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Pou2af1 encodes for OCA-B, a coactivator of OCT-1/2 transcription factors, which plays a key role in B-cell maturation. The function of OCA-B has also been studied in T cells, where T cells from Pou2af1−/− mice have impaired functions, such as cytokine production and T follicular helper (Tfh) differentiation. Arguably, some of these T-cell phenotypes may result from impaired T–B interactions, secondary to the well-documented B-cell defects in Pou2af1−/− mice. Yet, Pou2af1 is actively transcribed in activated T cells, suggesting a T-cell-intrinsic role. To isolate the T-cell-intrinsic impact of Pou2af1, we generated Pou2af1fl/fl mice with specific genetic disruption of Pou2af1 either in all hematopoietic cells or exclusively in T cells. While we confirm that Pou2af1 is expressed in activated T cells, we surprisingly find that T-cell cytokine production is not impaired in Pou2af1-deficient T cells. Moreover, Pou2af1-sufficient and Pou2af1-deficient T cells have comparable transcriptome profiles, arguing against a T-cell-intrinsic role for Pou2af1. In line with these observations, we demonstrate that Tfh maturation is influenced by T-cell-extrinsic deletion of Pou2af1, as observed both in competitive bone marrow chimeras and in Pou2af1fl/fl mice with specific deletion in B cells. Overall, this study provides strong evidence that Pou2af1 does not act as a transcriptional coactivator in T cells, and conclusively demonstrates that loss of OCA-B in B cells indirectly impacts Tfh differentiation, clarifying the role of OCA-B in the immune system.

Osteocalcin (OCN) is an osteoblast-derived hormone with pleiotropic physiological functions. Like many peptide hormones, OCN is subjected to post-translational modifications (PTMs) which control its activity. Here, we uncover O-glycosylation as a novel PTM present on mouse OCN and occurring on a single serine (S8) independently of its carboxylation and endoproteolysis, two other PTMs regulating this hormone. We also show that O-glycosylation increases OCN half-life in plasma ex vivo and in the circulation in vivo. Remarkably, in human OCN (hOCN), the residue corresponding to S8 is a tyrosine (Y12), which is not O-glycosylated. Yet, the Y12S mutation is sufficient to O-glycosylate hOCN and to increase its half-life in plasma compared to wildtype hOCN. These findings reveal an important species difference in OCN regulation, which may explain why serum concentrations of OCN are higher in mouse than in human., eLife digest Bones provide support and protection for organs in the body. However, over the last 15 years researchers have discovered that bones also release chemicals known as hormones, which can travel to other parts of the body and cause an effect. The cells responsible for making bone, known as osteoblasts, produce a hormone called osteocalcin which communicates with a number of different organs, including the pancreas and brain. When osteocalcin reaches the pancreas, it promotes the release of another hormone called insulin which helps regulate the levels of sugar in the blood. Osteocalcin also travels to other organs such as muscle, where it helps to degrade fats and sugars that can be converted into energy. It also has beneficial effects on the brain, and has been shown to aid memory and reduce depression. Osteocalcin has largely been studied in mice where levels are five to ten times higher than in humans. But it is unclear why this difference exists or how it alters the role of osteocalcin in humans. To answer this question, Al Rifai et al. used a range of experimental techniques to compare the structure and activity of osteocalcin in mice and humans. The experiments showed that mouse osteocalcin has a group of sugars attached to its protein structure, which prevent the hormone from being degraded by an enzyme in the blood. Human osteocalcin has a slightly different protein sequence and is therefore unable to bind to this sugar group. As a result, the osteocalcin molecules in humans are less stable and cannot last as long in the blood. Al Rifai et al. showed that when human osteocalcin was modified so the sugar group could attach, the hormone was able to stick around for much longer and reach higher levels when added to blood in the laboratory. These findings show how osteocalcin differs between human and mice. Understanding this difference is important as the effects of osteocalcin mean this hormone can be used to treat diabetes and brain disorders. Furthermore, the results reveal how the stability of osteocalcin could be improved in humans, which could potentially enhance its therapeutic effect.

