Teleost Growth Factor Independence (gfi) Genes Differentially Regulate Successive Waves Of Hematopoiesis

Growth Factor Independence (Gfi) transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic stem/progenitor cell (HSPC) development and lineage specification. In mammals, Gfi1. a regulates hematopoietic stem cells (HSC), myeloid and lymphoid populations, while its paralog, Gfi1. b, regulates HSC, megakaryocyte and erythroid development. In zebrafish, gfi1. aa is essential for primitive hematopoiesis; however, little is known about the role of gfi1. aa in definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we report the isolation and characterization of an additional hematopoietic gfi factor, gfi1. b. We show that gfi1. aa and gfi1. b are expressed in the primitive and definitive sites of hematopoiesis in zebrafish. Our functional analyses demonstrate that gfi1. aa and gfi1. b have distinct roles in regulating primitive and definitive hematopoietic progenitors, respectively. Loss of gfi1. aa silences markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1. b silences runx-1, c-myb, ikaros and cd41, indicating that gfi1. b is required for definitive hematopoiesis. We determine the epistatic relationships between the gfi factors and key hematopoietic transcription factors, demonstrating that gfi1. aa and gfi1. b join lmo2, scl, runx-1 and c-myb as critical regulators of teleost HSPC. Our studies establish a comparative paradigm for the regulation of hematopoietic lineages by gfi transcription factors. © 2012 Elsevier Inc.3732431441Amigo, J.D., Yu, M., Troadec, M.-B., Gwynn, B., Cooney, J.D., Lambert, A.J., Chi, N.C., Paw, B.H., Identification of distal cis-regulatory elements at mouse mitoferrin loci using zebrafish transgenesis (2011) Mol. Cell. Biol., 31, pp. 1344-1356Amigo, J.D., Ackermann, G.E., Cope, J.J., Yu, M., Cooney, J.D., Ma, D., Langer, N.B., Paw, B.H., The role and regulation of friend of GATA-1 (FOG-1) during blood development in the zebrafish (2009) Blood, 114, pp. 4654-4663Bolli, N., Payne, E.M., Rhodes, J., Gjini, E., Johnston, A.B., Guo, F., Lee, J.-S., Look, A.T., Cpsf1 is required for definitive HSC survival in zebrafish (2011) Blood, 117, pp. 3996-4007Burns, C.E., Traver, D., Mayhall, E., Shepard, J.L., Zon, L.I., Hematopoietic stem cell fate is established by the Notch-Runx pathway (2005) Genes Dev., 19, pp. 2331-2342Bussmann, J., Bakkers, J., Schulte-Merker, S., Early endocardial morphogenesis requires Scl/Tal1 (2007) PLoS Genet., 3, pp. e140Dan, K., Thrombocytosis in iron deficiency anemia (2005) Intern. Med., 44, pp. 1025-1026Davidson, A.J., Zon, L.I., The 'definitive' (and "primitive") guide to zebrafish hematopoiesis (2004) Oncogene, 23, pp. 7233-7246Dooley, K.A., Davidson, A.J., Zon, L.I., Zebrafish scl functions independently in hematopoietic and endothelial development (2005) Dev. Biol., 277, pp. 522-536Dufourcq, P., Rastegar, S., Strähle, U., Blader, P., Parapineal specific expression of gfi1 in the zebrafish epithalamus (2004) Gene. Expr. Patterns, 4, pp. 53-57Farr, C.J., Saiki, R.K., Erlich, H.A., McCormick, F., Marshall, C.J., Analysis of RAS gene mutations in acute myeloid leukemia by polymerase chain reaction and oligonucleotide probes (1988) Proc. Natl. Acad. Sci. USA, 85, pp. 1629-1633Ganis, J.J., Hsia, N., Trompouki, E., de Jong, J.L.O., Dibiase, A., Lambert, J.S., Jia, Z., Zon, L.I., Zebrafish globin switching occurs in two developmental stages and is controlled by the LCR (2012) Dev. Biol., 366, pp. 185-194Gering, M., Rodaway, A.R., Göttgens, B., Patient, R.K., Green, A.R., The SCL gene specifies haemangioblast