MicroRNA-15a and -16-1 act via MYB to elevate fetal hemoglobin expression in 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 . In human trisomy 13, there is delayed switching and up-regulation of the adult β-globin (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 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. GENETICS 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 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 compendium to search for – genes with preferential expression in erythroid precursors 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 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 Early Edition | 1of6 Downloaded by guest on September 29, 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 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 , 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. The association with chromosomal band 13q14 finding is supported using Bayesian probabilistic models (SI Ap- pendix). (C) A diagram showing all of the genes on chromosomal band 13q14. Of these, using an integrative genomic approach, miR-15a and -16-1 appear as top candidates (highlighted with a red box). (D) Pattern of miR-15a expression during human adult erythroid progenitor differentiation (shown relative to RNU19 expression; n = 4 per time point). The range of stages covers the cells from early CFU-E progenitors (day 1 of differentiation) to polychromatophilic erythroblasts (day 9 of differentiation), as described previously (7). (E) Pattern of miR-16 expression during human adult erythroid progenitor differentiation (shown relative to RNU19 expression; n = 4 per time point).

transcription, was present in the proximal of the genes play in modulating various aspects of hematopoiesis and from chromosomal band 13q14 in erythroid cell lines (defined as erythropoiesis specifically (17, 18). MiR-15a and -16 are expres- having a peak of H3K4me3 within 1.5 kb upstream from the sed throughout human erythroid maturation in adult bone mar- transcription start site). Using data derived from the ENCODE row cells and increase modestly with terminal differentiation project (16), we were able to find a number of unique peaks (SI (Fig. 1 D and E), consistent with prior observations in cord blood Appendix, Table S2) and of the gene list established from the and erythroid cell lines (19, 20). expression data, we could focus on 9 candidate genes (SI Ap- These observations suggest that increased expression of miR- pendix, Table S3). Of these, we noted that a top candidate in this 15a/16-1 in trisomy 13 could potentially result in elevated levels region was a precursor RNA (DLEU2) for two microRNAs, 15a of HbF expression. To directly test this hypothesis, we used and 16-1, which have identical seed targeting sequences (Fig. a lentiviral vector to increase expression of these microRNAs in 1C). This was of particular interest, given the role that micro- adult bone marrow-derived hematopoietic progenitors that sub-

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1018384108 Sankaran et al. Downloaded by guest on September 29, 2021 sequently underwent synchronous differentiation toward the ery- equivalent to controls at the later stages that represent more ma- throid lineage (7). By increasing expression of miR-15a and -16 ture (basophilic) erythroblasts (Fig. 2 E and F) (7). Such findings by an average of 1.5-fold (SI Appendix, Fig. S3), similar to what are reminiscent of the observations made using S-phase inhibitors would be expected in the context of a trisomy, we found that the for HbF induction in primate models, where the most responsive levels of γ-globin gene expression were robustly increased by an stages appeared to be the CFU-Es and proerythroblasts (22, 23). average of 2.4-fold (Fig. 2A). Because trisomy 13 can result in To gain further insight into the molecular etiology for these elevated γ-globin synthesis even in newborns with elevated HbF observations, we examined whether specific miR-15a/16-1 targets levels at baseline (21), we increased expression of these micro- may be potential mediators of the increased HbF expression. RNAs by 2- to 3-fold in human erythroleukemia K562 cells (SI Because evolutionary conservation of seed-matched targets shows Appendix, Fig. S4), which endogenously express high levels of great utility in identifying bona fide microRNA targets (24), we γ γ -globin, and found that the -globin levels could be further in- used a metric of target context and conservation (aggregate P , creased by 50% (Fig. 2B). Newborns with trisomy 13 also show CT ε which is a Bayesian estimate of the probability that a site is con- elevated expression of the embryonically expressed -globin,as served due to selective maintenance of miRNA targeting rather illustrated by the persistence of low level hemoglobin Gower 2 than by chance) and compared this with relative expression of (α ε ) expression (4). This increase was recapitulated in the pri- 2 2 mRNAs in the erythroid target tissues of interest relative to a large mary bone marrow-derived cells with increased miR-15a/16-1 number of other tissues (Fig. 3A and SI Appendix, Fig. S6). Using expression (Fig. 2C). Since alterations in γ-globin expression may accompany altered differentiation of cells, we examined the len- this approach, MYB was one miR-15a/16-1 target that appeared to tivirally transduced primary adult erythroid progenitors and found be of great interest, given that it was highly expressed in erythroid no major differences in the morphology or phenotype of cells with progenitors and had two conserved 8mer miR-15a/16-1 targeting increased miR-15a/16-1 expression compared with controls (Fig. sites (Fig. 3B). This was particularly notable, because common 2D and SI Appendix, Fig. S5). To gain further insight into the genetic variants from genomewide association studies have sug- mechanism by which these microRNAs may be acting to elevate gested that polymorphisms in the MYB locus are important me- γ-globin expression, we assessed cell cycle progression in the syn- diators of variation in HbF levels in (25), which is fur- chronously differentiating primary erythroid cells (7). Interest- ther supported by the finding that overexpression of MYB in cell ingly, we noted that cell cycle progression was slowed by miR-15a/ lines causes a decrease in γ-globin expression (26). We found that 16-1 overexpression at the early stages of differentiation that MYB levels were reduced with even a modest (two- to GENETICS represent colony-forming unit erythroid cells (CFU-Es) or pro- threefold) increase in miR-15a/16-1 expression in erythroid cell erythroblasts (G1- and S-phase difference, P < 0.001), but was lines (Fig. 3C) and a luciferase reporter assay confirmed that

