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Open Access 01-12-2018 | Research

SF3B1 deficiency impairs human erythropoiesis via activation of p53 pathway: implications for understanding of ineffective erythropoiesis in MDS

Authors: Yumin Huang, John Hale, Yaomei Wang, Wei Li, Shijie Zhang, Jieying Zhang, Huizhi Zhao, Xinhua Guo, Jing Liu, Hongxia Yan, Karina Yazdanbakhsh, Gang Huang, Christopher D. Hillyer, Narla Mohandas, Lixiang Chen, Ling Sun, Xiuli An

Published in: Journal of Hematology & Oncology | Issue 1/2018

Abstract

Background

SF3B1 is a core component of splicing machinery. Mutations in SF3B1 are frequently found in myelodysplastic syndromes (MDS), particularly in patients with refractory anemia with ringed sideroblasts (RARS), characterized by isolated anemia. SF3B1 mutations have been implicated in the pathophysiology of RARS; however, the physiological function of SF3B1 in erythropoiesis remains unknown.

Methods

shRNA-mediated approach was used to knockdown SF3B1 in human CD34⁺ cells. The effects of SF3B1 knockdown on human erythroid cell differentiation, cell cycle, and apoptosis were assessed by flow cytometry. RNA-seq, qRT-PCR, and western blot analyses were used to define the mechanisms of phenotypes following knockdown of SF3B1.

Results

We document that SF3B1 knockdown in human CD34⁺ cells leads to increased apoptosis and cell cycle arrest of early-stage erythroid cells and generation of abnormally nucleated late-stage erythroblasts. RNA-seq analysis of SF3B1-knockdown erythroid progenitor CFU-E cells revealed altered splicing of an E3 ligase Makorin Ring Finger Protein 1 (MKRN1) and subsequent activation of p53 pathway. Importantly, ectopic expression of MKRN1 rescued SF3B1-knockdown-induced alterations. Decreased expression of genes involved in mitosis/cytokinesis pathway including polo-like kinase 1 (PLK1) was noted in SF3B1-knockdown polychromatic and orthochromatic erythroblasts comparing to control cells. Pharmacologic inhibition of PLK1 also led to generation of abnormally nucleated erythroblasts.

Conclusions

These findings enabled us to identify novel roles for SF3B1 in human erythropoiesis and provided new insights into its role in regulating normal erythropoiesis. Furthermore, these findings have implications for improved understanding of ineffective erythropoiesis in MDS patients with SF3B1 mutations.

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Ginzburg Y, Rivella S. β-thalassemia: a model for elucidating the dynamic regulation of ineffective erythropoiesis and iron metabolism. Blood. 2011;118:4321–30.CrossRefPubMedPubMedCentral

Wickramasinghe SN, Wood WG. Advances in the understanding of the congenital dyserythropoietic anaemias. Br J Haematol. 2005;131:431–46.CrossRefPubMed

Lipton JM, Ellis SR. Diamond-Blackfan anemia: diagnosis, treatment, and molecular pathogenesis. Hematol Oncol Clin North Am. 2009;23:261–82.CrossRefPubMedPubMedCentral

Haldar K, Mohandas N. Malaria, erythrocytic infection, and anemia. Hematol Am Soc Hematol Educ Progr. 2009;2009:87–93.

Ebert BL, Galili N, Tamayo P, Bosco J, Mak R, Pretz J, et al. An erythroid differentiation signature predicts response to lenalidomide in myelodysplastic syndrome. Appelbaum FR, editor. PLoS Med. 2008;5:e35.

Lin FK, Suggs S, Lin CH, Browne JK, Smalling R, Egrie JC, et al. Cloning and expression of the human erythropoietin gene. Proc Natl Acad Sci U S A. 1985;82:7580–4.CrossRefPubMedPubMedCentral

D’Andrea AD, Lodish HF, Wong GG. Expression cloning of the murine erythropoietin receptor. Cell. 1989;57:277–85.CrossRefPubMed

Wu H, Liu X, Jaenisch R, Lodish HF. Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor. Cell. 1995;83:59–67.CrossRefPubMed

Lin CS, Lim SK, D’Agati V, Costantini F. Differential effects of an erythropoietin receptor gene disruption on primitive and definitive erythropoiesis. Genes Dev. 1996;10:154–64.CrossRefPubMed

10.

Cantor AB, Orkin SH. Transcriptional regulation of erythropoiesis: an affair involving multiple partners. Oncogene. 2002;21:3368–76.CrossRefPubMed

11.

Siatecka M, Bieker JJ. The multifunctional role of EKLF/KLF1 during erythropoiesis. Blood. 2011;118:2044–54.CrossRefPubMedPubMedCentral

12.