development from early mesoderm (1998) EMBO J., 17, pp. 4029-4045Gilks, C.B., Bear, S.E., Grimes, H.L., Tsichlis, P.N., Progression of interleukin-2 (IL-2)-dependent rat T cell lymphoma lines to IL-2-independent growth following activation of a gene (Gfi-1) encoding a novel zinc finger protein (1993) Mol. Cell. Biol., 13, pp. 1759-1768Grimes, H.L., Chan, T.O., Zweidler-Mckay, P.A., Tong, B., Tsichlis, P.N., The Gfi-1 proto-oncoprotein contains a novel transcriptional repressor domain, SNAG, and inhibits G1 arrest induced by interleukin-2 withdrawal (1996) Mol. Cell. Biol., 16, pp. 6263-6272Hock, H., Hamblen, M.J., Rooke, H.M., Schindler, J.W., Saleque, S., Fujiwara, Y., Orkin, S.H., Gfi-1 restricts proliferation and preserves functional integrity of haematopoietic stem cells (2004) Nature, 431, pp. 1002-1007Hock, H., Hamblen, M.J., Rooke, H.M., Traver, D., Bronson, R.T., Cameron, S., Orkin, S.H., Intrinsic requirement for zinc finger transcription factor Gfi-1 in neutrophil differentiation (2003) Immunity, 18, pp. 109-120Hsu, K., Traver, D., Kutok, J.L., Hagen, A., Liu, T.-X., Paw, B.H., Rhodes, J., Look, A.T., The pu.1 promoter drives myeloid gene expression in zebrafish (2004) Blood, 104, pp. 1291-1297Karsunky, H., Zeng, H., Schmidt, T., Zevnik, B., Kluge, R., Schmid, K.W., Dührsen, U., Möröy, T., Inflammatory reactions and severe neutropenia in mice lacking the transcriptional repressor Gfi1 (2002) Nat. Genet., 30, pp. 295-300Khandanpour, C., Sharif-Askari, E., Vassen, L., Gaudreau, M.-C., Zhu, J., Paul, W.E., Okayama, T., Möröy, T., Evidence that growth factor independence 1b regulates dormancy and peripheral blood mobilization of hematopoietic stem cells (2010) Blood, 116, pp. 5149-5161Liao, E.C., Trede, N.S., Ransom, D., Zapata, A., Kieran, M., Zon, L.I., Non-cell autonomous requirement for the bloodless gene in primitive hematopoiesis of zebrafish (2002) Development, 129, pp. 649-659Lieschke, G.J., Oates, A.C., Paw, B.H., Thompson, M.A., Hall, N.E., Ward, A.C., Ho, R.K., Layton, J.E., Zebrafish SPI-1 (PU.1) marks a site of myeloid development independent of primitive erythropoiesis: implications for axial patterning (2002) Dev. Biol., 246, pp. 274-295Lin, H.-F., Traver, D., Zhu, H., Dooley, K., Paw, B.H., Zon, L.I., Handin, R.I., Analysis of thrombocyte development in CD41-GFP transgenic zebrafish (2005) Blood, 106, pp. 3803-3810Liu, F., Walmsley, M., Rodaway, A., Patient, R., Fli1 acts at the top of the transcriptional network driving blood and endothelial development (2008) Curr. Biol., 18, pp. 1234-1240Long, Q., Meng, A., Wang, H., Jessen, J.R., Farrell, M.J., Lin, S., GATA-1 expression pattern can be recapitulated in living transgenic zebrafish using GFP reporter gene (1997) Development, 124, pp. 4105-4111Lyons, S.E., Lawson, N.D., Lei, L., Bennett, P.E., Weinstein, B.M., Liu, P.P., A nonsense mutation in zebrafish gata1 causes the bloodless phenotype in vlad tepes (2002) Proc. Natl. Acad. Sci. USA, 99, pp. 5454-5459Ma, D., Zhang, J., Lin, H.-F., Italiano, J., Handin, R.I., The identification and characterization of zebrafish hematopoietic stem cells (2011) Blood, 118, pp. 289-297Meeker, N.D., Hutchinson, S.A., Ho, L., Trede, N.S., Method for isolation of PCR-ready genomic DNA from zebrafish tissues (2007) BioTechniques, 43, pp. 610-614Nilsson, R., Schultz, I.J., Pierce, E.L., Soltis, K.A., Naranuntarat, A., Ward, D.M., Baughman, J.M., Mootha, V.K., Discovery of genes essential for heme biosynthesis through large-scale gene expression analysis (2009) Cell Metab., 10, pp. 119-130Patterson, L.J., Gering, M., Patient, R., Scl is required for dorsal aorta