A B C 40 2.0 0.00015 n i b ** 30 1.5 γ

ε 0.00010 f-Glo

20 o 1.0 e

tag 0.00005 n γ 10 0.5 e rc Percentage of -Globin Pe 0 0.0 0.00000 Relative Expression -Globin l 1 l o -1 o -1 tr 6 tr 6 n -1 -1 o a Control 5a-16- on C -1 C R -15a i R m miR-15 mi

D E 120 F 120

Control 80 80 of Cells of G1

age S nt 40 40 G2/M

miR Perce Percentage Cells of 15a-16 0 0 l o 6 r -1 rol t a t a-16 on 5 C -15 Con iR iR-1 m m

Fig. 2. Increased expression of miR-15a/16-1 in human erythroid cells results in elevated HbF and embryonic globin gene expression. (A) Percentage of γ-globin gene expression (as a percentage of all human β-like globin genes) in cells transduced with pLVX-puro control or pLVX-miR-15a/16-1 lentivirus (n =3 per group; ***P < 0.001). Measurement was at the basophilic erythroblast stage of differentiation (days 6–7 of differentiation). (B) Relative amount of γ-globin gene expression in K562 cells transduced with pSMPUW control or miR-15a/16-1 containing lentivirus, following selection (n = 3 per group; *P < 0.02). (C) Percentage of ε-globin gene expression (as a percentage of all human β-like globin genes) in primary bone marrow CD34-derived cells transduced with pLVX-puro control or pLVX/miR-15a/16-1 lentivirus (n = 3 per group; **P < 0.01). (D) Representative cytospin images of primary bone marrow CD34-derived cells transduced with pLVX-puro control or pLVX/miR-15a/16-1 lentivirus (taken with a 63× objective lens). All cells show similar size and morphological distribution on days 5–6 of differentiation. At other stages of differentiation the control and miR-15a/16-1 transduced cells also had the same morphology. (E and F) Cell cycle analysis of primary bone marrow CD34+-derived cells transduced with pLVX-puro control or pLVX/miR-15a/16-1 lentivirus on day 4 (E) and day 7(F) of differentiation (n =3–4 per group). All data are shown as the mean ± the SD.

Sankaran et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 C A 10 MYB

5 Control miR-15a-16 MYB

0 Expression Ratio 2 1.1 GAPDH Log -5 P Aggregate CT B D 1.2

0.8 MYB 3’UTR 654-661 5’...AUGAAAAACGUUUUUUGCUGCUA...3’ *** MYB 3’UTR 681-688 5’...CUUAGCCUGUAGACAUGCUGCUA...3’ ||||||| hsa-miR-15a GUGUUUGGUAAUACACGACGAU-5’ 0.4 hsa-miR-16-1 GCGGUGAUAAAUGCACGACGAU-5’

Relative Luciferase Activity 0.0 l 1 tro n o -16- 5a C 1 iR- E F G m 80 0.0008 *** *** 1.0 ***

ession 60 0.0006 *** pr ε γ Ex

B 40 0.0004 Y 0.5 ***

M ***

ive 20 0.0002 t Percentage of -Globin Percentage of -Globin Rela 0.0 0 0.0000 l l o 1 2 ol 1 2 o 1 2 tr B tr n Y YB YB MYB o M MYB M Contr hMYB h C h Con hM h s s sh s s s H I 0.8 100 Cells in S-phase 0.6 80 Control - 68.65% shMYB 1 - 39.94 % 0.4 60 shMYB 2 - 36.43 % 0.2 40

