The Cytosolic Protein G0S2 Maintains Quiescence in Hematopoietic Stem Cells

Bone marrow hematopoietic stem cells (HSCs) balance proliferation and differentiation by integrating complex transcriptional and post-translational mechanisms regulated by cell intrinsic and extrinsic factors. We found that transcripts of G0/G1 switch gene 2 (G0S2) are enriched in lineage− Sca-1+ c-kit+ (LSK) CD150+ CD48− CD41− cells, a population highly enriched for quiescent HSCs, whereas G0S2 expression is suppressed in dividing LSK CD150+ CD48− cells. Gain-of-function analyses using retroviral expression vectors in bone marrow cells showed that G0S2 localizes to the mitochondria, endoplasmic reticulum, and early endosomes in hematopoietic cells. Co-transplantation of bone marrow cells transduced with the control or G0S2 retrovirus led to increased chimerism of G0S2-overexpressing cells in femurs, although their contribution to the blood was reduced. This finding was correlated with increased quiescence in G0S2-overexpressing HSCs (LSK CD150+ CD48−) and progenitor cells (LS−K). Conversely, silencing of endogenous G0S2 expression in bone marrow cells increased blood chimerism upon transplantation and promoted HSC cell division, supporting an inhibitory role for G0S2 in HSC proliferation. A proteomic study revealed that the hydrophobic domain of G0S2 interacts with a domain of nucleolin that is rich in arginine-glycine-glycine repeats, which results in the retention of nucleolin in the cytosol. We showed that this cytosolic retention of nucleolin occurs in resting, but not proliferating, wild-type LSK CD150+ CD48− cells. Collectively, we propose a novel model of HSC quiescence in which elevated G0S2 expression can sequester nucleolin in the cytosol, precluding its pro-proliferation functions in the nucleolus

Silencing of endogenous G0S2 expression in BM cells increases blood chimerism upon transplantation.

<p>Expression of the endogenous G0S2 gene was silenced in BM cells using two G0S2-specific pSIREN-shRNA retroviruses (Sh1, Sh2). Luciferase pSIREN-shRNA (Luc) was used as a control. (A) Knockdown efficiency was determined by quantitative real-time PCR in transduced BM cells (<i>n</i> = 4). (B) Sixteen weeks after transplantation, blood chimerism was measured by flow cytometry (<i>n</i> = 3–4). (C) The contribution of donor-derived cells to the Gr-1⁺ CD11b⁺ and CD3⁺ T cell populations was analyzed at different times after the transplant (<i>n</i> = 3–4). *, <i>P</i><0.05, **, <i>P</i><0.01, and ***, <i>P</i><0.001 (two-tailed Student's <i>t</i>-test).</p

Ectopic G0S2 expression reduces hematological reconstitution after BM transplantation.

<p>BM cells were transduced with control MIGR1 or MIGR1-G0S2 (V5-tagged) retroviruses to study the effect of G0S2 on hematopoiesis. (A) Retroviral expression of G0S2 was measured by quantitative real-time PCR and immunoblotting (<i>n</i> = 3). (B) Transduced BM cells were used to study subcellular localization using anti-V5 (red), DAPI (blue) and anti-Nup98, COX IV, Calnexin or Rab5 (green) antibodies. The data represent three independent experiments. (C) The cell-cycle distribution of transduced BM cells was analyzed using nuclear staining with propidium iodine and flow cytometry (<i>n</i> = 3). (D) Transduced BM cells were transplanted into lethally irradiated mice and the frequency (CFU) and colony cell number were enumerated in methylcellulose culture (<i>n</i> = 3) after three months of hematologic reconstitution. (E) The contribution of donor-derived cells to myeloid and T cell populations in the peripheral blood was analyzed after transplantation by flow cytometry at different times post-transplant (<i>n</i> = 3–4). The data are representative of two independent experiments. *, <i>P</i><0.05, **, <i>P</i><0.01, and ***, <i>P</i><0.001 (two-tailed Student's <i>t</i>-test).</p

Quiescent HSCs exhibit cytosolic sequestration of nucleolin.

