Mitofusin 2 maintains haematopoietic stem cells with extensive lymphoid potential.

Haematopoietic stem cells (HSCs), which sustain production of all blood cell lineages, rely on glycolysis for ATP production, yet little attention has been paid to the role of mitochondria. Here we show in mice that the short isoform of a critical regulator of HSCs, Prdm16 (refs 4, 5), induces mitofusin 2 (Mfn2), a protein involved in mitochondrial fusion and in tethering of mitochondria to the endoplasmic reticulum. Overexpression and deletion studies, including single-cell transplantation assays, revealed that Mfn2 is specifically required for the maintenance of HSCs with extensive lymphoid potential, but not, or less so, for the maintenance of myeloid-dominant HSCs. Mfn2 increased buffering of intracellular Ca(2+), an effect mediated through its endoplasmic reticulum-mitochondria tethering activity, thereby negatively regulating nuclear translocation and transcriptional activity of nuclear factor of activated T cells (Nfat). Nfat inhibition rescued the effects of Mfn2 deletion in HSCs, demonstrating that negative regulation of Nfat is the prime downstream mechanism of Mfn2 in the maintenance of HSCs with extensive lymphoid potential. Mitochondria therefore have an important role in HSCs. These findings provide a mechanism underlying clonal heterogeneity among HSCs and may lead to the design of approaches to bias HSC differentiation into desired lineages after transplantation.

PRDM16 isoforms differentially regulate normal and leukemic hematopoiesis and inflammatory gene signature.

PRDM16 is a transcriptional coregulator involved in translocations in acute myeloblastic leukemia (AML), myelodysplastic syndromes, and T acute lymphoblastic leukemia that is highly expressed in and required for the maintenance of hematopoietic stem cells (HSCs), and can be aberrantly expressed in AML. Prdm16 is expressed as full-length (fPrdm16) and short (sPrdm16) isoforms, the latter lacking the N-terminal PR domain. The role of both isoforms in normal and malignant hematopoiesis is unclear. We show here that fPrdm16 was critical for HSC maintenance, induced multiple genes involved in GTPase signaling, and repressed inflammation, while sPrdm16 supported B cell development biased toward marginal zone B cells and induced an inflammatory signature. In a mouse model of human MLL-AF9 leukemia, fPrdm16 extended latency, while sPrdm16 shortened latency and induced a strong inflammatory signature, including several cytokines and chemokines that are associated with myelodysplasia and with a worse prognosis in human AML. Finally, in human NPM1-mutant and in MLL-translocated AML, high expression of PRDM16, which negatively impacts outcome, was associated with inflammatory gene expression, thus corroborating the mouse data. Our observations demonstrate distinct roles for Prdm16 isoforms in normal HSCs and AML, and identify sPrdm16 as one of the drivers of prognostically adverse inflammation in leukemia.

Dye-Independent Methods Reveal Elevated Mitochondrial Mass in Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) produce most cellular energy through glycolysis rather than through mitochondrial respiration. Consistent with this notion, mitochondrial mass has been reported to be low in HSCs. However, we found that staining with MitoTracker Green, a commonly used dye to measure mitochondrial content, leads to artefactually low fluorescence specifically in HSCs because of dye efflux. Using mtDNA quantification, enumeration of mitochondrial nucleoids, and fluorescence intensity of a genetically encoded mitochondrial reporter, we unequivocally show here that HSCs and multipotential progenitors (MPPs) have higher mitochondrial mass than lineage-committed progenitors and mature cells. Despite similar mitochondrial mass, respiratory capacity of MPPs exceeds that of HSCs. Furthermore, although elevated mitophagy has been invoked to explain low mitochondrial mass in HSCs, we observed that mitochondrial turnover capacity is comparatively low in HSCs. We propose that the role of mitochondria in HSC biology may have to be revisited in light of these findings.

Analysis of hypoxia and hypoxia-like states through metabolite profiling.

BACKGROUND: In diverse organisms, adaptation to low oxygen (hypoxia) is mediated through complex gene expression changes that can, in part, be mimicked by exposure to metals such as cobalt. Although much is known about the transcriptional response to hypoxia and cobalt, little is known about the all-important cell metabolism effects that trigger these responses. METHODS AND FINDINGS: Herein we use a low molecular weight metabolome profiling approach to identify classes of metabolites in yeast cells that are altered as a consequence of hypoxia or cobalt exposures. Key findings on metabolites were followed-up by measuring expression of relevant proteins and enzyme activities. We find that both hypoxia and cobalt result in a loss of essential sterols and unsaturated fatty acids, but the basis for these changes are disparate. While hypoxia can affect a variety of enzymatic steps requiring oxygen and heme, cobalt specifically interferes with diiron-oxo enzymatic steps for sterol synthesis and fatty acid desaturation. In addition to diiron-oxo enzymes, cobalt but not hypoxia results in loss of labile 4Fe-4S dehydratases in the mitochondria, but has no effect on homologous 4Fe-4S dehydratases in the cytosol. Most striking, hypoxia but not cobalt affected cellular pools of amino acids. Amino acids such as aromatics were elevated whereas leucine and methionine, essential to the strain used here, dramatically decreased due to hypoxia induced down-regulation of amino acid permeases. CONCLUSIONS: These studies underscore the notion that cobalt targets a specific class of iron proteins and provide the first evidence for hypoxia effects on amino acid regulation. This research illustrates the power of metabolite profiling for uncovering new adaptations to environmental stress.