A role for GPx3 in activity of normal and leukemia stem cells

The determinants of normal and leukemic stem cell self-renewal remain poorly characterized. We report that expression of the reactive oxygen species (ROS) scavenger glutathione peroxidase 3 (GPx3) positively correlates with the frequency of leukemia stem cells (LSCs) in Hoxa9+Meis1-induced leukemias. Compared with a leukemia with a low frequency of LSCs, a leukemia with a high frequency of LSCs showed hypomethylation of the Gpx3 promoter region, and expressed high levels of Gpx3 and low levels of ROS. LSCs and normal hematopoietic stem cells (HSCs) engineered to express Gpx3 short hairpin RNA (shRNA) were much less competitive in vivo than control cells. However, progenitor cell proliferation and differentiation was not affected by Gpx3 shRNA. Consistent with this, HSCs overexpressing Gpx3 were significantly more competitive than control cells in long-term repopulation experiments, and overexpression of the self-renewal genes Prdm16 or Hoxb4 boosted Gpx3 expression. In human primary acute myeloid leukemia samples, GPX3 expression level directly correlated with adverse prognostic outcome, revealing a potential novel target for the eradication of LSCs

CLEC-2 and Syk in the megakaryocytic/platelet lineage are essential for development

The C-type lectin receptor CLEC-2 signals through a pathway that is critically dependent on the tyrosine kinase Syk. We show that homozygous loss of either protein results in defects in brain vascular and lymphatic development, lung inflation and perinatal lethality. Furthermore, we find that conditional deletion of Syk in the haematopoietic lineage, or conditional deletion of CLEC-2 or Syk in the megakaryocyte/platelet lineage, also causes defects in brain vascular and lymphatic development, although the mice are viable. In contrast, conditional deletion of Syk in other haematopoietic lineages had no effect on viability or brain vasculature and lymphatic development. We show that platelets, but not platelet releasate, modulate the migration and intercellular adhesion of lymphatic endothelial cells through a pathway that is dependent on CLEC-2 and Syk. These studies demonstrate that megakaryocyte/platelet expression of CLEC-2 and Syk is required for normal brain vasculature and lymphatic development and that platelet CLEC-2 and Syk directly modulate lymphatic endothelial cell behaviour in vitro

Myelofibrosis: molecular and cell biological aspects

A subset of myeloproliferative disorders (MPN) and myelodyplastic syndromes (MDS) evolves to fibrosis of the bone marrow associated with haematopoietic insufficiency. We have been interested in chemokines involved in fibrogenesis within the bone marrow. Besides TGFβ we could identify a number of additional mediators including osteoprotegerin and bone morphogenic proteins. In MPN JAK2 or MPL mutation are not linked to the propensity for bone marrow fibrosis. The hypothesis that an increased intramedullary decay of megakaryocytes undergoing appotosis takes place within the marrow, thus liberating fibrogenic cytokines, could not be confirmed. On the contrary, megakaryocytes in primary fibrosis revealed low expression of proapoptotic genes such as BNIP3. Interestingly, BNIP 3 expression was down regulated in megakaryocytic cell lines kept in hypoxic conditions. Furthermore, expression arrays revealed hypoxia inducible genes to be up-regulated in primary myelofibrosis. Fibrotic MPN are characterized by aberrant proplatelet formation which represent cytoplasmic pseudopodia and normally extend into the sinus. In fibrotic MPN orientation of proplatelet growth appears to be disturbed, which could lead to an aberrant deposition of platelets in the marrow with consecutive liberation of fibrogenic cytokines

