Microrna-15A and -16-1 Act Via MYB to Elevate Fetal Hemoglobin Expression in Human Trisomy 13
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MicroRNA-15a and -16-1 act via MYB to elevate fetal hemoglobin expression in human trisomy 13 Vijay G. Sankarana,b,c,d, Tobias F. Mennee,f, Danilo Šcepanovicg, Jo-Anne Vergilioh, Peng Jia, Jinkuk Kima,g,i, Prathapan Thirua, Stuart H. Orkind,e,f,j, Eric S. Landera,b,k,l, and Harvey F. Lodisha,b,l,1 aWhitehead Institute for Biomedical Research, Cambridge, MA 02142; bBroad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, MA 02142; Departments of cMedicine and hPathology and eDivision of Hematology/Oncology, Children’s Hospital Boston, Boston, MA 02115; Departments of dPediatrics and kSystems Biology, Harvard Medical School, Boston, MA 02115; lDepartment of Biology and iHoward Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02142; gHarvard–Massachusetts Institute of Technology Division of Health Sciences and Technology, Cambridge, MA 02142; fDepartment of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115; and jThe Howard Hughes Medical Institute, Boston, MA 02115 Contributed by Harvey F. Lodish, December 13, 2010 (sent for review November 8, 2010) Many human aneuploidy syndromes have unique phenotypic other hematopoietic cells shifts to the bone marrow, the pre- consequences, but in most instances it is unclear whether these dominant postnatal site for hematopoiesis, another switch phenotypes are attributable to alterations in the dosage of specific occurs, resulting in down-regulation of γ-globin and concomitant genes. In human trisomy 13, there is delayed switching and up-regulation of the adult β-globin gene (6, 8, 10). There is persistence of fetal hemoglobin (HbF) and elevation of embryonic a limited understanding of the molecular control of these globin hemoglobin in newborns. Using partial trisomy cases, we mapped gene switches that occur in human ontogeny, particularly with this trait to chromosomal band 13q14; by examining the genes in regard to the fetal-to-adult hemoglobin switch within the de- this region, two microRNAs, miR-15a and -16-1, appear as top finitive erythroid lineage. Recent insight into these mechanisms candidates for the elevated HbF levels. Indeed, increased expres- has come from the field of human genetics (6) and resulted in the sion of these microRNAs in primary human erythroid progenitor identification of the transcription factor BCL11A as a major cells results in elevated fetal and embryonic hemoglobin gene regulator of this process (7). However, it is apparent that this expression. Moreover, we show that a direct target of these factor cannot solely be responsible for this switch in ontogeny. microRNAs, MYB, plays an important role in silencing the fetal and embryonic hemoglobin genes. Thus we demonstrate how the de- Results velopmental regulation of a clinically important human trait can Mapping of partial trisomy cases provides an opportunity to be better understood through the genetic and functional study of deduce genotype–phenotype relationships (1, 11). Given the aneuploidy syndromes and suggest that miR-15a, -16-1, and MYB dramatic reduction in births with trisomy 13 following the may be important therapeutic targets to increase HbF levels in availability of prenatal diagnosis (12), mapping of such traits patients with sickle cell disease and β-thalassemia. must rely on cases that have previously been cytogenetically mapped. Analysis of partial trisomy 13 cases has suggested that erythropoiesis | globin gene regulation specific regions on the proximal part of chromosome 13 may be associated with elevations in HbF (1). Using eight well-anno- uman syndromes that are attributable to chromosomal tated cases with detailed cytogenetic mapping data available Himbalances or aneuploidy provide a unique opportunity to (11), chromosomal band 13q14 appears to be unambiguously understand the phenotypic consequences of altered gene dosage associated with elevated HbF levels (Fig. 1A). By accounting for (1, 2). Such observations also provide the prospect of gaining all 57 partial trisomy cases that have been reported with HbF insight into the mechanisms mediating normal human de- measurements (SI Appendix, Fig. S1) (13, 14), with varying velopment and physiology. However, in the vast majority of degrees of detail reported for cytogenetic mapping, a clear as- fi instances there is a limited understanding of how alterations in sociation with 13q14 is again deduced (Fig. 1B). This nding is specific genetic loci contribute to the consequent phenotypic strongly supported by Bayesian chromosomal region association features seen in aneuploidy syndromes. Trisomy of chromosome models we developed (SI Appendix). 