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Trim58 Degrades Dynein and Regulates Terminal Erythropoiesis

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Trim58 Degrades Dynein and Regulates Terminal Erythropoiesis Developmental Cell Article Trim58 Degrades Dynein and Regulates Terminal Erythropoiesis Christopher S. Thom,1,2,6 Elizabeth A. Traxler,1,2,6 Eugene Khandros,1,2 Jenna M. Nickas,1 Olivia Y. Zhou,1 Jacob E. Lazarus,2,3 Ana P.G. Silva,4 Dolly Prabhu,1 Yu Yao,1 Chiaka Aribeana,1 Serge Y. Fuchs,5 Joel P. Mackay,4 Erika L.F. Holzbaur,3 and Mitchell J. Weiss1,7,* 1Division of Hematology, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA 2Cell and Molecular Biology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA 3Department of Physiology and Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA 4School of Molecular Bioscience, The University of Sydney, Sydney NSW 2006, Australia 5Department of Animal Biology and Mari Lowe Comparative Oncology Center, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA 6Co-first author 7Present address: Department of Hematology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, MS #355, Memphis, TN 38105, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2014.07.021 SUMMARY Welch et al., 2004), global DNA demethylation (Shearstone et al., 2011), reduced cell volume (Dolznig et al., 1995), and ejec- TRIM58 is an E3 ubiquitin ligase superfamily member tion of the nucleus to yield a reticulocyte, which develops further implicated by genome-wide association studies to into a mature red blood cell (Liu et al., 2010). The mechanisms that regulate human erythrocyte traits. Here, we show govern these processes are incompletely understood. that Trim58 expression is induced during late eryth- Expulsion of nuclei from erythroblasts (Keerthivasan et al., ropoiesis and that its depletion by small hairpin 2011; Konstantinidis et al., 2012) occurs exclusively in mammals RNAs (shRNAs) inhibits the maturation of late-stage and may represent an evolutionary adaptation to optimize erythrocyte rheology for transport through small capillary beds nucleated erythroblasts to anucleate reticulocytes. (Gaehtgens et al., 1981; Mueller et al., 2008). Erythroblast Imaging flow cytometry studies demonstrate that enucleation is preceded by nuclear condensation and requires Trim58 regulates polarization and/or extrusion of histone deacetylation (Ji et al., 2010; Popova et al., 2009) and erythroblast nuclei. In vitro, Trim58 directly binds suppression of the Myc proto-oncogene (Jayapal et al., 2010). and ubiquitinates the intermediate chain of the The condensed nucleus polarizes to one side of the cell via microtubule motor dynein. In cells, Trim58 stimulates transport mechanisms that require microtubules and phosphoi- proteasome-dependent degradation of the dynein nositide 3-kinase (Wang et al., 2012), followed by Rac GTPase- holoprotein complex. During erythropoiesis, Trim58 mediated formation of a contractile actomyosin ring between expression, dynein loss, and enucleation occur the incipient reticulocyte and nucleus (Ji et al., 2008; Konstanti- concomitantly, and all are inhibited by Trim58 nidis et al., 2012). Forces generated by the contractile ring pro- shRNAs. Dynein regulates nuclear positioning and mote further nuclear extrusion (Ji et al., 2008; Wang et al., 2012). Final separation between the reticulocyte and nucleus is microtubule organization, both of which undergo facilitated by transport of lipid vesicles to the interface, which dramatic changes during erythroblast enucleation. promotes remodeling and resolution of the plasma membrane Thus, we propose that Trim58 promotes this pro- surrounding both structures (Keerthivasan et al., 2010; Keerthi- cess by eliminating dynein. Our findings identify an vasan et al., 2011). In vivo, the ejected nucleus with surrounding erythroid-specific regulator of enucleation and eluci- plasma membrane, termed a pyrenocyte (McGrath et al., 2008), date a previously unrecognized mechanism for con- is scavenged by macrophages (Soni et al., 2006; Yoshida et al., trolling dynein activity. 