Polycomb repressive complex 1.1 coordinates homeostatic and emergency myelopoiesis
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The authors present a manuscript aiming to understand the mechanism(s) underlying myeloid bias in HSCs, specifically focused on the role of Pcgf1, and therefore PRC1.1, in the regulation of hematopoiesis. This important work is of interest to the community of researchers interested in myeloid differentiation, lineage fate decisions in hematopoietic stem cells, and the molecular mechanisms that contribute to the initiation of myeloid malignancies. The methods are rigorous and the results convincingly support the authors' conclusions.
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Abstract
Polycomb repressive complex (PRC) 1 regulates stem cell fate by mediating mono-ubiquitination of histone H2A at lysine 119. While canonical PRC1 is critical for hematopoietic stem and progenitor cell (HSPC) maintenance, the role of non-canonical PRC1 in hematopoiesis remains elusive. PRC1.1, a non-canonical PRC1, consists of PCGF1, RING1B, KDM2B, and BCOR. We recently showed that PRC1.1 insufficiency induced by the loss of PCGF1 or BCOR causes myeloid-biased hematopoiesis and promotes transformation of hematopoietic cells in mice. Here we show that PRC1.1 serves as an epigenetic switch that coordinates homeostatic and emergency hematopoiesis. PRC1.1 maintains balanced output of steady-state hematopoiesis by restricting C/EBPα-dependent precocious myeloid differentiation of HSPCs and the HOXA9- and β-catenin-driven self-renewing network in myeloid progenitors. Upon regeneration, PRC1.1 is transiently inhibited to facilitate formation of granulocyte-macrophage progenitor (GMP) clusters, thereby promoting emergency myelopoiesis. Moreover, constitutive inactivation of PRC1.1 results in unchecked expansion of GMPs and eventual transformation. Collectively, our results define PRC1.1 as a novel critical regulator of emergency myelopoiesis, dysregulation of which leads to myeloid transformation.
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eLife assessment
The authors present a manuscript aiming to understand the mechanism(s) underlying myeloid bias in HSCs, specifically focused on the role of Pcgf1, and therefore PRC1.1, in the regulation of hematopoiesis. This important work is of interest to the community of researchers interested in myeloid differentiation, lineage fate decisions in hematopoietic stem cells, and the molecular mechanisms that contribute to the initiation of myeloid malignancies. The methods are rigorous and the results convincingly support the authors' conclusions.
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Reviewer #1 (Public Review):
This interesting manuscript by Nakajima-Takagi et al describes the roles of the PRC 1.1 member Pcgf1 in myeloid lineage commitment in hematopoiesis and in regulating myeloid differentiation and self-renewal during emergency myelopoiesis. The roles of Pcgf1 have been explored previously in the context of Runx1 depletion or in the context of myelofibrosis together with the JAK2V617F mutation, but this is the first report of the specific roles of Pcgf1 in HSCs and in myelopoiesis. The authors convincingly demonstrate that conditional deletion of Pcgf1 in hematopoietic cells causes a lineage switch in HSCs from lymphoid to myeloid fates and that a key mechanism for this lineage switch is regulation of the H2AK119ub1 chromatin mark, leading to de-repression of CEBPalpha, a key transcription factor that promotes …
Reviewer #1 (Public Review):
This interesting manuscript by Nakajima-Takagi et al describes the roles of the PRC 1.1 member Pcgf1 in myeloid lineage commitment in hematopoiesis and in regulating myeloid differentiation and self-renewal during emergency myelopoiesis. The roles of Pcgf1 have been explored previously in the context of Runx1 depletion or in the context of myelofibrosis together with the JAK2V617F mutation, but this is the first report of the specific roles of Pcgf1 in HSCs and in myelopoiesis. The authors convincingly demonstrate that conditional deletion of Pcgf1 in hematopoietic cells causes a lineage switch in HSCs from lymphoid to myeloid fates and that a key mechanism for this lineage switch is regulation of the H2AK119ub1 chromatin mark, leading to de-repression of CEBPalpha, a key transcription factor that promotes myeloid cell fate. They also perform a single-cell RNAseq experiment and demonstrate an increase in the population of "self-renewing GMPs", and they attribute this increase to an upregulation in HoxA9 expression and beta-catenin activation. They also demonstrate that HoxA9 overexpression promotes beta-catenin activation, which has been observed in emergency myelopoiesis in other studies, though the mechanism for this is unclear. The authors also demonstrate that deletion of Pcgf1 in hematopoietic cells can also lead to deregulated myelopoiesis, leading to a lethal MPN in a subset of animals. They conclude that Pcgf1 plays a critical role to regulate emergency myelopoiesis, and to prevent the malignant transformation of myeloid progenitors.
Overall, the methods are highly rigorous and the results support the authors' conclusions. The only conclusion that would require further clarification is that Pcgf1 promotes emergency myelopoiesis. Emergency myelopoiesis typically starts with a proliferative burst of myeloid progenitors in response to a stress stimulus, followed by enhanced myeloid differentiation into mature functional myeloid cells. In this Pcgf1 KO mouse model, it is clear that there is an increase in the production of myeloid progenitors, and prolonged survival of myeloid progenitors in culture, but there is no demonstration that this results in the generation of mature functional myeloid cells. It appears that there may also be a differentiation block, likely due to the increase in "self-renewing progenitors", which is likely a consequence of HoxA9 upregulation, and possibly the beta-catenin activation in myeloid progenitors. Therefore, if there is also a differentiation block due to Pcgf1 deletion, the statement that emergency myelopoiesis is enhanced may be an oversimplification. What appears to occur is an expansion of a pool of self-renewing transformed or pre-transformed myeloid progenitors, and the relevance of this event to emergency myelopoiesis is not entirely clear. However, there is a clear significance of these findings and this new mouse model for studying the pathogenesis of myeloid malignancies, such as MPN, MDS, or AML, in which mutations in other components of PRC1.1 are frequently mutated, so this study is likely to have a significant impact in the field.