13 is one of the few viable human aneuploidies and is associated We then used an integrative genomic approach to identify with a number of unique features (1), including a delayed switch candidates within the region implicated from the partial trisomy from fetal to adult hemoglobin and persistently elevated levels of cases, by analyzing a gene expression compendium to search for – genes with preferential expression in erythroid precursors GENETICS fetal hemoglobin (HbF) (1, 3 5). This trait is of considerable + interest given that it is one of the few quantitative and objective (CD71 ) relative to other cell types (SI Appendix) (15). Of the 76 genes in the region, 14 (18%) passed this test (SI Appendix, Fig. biochemical phenotypes described in such syndromes. Addi- fi tionally, the regulation of HbF is of great interest given the well- S2 and Table S1). We could further lter potential candidates by examining whether the histone 3 lysine 4 trimethylation characterized role of elevated HbF in ameliorating clinical se- fi verity in sickle cell disease and β-thalassemia (6, 7). (H3K4me3) modi cation, a well-characterized marker of active During human development a series of switches occurs in- volving the transcription of the globin genes residing within the β-globin locus on human chromosome 11. A transient lineage of Author contributions: V.G.S. designed research; V.G.S., T.F.M., J.-A.V., and P.J. performed research; V.G.S., T.F.M., D.S., P.T., S.H.O., E.S.L., and H.F.L. contributed new reagents/ red blood cells, the primitive erythroid lineage, is produced in analytic tools; V.G.S., D.S., J.-A.V., J.K., P.T., S.H.O., E.S.L., and H.F.L. analyzed data; and the first few weeks of human gestation (8). These cells produce V.G.S., E.S.L., and H.F.L. wrote the paper. a unique embryonic β-like globin chain, ε-globin (9). Additionally The authors declare no conflict of interest. small amounts of other β-like globin genes are expressed in this Freely available online through the PNAS open access option. fi lineage (8, 9). Subsequently, de nitive erythroid cells are pro- Data deposition: The data reported in this paper have been deposited in the Gene Ex- duced from long-term self-renewing hematopoietic stem cells. pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE25678). Initially these cells predominantly express the β-like fetal he- 1To whom correspondence should be addressed. E-mail: [email protected]. γ moglobin gene, -globin, and are produced in the fetal liver (10). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Around the time of birth, when production of erythroid and 1073/pnas.1018384108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1018384108 PNAS | January 25, 2011 | vol. 108 | no. 4 | 1519–1524 Downloaded by guest on September 27, 2021 1 A 13 Partial Trisomy Cases B Elevated HbF p 12 0.9 Normal HbF 11.2 Elevated HbF 11.1 0.8 11 Normal HbF 12 0.7 Chr. 13 0.6 13 14 0.5 q 21 0.4 22 Portion with Trisomy 0.3 31 0.2 32 33 0.1 34 0 pter−q11 q12 q13 q14 q21 q22 q31 q32−qter Chr. 13 Band Chromosome Bands Localized by FISH Mapping Clones C Chromosome Band 13q14.11 13q14.12 13q14.2 13q14.3 RefSeq Genes LOC646982 KIAA0564 EPSTI1 SERP2 KCTD4 CPB2 ESD SUCLA2 FNDC3A KCNRG FAM124A NEK5 LOC646982 DGKH ENOX1 NUFIP1 SIAH3 HTR2A NUDT15 FNDC3A KCNRG SERPINE3 NEK3 LOC646982 DGKH ENOX1 KIAA1704 ZC3H13 HTR2A MED4 MLNR DLEU1 INTS6 NEK3 FOXO1 AKAP11 CCDC122 GTF2F2 CPB2 ITM2B CDADC1 DLEU7 DHRS12 SUGT1 MRPS31 TNFSF11 C13orf31 TPT1 LCP1 RB1 SETDB2 RNASEH2B ATP7B SLC25A15 TNFSF11 C13orf31 SNORA31 LRCH1 LPAR6 SETDB2 RNASEH2B ATP7B SUGT1L1 C13orf30 TSC22D1 COG3 LRCH1 LPAR6 PHF11 GUCY1B2 ALG11 MIR621 EPSTI1 TSC22D1 FAM194B LRCH1 LPAR6 PHF11 INTS6 NEK3 ELF1 DNAJC15 LOC100190939 RCBTB2 RCBTB1 INTS6 NEK3 ELF1 LOC121838 SLC25A30 CYSLTR2 ARL11 WDFY2 SUGT1 WBP4 SPERT CAB39L DHRS12 LECT1 KBTBD6 C13orf18 CAB39L FLJ37307 LECT1 KBTBD7 EBPL FLJ37307 MTRF1 KPNA3 CCDC70 NARG1L LOC220429 UTP14C NARG1L C13orf1 THSD1P NARG1L C13orf1 THSD1 OR7E37P C13orf1 THSD1 OR7E37P DLEU2 VPS36 C13orf15 TRIM13 CKAP2 KIAA0564 TRIM13 CKAP2 TRIM13 LOC220115 TRIM13 HNRNPA1L2 MIR16-1 HNRNPA1L2 MIR15A FAM10A4 D 1.0 E 80 n 0.8 60 essio miR-15a 0.6 miR-16 r xp 40 0.4 E ve i 20 0.2 Relative Expression Relat 0.0 0 1 3 5 7 9 1 3 5 7 9 Days of Differentation Days of Differentation Fig. 1. Identification of miR-15a/16-1 as candidates for causing the elevated HbF levels in trisomy 13. (A) A set of eight well-annotated cases, along with the full trisomy of chromosome 13, demonstrates that chromosomal band 13q14 is umambiguously associated with the trait of elevated HbF levels (11). Cases with and without elevated HbF levels are labeled in purple and blue, respectively. Cases are considered to have elevated HbF levels when the measured level is greater than two SDs above the mean level for age (3, 21). (B) A compilation of all 57 partial trisomy cases reported shows that chromosomal band 13q14 is most frequently associated with elevated HbF (13, 14). The proportion of cases with elevated or normal HbF levels in patients with trisomy of each chro- mosomal band is shown in this diagram.