Pevny L, Lin CS, D’Agati V, Simon MC, Orkin SH, Costantini F. Development of hematopoietic cells lacking transcription factor GATA-1. Development. 1995;121:163–72.PubMed

13.

Dore LC, Amigo JD, Dos Santos CO, Zhang Z, Gai X, Tobias JW, et al. A GATA-1-regulated microRNA locus essential for erythropoiesis. Proc Natl Acad Sci U S A. 2008;105:3333–8.CrossRefPubMedPubMedCentral

14.

Zhu Y, Wang D, Wang F, Li T, Dong L, Liu H, et al. A comprehensive analysis of GATA-1-regulated miRNAs reveals miR-23a to be a positive modulator of erythropoiesis. Nucleic Acids Res. 2013;41:4129–43.CrossRefPubMedPubMedCentral

15.

Zhao G, Yu D, Weiss MJ. MicroRNAs in erythropoiesis. Curr Opin Hematol. 2010;17:155–62.PubMed

16.

Ji P, Yeh V, Ramirez T, Murata-Hori M, Lodish HF. Histone deacetylase 2 is required for chromatin condensation and subsequent enucleation of cultured mouse fetal erythroblasts. Haematologica. 2010;95:2013–21.CrossRefPubMedPubMedCentral

17.

Yan H, Wang Y, Qu X, Li J, Hale J, Huang Y, et al. Distinct roles for TET family proteins in regulating human erythropoiesis. Blood. 2017;129:2002–12.CrossRefPubMed

18.

Gascard P, Lee G, Coulombel L, Auffray I, Lum M, Parra M, et al. Characterization of multiple isoforms of protein 4.1R expressed during erythroid terminal differentiation. Blood. 1998;92:4404–14.PubMed

19.

Horne WC, Huang SC, Becker PS, Tang TK, Benz EJ. Tissue-specific alternative splicing of protein 4.1 inserts an exon necessary for formation of the ternary complex with erythrocyte spectrin and F-actin. Blood. 1993;82:2558–63.PubMed

20.

Conboy JG, Shitamoto R, Parra M, Winardi R, Kabra A, Smith J, et al. Hereditary elliptocytosis due to both qualitative and quantitative defects in membrane skeletal protein 4.1. Blood. 1991;78:2438–43.PubMed

21.

Yamamoto ML, Clark TA, Gee SL, Kang J-A, Schweitzer AC, Wickrema A, et al. Alternative pre-mRNA splicing switches modulate gene expression in late erythropoiesis. Blood. 2009;113:3363–70.CrossRefPubMedPubMedCentral

22.

Pimentel H, Parra M, Gee S, Ghanem D, An X, Li J, et al. A dynamic alternative splicing program regulates gene expression during terminal erythropoiesis. Nucleic Acids Res. 2014;42:4031–42.CrossRefPubMedPubMedCentral

23.

Cheng AW, Shi J, Wong P, Luo KL, Trepman P, Wang ET, et al. Muscleblind-like 1 (Mbnl1) regulates pre-mRNA alternative splicing during terminal erythropoiesis. Blood. 2014;124:598–610.CrossRefPubMedPubMedCentral

24.

Wahl MC, Will CL, Lührmann R. The spliceosome: design principles of a dynamic RNP machine. Cell. 2009;136:701–18.CrossRefPubMed

25.

Yoshida K, Sanada M, Shiraishi Y, Nowak D, Nagata Y, Yamamoto R, et al. Frequent pathway mutations of splicing machinery in myelodysplasia. Nature. 2011;478:64–9.CrossRefPubMed

26.

Haferlach T, Nagata Y, Grossmann V, Okuno Y, Bacher U, Nagae G, et al. Landscape of genetic lesions in 944 patients with myelodysplastic syndromes. Leukemia. 2014;28:241–7.CrossRefPubMed

27.

Malcovati L, Karimi M, Papaemmanuil E, Ambaglio I, Jädersten M, Jansson M, et al. SF3B1 mutation identifies a distinct subset of myelodysplastic syndrome with ring sideroblasts. Blood. 2015;126:233–41.CrossRefPubMedPubMedCentral

28.

Papaemmanuil E, Cazzola M, Boultwood J, Malcovati L, Vyas P, Bowen D, et al. Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts. N Engl J Med. 2011;365:1384–95.CrossRefPubMedPubMedCentral

29.

Hu J, Liu J, Xue F, Halverson G, Reid M, Guo A, et al. Isolation and functional characterization of human erythroblasts at distinct stages: implications for understanding of normal and disordered erythropoiesis in vivo. Blood. 2013;121:3246–53.CrossRefPubMedPubMedCentral

30.