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Reviewer #2 (Public Review):
The work from Nakajima-Takagi et al describes the phenotypes and study of a PCGF1 mutant mouse model. PCGF1 is a core component of the non-canonical PRC1.1 complex and specific functions of this complex in hematopoiesis. Using somatic inactivation models, the authors demonstrate that the acute deletion of PCGF1 from adult hematopoiesis leads to a progressive myeloid bias in the bone marrow and peripheral blood. This occurs at the expense of the HSPC compartment, with a reduction in all populations and of the lymphoid committed populations. The myeloid bias is cell intrinsic, as competitive transplant of the PGCF1 deficient bone marrow recapitulates the phenotype. The effect is not due to exhaustion or loss of self-renewal of the HSCs.
To understand the basis for the myeloid bias, the authors first assessed …Reviewer #2 (Public Review):
The work from Nakajima-Takagi et al describes the phenotypes and study of a PCGF1 mutant mouse model. PCGF1 is a core component of the non-canonical PRC1.1 complex and specific functions of this complex in hematopoiesis. Using somatic inactivation models, the authors demonstrate that the acute deletion of PCGF1 from adult hematopoiesis leads to a progressive myeloid bias in the bone marrow and peripheral blood. This occurs at the expense of the HSPC compartment, with a reduction in all populations and of the lymphoid committed populations. The myeloid bias is cell intrinsic, as competitive transplant of the PGCF1 deficient bone marrow recapitulates the phenotype. The effect is not due to exhaustion or loss of self-renewal of the HSCs.
To understand the basis for the myeloid bias, the authors first assessed transcriptome signatures and see a shift in gene expression programs related to myeloid development and targets of the key myeloid transcription factor C/EBPa. Further analysis demonstrated an increased expression of Cebp1 in the PCGF1-deficient LSK cells. Reducing the expression of Cebpa could modify the myeloid skewing of Pcgf1 deficient cells in culture. This de-repression of Cebpa correlates with changed local H2AK119ub1 levels in the HPSC populations.Additional studies assessed how the loss of Pcgf1 changed the response to hemoablation, in this instance with a single dose of 5-FU. This study coupled with scRNA-seq suggested that PRC1.1 was important in regulating the GMP populations, potentially through a self-renewal program. This led to a focussed analysis of the GMPs, with evidence for altered Hoxa9 and b-catenin levels contributing to the altered GMP behaviours. Both have been implicated and demonstrated to have functional roles in these programs in other studies.
Finally, ageing of Pcgf1 deficient mice demonstrated that these mice were predisposed to developing T-ALL and MPN. The authors provide a characterisation of these moribund states and their phenotypes are consistent with the diagnosis.
Overall the work demonstrates a specific requirement for Pcgf1, and therefore PRC1.1, in the regulation of hematopoiesis. I think the authors largely achieved the aims and the results are supportive of the conclusions. The work shows myeloid bias, experimental evidence that this is due to a derepression of a myeloid lineage program in the HPSC and associated chromatin changes, and functions for Pcgf1 in both hematopoietic regeneration and malignancy. This suggests a unique role for non-canonical PRC1.1 compared to canonical PRC 1.
Strengths:
- in vivo experiments and evidence;
- multiple lines of evidence supporting the conclusion;
- mechanistic studies provide direct evidence of the proposed mechanism.Weaknesses:
- can the authors demonstrate normal maturation of the myeloid lineages as this would be important to differentiate between myeloid bias and a block in myeloid differentiation? This is important to distinguish between.
- include analysis of mature myeloid cells and FACS plots to allow assessment of maturation. -
Reviewer #3 (Public Review):
The myeloid bias in hematopoietic stem and progenitor cells upon genetic inactivation of Pcgf1, a component of the PRC1 complex is convincingly demonstrated by a Pcgf1 conditional allele crossed with a tamoxifen-inducible Are, combined with transplantation. The overproduction of myeloid cells may be contributed to by the derepression of two PRC target genes Cebpa and Hoxa9 at the multipotent HSPC and lineage-committed GMP levels. The involvement of these two genes is demonstrated by decreased H2AK119ub1, elevated CEBPa expression, and increased CEBPa binding motifs in KO HSCs. Functional rescue by manipulating CEBPa levels on the background of Pcgf1 KO is attempted in vitro. The derepression of Hoxa9 is shown in Pcgf1 KO GMPs, which expanded in the KO mice. Finally, constitutive inactivation of Pcgf1 results …
Reviewer #3 (Public Review):
The myeloid bias in hematopoietic stem and progenitor cells upon genetic inactivation of Pcgf1, a component of the PRC1 complex is convincingly demonstrated by a Pcgf1 conditional allele crossed with a tamoxifen-inducible Are, combined with transplantation. The overproduction of myeloid cells may be contributed to by the derepression of two PRC target genes Cebpa and Hoxa9 at the multipotent HSPC and lineage-committed GMP levels. The involvement of these two genes is demonstrated by decreased H2AK119ub1, elevated CEBPa expression, and increased CEBPa binding motifs in KO HSCs. Functional rescue by manipulating CEBPa levels on the background of Pcgf1 KO is attempted in vitro. The derepression of Hoxa9 is shown in Pcgf1 KO GMPs, which expanded in the KO mice. Finally, constitutive inactivation of Pcgf1 results in lethal myeloproliferation. Together, this paper demonstrates another HSPC regulator, whose loss of function leads to myeloid-biased hematopoiesis, which in extreme cases could end in myeloid transformation.
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