Cell-specific gene inactivation experiments delineate the functions of the enzymes required for osteocalcin modification and demonstrate that it is its uncarboxylated form that acts as a hormone., Osteocalcin (OCN) is an osteoblast-derived hormone favoring glucose homeostasis, energy expenditure, male fertility, brain development, and cognition. Before being secreted by osteoblasts in the bone extracellular matrix, OCN is γ-carboxylated by the γ-carboxylase (GGCX) on three glutamic acid residues, a cellular process requiring reduction of vitamin K (VK) by a second enzyme, a reductase called VKORC1. Although circumstantial evidence suggests that γ-carboxylation may inhibit OCN endocrine functions, genetic evidence that it is the case is still lacking. Here we show using cell-specific gene inactivation models that γ-carboxylation of OCN by GGCX inhibits its endocrine function. We further show that VKORC1 is required for OCN γ-carboxylation in osteoblasts, whereas its paralogue, VKORC1L1, is dispensable for this function and cannot compensate for the absence of VKORC1 in osteoblasts. This study genetically and biochemically delineates the functions of the enzymes required for OCN modification and demonstrates that it is the uncarboxylated form of OCN that acts as a hormone.

Osteocalcin (OCN) is an osteoblast-derived hormone that increases energy expenditure, insulin sensitivity, insulin secretion, and glucose tolerance. The cDNA sequence of OCN predicts that, like many other peptide hormones, OCN is first synthesized as a prohormone (pro-OCN). The importance of pro-OCN maturation in regulating OCN and the identity of the endopeptidase responsible for pro-OCN cleavage in osteoblasts are still unknown. Here, we show that the proprotein convertase furin is responsible for pro-OCN maturation in vitro and in vivo. Using pharmacological and genetic experiments, we also determined that furin-mediated pro-OCN cleavage occurred independently of its γ-carboxylation, a posttranslational modification that is known to hamper OCN endocrine action. However, because pro-OCN is not efficiently decarboxylated and activated during bone resorption, inactivation of furin in osteoblasts in mice resulted in decreased circulating levels of undercarboxylated OCN, impaired glucose tolerance, and reduced energy expenditure. Furthermore, we show that Furin deletion in osteoblasts reduced appetite, a function not modulated by OCN, thus suggesting that osteoblasts may secrete additional hormones that regulate different aspects of energy metabolism. Accordingly, the metabolic defects of the mice lacking furin in osteoblasts became more apparent under pair-feeding conditions. These findings identify furin as an important regulator of bone endocrine function. ispartof: Journal of Clinical Investigation vol:127 issue:11 pages:4104-4117 ispartof: location:United States status: published

Vol. 208, No. 6, March 16, 2015. [10.1083/jcb.201409111][1]. The authors noticed a mistake in the labeling of the genotyping primers in Fig. S1 A and Table S1. In Fig. S1 A, the primers P1 and P2 are actually flanking the 5′loxP site region. In addition, the P3 primer should appear downstream of

Osteocalcin is a hormone produced in bones by osteoblasts and regulating energy metabolism. While osteocalcin exists in two forms, γ-carboxylated and undercarboxylated only the latter appears to function as a hormone in vivo. It has been proposed recently that osteoclasts, the bone-resorbing cells, are responsible of decarboxylating, i.e. activating osteocalcin. To address the role of osteoclasts in the maintenance of energy metabolism we analyzed mutant mouse strains harboring either an increase or a decrease in osteoclasts number. Osteoprotegerin-deficient mice that are characterized by an increase in the number of osteoclasts demonstrate an increase in serum levels of undercarboxylated osteocalcin and are significantly more glucose tolerant than WT animals. Conversely, osteoclasts ablation in mice results in a decrease in serum undercarboxylated osteocalcin levels and in reduced glucose tolerance. These results support the notion that osteoclasts are controlling glucose metabolism at least in part through the regulation of osteocalcin decarboxylation.

In order to resorb the mineralized bone extracellular matrix, the osteoclast relies on the generation of a resorption lacuna characterized by the presence of specific proteases and a low pH. Hence, bone resorption by osteoclasts is highly dependent on lysosomes, the organelles specialized in intra- and extracellular material degradation. This is best illustrated by the fact that multiple forms of human osteopetrosis are caused by mutations in genes encoding for lysosomal proteins. Yet, until recently, the molecular mechanisms regulating lysosomal biogenesis and function in osteoclasts were poorly understood. Here we review the latest developments in the study of lysosomal biogenesis and function in osteoclasts with an emphasis on the transcriptional control of these processes.

