A human-specific VNTR in the TRIB3 promoter causes gene expression variation between individuals


Autoři: Tiit Örd aff001;  Tarmo Puurand aff002;  Daima Örd aff001;  Tarmo Annilo aff003;  Märt Möls aff002;  Maido Remm aff002;  Tõnis Örd aff001
Působiště autorů: Estonian Biocentre, Institute of Genomics, University of Tartu, Tartu, Estonia aff001;  Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia aff002;  Estonian Genome Center, Institute of Genomics, University of Tartu, Tartu, Estonia aff003;  Institute of Mathematics and Statistics, University of Tartu, Tartu, Estonia aff004
Vyšlo v časopise: A human-specific VNTR in the TRIB3 promoter causes gene expression variation between individuals. PLoS Genet 16(8): e1008981. doi:10.1371/journal.pgen.1008981
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008981

Souhrn

Tribbles homolog 3 (TRIB3) is pseudokinase involved in intracellular regulatory processes and has been implicated in several diseases. In this article, we report that human TRIB3 promoter contains a 33-bp variable number tandem repeat (VNTR) and characterize the heterogeneity and function of this genetic element. Analysis of human populations around the world uncovered the existence of alleles ranging from 1 to 5 copies of the repeat, with 2-, 3- and 5-copy alleles being the most common but displaying considerable geographical differences in frequency. The repeated sequence overlaps a C/EBP-ATF transcriptional regulatory element and is highly conserved, but not repeated, in various mammalian species, including great apes. The repeat is however evident in Neanderthal and Denisovan genomes. Reporter plasmid experiments in human cell culture reveal that an increased copy number of the TRIB3 promoter 33-bp repeat results in increased transcriptional activity. In line with this, analysis of whole genome sequencing and RNA-Seq data from human cohorts demonstrates that the copy number of TRIB3 promoter 33-bp repeats is positively correlated with TRIB3 mRNA expression level in many tissues throughout the body. Moreover, the copy number of the TRIB3 33-bp repeat appears to be linked to known TRIB3 eQTL SNPs as well as TRIB3 SNPs reported in genetic association studies. Taken together, the results indicate that the promoter 33-bp VNTR constitutes a causal variant for TRIB3 expression variation between individuals and could underlie the results of SNP-based genetic studies.

Klíčová slova:

Gene expression – Genome sequencing – Human genomics – Luciferase – Mammalian genomics – Plasmid construction – Polymerase chain reaction – Single nucleotide polymorphisms


Zdroje

1. Örd T, Örd T. Mammalian Pseudokinase TRIB3 in Normal Physiology and Disease: Charting the Progress in Old and New Avenues. Curr Protein Pept Sci. 2017;18(8):819–42. doi: 10.2174/1389203718666170406124547 MEDLINE:28393700. 28393700

2. Jousse C, Deval C, Maurin A-C, Parry L, Cherasse Y, Chaveroux C, et al. TRB3 inhibits the transcriptional activation of stress-regulated genes by a negative feedback on the ATF4 pathway. J Biol Chem. 2007;282(21):15851–61. doi: 10.1074/jbc.M611723200 MEDLINE:17369260. 17369260

3. Örd D, Meerits K, Örd T. TRB3 protects cells against the growth inhibitory and cytotoxic effect of ATF4. Exp Cell Res. 2007;313(16):3556–67. doi: 10.1016/j.yexcr.2007.07.017 MEDLINE:17707795. 17707795

4. Örd T, Örd D, Adler P, Vilo J, Örd T. TRIB3 enhances cell viability during glucose deprivation in HEK293-derived cells by upregulating IGFBP2, a novel nutrient deficiency survival factor. Biochim Biophys Acta Mol Cell Res. 2015;1853(10 Pt A):2492–505. doi: 10.1016/j.bbamcr.2015.06.006 MEDLINE:26094770. 26094770

5. Örd D, Örd T, Biene T, Örd T. TRIB3 increases cell resistance to arsenite toxicity by limiting the expression of the glutathione-degrading enzyme CHAC1. Biochim Biophys Acta Mol Cell Res. 2016;1863(11):2668–80. doi: 10.1016/j.bbamcr.2016.08.003 MEDLINE:27526673. 27526673