Bmi1 Confers Resistance to Oxidative Stress on Hematopoietic Stem Cells

The polycomb-group (PcG) proteins function as general regulators of stem cells. We previously reported that retrovirus-mediated overexpression of Bmi1, a gene encoding a core component of polycomb repressive complex (PRC) 1, maintained self-renewing hematopoietic stem cells (HSCs) during long-term culture. However, the effects of overexpression of Bmi1 on HSCs in vivo remained to be precisely addressed.In this study, we generated a mouse line where Bmi1 can be conditionally overexpressed under the control of the endogenous Rosa26 promoter in a hematopoietic cell-specific fashion (Tie2-Cre;R26Stop(FL)Bmi1). Although overexpression of Bmi1 did not significantly affect steady state hematopoiesis, it promoted expansion of functional HSCs during ex vivo culture and efficiently protected HSCs against loss of self-renewal capacity during serial transplantation. Overexpression of Bmi1 had no effect on DNA damage response triggered by ionizing radiation. In contrast, Tie2-Cre;R26Stop(FL)Bmi1 HSCs under oxidative stress maintained a multipotent state and generally tolerated oxidative stress better than the control. Unexpectedly, overexpression of Bmi1 had no impact on the level of intracellular reactive oxygen species (ROS).Our findings demonstrate that overexpression of Bmi1 confers resistance to stresses, particularly oxidative stress, onto HSCs. This thereby enhances their regenerative capacity and suggests that Bmi1 is located downstream of ROS signaling and negatively regulated by it

CBX4-mediated SUMO modification regulates BMI1 recruitment at sites of DNA damage

Polycomb group (PcG) proteins are involved in epigenetic silencing where they function as major determinants of cell identity, stem cell pluripotency and the epigenetic gene silencing involved in cancer development. Recently numerous PcG proteins, including CBX4, have been shown to accumulate at sites of DNA damage. However, it remains unclear whether or not CBX4 or its E3 sumo ligase activity is directly involved in the DNA damage response (DDR). Here we define a novel role for CBX4 as an early DDR protein that mediates SUMO conjugation at sites of DNA lesions. DNA damage stimulates sumoylation of BMI1 by CBX4 at lysine 88, which is required for the accumulation of BMI1 at DNA damage sites. Moreover, we establish that CBX4 recruitment to the sites of laser micro-irradiation-induced DNA damage requires PARP activity but does not require H2AX, RNF8, BMI1 nor PI-3-related kinases. The importance of CBX4 in the DDR was confirmed by the depletion of CBX4, which resulted in decreased cellular resistance to ionizing radiation. Our results reveal a direct role for CBX4 in the DDR pathway

SCL/TAL1 cooperates with Polycomb RYBP-PRC1 to suppress alternative lineages in blood-fated cells

During development, it is unclear if lineage-fated cells derive from multilineage-primed progenitors and whether active mechanisms operate to restrict cell fate. Here we investigate how mesoderm specifies into blood-fated cells. We document temporally restricted co-expression of blood (Scl/Tal1), cardiac (Mesp1) and paraxial (Tbx6) lineage-affiliated transcription factors in single cells, at the onset of blood specification, supporting the existence of common progenitors. At the same time-restricted stage, absence of SCL results in expansion of cardiac/paraxial cell populations and increased cardiac/paraxial gene expression, suggesting active suppression of alternative fates. Indeed, SCL normally activates expression of co-repressor ETO2 and Polycomb-PRC1 subunits (RYBP, PCGF5) and maintains levels of Polycomb-associated histone marks (H2AK119ub/H3K27me3). Genome-wide analyses reveal ETO2 and RYBP co-occupy most SCL target genes, including cardiac/paraxial loci. Reduction of Eto2 or Rybp expression mimics Scl-null cardiac phenotype. Therefore, SCL-mediated transcriptional repression prevents mis-specification of blood-fated cells, establishing active repression as central to fate determination processes

Genome-Wide Interrogation of Mammalian Stem Cell Fate Determinants by Nested Chromosome Deletions