SCL/TAL1, a tissue-specific transcription factor of the basic helix-loop-helix family, and c-Kit, a tyrosine kinase receptor, control hematopoietic stem cell survival and quiescence. Here we report that SCL levels are limiting for the clonal expansion of Kit⁺ multipotent and erythroid progenitors. In addition, increased SCL expression specifically enhances the sensitivity of these progenitors to steel factor (KIT ligand) without affecting interleukin-3 response, whereas a DNA-binding mutant antagonizes KIT function and induces apoptosis in progenitors. Furthermore, a twofold increase in SCL levels in mice bearing a hypomorphic Kit allele (W41/41) corrects their hematocrits and deficiencies in erythroid progenitor numbers. At the molecular level, we found that SCL and c-Kit signaling control a common gene expression signature, of which 19 genes are associated with apoptosis. Half of those were decreased in purified megakaryocyte/erythroid progenitors (MEPs) from W41/41 mice and rescued by the SCL transgene. We conclude that Scl operates downstream of Kit to support the survival of MEPs. Finally, higher SCL expression upregulates Kit in normal bone marrow cells and increases chimerism after bone marrow transplantation, indicating that Scl is also upstream of Kit. We conclude that Scl and Kit establish a positive feedback loop in multipotent and MEPs.

Abstract 2520 Poster Board II-497 The life-long production of blood cells depends on the regenerative capacity of a rare bone marrow population, the hematopoietic stem cells (HSCs). In the adult, the majority of HSCs are quiescent while a large proportion of progenitors are more cycling. The state of quiescence in HSCs is reversible and these cells can be triggered into cycle by chemotoxic injuries, exposure to cytokines in vitro, as well as transplantation in vivo. SCL/TAL1 is a bHLH transcription factor that has a critical role in generating HSCs during development. However, the role of SCL in adult HSCs is still a matter of debate. In the present study, we took several approaches to address this question. Scl expression was monitored by quantitative PCR analysis in a population that contains adult long-term reconstituting HSCs (LT-HSCs) at a frequency of 20–50%: Kit+Sca+Lin-CD150+CD48-. RT-PCR results were confirmed by β-galactosidase staining of these cells in Scl-LacZ mice. We show that Scl is highly expressed in LT-HSC and that its expression correlates with quiescence, i.e. Scl levels decrease when LT-HSCs exit the G0 state. In order to assess stem cell function, we performed several transplantation assays with adult bone marrow cells in which SCL protein levels were decreased at least two-fold by gene targeting or by RNA interference. 1) The mean stem cell activity of HSCs transplanted at ∼1 CRU was two-fold decreased in Scl heterozygous (Scl+/−) mice. 2) In competitive transplantation, the contribution of Scl+/− cells to primitive populations as well mature cells in the bone marrow was significantly decreased 8 months after transplantation. 3) In secondary transplantation assays, Scl+/− HSCs were severely impaired in their ability to reconstitute secondary recipient in stem cells and progenitor populations and in almost all mature lineages. 4) Reconstitution of the stem cell pool by adult HSCs expressing Scl-directed shRNAs was significantly decreased compared to controls. We therefore conclude that SCL levels regulate HSC long term competence. Since Scl levels decrease when LT-HSCs exit the G0 state, we addressed the question whether the cell cycle state of LT-HSCs is sensitive to Scl gene dosage. We stained bone marrow cell populations with Hoechst and Pyronin Y. At steady state, percentage LT-HSCs in G1 fraction appears to be significantly increased in mice lacking one allele of Scl. Furthermore, a three-fold increase in G1 fraction was also observed when cells were infected with Scl-directed shRNA, suggesting that a decrease in Scl levels facilitates G0-G1 transition. At the molecular level, we show by chromatin immunoprecipitation that SCL occupies the Cdkn1a and Id1 loci. Furthermore, in purified Kit+Sca+Lin-CD150+CD48- cells, the expression levels of these two regulators of HSC cell cycle and long-term functions are sensitive to Scl gene dosage. Together, our observations suggest that SCL impedes G0-G1 transition in HSCs and regulates their long-term competence. Disclosures: No relevant conflicts of interest to declare.