6. Butcher L, Ahluwalia M, Örd T, Johnston J, Morris RH, Kiss-Toth E, et al. Evidence for a role of TRIB3 in the regulation of megakaryocytopoiesis. Sci Rep. 2017;7(1):6684. doi: 10.1038/s41598-017-07096-w MEDLINE:28751721. 28751721

7. Örd T, Örd D, Örd T. TRIB3 limits FGF21 induction during in vitro and in vivo nutrient deficiencies by inhibiting C/EBP-ATF response elements in the Fgf21 promoter. Biochim Biophys Acta Gene Regul Mech. 2018;1861(3):271–81. doi: 10.1016/j.bbagrm.2018.01.014 MEDLINE:29378327. 29378327

8. Erazo T, Lorente M, Lopez-Plana A, Munoz-Guardiola P, Fernandez-Nogueira P, Garcia-Martinez JA, et al. The New Antitumor Drug ABTL0812 Inhibits the Akt/mTORC1 Axis by Upregulating Tribbles-3 Pseudokinase. Clin Cancer Res. 2016;22(10):2508–19. doi: 10.1158/1078-0432.CCR-15-1808 WOS:000375839200023. 26671995

9. Felip I, Moiola CP, Megino-Luque C, Lopez-Gil C, Cabrera S, Sole-Sanchez S, et al. Therapeutic potential of the new TRIB3-mediated cell autophagy anticancer drug ABTL0812 in endometrial cancer. Gynecol Oncol. 2019;153(2):425–35. doi: 10.1016/j.ygyno.2019.03.002 MEDLINE:30853360. 30853360

10. López-Plana A, Fernández-Nogueira P, Muñoz-Guardiola P, Solé-Sánchez S, Megías-Roda E, Pérez-Montoyo H, et al. The novel pro-autophagy anticancer drug ABTL0812 potentiates chemotherapy in adenocarcinoma and squamous Non-Small Cell Lung Cancer. Int J Cancer. 2020. doi: 10.1002/ijc.32865 31943158

11. Salazar M, Carracedo A, Salanueva IJ, Hernandez-Tiedra S, Lorente M, Egia A, et al. Cannabinoid action induces autophagy-mediated cell death through stimulation of ER stress in human glioma cells. J Clin Invest. 2009;119(5):1359–72. doi: 10.1172/jci37948 WOS:000265843400034. 19425170

12. Salazar M, Lorente M, Garcia-Taboada E, Hernandez-Tiedra S, Davila D, Francis SE, et al. The pseudokinase tribbles homologue-3 plays a crucial role in cannabinoid anticancer action. Biochim Biophys Acta Mol Cell Biol Lipids. 2013;1831(10):1573–8. doi: 10.1016/j.bbalip.2013.03.014 MEDLINE:23567453. 23567453

13. Salazar M, Lorente M, Garcia-Taboada E, Perez Gomez E, Davila D, Zuniga-Garcia P, et al. Loss of Tribbles pseudokinase-3 promotes Akt-driven tumorigenesis via FOXO inactivation. Cell Death Differ. 2015;22(1):131–44. doi: 10.1038/cdd.2014.133 MEDLINE:25168244. 25168244

14. Borsting E, Patel SV, Decleves AE, Lee SJ, Rahman QM, Akira S, et al. Tribbles Homolog 3 Attenuates Mammalian Target of Rapamycin Complex-2 Signaling and Inflammation in the Diabetic Kidney. J Am Soc Nephrol. 2014;25(9):2067–78. doi: 10.1681/ASN.2013070811 WOS:000341059200021. 24676635

15. Du KY, Herzig S, Kulkarni RN, Montminy M. TRB3: A tribbles homolog that inhibits Akt/PKB activation by insulin in liver. Science. 2003;300(5625):1574–7. doi: 10.1126/science.1079817 WOS:000183333100056. 12791994

16. Liew CW, Bochenski J, Kawamori D, Hu J, Leech CA, Wanic K, et al. The pseudokinase tribbles homolog 3 interacts with ATF4 to negatively regulate insulin exocytosis in human and mouse beta cells. J Clin Invest. 2010;120(8):2876–88. doi: 10.1172/JCI36849 WOS:000280492100023. 20592469