as well as blood formation in zebrafish embryos (2005) Blood, 105, pp. 3502-3511Patterson, L.J., Gering, M., Eckfeldt, C.E., Green, A.R., Verfaillie, C.M., Ekker, S.C., Patient, R., The transcription factors Scl and Lmo2 act together during development of the hemangioblast in zebrafish (2007) Blood, 109, pp. 2389-2398Paw, B.H., Moskowitz, S.M., Uhrhammer, N., Wright, N., Kaback, M.M., Neufeld, E.F., Juvenile GM2 gangliosidosis caused by substitution of histidine for arginine at position 499 or 504 of the alpha-subunit of beta-hexosaminidase (1990) J. Biol. Chem., 265, pp. 9452-9457Pelster, B., Burggren, W.W., Disruption of hemoglobin oxygen transport does not impact oxygen-dependent physiological processes in developing embryos of zebra fish (Danio rerio) (1996) Circ. Res., 79, pp. 358-362Person, R.E., Li, F.-Q., Duan, Z., Benson, K.F., Wechsler, J., Papadaki, H.A., Eliopoulos, G., Horwitz, M., Mutations in proto-oncogene GFI1 cause human neutropenia and target ELA2 (2003) Nat. Genet., 34, pp. 308-312Postlethwait, J., Woods, I., Ngo-Hazelett, P., Yan, Y., Kelly, P., Chu, F., Huang, H., Talbot, W., Zebrafish comparative genomics and the origins of vertebrate chromosomes (2000) Genome Res., 10, p. 1890Randrianarison-Huetz, V., Laurent, B., Bardet, V., Blobe, G.C., Huetz, F., Duménil, D., Gfi-1B controls human erythroid and megakaryocytic differentiation by regulating TGF-beta signaling at the bipotent erythro-megakaryocytic progenitor stage (2010) Blood, 115, pp. 2784-2795Rhodes, J., Hagen, A., Hsu, K., Deng, M., Liu, T.-X., Look, A.T., Kanki, J.P., Interplay of pu.1 and gata1 determines myelo-erythroid progenitor cell fate in zebrafish (2005) Dev. Cell, 8, pp. 97-108Saleque, S., Cameron, S., Orkin, S.H., The zinc-finger proto-oncogene Gfi-1b is essential for development of the erythroid and megakaryocytic lineages (2002) Genes Dev., 16, pp. 301-306Schmidt, T., Karsunky, H., Gau, E., Zevnik, B., Elsässer, H.P., Möröy, T., Zinc finger protein GFI-1 has low oncogenic potential but cooperates strongly with pim and myc genes in T-cell lymphomagenesis (1998) Oncogene, 17, pp. 2661-2667Schmittgen, T.D., Livak, K.J., Analyzing real-time PCR data by the comparative C(T) method (2008) Nat. Protoc., 3, pp. 1101-1108Shaw, G.C., Cope, J.J., Li, L., Corson, K., Hersey, C., Ackermann, G.E., Gwynn, B., Paw, B.H., Mitoferrin is essential for erythroid iron assimilation (2006) Nature, 440, pp. 96-100Sood, R., English, M.A., Belele, C.L., Jin, H., Bishop, K., Haskins, R., McKinney, M.C., Liu, P.P., Development of multilineage adult hematopoiesis in the zebrafish with a runx1 truncation mutation (2010) Blood, 115, pp. 2806-2809Spooner, C.J., Cheng, J.X., Pujadas, E., Laslo, P., Singh, H., A recurrent network involving the transcription factors PU.1 and Gfi1 orchestrates innate and adaptive immune cell fates (2009) Immunity, 31, pp. 576-586Stainier, D.Y., Weinstein, B.M., Detrich, H.W., Zon, L.I., Fishman, M.C., Cloche, an early acting zebrafish gene, is required by both the endothelial and hematopoietic lineages (1995) Development, 121, pp. 3141-3150Thompson, M.A., Ransom, D.G., Pratt, S.J., MacLennan, H., Kieran, M.W., Detrich, H.W., Vail, B., Zon, L.I., The cloche and spadetail genes differentially affect hematopoiesis and vasculogenesis (1998) Dev. Biol., 197, pp. 248-269van der Meer, L.T., Jansen, J.H., van der Reijden, B.A., Gfi1 and Gfi1b: key regulators of hematopoiesis (2010) Leukemia, 24, pp. 1834-1843Wallis, D., Hamblen, M., Zhou, Y., Venken, K.J.T., Schumacher, A., Grimes, H.L., Zoghbi, H.Y., Bellen, H.J., The zinc finger transcription factor Gfi1, implicated in lymphomagenesis, is required for inner ear hair cell differentiation and survival (2003) Development, 130, pp. 221-232Wei, W., Wen, L., Huang, P., Zhang, Z., Chen, Y., Xiao, A., Huang, H., Lin, S., Gfi1.1 regulates hematopoietic lineage differentiation during zebrafish embryogenesis (2008) Cell Res., 18, pp. 677-685Wilson, N.K., Timms, R.T., Kinston, S.J., Cheng, Y.