<p>(A) Nucleolin colocalizes with the overexpressed G0S2 protein in LSK CD150⁺ CD48⁻ cells purified from mice transplanted with BM cells transduced with the MIGR1-G0S2 V5-tagged retrovirus. (B) Expression of nucleolin and Ki67 was determined in wild-type LS⁻K (proliferative progenitors), LSK CD150⁺ CD48⁻ cells purified from wild-type mice (dormant HSCs), and LSK CD150⁺ CD48⁻ cells purified from wild-type mice injected 6 days earlier with a single dose of 5-FU (proliferative HSCs). Images of DAPI, nucleolin, and Ki67 staining are shown for a representative cell. The data represent two independent experiments.</p

The hydrophobic domain of G0S2 interacts with the RGG domain of nucleolin.

<p>Co-immunoprecipitation and proteomic analyses were used to identify G0S2 protein partners. (A) G0S2-interacting proteins were pulled down with anti-V5 antibody in EL4 cells transduced with the empty or G0S2-V5 tagged retrovirus. Bands from a SDS-PAGE gel stained with Coomassie Blue were cut out for identification by mass spectrometry. (B) Reciprocal co-immunoprecipitation of V5-tagged G0S2 and nucleolin in EL4 and BM cells transduced with the MIGR1 (Ctrl) or MIGR1-G0S2 (G0S2) retrovirus. (C) Diagram depicting the domains in the wild-type G0S2 protein and the deletion mutants G0S2-ΔHD, G0S2^1–26 and G0S2^43–103. (D) The interaction between endogenous nucleolin and ectopic V5-tagged G0S2 was analyzed in NIH3T3 cells transduced with the empty retrovirus (Ctrl) or retroviruses bearing wild-type G0S2 (G0S2-wt) or G0S2 mutant constructs (G0S2-ΔHD, G0S2^1–26 and G0S2^43–103). (E) Diagram depicting the domains of the wild-type nucleolin (Ncl) protein and the deletion mutants Ncl^1–286, Ncl^287–707, Ncl-ΔRGG and Ncl-ΔRBDs (FLAG-tagged). RGG, arginine-glycine-glycine-rich domain; RBD, RNA-binding domain. (F) Interactions between V5-tagged G0S2 and FLAG-tagged nucleolin constructs (Ncl-wt, Ncl^1–286, Ncl^287–707, Ncl-ΔRGG and Ncl-ΔRBDs). The data represent two independent experiments.</p

G0S2 is expressed in dormant hematopoietic stem cells.

<p>(A) G0S2 transcripts were measured by quantitative real-time PCR in bone marrow hematopoietic stem and progenitor cells based on SLAM markers and mature myeloid and lymphoid cells purified from the spleen (<i>n</i> = 3). Statistical significance is indicated between HSCs and progenitor cells (MPP, CMP, GMP, MEP). (B) Expression of G0S2 and cyclin E2 in BM cells isolated at different times after administration of a single dose of 5-FU in C57BL/6 mice. The relative expression levels of G0S2 and cyclin E2 are shown as percentages of basal levels (<i>n</i> = 3–4). (C) Transcript levels of G0S2 and cyclin E2 were measured in LSK CD150⁺ CD48⁻ cells purified from untreated or 5-FU-treated (day 6) mice (<i>n</i> = 3). The data represent the mean and standard deviation of each experiment. *, <i>P</i><0.05 and **, <i>P</i><0.01 (two-tailed Student's <i>t</i>-test).</p

Perspectives from the Society for Pediatric Research: advice on sustaining science and mentoring during COVID-19.

The COVID-19 pandemic will leave an indelible mark on the careers of current medical trainees. Given the disruptions to medical education, economic impact on institutions, and the uncertainties around future job prospects, trainees are facing unprecedented challenges. This situation is especially concerning for futures of pediatric physician-scientist trainees, where concerns regarding maintaining the pipeline were well documented prior to the emergence of COVID-19. In this Perspectives article, we leverage the unique expertise of our workgroup to address concerns of physician-scientist trainees and to provide suggestions on how to navigate career trajectories in the post-COVID-19 era. We identified and addressed four major areas of concern: lack of in-person conferences and the associated decrease access to mentors and networking activities, decreased academic productivity, diminished job prospects, and mental health challenges. We also suggest actions for trainees, mentors and educational leaders, and institutions to help support trainees during the pandemic, with a goal of maintaining the pediatric physician-scientist pipeline