17. Prudente S, Hribal ML, Flex E, Turchi F, Morini E, De Cosmo S, et al. The functional Q84R polymorphism of mammalian Tribbles homolog TRB3 is associated with insulin resistance and related cardiovascular risk in Caucasians from Italy. Diabetes. 2005;54(9):2807–11. doi: 10.2337/diabetes.54.9.2807 MEDLINE:16123373. 16123373

18. Prudente S, Baratta R, Andreozzi F, Morini E, Farina MG, Nigro A, et al. TRIB3 R84 variant affects glucose homeostasis by altering the interplay between insulin sensitivity and secretion. Diabetologia. 2010;53(7):1354–61. doi: 10.1007/s00125-010-1749-1 MEDLINE:20393693. 20393693

19. Aimé P, Sun X, Zareen N, Rao A, Berman Z, Volpicelli-Daley L, et al. Trib3 Is Elevated in Parkinson's Disease and Mediates Death in Parkinson's Disease Models. J Neurosci. 2015;35(30):10731–49. doi: 10.1523/JNEUROSCI.0614-15.2015 MEDLINE:26224857. 26224857

20. Aimé P, Karuppagounder SS, Rao A, Chen Y, Burke RE, Ratan RR, et al. The drug adaptaquin blocks ATF4/CHOP-dependent pro-death Trib3 induction and protects in cellular and mouse models of Parkinson's disease. Neurobiol Dis. 2020;136. doi: 10.1016/j.nbd.2019.104725 31911115

21. Hua F, Li K, Yu JJ, Lv XX, Yan J, Zhang XW, et al. TRB3 links insulin/IGF to tumour promotion by interacting with p62 and impeding autophagic/proteasomal degradations. Nat Commun. 2015;6. doi: 10.1038/ncomms8951 WOS:000360346700006. 26268733

22. Izrailit J, Berman HK, Datti A, Wrana JL, Reedijk M. High throughput kinase inhibitor screens reveal TRB3 and MAPK-ERK/TGFbeta pathways as fundamental Notch regulators in breast cancer. Proc Natl Acad Sci U S A. 2013;110(5):1714–9. doi: 10.1073/pnas.1214014110 MEDLINE:23319603. 23319603

23. Li K, Wang F, Cao W-B, Lv X-X, Hua F, Cui B, et al. TRIB3 Promotes APL Progression through Stabilization of the Oncoprotein PML-RARalpha and Inhibition of p53-Mediated Senescence. Cancer cell. 2017;31(5):697–710.e7. doi: 10.1016/j.ccell.2017.04.006 MEDLINE:28486108. 28486108

24. Lorenzi M, Altmann A, Gutman B, Wray S, Arber C, Hibar DP, et al. Susceptibility of brain atrophy to TRIB3 in Alzheimer's disease, evidence from functional prioritization in imaging genetics. Proc Natl Acad Sci U S A. 2018;115(12):3162–7. doi: 10.1073/pnas.1706100115 MEDLINE:29511103. 29511103

25. Yamada Y, Kato K, Oguri M, Horibe H, Fujimaki T, Yasukochi Y, et al. Identification of nine genes as novel susceptibility loci for early-onset ischemic stroke, intracerebral hemorrhage, or subarachnoid hemorrhage. Biomed Rep. 2018;9(1):8–20. doi: 10.3892/br.2018.1104 WOS:000440677300002. 29930801

26. Sulovari A, Li RY, Audano PA, Porubsky D, Vollger MR, Logsdon GA, et al. Human-specific tandem repeat expansion and differential gene expression during primate evolution. Proc Natl Acad Sci U S A. 2019;116(46):23243–53. doi: 10.1073/pnas.1912175116 WOS:000496506600056. 31659027

27. Bellizzi D, Rose G, Cavalcante P, Covello G, Dato S, De Rango F, et al. A novel VNTR enhancer within the SIRT3 gene, a human homologue of SIR2, is associated with survival at oldest ages. Genomics. 2005;85(2):258–63. doi: 10.1016/j.ygeno.2004.11.003 WOS:000226843300011. 15676284

28. Song JHT, Lowe CB, Kingsley DM. Characterization of a Human-Specific Tandem Repeat Associated with Bipolar Disorder and Schizophrenia. Am J Hum Genet. 2018;103(3):421–30. doi: 10.1016/j.ajhg.2018.07.011 WOS:000443819500011. 30100087