-H., Oram, S.H., Landry, J.-R., Mullender, J., Gottgens, B., Gfi1 expression is controlled by five distinct regulatory regions spread over 100 kilobases, with Scl/Tal1, Gata2, PU.1, Erg, Meis1, and Runx1 acting as upstream regulators in early hematopoietic cells (2010) Mol. Cell. Biol., 30, pp. 3853-3863Woods, I., Kelly, P., Chu, F., Ngo-Hazelett, P., Yan, Y., Huang, H., Postlethwait, J., Talbot, W., A comparative map of the zebrafish genome (2000) Genome Res., 10, p. 1903Zeng, H., Yücel, R., Kosan, C., Klein-Hitpass, L., Möröy, T., Transcription factor Gfi1 regulates self-renewal and engraftment of hematopoietic stem cells (2004) EMBO J., 23, pp. 4116-412

Bacterial chitobiase structure provides insight into catalytic mechanism and the basis of Tay–Sachs disease

Chitin, the second most abundant polysaccharide on earth, is degraded by chitinases and chitobiases. The structure of Serratia marcescens chitobiase has been refined at 1.9 A resolution. The mature protein is folded into four domains and its active site is situated at the C-terminal end of the central (beta alpha)8-barrel. Based on the structure of the complex with the substrate disaccharide chitobiose, we propose an acid-base reaction mechanism, in which only one protein carboxylate acts as catalytic acid, while the nucleophile is the polar acetamido group of the sugar in a substrate-assisted reaction. The structural data lead to the hypothesis that the reaction proceeds with retention of anomeric configuration. The structure allows us to model the catalytic domain of the homologous hexosaminidases to give a structural rationale to pathogenic mutations that underlie Tay-Sachs and Sandhoff disease

Macrocytic anemia and mitochondriopathy resulting from a defect in sideroflexin 4.

We used exome sequencing to identify mutations in sideroflexin 4 (SFXN4) in two children with mitochondrial disease (the more severe case also presented with macrocytic anemia). SFXN4 is an uncharacterized mitochondrial protein that localizes to the mitochondrial inner membrane. sfxn4 knockdown in zebrafish recapitulated the mitochondrial respiratory defect observed in both individuals and the macrocytic anemia with megaloblastic features of the more severe case. In vitro and in vivo complementation studies with fibroblasts from the affected individuals and zebrafish demonstrated the requirement of SFXN4 for mitochondrial respiratory homeostasis and erythropoiesis. Our findings establish mutations in SFXN4 as a cause of mitochondriopathy and macrocytic anemia

Macrocytic anemia and mitochondriopathy resulting from a defect in sideroflexin 4

We used exome sequencing to identify mutations in sideroflexin 4 (SFXN4) in two children with mitochondrial disease (the more severe case also presented with macrocytic anemia). SFXN4 is an uncharacterized mitochondrial protein that localizes to the mitochondrial inner membrane. sfxn4 knockdown in zebrafish recapitulated the mitochondrial respiratory defect observed in both individuals and the macrocytic anemia with megaloblastic features of the more severe case. In vitro and in vivo complementation studies with fibroblasts from the affected individuals and zebrafish demonstrated the requirement of SFXN4 for mitochondrial respiratory homeostasis and erythropoiesis. Our findings establish mutations in SFXN4 as a cause of mitochondriopathy and macrocytic anemia. \ua9 2013 by The American Society of Human Genetics. All rights reserved