2005), while reticulocytes are released into the circulation and undergo further maturation (Gifford et al., 2006). Enucleation shares several features with cytokinesis (Keerthivasan et al., INTRODUCTION 2010; Konstantinidis et al., 2012). All of the proteins currently known to participate in erythroblast enucleation are ubiquitously Humans produce about 2 million red blood cells (erythrocytes) expressed, with functional roles in mitosis. How erythroblasts each second to replace those lost by senescence (McGrath and co-opt generalized mitotic machinery for enucleation is un- Palis, 2008). Erythropoiesis is accompanied by several special- known. Presumably, this is mediated in part by the expression ized cell divisions associated with activation of erythroid-specific of one or more erythroid-restricted proteins. genes (Cheng et al., 2009; Welch et al., 2004), repression of alter- Here, we identify a role for the erythroid protein Trim58 in nate lineage genes (Cheng et al., 2009; Kingsley et al., 2013; erythroblast enucleation. Trim58 is a member of the tripartite 688 Developmental Cell 30, 688–700, September 29, 2014 ª2014 Elsevier Inc. Developmental Cell Investigating the Role of Trim58 in Erythropoiesis ACB Figure 1. Trim58 Is Expressed during Late-Stage Erythropoiesis and Regulates Erythroid Maturation * (A) Composite of three images showing Trim58 Trim58 Gene Locus mRNA (red) and DNA (DAPI, blue) in an E14.5 Scale 5 kb mm9 chr11: 58,452,000 58,453,000 58,454,000 58,455,000 58,456,000 58,457,000 58,458,000 58,459,000 58,460,000 58,461000, 58 462, 000, 58 463, 000, 58 464, 000, 58 465, 000, 58466 000, , 58 467 000, , 58 468 , ,000 UCSC Genes Based on RefSeq, UniProt, GenBank, CCDS and Comparative Genomics UCSC Genes 98 _ Erythroblast GATA1 TFBS ChIP-seq Signal from ENCODE/PSU Erythrobl GATA1 Gata1 mouse embryo. Note strong Trim58 expression in 1 _ 245 _ Erythroblast TAL1 TFBS ChIP-seq Signal from ENCODE/PSU Erythrobl TAL1 fetal liver, the site of definitive erythropoiesis. 1 _ SCL/Tal1 (B) E14.5 fetal liver erythroblasts were flow cy- mRNA Expression mRNA ChIP-Seq (ery) tometry purified (Pop et al., 2010), and Trim58 S0 S1 S3 S4/5 mRNA was analyzed by semiquantitative real-time Erythroblast Stage PCR. The y axis shows relative mRNA expression, Trim58 E normalized to S0 cells, which were assigned a D S4/5 value of 1. The x axis shows progressive devel- Mock VectorV shLucshScrsh58 #3sh58 #4sh58 #5sh58 kD#7 opmental stages from less to more mature. The * -75 results represent mean ± SEM for three biological (long) replicates. *p < 0.05. +shRNA enucleation Trim58 -50 (C) ChIP-seq analysis of transcription factor Expansion Maturation Trim58 (short) binding to the Trim58 locus in E14.5 fetal liver (24-72 hrs) (up to 48 hrs) erythroblasts (M. Pimkin, A.V. Kossenkov, T. Dex SCF Epo Actin Mishra, C.S. Morrissey, W. Wu, C.A. Keller, G.A. Epo Puro Control shRNAs Trim58 shRNAs Blobel, D. Lee, M.A. Beer, R.C. Hardison, and Maturation (hrs) M.J.W., unpublished data). The blue line depicts FGthe Trim58 gene, with exons shown as rectangles. 024283248 shLuc ** Transcription factor binding sites are indicated shLuc shTrim58 in red. ** (D) Trim58 knockdown studies. E14.5 murine fetal ** liver erythroid precursors were purified, infected ns ** sh58 with retroviruses encoding Trim58 or control % enucleated shRNAs, and cultured for 24–72 hr in expansion Hoechst Forward Scatter 024283248 medium with puromycin (Puro) to inhibit terminal Maturation (hrs) maturation and select for infected cells. The cells were then switched to maturation medium, which HIshLuc shTrim58 36 hrs maturation facilitates development to the reticulocyte stage over 48 hr. Late-stage S4/5 cells are small and En hemoglobinized with condensed nuclei (see B) R (Pop et al., 2010). Dex, dexamethasone. Scale bar, 10 mm. (E) Western blot for Trim58 in erythroblasts ex- N E En % reticulocytes pressing Trim58 or control shRNAs after 48 hr R E maturation. Luc, luciferase; Scr, scrambled. The sh58 asterisk represents a nonspecific band. ‘‘Long’’ and ‘‘short’’ exposures are from the same blot. (F) Enucleation of control (shLuc) and Trim58-deficient (shTrim58 #4) erythroblasts measured by flow cytometry the indicated time points. During maturation, erythroblasts become smaller, indicated by decreased forward scatter, and ultimately enucleate to become HoechstÀ reticulocytes (boxed regions). (G) Percent enucleation over time in cultures treated with control (shLuc) or Trim58 shRNA #4. Mean ± SD for three biological replicates. **p < 0.01; ns, not significant. (H) Representative histology of control (shLuc) and Trim58-deficient (shTrim58 #4) erythroid cultures after 36 hr maturation. Examples of anucleate reticulocytes (R), nucleated erythroblasts (E), extruded nuclei (N), and erythroblasts in the process of enucleation (En) are shown. Scale bar, 20 mm. (I) Summary of reticulocyte fractions from (H). Six hundred cells were counted on each slide. See also Figures S1 and S2. motif-containing family of proteins that frequently possess E3 uitous protein complex that is essential for most other eukary- ubiquitin ligase activities and function broadly in physiology otic cells. and disease (Napolitano and Meroni, 2012). Genome-wide association studies (GWAS) show that SNPs linked to the hu- RESULTS man TRIM58 gene associate with variations in the size and/ or number of circulating erythrocytes (Kamatani et al., 2010; Trim58 Is Induced during Late-Stage Erythropoiesis van der Harst
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