29. Chaisson MJP, Wilson RK, Eichler EE. Genetic variation and the de novo assembly of human genomes. Nat Rev Genet. 2015;16(11):627–40. doi: 10.1038/nrg3933 WOS:000362909300006. 26442640

30. Örd D, Örd T. Characterization of human NIPK (TRB3, SKIP3) gene activation in stressful conditions. Biochem Biophys Res Commun. 2005;330(1):210–8. doi: 10.1016/j.bbrc.2005.02.149 MEDLINE:15781252. 15781252

31. Örd T, Örd D, Kõivomägi M, Juhkam K, Örd T. Human TRB3 is upregulated in stressed cells by the induction of translationally efficient mRNA containing a truncated 5'-UTR. Gene. 2009;444(1–2):24–32. doi: 10.1016/j.gene.2009.06.001 MEDLINE:19505541. 19505541

32. Leitsalu L, Alavere H, Tammesoo ML, Leego E, Metspalu A. Linking a Population Biobank with National Health Registries-The Estonian Experience. J Pers Med. 2015;5(2):96–106. doi: 10.3390/jpm5020096 WOS:000457944500005. 25894366

33. Mitt M, Kals M, Parn K, Gabriel SB, Lander ES, Palotie A, et al. Improved imputation accuracy of rare and low-frequency variants using population-specific high-coverage WGS-based imputation reference panel. Eur J Hum Genet. 2017;25(7):869–76. doi: 10.1038/ejhg.2017.51 WOS:000403061300016. 28401899

34. Siepel A, Bejerano G, Pedersen JS, Hinrichs AS, Hou MM, Rosenbloom K, et al. Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 2005;15(8):1034–50. doi: 10.1101/gr.3715005 WOS:000231032000002. 16024819

35. Pollard KS, Hubisz MJ, Rosenbloom KR, Siepel A. Detection of nonneutral substitution rates on mammalian phylogenies. Genome Res. 2010;20(1):110–21. doi: 10.1101/gr.097857.109 WOS:000273249500012. 19858363

36. Meyer M, Kircher M, Gansauge MT, Li H, Racimo F, Mallick S, et al. A High-Coverage Genome Sequence from an Archaic Denisovan Individual. Science. 2012;338(6104):222–6. doi: 10.1126/science.1224344 WOS:000309712300037. 22936568

37. Prüfer K, de Filippo C, Grote S, Mafessoni F, Korlević P, Hajdinjak M, et al. A high-coverage Neandertal genome from Vindija Cave in Croatia. Science. 2017;358(6363):655–8. doi: 10.1126/science.aao1887 WOS:000414240500039. 28982794

38. Karczewski KJ, Francioli LC, Tiao G, Cummings BB, Alföldi J, Wang Q, et al. Variation across 141,456 human exomes and genomes reveals the spectrum of loss-of-function intolerance across human protein-coding genes. bioRxiv. 2019:531210. doi: 10.1101/531210

39. Bergström A, McCarthy SA, Hui RY, Almarri MA, Ayub Q, Danecek P, et al. Insights into human genetic variation and population history from 929 diverse genomes. Science. 2020;367(6484):1339–+. doi: 10.1126/science.aay5012 WOS:000522167400043. 32193295

40. Slatkin M, Barton NH. A COMPARISON OF 3 INDIRECT METHODS FOR ESTIMATING AVERAGE LEVELS OF GENE FLOW. Evolution. 1989;43(7):1349–68. doi: 10.1111/j.1558-5646.1989.tb02587.x WOS:A1989AX63000001. 28564250

41. Huggins CJ, Mayekar MK, Martin N, Saylor KL, Gonit M, Jailwala P, et al. C/EBP gamma Is a Critical Regulator of Cellular Stress Response Networks through Heterodimerization with ATF4. Mol Cell Biol. 2016;36(5):693–713. doi: 10.1128/mcb.00911-15 WOS:000372328600004. 26667036