Macrocytic Anemia And Mitochondriopathy Resulting From A Defect In Sideroflexin 4

We used exome sequencing to identify mutations in sideroflexin 4 (SFXN4) in two children with mitochondrial disease (the more severe case also presented with macrocytic anemia). SFXN4 is an uncharacterized mitochondrial protein that localizes to the mitochondrial inner membrane. sfxn4 knockdown in zebrafish recapitulated the mitochondrial respiratory defect observed in both individuals and the macrocytic anemia with megaloblastic features of the more severe case. In vitro and in vivo complementation studies with fibroblasts from the affected individuals and zebrafish demonstrated the requirement of SFXN4 for mitochondrial respiratory homeostasis and erythropoiesis. Our findings establish mutations in SFXN4 as a cause of mitochondriopathy and macrocytic anemia. © 2013 by The American Society of Human Genetics. All rights reserved.935906914AHA; American Heart Association; American Society of Hematology; 6-FY09-289; March of Dimes Foundation; DK085217; NIH; National Institutes of Health; T32 HL007574; NIH; National Institutes of Health; F32 DK098866; NIH; National Institutes of Health; R01 GM61721; NIH; National Institutes of Health; R01 GM097136; NIH; National Institutes of Health; P01 HD032062; NIH; National Institutes of Health; R01 DK070838; NIH; National Institutes of Health; P01 HL032262; NIH; National Institutes of HealthVafai, S.B., Mootha, V.K., Mitochondrial disorders as windows into an ancient organelle (2012) Nature, 491, pp. 374-383Aslinia, F., Mazza, J.J., Yale, S.H., Megaloblastic anemia and other causes of macrocytosis (2006) Clin. Med. Res., 4, pp. 236-241Dimauro, S., Servidei, S., Zeviani, M., Dirocco, M., Devivo, D.C., Didonato, S., Uziel, G., Johnsen, S.D., Cytochrome c oxidase deficiency in Leigh syndrome (1987) Ann. Neurol., 22, pp. 498-506Yu, H.C., Sloan, J.L., Scharer, G., Brebner, A., Quintana, A.M., Achilly, N.P., Manoli, I., Schneck, U., An X-linked cobalamin disorder caused by mutations in transcriptional coregulator HCFC1 (2013) Am. J. Hum. Genet., 93, pp. 506-514Gherasim, C., Lofgren, M., Banerjee, R., Navigating the B(12) road: Assimilation, delivery, and disorders of cobalamin (2013) J. Biol. Chem., 288, pp. 13186-13193Nyhan, W.L., Disorders of purine and pyrimidine metabolism (2005) Mol. Genet. Metab., 86, pp. 25-33Lieber, D.S., Calvo, S.E., Shanahan, K., Slate, N.G., Liu, S., Hershman, S.G., Gold, N.B., Berry, G.T., Targeted exome sequencing of suspected mitochondrial disorders (2013) Neurology, 80, pp. 1762-1770Calvo, S.E., Compton, A.G., Hershman, S.G., Lim, S.C., Lieber, D.S., Tucker, E.J., Laskowski, A., Jaffe, D.B., Molecular diagnosis of infantile mitochondrial disease with targeted next-generation sequencing (2012) Sci. Transl. Med., 4, pp. 18r-10Mayr, J.A., Haack, T.B., Graf, E., Zimmermann, F.A., Wieland, T., Haberberger, B., Superti-Furga, A., Baumgartner, M.R., Lack of the mitochondrial protein acylglycerol kinase causes Sengers syndrome (2012) Am. J. Hum. Genet., 90, pp. 314-320Haack, T.B., Haberberger, B., Frisch, E.M., Wieland, T., Iuso, A., Gorza, M., Strecker, V., Herberg, U., Molecular diagnosis in mitochondrial complex 1 deficiency using exome sequencing (2012) J. Med. Genet., 49, pp. 277-283Isken, O., Maquat, L.E., The multiple lives of NMD factors: Balancing roles in gene and genome regulation (2008) Nat. Rev. Genet., 9, pp. 