Li J, Hale J, Bhagia P, Xue F, Chen L, Jaffray J, et al. Isolation and transcriptome analyses of human erythroid progenitors: BFU-E and CFU-E. Blood American Society of Hematology. 2014;124:3636–45.

31.

Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 2013;14:R36.CrossRefPubMedPubMedCentral

32.

Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 2012;7:562–78.CrossRefPubMedPubMedCentral

33.

Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102:15545–50.CrossRefPubMedPubMedCentral

34.

Mootha VK, Lindgren CM, Eriksson K-F, Subramanian A, Sihag S, Lehar J, et al. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet. 2003;34:267–73.CrossRefPubMed

35.

Vitting-Seerup K, Porse BT, Sandelin A, Waage J. spliceR: an R package for classification of alternative splicing and prediction of coding potential from RNA-seq data. BMC Bioinformatics. 2014;15:81.CrossRefPubMedPubMedCentral

36.

Gautier E-F, Ducamp S, Leduc M, Salnot V, Guillonneau F, Dussiot M, et al. Comprehensive proteomic analysis of human erythropoiesis. Cell Rep. 2016;16:1470–84.CrossRefPubMedPubMedCentral

37.

Enge M, Bao W, Hedström E, Jackson SP, Moumen A, Selivanova G. MDM2-dependent downregulation of p21 and hnRNP K provides a switch between apoptosis and growth arrest induced by pharmacologically activated p53. Cancer Cell. 2009;15:171–83.CrossRefPubMed

38.

Vousden KH, Prives C. Blinded by the light: the growing complexity of p53. Cell. 2009;137:413–31.CrossRefPubMed

39.

Follis AV, Llambi F, Merritt P, Chipuk JE, Green DR, Kriwacki RW. Pin1-induced proline isomerization in cytosolic p53 mediates BAX activation and apoptosis. Mol Cell. 2015;59:677–84.CrossRefPubMedPubMedCentral

40.

Chipuk JE, Kuwana T, Bouchier-Hayes L, Droin NM, Newmeyer DD, Schuler M, et al. Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis. Science (80- ). 2004;303:1010–4.CrossRef

41.

Spender LC, Carter MJ, O’Brien DI, Clark LJ, Yu J, Michalak EM, et al. Transforming growth factor-β directly induces p53-up-regulated modulator of apoptosis (PUMA) during the rapid induction of apoptosis in myc-driven B-cell lymphomas. J Biol Chem. 2013;288:5198–209.CrossRefPubMed

42.

Darman RB, Seiler M, Agrawal AA, Lim KH, Peng S, Aird D, et al. Cancer-associated SF3B1 hotspot mutations induce cryptic 3’ splice site selection through use of a different branch point. Cell Rep. 2015;13:1033–45.CrossRefPubMed

43.

Obeng EA, Chappell RJ, Seiler M, Chen MC, Campagna DR, Schmidt PJ, et al. Physiologic expression of Sf3b1(K700E) causes impaired erythropoiesis, aberrant splicing, and sensitivity to therapeutic spliceosome modulation. Cancer Cell. 2016;30:404–17.CrossRefPubMedPubMedCentral

44.

Lee E-W, Lee M-S, Camus S, Ghim J, Yang M-R, Oh W, et al. Differential regulation of p53 and p21 by MKRN1 E3 ligase controls cell cycle arrest and apoptosis. EMBO J. 2009;28:2100–13.CrossRefPubMedPubMedCentral

45.

Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, et al. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science (80-. ). 2004;303:844–8.CrossRef

46.

Haupt Y, Maya R, Kazaz A, Oren M. Mdm2 promotes the rapid degradation of p53. Nature. 1997;387:296–9.CrossRefPubMed

47.

Chen K, Liu J, Heck S, Chasis JA, An X, Mohandas N. Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression during erythropoiesis. Proc Natl Acad Sci U S A. 2009;106:17413–8.CrossRefPubMedPubMedCentral

48.

Dussiot M, Maciel TT, Fricot A, Chartier C, Negre O, Veiga J, et al. An activin receptor IIA ligand trap corrects ineffective erythropoiesis in β-thalassemia. Nat Med. 2014;20:398–407.CrossRefPubMed

49.

Suragani RNVS, Cadena SM, Cawley SM, Sako D, Mitchell D, Li R, et al. Transforming growth factor-β superfamily ligand trap ACE-536 corrects anemia by promoting late-stage erythropoiesis. Nat Med. 2014;20:408–14.CrossRefPubMed

50.

Visconte V, Makishima H, Maciejewski JP, Tiu RV. Emerging roles of the spliceosomal machinery in myelodysplastic syndromes and other hematological disorders. Leukemia. 2012;26:2447–54.CrossRefPubMedPubMedCentral

51.

Hahn CN, Scott HS. Spliceosome mutations in hematopoietic malignancies. Nat Genet. 2011;44:9–10.CrossRefPubMed

52.

Dolatshad H, Pellagatti A, Fernandez-Mercado M, Yip BH, Malcovati L, Attwood M, et al. Disruption of SF3B1 results in deregulated expression and splicing of key genes and pathways in myelodysplastic syndrome hematopoietic stem and progenitor cells. Leukemia. 2015;29:1798.CrossRefPubMedPubMedCentral

53.

Alsafadi S, Houy A, Battistella A, Popova T, Wassef M, Henry E, et al. Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage. Nat Commun. 2016;7:10615.CrossRefPubMedPubMedCentral

54.

Wang C, Sashida G, Saraya A, Ishiga R, Koide S, Oshima M, et al. Depletion of Sf3b1 impairs proliferative capacity of hematopoietic stem cells but is not sufficient to induce myelodysplasia. Blood. 2014;123:3336–43.CrossRefPubMed

55.

Matsunawa M, Yamamoto R, Sanada M, Sato-Otsubo A, Shiozawa Y, Yoshida K, et al. Haploinsufficiency of Sf3b1 leads to compromised stem cell function but not to myelodysplasia. Leukemia. 2014;28:1844–50.CrossRefPubMed

56.

Matsson H, Davey EJ, Draptchinskaia N, Hamaguchi I, Ooka A, Levéen P, et al. Targeted disruption of the ribosomal protein S19 gene is lethal prior to implantation. Mol Cell Biol. 2004;24:4032–7.CrossRefPubMedPubMedCentral

57.

Khoriaty R, Vasievich MP, Jones M, Everett L, Chase J, Tao J, et al. Absence of a red blood cell phenotype in mice with hematopoietic deficiency of SEC23B. Mol Cell Biol. 2014;34:3721–34.CrossRefPubMedPubMedCentral

58.

Cazzola M, Invernizzi R, Bergamaschi G, Levi S, Corsi B, Travaglino E, et al. Mitochondrial ferritin expression in erythroid cells from patients with sideroblastic anemia. Blood. 2003;101:1996–2000.CrossRefPubMed

59.

Levi S, Corsi B, Bosisio M, Invernizzi R, Volz A, Sanford D, et al. A human mitochondrial ferritin encoded by an intronless gene. J Biol Chem. 2001;276:24437–40.CrossRefPubMed

60.

Visconte V, Tabarroki A, Zhang L, Parker Y, Hasrouni E, Mahfouz R, et al. Splicing factor 3b subunit 1 (Sf3b1) haploinsufficient mice display features of low risk Myelodysplastic syndromes with ring sideroblasts. J Hematol Oncol. 2014;7:89.CrossRefPubMedPubMedCentral

61.

Danilova N, Sakamoto KM, Lin S. Ribosomal protein S19 deficiency in zebrafish leads to developmental abnormalities and defective erythropoiesis through activation of p53 protein family. Blood. 2008;112:5228–37.CrossRefPubMed

62.

Jaako P, Flygare J, Olsson K, Quere R, Ehinger M, Henson A, et al. Mice with ribosomal protein S19 deficiency develop bone marrow failure and symptoms like patients with Diamond-Blackfan anemia. Blood. 2011;118:6087–96.CrossRefPubMed

63.

Dutt S, Narla A, Lin K, Mullally A, Abayasekara N, Megerdichian C, et al. Haploinsufficiency for ribosomal protein genes causes selective activation of p53 in human erythroid progenitor cells. Blood. 2011;117:2567–76.CrossRefPubMedPubMedCentral

64.

Furney SJ, Pedersen M, Gentien D, Dumont AG, Rapinat A, Desjardins L, et al. SF3B1 mutations are associated with alternative splicing in uveal melanoma. Cancer Discov. 2013;3:1122–9.CrossRefPubMedPubMedCentral

Title: SF3B1 deficiency impairs human erythropoiesis via activation of p53 pathway: implications for understanding of ineffective erythropoiesis in MDS
Authors: Yumin Huang
John Hale
Yaomei Wang
Wei Li
Shijie Zhang
Jieying Zhang
Huizhi Zhao
Xinhua Guo
Jing Liu
Hongxia Yan
Karina Yazdanbakhsh
Gang Huang
Christopher D. Hillyer
Narla Mohandas
Lixiang Chen
Ling Sun
Xiuli An
Publication date: 01-12-2018
Publisher: BioMed Central
Published in: Journal of Hematology & Oncology / Issue 1/2018
Electronic ISSN: 1756-8722
DOI: https://doi.org/10.1186/s13045-018-0558-8

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