The functional longevity of HSCs relies on their ability to balance proliferation, differentiation, and quiescence. Stem-cell intrinsic activators of quiescence can prevent the loss of stemness by modulating responses to differentiation-inducing signals. In this study, we found that G0S2 restricts the steady-state number of HSCs entering the cell cycle in a cell-autonomous manner. Furthermore, we showed that elevated levels of G0S2 in HSCs cause retention of nucleolin in the cytosol and inhibition of cell proliferation.
The initial finding that G0S2 expression is inversely correlated with cyclin E2 levels in LSK CD150+
cells, a population highly enriched for HSCs 
, suggested that G0S2 may be involved in the maintenance of stem cell quiescence. Competitive BM transplants revealed that HSCs overexpressing G0S2 were able to contribute to the hematopoietic system over the long term, although with lower efficiency than wild-type HSCs. Conversely, retroviral silencing of the endogenous G0S2 gene in BM cells resulted in increased contributions to the peripheral myeloid and lymphoid cell populations upon transplantation. We hypothesized that G0S2 primarily regulates the decision to proliferate or remain quiescent in HSCs: ectopic G0S2 expression led to an increased percentage of quiescent HSCs, whereas silencing resulted in increased HSC proliferation. We also observed that G0S2 modulates at some extent the proliferation of hematopoietic progenitor cells although myeloid committed progenitor cells appear to be less dependent on G0S2 regulation. Consistent with increased homing of quiescent HSCs 
, ectopic G0S2 expression led to increased chimerism in the BM but lowered the contribution to blood in competitive transplants. This result is more likely attributable to reduced HSC proliferation than decreased differentiation because no differences were observed in the number of myeloid colonies in clonogenic assays. In support of our findings, a gene expression analysis in fibroblasts subjected to prolonged quiescence revealed that upregulation of G0S2 was part of a gene signature that suppresses proliferation 
Other factors known to induce HSC quiescence are p21, Gfi1, Pten, and FoxO1, 3 and 4 
. Knockout mice deficient in these factors showed increased homeostatic cycling of HSCs and eventual stem cell exhaustion, although augmented HSC proliferation does not always cause a loss of stem cell function 
. For instance, overexpression of HoxB4 uncouples cell division from differentiation in HSCs, leading to ex vivo
HSC expansion 
. Our data suggest that modulations of G0S2 expression might be applied to preserve HSC stemness during ex vivo
culture, without the side effect of promoting differentiating cell division events that might compromise long-term stem cell function. However, the upstream signaling pathways and transcription factors that regulate G0S2 expression in HSCs are largely unknown. The only activator of G0S2 described in hematopoietic cells is retinoic acid 
. Interestingly, retinoic acid treatment enhances ex vivo
maintenance of HSCs 
. Similar to G0S2, Stat5 activity promotes HSC quiescence 
, raising the possibility that G0S2 could be regulated downstream of c-kit and Mpl signaling. However, inhibition of calcium-dependent calcineurin with cyclosporin A suppresses G0S2 transcription in human blood mononuclear cells 
, and deletion of calmodulin-dependent protein kinase IV resulted in hematopoietic failure due to a numeric and functional reduction of HSCs 
. Future studies are required to define the regulation of G0S2 expression in HSCs.
The cytosolic localization of G0S2 suggested that the G0S2-mediated inhibition of HSC proliferation likely comprises a non-transcriptional mechanism involving protein-protein interactions. In a proteomic analysis, we identified nucleolin as a new protein partner of G0S2 and further showed that the hydrophobic domain of G0S2 is required for this interaction. Nucleolin is a phosphonucleolar protein involved in several phases of ribosome biosynthesis: transcription of ribosomal DNA in nucleoli, maturation of pre-ribosomal RNA, and transport of ribonucleoproteins and pre-ribosomal particles for ribosome assembly of ribosomes in the cytosol 
. However, the role of nucleolin in the HSC proliferation has not been well studied, although nucleolin's function is generally thought to be associated to cell growth and cell division. Nucleolin expression is elevated in rapidly dividing cells and tumor cells, supporting a role for this protein in cell proliferation 
. Conversely, silencing of nucleolin expression in HeLa cells and human fibroblasts reduced both the percentage of cells in S phase and cell growth 
. In hematopoietic cells, retinoblastoma protein antagonizes the nucleolin-mediated activation of the CD34 promoter in KG1 acute myelogenous leukemia cells 
. Finally, nucleolin may contribute to the IFNα-mediated release of HSCs from quiescence due to its ability to transport Stat1 proteins into the nucleus 
. Therefore, G0S2 might modulate proliferation by interfering with the pro-proliferation functions of nucleolin. In addition to nucleolin, we also determined that ribonucleoproteins L3, L6, and L13 bind to G0S2. Several ribosomal proteins, including L3, L6 and L13, interact with nucleolin via the RGG domain in the C-terminal moiety 
. The fact that nucleolin also binds to the ribonucleoproteins found in our screen of G0S2-interacting proteins indicates that G0S2 either binds directly to the RGG domain or to a complex containing ribonucleoproteins and nucleolin.
Our overexpression study suggests that G0S2 interacts with nucleolin when its cellular level reaches a certain threshold. This interaction leads to the retention of nucleolin in the cytosol and suppression of cell division. In support of the physiological relevance of our findings, we demonstrated that wild-type LSK CD150+
cells, a population with high levels of endogenous G0S2 gene expression, displayed a perinuclear distribution of nucleolin. In contrast to the predominantly ‘ring-like’ distribution observed in HSCs, progenitor cells (LS−
K) showed both perinuclear and nucleolar localization of nucleolin, suggesting a lower dependency on the G0S2 and Nucleolin interaction. This difference might be related to the levels of G0S2 expression in stem versus progenitor cells. The ring-like extranuclear distribution of nucleolin was previously described in nonproliferating leukemic cells of chronic lymphocytic leukemia patients 
. Similarly, T lymphocytes from HIV-positive patients show a cytosolic expression of nucleolin that is not observed in healthy donors 
; however, this subcellular localization is associated with apoptosis in CD4 T cells. Our work is the first report to describe a mechanism for the subcellular redistribution of nucleolin and the association of G0S2 with proliferation in hematopoietic cells.
Most research in the field has focused on identifying the transcriptional machinery that controls stem cell proliferation. Our work supports a novel role for G0S2 in the regulation of nucleolin function by its sequestration of nucleolin in the cytosol in quiescent HSCs. This mechanism could potentially be targeted to reduce cell-cycle-dependent cytotoxicity and improve engraftment in bone marrow transplants.