The Saccharomyces cerevisiae prohormone-processing enzyme Kex2p is biosynthesized as an inactive precursor extended by its N-terminal proregion. Here we show that deletion of the proregion renders Kex2p inactive both in vivo and in vitro. Absence of the proregion impaired glycosylation and stability and resulted in the retention of the enzyme in the endoplasmic reticulum. These phenotypes were partially complemented by expression of the proregion intrans. Trans complementation was specific to Kex2p proregion because expression of any of the seven mammalian prohormone convertase propeptides had no effect. These data are consistent with a model whereby Kex2p proregion functions as an intramolecular chaperone and indicate that covalent linkage to the protein is not an absolute requirement for proregion function. Furthermore, extensive mutagenesis revealed that, in addition to their function as proteolytic recognition sites, C-terminal basic residues play an active role in proregion-dependent Kex2p activation.

Hematopoietic stem cells (HSC) reside in specific bone marrow niches comprised of perisinusoidal Nestin-GFP+ (Nes-GFP+) and leptin receptor (LepR)+ stromal cells which highly overlap with each other, as well as osteolineage and endothelial cells. These different cellular constituents regulate HSC maintenance and retention in the bone marrow (BM). Recently, our laboratory further identified rare periarteriolar Nes-GFPbright cells which have been identified to be pericytes and functionally crucial for HSC quiescence and maintenance, from the more abundant reticular Nes-GFPdim population (Kunisaki et al, unpublished data). The function of Nes-GFP+ niche cells is tightly regulated by the sympathetic nervous system (SNS) via β2- and β3-adrenergic receptors. In acute myeloid leukemia (AML), BM infiltration by leukemic blasts is known to lead to hematopoietic failure. Although cytopenias are thought to result from the BM occupation by AML, the mechanisms remain unclear. In this study, we investigated the impact of AML on BM niche constituents, and evaluated the influence of SNS signals in AML progression using a syngeneic murine MLL-AF9 transplantation model. We observed a ∼4.4-fold expansion of Nes-GFP+ stromal cells (p Disclosures: Armstrong: Epizyme Inc.: Has consulted for Epizyme Inc. Other.

Abstract 505 Few markers have thus far been identified on native mesenchymal stem cells (MSCs), both in the mouse and human systems. Most markers cited in the literature are indeed based on expression analyses on heterogeneous cultured cell populations, which may not have self-renewal properties if rigorously tested by transplantation assays. Previous studies using Nestin (Nes)-Gfp transgenic mice showed that Nes-GFP+ cells are self-renewing MSCs, a major constituent of the hematopoietic stem cell (HSC) niche in the bone marrow (BM) (Nature 2010; 466:829). However, the cytoplasmic location of Nestin precludes prospective live cell isolation outside of the transgenic mice. Hence, finding a combination of surface markers labeling Nestin+ cells in situ would be valuable to isolate bona fide MSCs and characterize niche cells. Screening analyses toward this end revealed that PDGFRα and CD51 expression among CD45− Ter119− CD31− BM stromal cells comprised a large fraction (∼60%) of Nes-GFP+ cells. Upon gating first on PDGFRα+ and CD51+, double-positive cells were also highly enriched in Nes-GFP+ cells (∼75%), and represented a rare fraction (∼2%) of the stromal population. Endogenous Nestin expression was also enriched in PDGFRα+ CD51+ cells, compared to single-positive or double-negative stromal cells (control subsets). Cell sorting of BM PDGFRα+ CD51+ and control subsets revealed that PDGFRα+ CD51+ significantly enriched (> 10-fold, p 7-fold, p Disclosures: No relevant conflicts of interest to declare.

SCL/TAL1 is a member of the basic helix-loop-helix family of transcription factors that also includes E2A and HEB. SCL is essential at the onset of hematopoiesis and activates transcription through heterodimerization with E2A. Both genes are expressed in long-term and short-term repopulating hematopoietic stem cells (HSCs) as assessed by expression profiling. To determine whether these genes regulate lineage commitment in HSCs and possibly other HSCs functions, we proceeded to serial transplantation of E2A-deficient or SCL-impaired HSCs. All mice were on a C57Bl/6 background and transplanted donor cells were discriminated from host cells by differential expression of CD45 isoforms. Since SCL function is dosage-sensitive, we used bone marrow cells from SCL LacZ/wt mice in which one SCL allele has been invalidated. When transplanted mice were analyzed at 4 months for reconstitution in all hematopoietic organs, SCLLacZ/wt HSCs (CD45.2) showed normal repopulating capacity in competition with wild type cells (CD45.1) as expected. Strikingly, analysis at 8 months revealed that SCLLacZ/wt HSCs were markedly impaired compared to normal HSCs. The contribution of SCLLacZ/wt cells to the Kit+Sca+Lin− (KSL) and all myeloid progenitor (CMP, MEP and GMP) populations was less than 5%. This long term defect was also observed in secondary transplantation assays. Despite this near absence of HSCs and CMPs, common lymphoid progenitors (CLPs) were almost equally composed of SCLLacZ/wt and wild type cells, and the generation of B22O positive cells was expanded when SCL gene dosage was reduced. Together, our results indicate that long-term stem cell activity requires full SCL gene dosage. Furthermore, SCL operates at the branchpoint of the myeloid and lymphoid lineages to favor the myeloid lineages. Since E2A is an obligate SCL partner, we next addressed E2A function in HSCs. As E2A−/− embryos die before birth, we therefore transplanted E14.5 fetal liver cells. E2A deficiency did not affect the number of HSCs, as assessed by limiting dilution analysis. Nonetheless, serial transplantation assays revealed that HSCs from wild type littermates could be serially transplanted beyond the fourth passage while there was an early exhaustion of E2A-deficient HSCs at the third transplantation. Together, our analyses are consistent with the view that E2A is the functional partner of SCL in HSCs and that SCL/E2A does not control the size of the HSC pool but is required for long-term HSC activity. Similarly to SCL impairment, all progenitors were decreased by E2A deficiency. There were, however, two major differences. First, CLPs were severely impaired indicating that E2A activity is required for specification of the lymphoid lineages. Second, CD11b positive cells were expanded by E2A-deficiency and this was gene dosage-dependent, consistent with the view that E2A inhibits the monocyte-granulocyte lineage. Taken together, our results indicate that SCL collaborates with E2A in HSCs to maintain their long-term activities. In contrast, at the myeloid-lymphoid branchpoint, the functions of SCL and E2A are opposite: SCL favors the myeloid lineage at the expense of the lymphoid lineage, while E2A specifies the lymphoid lineage and inhibits granulocyte-macrophage differentiation.

The c-Kit tyrosine kinase receptor and the SCL/Tal1 (Stem Cell Leukemia) transcription factor are crucial to hematopoiesis and are co-expressed by hematopoietic progenitors. In the present study, we directly address the question whether SCL and c-Kit establish a code for the fate of multipotent progenitors in the adult. We therefore proceeded to loss and gain of function experiments, via retroviral mediated gene transfer in primary hematopoietic cells. We show that SCL is functionally required to sustain the survival of c-Kit+ cells in response to Steel Factor (Kit ligand), but not to GM-CSF and IL-3. Interfering with SCL function using a DNA-binding defective SCL mutant (ΔbSCL) caused a 100-fold loss of multipotent progenitors, due to massive apoptotic death in the c-Kit+ fraction, indicating that the DNA binding function of SCL is essential in cell survival. An antisense SCL also caused apoptosis in c-Kit+ cells, which was rescued by co-delivering SCL in the sense orientation. Furthermore, SCL sets threshold for the response of multipotent progenitors to Steel factor without affecting their response to IL-3 or GM-CSF, demonstrating the specificity of SCL for c-Kit-dependent pathway. Moreover, SCL levels in purified hematopoietic progenitors (HSC, CMP, MEP, CLP) were 3.5 to 15-fold decreased in W41 W41 mice that have a hypomorphic c-Kit allele when compared to age-matched wild type controls. Conversely, ectopic SCL expression is sufficient to bypass c-Kit signalling to suppress apoptosis and increase the proliferative potential of multipotent progenitors, as anemia and other hematopoietic defects caused by a hypomorphic c-Kit allele were rescued by introducing an SCL transgene into the W41 W41 background. We conclude that SCL operates within the c-Kit pathway to suppress apoptosis and determine the clonal output of adult multipotent progenitors. To define the pathway through which SCL suppresses apoptosis in hematopoietic progenitors, we performed microarrays analysis on Δb-SCL expressing cells and identified novel survival genes that were confirmed to be direct SCL targets by chromatin immunoprecipitation. Finally, the expression of these target genes was decreased in purified progenitors from W41 W41 mice as assessed by quantitative PCR. Together, this study reveals a new and essential genetic pathway involving SCL downstream of c-Kit signalling for the survival of hematopoietic progenitors.