699-712Paw, B.H., Tieu, P.T., Kaback, M.M., Lim, J., Neufeld, E.F., Frequency of three Hex A mutant alleles among Jewish and non-Jewish carriers identified in a Tay-Sachs screening program (1990) Am. J. Hum. Genet., 47, pp. 698-705Farr, C.J., Saiki, R.K., Erlich, H.A., McCormick, F., Marshall, C.J., Analysis of RAS gene mutations in acute myeloid leukemia by polymerase chain reaction and oligonucleotide probes (1988) Proc. Natl. Acad. Sci. USA, 85, pp. 1629-1633Paw, B.H., Moskowitz, S.M., Uhrhammer, N., Wright, N., Kaback, M.M., Neufeld, E.F., Juvenile GM2 gangliosidosis caused by substitution of histidine for arginine at position 499 or 504 of the alpha-subunit of beta-hexosaminidase (1990) J. Biol. Chem., 265, pp. 9452-9457Pagliarini, D.J., Calvo, S.E., Chang, B., Sheth, S.A., Vafai, S.B., Ong, S.E., Walford, G.A., Chen, W.K., A mitochondrial protein compendium elucidates complex 1 disease biology (2008) Cell, 134, pp. 112-123Chen, W., Paradkar, P.N., Li, L., Pierce, E.L., Langer, N.B., Takahashi- Makise, N., Hyde, B.B., Paw, B.H., Abcb10 physically interacts with mitoferrin-1 (Slc25a37) to enhance its stability and function in the erythroid mitochondria (2009) Proc. Natl. Acad. Sci. USA, 106, pp. 16263-16268Fay, F.S., Taneja, K.L., Shenoy, S., Lifshitz, L., Singer, R.H., Quantitative digital analysis of diffuse and concentrated nuclear distributions of nascent transcripts, SC35 and poly(A) (1997) Exp. Cell Res., 231, pp. 27-37Chen, H.W., Rainey, R.N., Balatoni, C.E., Dawson, D.W., Troke, J.J., Wasiak, S., Hong, J.S., French, S.W., Mammalian polynucleotide phosphorylase is an intermembrane space Nase that maintains mitochondrial homeostasis (2006) Mol. Cell. Biol., 26, pp. 8475-8487Lieschke, G.J., Currie, P.D., Animal models of human disease: Zebrafish swim into view (2007) Nat. Rev. Genet., 8, pp. 353-367Cooney, J.D., Hildick-Smith, G.J., Shafizadeh, E., McBride, P.F., Carroll, K.J., Anderson, H., Shaw, G.C., Dalton, A.J., Teleost growth factor independence (gfi) genes differentially regulate successive waves of hematopoiesis (2013) Dev. Biol., 373, pp. 431-441Amigo, J.D., Yu, M., Troadec, M.B., Gwynn, B., Cooney, J.D., Lambert, A.J., Chi, N.C., Kaplan, J., Identification of distal cis- regulatory elements at mouse mitoferrin loci using zebrafish transgenesis (2011) Mol. Cell. Biol., 31, pp. 1344-1356Thon, J.N., Macleod, H., Begonja, A.J., Zhu, J., Lee, K.C., Mogilner, A., Hartwig, J.H., Italiano, Jr.J.E., Microtubule and cortical forces determine platelet size during vascular platelet production (2012) Nat Commun, 3, p. 852Pase, L., Layton, J.E., Kloosterman, W.P., Carradice, D., Waterhouse, P.M., Lieschke, G.J., MiR-451 regulates zebrafish erythroid maturation in vivo via its target gata2 (2009) Blood, 113, pp. 1794-1804Zhao, Y., Qin, W., Zhang, J.P., Hu, Z.Y., Tong, J.W., Ding, C.B., Peng, Z.G., Jiang, J.D., HCV IRES-mediated core expression in zebrafish (2013) PLoS ONE, 8, pp. e56985Ma, Y., Wu, M., Li, D., Li, X.Q., Li, P., Zhao, J., Luo, M.N., Ma, X., Embryonic developmental toxicity of selenite in zebrafish (Danio rerio) and prevention with folic acid (2012) Food Chem. Toxicol., 50, pp. 2854-2863Amigo, J.D., Ackermann, G.E., Cope, J.J., Yu, M., Cooney, J.D., Ma, D., Langer, N.B., Horsely, W., The role and regulation of friend of GATA-1 (FOG-1) during blood development in the zebrafish (2009) Blood, 114, pp. 4654-4663Bergmann, A.K., Campagna, D.R., McLoughlin, E.M., Agarwal, S., Fleming, M.D., Bottomley, S.S., Neufeld, E.J., Systematic molecular genetic analysis of congenital sideroblastic anemia: Evidence for genetic heterogeneity and identification of novel mutations (2010) Pediatr. Blood Cancer, 54, pp. 273-278Ganis, J.J., Hsia, N., Trompouki, E., De Jong, J.L., Dibiase, A., Lambert, J.S., Jia, Z., Sandstrom, R., Zebrafish globin switching occurs in two developmental stages and is controlled by the LCR (2012) Dev. Biol., 366, pp. 185-194Yu, D., Dos Santos, C.O., Zhao, G., Jiang, J., Amigo, J.D., Khandros, E., Dore, L.C., Zhang, Z., MiR-451 protects against erythroid oxidant stress by repressing 14-3-3zeta (2010) Genes Dev, 24, pp. 1620-1633Blanc, L., Ciciotte, S.L., Gwynn, B., Hildick-Smith, G.J., Pierce, E.L., Soltis, K.A., Cooney, J.D., Peters, L.L., Critical function for the Ras-GTPase activating protein RASA3 in vertebrate erythropoiesis and megakaryopoiesis (2012) Proc. Natl. Acad. Sci. USA, 109, pp. 12099-12104Shah, D.I., Takahashi-Makise, N., Cooney, J.D., Li, L., Schultz, I.J., Pierce, E.L., Narla, A., Medlock, A.E., Mitochondrial Atpif1 regulates haem synthesis in developing erythroblasts (2012) Nature, 491, pp. 608-612Kornblum, C., Nicholls, T.J., Haack, T.B., Schöler, S., Peeva, V., Danhauser, K., Hallmann, K., Iuso, A., Loss-of-function mutations in MGME1 impair mtDNA replication and cause multisystemic mitochondrial disease (2013) Nat. Genet., 45, pp. 214-219Finsterer, J., Hematological manifestations of primary mitochondrial disorders (2007) Acta Haematol, 118, pp. 88-98Riley, L.G., Cooper, S., Hickey, P., Rudinger-Thirion, J., McKenzie, M., Compton, A., Lim, S.C., Giegé, R., Mutation of the mitochondrial tyrosyl- tRNA synthetase gene, YARS2, causes myopathy, lactic acidosis, and sideroblastic anemia - MLASA syndrome (2010) Am. J. Hum. Genet., 87, pp. 52-59Fellman, V., The GRACILE syndrome, a neonatal lethal metabolic disorder with iron overload (2002) Blood Cells Mol. Dis., 29, pp. 444-450Fleming, M.D., Campagna, D.R., Haslett, J.N., Trenor III, C.C., Andrews, N.C., A mutation in a mitochondrial transmembrane protein is responsible for the pleiotropic hematological and skeletal phenotype of flexed-tail (f/f) mice (2001) Genes Dev, 15, pp. 652-657Hegde, S., Lenox, L.E., Lariviere, A., Porayette, P., Perry, J.M., Yon, M., Paulson, R.F., An intronic sequence mutated in flexed-tail mice regulates splicing of Smad5 (2007) Mamm. Genome, 18, pp. 852-860Miyake, S., Yamashita, T., Taniguchi, M., Tamatani, M., Sato, K., Tohyama, M., Identification and characterization of a novel mitochondrial tricarboxylate carrier (2002) Biochem. Biophys. Res. Commun., 295, pp. 463-468Soranzo, N., Spector, T.D., Mangino, M., Kuhnel, B., Rendon, A., Teumer, A., Willenborg, C., Li, M., A genome-wide meta-analysis identifies 22 loci associated with eight hematological parameters in the HaemGen consortium (2009) Nat. Genet., 41, pp. 1182-1190Van Der Harst, P., Zhang, W., Mateo Leach, I., Rendon, A., Verweij, N., Sehmi, J., Paul, D.S., Li, X., Seventy-five genetic loci influencing the human red blood cell (2012) Nature, 492, pp. 369-375Ganesh, S.K., Zakai, N.A., Van Rooij, F.J., Soranzo, N., Smith, A.V., Nalls, M.A., Chen, M.H., Dehghan, A., Multiple loci influence erythrocyte phenotypes in the CHARGE Consortium (2009) Nat. Genet., 41, pp. 1191-1198Foo, J.N., Liu, J.J., Tan, E.K., Whole-genome and whole-exome sequencing in neurological diseases (2012) Nat Rev Neurol, 8, pp. 508-517Bras, J., Guerreiro, R., Hardy, J., Use of nextgeneration sequencing and other whole-genome strategies to dissect neurological disease (2012) Nat. Rev. Neurosci., 13, pp. 453-46

TMEM14C is required for erythroid mitochondrial heme metabolism

The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liverderived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias