Aging-like Phenotype and Defective Lineage Specification in SIRT1-Deleted Hematopoietic Stem and Progenitor Cells

Summary Aging hematopoietic stem cells (HSCs) exhibit defective lineage specification that is thought to be central to increased incidence of myeloid malignancies and compromised immune competence in the elderly. Mechanisms underlying these age-related defects remain largely unknown. We show that the deacetylase Sirtuin (SIRT)1 is required for homeostatic HSC maintenance. Differentiation of young SIRT1-deleted HSCs is skewed toward myeloid lineage associated with a significant decline in the lymphoid compartment, anemia, and altered expression of associated genes. Combined with HSC accumulation of damaged DNA and expression patterns of age-linked molecules, these have striking overlaps with aged HSCs. We further show that SIRT1 controls HSC homeostasis via the longevity transcription factor FOXO3. These findings suggest that SIRT1 is essential for HSC homeostasis and lineage specification. They also indicate that SIRT1 might contribute to delaying HSC aging

Evidence for AKT-independent regulation of FOXO1 and FOXO3 in haematopoietic stem and progenitor cells

<p>Transcription factors FOXOs (1, 3, 4) are essential for the maintenance of haematopoietic stem cells. FOXOs are evolutionary conserved substrates of the AKT serine threonine protein kinase that are also phosphorylated by several kinases other than AKT. Specifically, phosphorylation by AKT is known to result in the cytosolic localization of FOXO and subsequent inhibition of FOXO transcriptional activity. In addition to phosphorylation, FOXOs are regulated by a number of other post-translational modifications including acetylation, methylation, redox modulation, and ubiquitination that altogether determine these factors' output. Cumulating evidence raises the possibility that in stem cells, including in haematopoietic stem cells, AKT may not be the dominant regulator of FOXO. To address this question in more detail, we examined gene expression, subcellular localization, and response to AKT inhibition of FOXO1 and FOXO3, the main FOXO expressed in HSPCs (haematopoietic stem and progenitor cells). Here we show that while FOXO1 and FOXO3 transcripts are expressed at similar levels, endogenous FOXO3 protein is mostly nuclear compared to the cytoplasmic localization of FOXO1 in HSPCs. Furthermore, inhibition of AKT does not enhance nuclear localization of FOXO1 nor FOXO3. Nonetheless AKT inhibition in the context of loss of NAD-dependent SIRT1 deacetylase modulates FOXO3 localization in HSPCs. Together, these data suggest that FOXO3 is more active than FOXO1 in primitive haematopoietic stem and multipotent progenitor cells. In addition, they indicate that upstream regulators other than AKT, such as SIRT1, maintain nuclear FOXO localization and activity in HSPCs.</p

The Spi1/PU.1 transcription factor accelerates replication fork progression by increasing PP1 phosphatase in leukemia

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A Systems Approach Identifies Essential FOXO3 Functions at Key Steps of Terminal Erythropoiesis

<div><p>Circulating red blood cells (RBCs) are essential for tissue oxygenation and homeostasis. Defective terminal erythropoiesis contributes to decreased generation of RBCs in many disorders. Specifically, ineffective nuclear expulsion (enucleation) during terminal maturation is an obstacle to therapeutic RBC production <i>in vitro</i>. To obtain mechanistic insights into terminal erythropoiesis we focused on FOXO3, a transcription factor implicated in erythroid disorders. Using an integrated computational and experimental systems biology approach, we show that FOXO3 is essential for the correct temporal gene expression during terminal erythropoiesis. We demonstrate that the FOXO3-dependent genetic network has critical physiological functions at key steps of terminal erythropoiesis including enucleation and mitochondrial clearance processes. FOXO3 loss deregulated transcription of genes implicated in cell polarity, nucleosome assembly and DNA packaging-related processes and compromised erythroid enucleation. Using high-resolution confocal microscopy and imaging flow cytometry we show that cell polarization is impaired leading to multilobulated <i>Foxo3</i>^<i>-/-</i> erythroblasts defective in nuclear expulsion. Ectopic FOXO3 expression rescued <i>Foxo3</i>^<i>-/-</i> erythroblast enucleation-related gene transcription, enucleation defects and terminal maturation. Remarkably, FOXO3 ectopic expression increased wild type erythroblast maturation and enucleation suggesting that enhancing FOXO3 activity may improve RBCs production. Altogether these studies uncover FOXO3 as a novel regulator of erythroblast enucleation and terminal maturation suggesting FOXO3 modulation might be therapeutic in disorders with defective erythroid maturation.</p></div

Model.

<p><b>(A)</b> Depiction of expression of clusters Q and R genes in <i>Foxo3</i>^<i>-/-</i> versus wild type erythroblasts. Cluster Q is enriched for nucleosome assembly, heme biosynthesis, and DNA packaging-related processes while cluster R is enriched for autophagy and catabolic processes. <b>(B)</b> Model for gene expression in terminally maturing erythroblasts. Complexes of core erythroid transcription factors regulate the genetic programs required for maturation of the initial erythroblast stages. These transcription factor complexes may also induce <i>Foxo3</i> expression in immature erythroblasts. In turn, FOXO3 cooperates with these factors to sustain and/or enhance the erythroid transcriptional program during the later stages of terminal maturation.</p

Autophagy and mitochondrial removal are impaired in <i>Foxo3</i>^<i>-/-</i> erythroblasts

<p><b>(A)</b> Western blot analysis of LC3B protein of WT and <i>Foxo3</i>^<i>-/-</i> bone marrow TER119⁺ cells (n = 3 mice for each genotype). Quantification of the LC3BII/ LC3B-I ratio in one representative of two independent experiments is shown (bottom panel). <b>(B)</b> Western blot analysis of LC3B protein extracted from WT and <i>Foxo3</i>^<i>-/-</i> bone marrow erythroblasts Gates II to IV (insufficient Gate I cell numbers for Western blot). Quantification of the LC3B-II/LC3B-I ratio is shown (panel below). <b>(C)</b> Autophagic flux in WT and <i>Foxo3</i>^<i>-/-</i> bone marrow cells was analyzed by flow cytometry. Cells were cultured with chloroquine (50 μM) for the indicated time points and autophagosomes were detected by Cyto-ID in specific gates according to TER119, CD44 and FSC properties. Flux was calculated by subtracting the value obtained from the untreated sample to the value obtained at each of the different time points. Results are mean ± SEM of n = 3. One representative of three independent experiments is shown. <b>(D)</b> Aliquots of cell lysates from (<b>C</b>) at the indicated time points were subjected to Western blot analysis of LC3B showing two replicates. Quantification of the LC3B-II protein is normalized to total actin, and the relative accumulation of LC3B-II is quantified (bottom panel). <b>(E)</b> Flow cytometry analysis (left panels) and quantification (right panel, n = 4 in each genotype) of Mitotracker Red CMXRos in combination with CD71 surface expression of WT and <i>Foxo3</i>^<i>-/-</i> peripheral blood. *<i>P</i> < 0.05 **<i>P</i> < 0.01 ***<i>P <</i> 0.001, Student’s <i>t</i> test.</p

Defective enucleation in <i>Foxo3</i>^<i>-/-</i> bone marrow erythroblasts.

<p><b>(A)</b> Enucleation was analyzed by immunofluorescence of freshly isolated bone marrow cells from WT (n = 5) and <i>Foxo3</i>^<i>-/-</i> (n = 3) mice using anti-TER119 antibody (green), Rhodamine Phalloidin (red) and Hoechst (blue). Images were obtained by confocal microscopy and abnormal enucleating cells counted. Representative images of enucleating cells are shown, with white asterisks denoting abnormally enucleating cells. At least 10 enucleating cells were counted per bone marrow and the results indicate the percentage of abnormal enucleating cells in each bone marrow as mean ± SEM. <b>(B)</b> Quantification of abnormal nuclei within the orthochromatic faction of WT and <i>Foxo3</i>^<i>-/-</i> erythroblasts by imaging flow cytometry. Abnormal nuclei were defined as having high 3-fold symmetry of the nucleus [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005526#pgen.1005526.ref064" target="_blank">64</a>]. Representative images of normal and abnormal nuclei from <i>Foxo3</i> mutants are shown are shown. Results are mean ± SEM of n = 4. *<i>P</i> < 0.05 **<i>P <</i> 0.01 ***<i>P</i> < 0.001, Student’s <i>t</i> test. <b>(C)</b> Model for the impact of loss of FOXO3 on the enucleation process. <b>(D)</b> Heatmap of RNA-Seq data of CDC42-related gene cluster (both upstream and downstream of CDC42) implicated in polarity and actin polymerization in Gates I-II and III of <i>Foxo3</i> wild type and mutant erythroblasts.</p