42. Shan JX, Zhang F, Sharkey J, Tang TA, Ord T, Kilberg MS. The C/ebp-Atf response element (CARE) location reveals two distinct Atf4-dependent, elongation-mediated mechanisms for transcriptional induction of aminoacyl-tRNA synthetase genes in response to amino acid limitation. Nucleic Acids Res. 2016;44(20):9719–32. doi: 10.1093/nar/gkw667 WOS:000393817800023. 27471030

43. Shan JX, Örd D, Örd T, Kilberg MS. Elevated ATF4 Expression, in the Absence of Other Signals, Is Sufficient for Transcriptional Induction via CCAAT Enhancer-binding Protein-activating Transcription Factor Response Elements. J Biol Chem. 2009;284(32):21241–8. doi: 10.1074/jbc.M109.011338 WOS:000268564400017. 19509279

44. Lepik K, Annilo T, Kukuskina V, Kisand K, Kutalik Z, Peterson P, et al. C-reactive protein upregulates the whole blood expression of CD59—an integrative analysis. PLoS Comput Biol. 2017;13(9). doi: 10.1371/journal.pcbi.1005766 WOS:000411981000043. 28922377

45. Lonsdale J, Thomas J, Salvatore M, Phillips R, Lo E, Shad S, et al. The Genotype-Tissue Expression (GTEx) project. Nat Genet. 2013;45(6):580–5. doi: 10.1038/ng.2653 WOS:000319563900002. 23715323

46. Buniello A, MacArthur JAL, Cerezo M, Harris LW, Hayhurst J, Malangone C, et al. The NHGRI-EBI GWAS Catalog of published genome-wide association studies, targeted arrays and summary statistics 2019. Nucleic Acids Res. 2019;47(D1):D1005–D12. doi: 10.1093/nar/gky1120 WOS:000462587400139. 30445434

47. Canela-Xandri O, Rawlik K, Tenesa A. An atlas of genetic associations in UK Biobank. Nat Genet. 2018;50(11):1593–9. doi: 10.1038/s41588-018-0248-z WOS:000448398000015. 30349118

48. Werling DM, Lowe JK, Luo R, Cantor RM, Geschwind DH. Replication of linkage at chromosome 20p13 and identification of suggestive sex-differential risk loci for autism spectrum disorder. Mol Autism. 2014;5. doi: 10.1186/2040-2392-5-13 WOS:000334716000001. 24533643

49. Lambert JC, Ibrahim-Verbaas CA, Harold D, Naj AC, Sims R, Bellenguez C, et al. Meta-analysis of 74,046 individuals identifies 11 new susceptibility loci for Alzheimer's disease. Nat Genet. 2013;45(12):1452–U206. doi: 10.1038/ng.2802 WOS:000327715800010. 24162737

50. Luciano M, Hansell NK, Lahti J, Davies G, Medland SE, Raikkonen K, et al. Whole genome association scan for genetic polymorphisms influencing information processing speed. Biol Psychol. 2011;86(3):193–202. doi: 10.1016/j.biopsycho.2010.11.008 MEDLINE:21130836. 21130836

51. Shah T, Zabaneh D, Gaunt T, Swerdlow DI, Shah S, Talmud PJ, et al. Gene-Centric Analysis Identifies Variants Associated With Interleukin-6 Levels and Shared Pathways With Other Inflammation Markers. Circ Cardiovasc Genet. 2013;6(2):163–70. doi: 10.1161/CIRCGENETICS.112.964254 WOS:000317741400005. 23505291

52. Genovese LM, Geraci F, Corrado L, Mangano E, D'Aurizio R, Bordoni R, et al. A Census of Tandemly Repeated Polymorphic Loci in Genic Regions Through the Comparative Integration of Human Genome Assemblies. Front Genet. 2018;9. doi: 10.3389/fgene.2018.00155 WOS:000431218500001. 29770143

53. Carraro V, Maurin AC, Lambert-Langlais S, Averous J, Chaveroux C, Parry L, et al. Amino Acid Availability Controls TRB3 Transcription in Liver through the GCN2/eIF2 alpha/ATF4 Pathway. Plos One. 2010;5(12). doi: 10.1371/journal.pone.0015716 WOS:000285576900037.

54. Adams CM, Ebert SM, Dyle MC. Role of ATF4 in skeletal muscle atrophy. Curr Opin Clin Nutr Metab Care. 2017;20(3):164–8. doi: 10.1097/MCO.0000000000000362 WOS:000400991100004. 28376050

55. Karsenty G. Transcriptional control of skeletogenesis. Annu Rev Genomics Hum Genet. 2008;9:183–96. doi: 10.1146/annurev.genom.9.081307.164437 WOS:000259629000010. 18767962

56. Wortel IMN, van der Meer LT, Kilberg MS, van Leeuwen FN. Surviving Stress: Modulation of ATF4-Mediated Stress Responses in Normal and Malignant Cells. Trends Endocrinol Metab. 2017;28(11):794–806. WOS:000414064400004. doi: 10.1016/j.tem.2017.07.003 28797581

57. Perelman P, Johnson WE, Roos C, Seuanez HN, Horvath JE, Moreira MAM, et al. A Molecular Phylogeny of Living Primates. PLoS Genet. 2011;7(3). doi: 10.1371/journal.pgen.1001342 WOS:000288996600024. 21436896

58. Green RE, Krause J, Briggs AW, Maricic T, Stenzel U, Kircher M, et al. A Draft Sequence of the Neandertal Genome. Science. 2010;328(5979):710–22. doi: 10.1126/science.1188021 WOS:000277357100030. 20448178

59. Harvati K, Roding C, Bosman AM, Karakostis FA, Grun R, Stringer C, et al. Apidima Cave fossils provide earliest evidence of Homo sapiens in Eurasia. Nature. 2019;571(7766):500–+. doi: 10.1038/s41586-019-1376-z WOS:000477016700058. 31292546

60. Hershkovitz I, Weber GW, Quam R, Duval M, Grun R, Kinsley L, et al. The earliest modern humans outside Africa. Science. 2018;359(6374):456–9. doi: 10.1126/science.aap8369 WOS:000423283200046. 29371468

61. Eyers PA, Keeshan K, Kannan N. Tribbles in the 21st Century: The Evolving Roles of Tribbles Pseudokinases in Biology and Disease. Trends Cell Biol. 2017;27(4):284–98. doi: 10.1016/j.tcb.2016.11.002 WOS:000399431000005. 27908682

62. Qi L, Heredia JE, Altarejos JY, Screaton R, Goebel N, Niessen S, et al. TRB3 links the E3 ubiquitin ligase COP1 to lipid metabolism. Science. 2006;312(5781):1763–6. doi: 10.1126/science.1123374 WOS:000238452800048. 16794074

63. Saleem S, Biswas SC. Tribbles Pseudokinase 3 Induces Both Apoptosis and Autophagy in Amyloid-beta-induced Neuronal Death. J Biol Chem. 2017;292(7):2571–85. doi: 10.1074/jbc.M116.744730 WOS:000395535100002. 28011637

64. Zareen N, Biswas SC, Greene LA. A feed-forward loop involving Trib3, Akt and FoxO mediates death of NGF-deprived neurons. Cell Death Differ. 2013;20(12):1719–30. doi: 10.1038/cdd.2013.128 MEDLINE:24212932. 24212932

65. Liu GY, Jin SL, Hu Y, Jiang QH. Disease status affects the association between rs4813620 and the expression of Alzheimer's disease susceptibility gene TRIB3. Proc Natl Acad Sci U S A. 2018;115(45):E10519–E20. doi: 10.1073/pnas.1812975115 WOS:000449459000003. 30355771

66. Dev A, Asch R, Jachimowicz E, Rainville N, Johnson A, Greenfest-Allen E, et al. Governing roles for Trib3 pseudokinase during stress erythropoiesis. Exp Hematol. 2017;49:48–55. doi: 10.1016/j.exphem.2016.12.010 WOS:000399861000007. 28062363

67. Kaplinski L, Lepamets M, Remm M. GenomeTester4: a toolkit for performing basic set operations—union, intersection and complement on k-mer lists. Gigascience. 2015;4. doi: 10.1186/s13742-015-0097-y WOS:000365669500001. 26640690

68. Shin J-H, Blay S, McNeney B, Graham J. LDheatmap: An R Function for Graphical Display of Pairwise Linkage Disequilibria Between Single Nucleotide Polymorphisms. J Stat Softw. 2006;16(Code Snippet 3):1–9. doi: 10.18637/jss.v016.c03


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