Understanding the function of important DNA elements in mammalian stem cell genomes would be enhanced by the availability of deletion collections in which segmental haploidies are precisely characterized. Using a modified Cre-loxP–based system, we now report the creation and characterization of a collection of ∼1,300 independent embryonic stem cell (ESC) clones enriched for nested chromosomal deletions. Mapping experiments indicate that this collection spans over 25% of the mouse genome with good representative coverage of protein-coding genes, regulatory RNAs, and other non-coding sequences. This collection of clones was screened for in vitro defects in differentiation of ESC into embryoid bodies (EB). Several putative novel haploinsufficient regions, critical for EB development, were identified. Functional characterization of one of these regions, through BAC complementation, identified the ribosomal gene Rps14 as a novel haploinsufficient determinant of embryoid body formation. This new library of chromosomal deletions in ESC (DelES: http://bioinfo.iric.ca/deles) will serve as a unique resource for elucidation of novel protein-coding and non-coding regulators of ESC activity

Relationships between Hematopoiesis and Hepatogenesis in the Midtrimester Fetal Liver Characterized by Dynamic Transcriptomic and Proteomic Profiles

In fetal hematopoietic organs, the switch from hematopoiesis is hypothesized to be a critical time point for organogenesis, but it is not yet evidenced. The transient coexistence of hematopoiesis will be useful to understand the development of fetal liver (FL) around this time and its relationship to hematopoiesis. Here, the temporal and the comparative transcriptomic and proteomic profiles were observed during the critical time points corresponding to the initiation (E11.5), peak (E14.5), recession (E15.5), and disappearance (3 ddp) of mouse FL hematopoiesis. We found that E11.5-E14.5 corresponds to a FL hematopoietic expansion phase with distinct molecular features, including the expression of new transcription factors, many of which are novel KRAB (Kruppel-associated box)-containing zinc finger proteins. This time period is also characterized by extensive depression of some liver functions, especially catabolism/utilization, immune and defense, classical complement cascades, and intrinsic blood coagulation. Instead, the other liver functions increased, such as xenobiotic and sterol metabolism, synthesis of carbohydrate and glycan, the alternate and lectin complement cascades and extrinsic blood coagulation, and etc. Strikingly, all of the liver functions were significantly increased at E14.5-E15.5 and thereafter, and the depression of the key pathways attributes to build the hematopoietic microenvironment. These findings signal hematopoiesis emigration is the key to open the door of liver maturation

Claudin 13, a Member of the Claudin Family Regulated in Mouse Stress Induced Erythropoiesis

Mammals are able to rapidly produce red blood cells in response to stress. The molecular pathways used in this process are important in understanding responses to anaemia in multiple biological settings. Here we characterise the novel gene Claudin 13 (Cldn13), a member of the Claudin family of tight junction proteins using RNA expression, microarray and phylogenetic analysis. We present evidence that Cldn13 appears to be co-ordinately regulated as part of a stress induced erythropoiesis pathway and is a mouse-specific gene mainly expressed in tissues associated with haematopoietic function. CLDN13 phylogenetically groups with its genomic neighbour CLDN4, a conserved tight junction protein with a putative role in epithelial to mesenchymal transition, suggesting a recent duplication event. Mechanisms of mammalian stress erythropoiesis are of importance in anaemic responses and expression microarray analyses demonstrate that Cldn13 is the most abundant Claudin in spleen from mice infected with Trypanosoma congolense. In mice prone to anaemia (C57BL/6), its expression is reduced compared to strains which display a less severe anaemic response (A/J and BALB/c) and is differentially regulated in spleen during disease progression. Genes clustering with Cldn13 on microarrays are key regulators of erythropoiesis (Tal1, Trim10, E2f2), erythrocyte membrane proteins (Rhd and Gypa), associated with red cell volume (Tmcc2) and indirectly associated with erythropoietic pathways (Cdca8, Cdkn2d, Cenpk). Relationships between genes appearing co-ordinately regulated with Cldn13 post-infection suggest new insights into the molecular regulation and pathways involved in stress induced erythropoiesis and suggest a novel, previously unreported role for claudins in correct cell polarisation and protein partitioning prior to erythroblast enucleation