assessed on a Bioanalyzer RNA pico chip. RNA (10ng) was rRNA-depleted and used for library preparation with the SMARTer Stranded RNA-Seq kit (Takara Clontech, Mountain View, CA, USA) designed for low input. The RNA-seq libraries were sequenced on an Illumina HiSeq 2500 sequencer in a paired-end 50-bp configuration. The number of reads obtained for each sample ranged between 50 and 95 millions.
The high quality of sequence reads was confirmed using the FastQC tool (Babraham Bioinformatics, Cambridge, UK). To ensure high mapability, the few low-quality bases were trimmed from read extremities using Trimmomatic v.0.33.57 Reads were then mapped to the mouse UCSC mm9 reference assembly using TopHat v2.0.9 in conjunction with the Bowtie 1.0.0 algorithm,58, 59, 60 and gene expression was quantified by counting the number of uniquely mapped reads with featureCounts using default parameters.61
Normalization and differential gene expression analysis was conducted using the edgeR Bioconductor package.62 We retained genes that had a minimum expression level of five counts per million reads in at least 3 of the 12 samples. Pairwise differential gene expression analysis was performed by comparing samples from mutant mice (Puma−/−, Mysm1−/−Puma+/−, Mysm1−/−Puma−/−) relative to wild-type samples. Genes with changes in expression ≥|2| and an adjusted P-value of <0.05 were considered significant.
For figure preparation and RNA-seq data visualization in IGV genome browser,63 triplicate data were combined and bigwig scaled per 10 million reads. Mapping into gene exons was generated using a succession of genomeCoverageBed and wigToBigWig tools.64 Unbiased clustering analysis of relative gene expression changes (Log2 fold change; Figure 8) and heatmap visualization were performed using Pearson correlation within MeV software.65
HEK293T cells were maintained in DMEM (Wisent) with 10% FCS and 2mM L-Glutamine (Wisent). Cells were passaged every 2–3 days and all transfections were performed within 10 passages from thawing. The cells were transiently transfected using Lipofectamine 2000 (Life Technologies) with pcDNA3.1(+) vector encoding N-terminal triple-Flag-tagged mouse Mysm1 (NM177239, Life Technologies), and/or a pMiG vector encoding mouse wild-type untagged p53.
Ba/F3 cells were maintained in RPMI-1640 (Wisent) with 10% FCS, 5% WEHI-conditioned media, 2mM L-Glutamine, 100μg/ml streptomycin, and 100U/ml penicillin (Wisent). The cells were maintained at 0.5–2 × 106 cells/ml at all times. Ba/F3 line stably expressing triple-Flag-tagged MYSM1 was generated by electroporation of 107 cells with 2.5μg of BglII-linearized Mysm1-pcDNA3.1(+) vector (Life Technologies) in GenePulser II (Bio-Rad, Hercules, CA, USA). Transfected cells were selected and maintained in G418-containg media (0.8mg/ml, EMD Millipore, Billerica, MA, USA).
For Mysm1 knockdown in Ba/F3 cells, six shRNA sequences were designed to target Mysm1 transcript as described by Robert FW et al.66 Oligonucleotides were purchased from BioCorp (DDO, QC, Canada), PCR-amplified, and cloned into the pMSCV-mir30-PIG (MLP) vector.67 MLP-shFF vector was used for off-target knockdown controls. Retrovirus was produced in Phoenix cells (ATCC, Manassas, VA, USA).68 Ba/F3 cells were infected, selected, and further maintained in 2μg/ml puromycin (Wisent). On the basis of knockdown efficiency, the following shRNA was chosen 5′-TGCTGTTGACAGTGAGCGACCGGGAAATGATGAAAGTACATAGTGAAGCCACAGATGTATGTACTTTCATCATTTCCCGGCTGCCTACTGCCTCGGA-3′, and six independent infections performed to generate six knockdown lines for experiments.
Independently infected shFF and shMysm1 lines (six each) were plated at 1 × 106 cells/ml in fresh media without IL-3 at the beginning of each experiment. As IL-3 has been shown to block apoptotic programs in this cell line,69, 70 it was excluded from the experiments to avoid masking of p53-mediated stress responses. Cells were irradiated with 3Gy in a RS2000 irradiator (Rad Source) at indicated time points and always harvested alongside untreated cells to control for endogenous stress. Cell death was assessed usingtrypan blue dye exclusion.
ChIP was performed as described previously,71 with minor modifications. Briefly, cells were fixed by addition of formaldehyde in the culture media to a final concentration of 1%, and was incubated for 11min at room temperature, followed by addition of 0.125M of glycine to stop fixation. Nuclei were then extracted with 5min lysis in 0.25% Triton buffer (10mM Tris-HCl pH 8, 10mM EDTA, 0.5mM EGTA), followed by 30min lysis in 200mM NaCl buffer (10mM Tris-HCl pH 8, 1mM EDTA, 0.5mM EGTA). Nuclei were resuspended in sonication buffer (10mM Tris pH 8, 140mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.5% SDS, 0.5% Triton, and 0.05% NaDOC) and sonicated 12 times for 30s in S450D Sonifier (Crystal Electronics, Newmarket, ON, Canada) at 80%, with 30s rest in a cooling water bath.
Beads were prepared overnight with 20μl of Dynabeads Protein G (Invitrogen, Life Technologies) conjugated with 2–3μg of test antibody (listed in Supplementary Table S3) or control IgG (Santa Cruz, Dallas, TX, USA). Immunoprecipitation was performed by overnight incubation of antibody–bead matrices with sheared chromatin from the equivalent of 5 × 106 cells. For transcription factor ChIP, six washes were performed with low stringency buffers, whereas four medium-stringency washes were used for histone ChIP. Samples were de-crosslinked by overnight incubation at 65°C in 1% SDS buffer (50mM Tris pH 8, 10mM EDTA), and following RNaseA and Protienase K enzymatic treatments, ChIP DNA was purified using the Qiaquick PCR cleanup kit (Qiagen, Venlo, Netherlands). ChIP enrichment was quantified using real-time qPCR analysis (primer sequences provided in Supplementary Table S2). All CT values were normalized to those of the pro-opiomelanocortin (Pomc) gene, which serves as a negative binding region. Enrichment was calculated relative to control IgG for transcription factors, and relative to total histone H3 for H3K27ac and H3K4me3.
For co-immunoprecipitation, cells were lysed in B450 buffer (25mM Tris pH 7.5, 2mM EDTA, 450mM NaCl, 0.2% NP40, 5% glycerol) 24–48h after transfection. Samples were dounce-homogenized and cleared using centrifugation. Cleared lysates were diluted 1/3 to 150mM NaCl and 0.1% NP40 and their protein content quantified with the BCA assay (Thermo Scientific, Waltham, MA, USA). Antibody-conjugated Dynabeads Protein G (Invitrogen, Life Technologies) was prepared by incubation of 25μl bead slurry with 2.5μg of Flag M2 (Sigma), p53 1C12 (Cell Signaling, Danvers, MA, USA), or control IgG (Santa Cruz) for at least 4h. Immunoprecipitation of the protein lysate with antibody-conjugated Dynabeads was performed with rotation at 4°C for 2h. For HEK co-transfection IP, 500μg total protein was used; for Ba/F3 endogenous IP, 5mg total protein was used. After four washes with IP buffer (25mM Tris pH 7.5, 1mM EDTA, 150mM NaCl, 0.1% NP40), protein complexes were eluted in 1 × SDS sample buffer with boiling for 10min at 95°C. Eluates were then separated from the beads and reduced by addition of 50mM DTT, followed by heating for further 10min at 95°C. For western blot analysis, cells were lysed in a modified RIPA buffer supplemented with DTT, EDTA, and protease and phosphatase inhibitors (Thermo Scientific). Protein concentration was assessed using the BCA assay (Thermo Scientific), and samples were prepared by boiling in Laemmli buffer. Commercial antibodies used for western blot analysis are listed in Supplementary Table S4.
This was performed according to previously described protocols.72 Briefly, mouse Mysm1 cDNA fragment encoding amino acids 162–363 was cloned into pGEX-2TK vector (GE Healthcare, Buckinghamshire, UK), expressed as a GST fusion protein in Escherichia coli strain BL21 (New England Biolabs, Ipswich, MA, USA) and purified using affinity chromatography against glutathione sepharose 4B (GE Healthcare). Rabbit immunization was performed according to SOP 406 of the McGill Comparative Medicine and Animal Resource Centre (CMARC, http://www.mcgill.ca/research/researchers/compliance/ animal/sop), with 0.1mg of protein injected subcutaneously in incomplete Freund's adjuvant, boosting every 4 weeks. For antibody purification, the same Mysm1 cDNA fragment was cloned into plasmid pQE40 (Qiagen), expressed as a DHFR fusion protein in E. coli strain M15(pREP4) (Qiagen), and was isolated using affinity chromatography against Ni-NTA agarose (Qiagen). Antibody purification from rabbit serum was performed by preparative immunoblotting, as previously described.72
Statistical comparisons were performed with Prism 5.0 (GraphPad, La Jolla, CA, USA), using Student's t-test for two data sets and ANOVA for multiple comparisons.
This work was funded by the Canadian Institutes of Health Research (CIHR, grant # 123403 to AN), the Canadian Foundation for Innovation (CFI, grant # 29838 to AN), Merck, Sharp & Dohme and McGill Faculty of Medicine Grant for Translational Research (grant #238371 to AN), and Startup Funds from the McGill Faculty of Medicine. AN is a Canada Research Chair Tier 2 in Hematopoiesis and Lymphocyte Differentiation (grant # 950–228977). DL was supported by a postdoctoral fellowship from the CIHR Neuroinflammation training program and from the Fonds de Recherche du Québec—Santé. We thank Eric Massicotte, Julie Lord, and Julien Leconte of the IRCM flow cytometry facility for cell sorting; Dr. Odile Neyret and Myriam Rondeau of the IRCM Molecular Biology and Functional Genomics Facility for whole-genome library preparation and RNA-Seq; Su-Jin Park, Karen Hope Stone, and McGill CMARC for technical assistance with anti-MYSM1 antibody production; Dr Anatoli Onichtchenko and Isabella Albanese for the production of FLAG-MYSM1 Ba/F3 cells; Dr. Peiman Shooshtarizadeh, and Professor Tarik Möröy for advice on HSPC transcriptional analysis; Tanya Koch, Geneviève Perreault, Patricia D'Arcy, and other staff of McGill CMARC for their technical assistance; Shiyang Shen and Su-Jin Park for genotyping.
Experiments were performed by JIB (Figures 1–8), JCP (Figures 1 and 4–8), SS (Figures 4 and 5), and AN (Figures 4–7). Experiments were designed by JIB (Figures 1–8), AN (Figures 1 and 4–8), and DL (Figures 2, 3, and 8). JIB, AN, DL, and JCP analyzed the data. DL performed RNA-Seq bioinformatics analysis. AN, JIB, and DL wrote the paper, with assistance from JCP in editing and revisions. AN, DL, FR, RC, PG, and JP supervised the work.
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on Cell Death and Differentiation website (http://www.nature.com/cdd)
Edited by M Oren