0.0 % of Maximum Enrichment Score 20

Up in shMYB Down in shMYB Propidium Iodide

Fig. 3. MYB is a target of miR-15a/16-1 and regulates HbF expression. (A) By comparing the aggregate PCT of a variety of miR-15 or -16 seed targets + (24) with the log2 normalized relative expression in early (CD34 ) hematopoietic/erythroid progenitors (relative to a panel of 78 other human cells and tissues), MYB appears to be a standout candidate target (highlighted in red with arrow). The x-axis plots aggregate PCT (24) on a linear scale, whereas the y-axis shows relative expression in the erythroid progenitors as a log2 ratio. (B) Two 8mer target sites for miR-15aand-16-1arelocatedinthe3′- UTR of MYB.(C) Ectopic expression of miR-15a/16-1 in K562 cells at a level two- to threefold of normal results in reduction in MYB protein levels; GADPH was used as a loading control for this Western blot. (D) Cotransfection of 293T cells with control or miR-15a/16-1 expression vector (in pLVX) and a MYB 3′-UTR construct luciferase reporter (in psiCHECK-2 vector) show reduced luciferase activity with elevated microRNA expression. (E) Relative MYB expression is shown on day 5 of differentiation in primary human CD34+-derived cells transduced with pLKO.1 control or pLKO.1 with shRNAs targeting MYB (n = 3 per group; ***P < 0.001). (F and G)Percentageofγ-globin (F)andε-globin (G)geneexpressionasapercentageofall human β-like globin genes on day 7 of differentiation in cells transduced with pLKO.1 control or pLKO.1 vector expressing two different shRNAs targeting MYB (n = 3 per group; ***P < 0.001). (H) Gene set enrichment analysis (30) of a monocyte gene expression signature, composed of 371 genes (31), evaluated using the expression array data of shMYB cells versus controls (n = 4 per group), demonstrates global up-regulation of the monocyte gene signature in the shMYB cells. Enrichment plot is shown above heat map (below) using a green line to show the running enrichment score. (I) Representative flow cytometry profiles of propidium iodide staining in K562 cells transduced with empty or shMYB containing pLKO.1 lentiviruses. Data are shown as an average of three experiments for each group (P < 0.001 for both shMYB experiments relative to controls). All data are shown as the mean ± SD.

the MYB 3′-UTR is a direct target of these microRNAs (Fig. 3D), occurs to a lesser extent with miR-15a/16-1 elevation (Figs. 2A consistent with previous studies (20, 27, 28). and 3F). Concomitantly, we found that expression of the em- To test whether MYB may be a critical mediator of γ-globin bryonic globin chain, ε-globin, was also dramatically increased by expression, we reduced MYB expression with two shRNAs in MYB knockdown (Fig. 3G). Our examination of expression data synchronously differentiating adult erythroid progenitors (Fig. from a recent study of MYB siRNA treatment of umbilical cord 3E). This knockdown robustly increased γ-globin expression, as blood erythroid progenitors (29) demonstrated robust elevations

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1018384108 Sankaran et al. Downloaded by guest on September 29, 2021 of both γ-globin and ε-globin gene expression, even with higher A C baseline levels of these globin genes at this stage of human on- togeny (SI Appendix, Fig. S7). Examination of cells with a knockdown of MYB demonstrated an increased presence of myeloid (primarily monocyte) cells and signs of precocious erythroid differentiation by morphological examination (SI Appendix, Fig. S8). To more precisely define the molecular basis of these changes, expression analysis was per- formed with cells from one of the MYB knockdowns along with a set of controls. We did not observe any significant difference in B D mRNA expression levels of known regulators of erythropoiesis and globin gene expression, including BCL11A, GATA1, KLF1 (EKLF), ZFPM1 (FOG-1), and SOX6, that we and others have previously described (7, 10) (SI Appendix, Fig. S9). Gene set enrichment analysis (30) was used with gene sets derived from lineage-specific and differentiation stage-specific expression datasets (SI Appendix). A marked increase in the expression of the monocyte gene set was notable in the shRNA-treated cells compared with controls (Fig. 3H) (31). In permissive conditions, Fig. 4. Pathology from trisomy 13 autopsy cases reveals normal erythro- a similar type of knockdown can also result in increased pro- poiesis and perturbed megakaryopoiesis. (A) Bone marrow (BM) section duction of megakaryocytes (19). Moreover, when gene sets were from a trisomy 13 case reveals normal erythroid maturation with a normal created from the MYB knockdown expression sets compared immature:mature progenitor ratio. (B) Abnormal and increased numbers of fi megakaryocytes that have hypolobulated nuclei with a “staghorn” ap- with controls, the up-regulated genes were found to be signi - pearance are seen in a BM section. (C) Normal erythropoiesis and increased cantly enriched in the later stages of erythroid differentiation, megakaryocytes with hypolobulated nuclei seen in a BM section. (D) Normal supporting the notion that precocious erythroid differentiation erythropoietic maturation and a megakaryocyte with a staghorn appear- was occurring in this context (SI Appendix, Fig. S10) (32). To- ance are seen on this BM section. Examples of some dysplastic mega- fi gether, these ndings suggest that MYB is necessary for the karyocytes with staghorn and hypolobulated nuclei are highlighted in the GENETICS normal differentiation kinetics of adult erythroid cells and re- images with cyan arrows. All images are shown at 400× magnification and duction in the level of this gene results in altered erythroid dif- slides were stained with hematoxylin and eosin. ferentiation kinetics and the increased presence of cells from other lineages. Consistent with the findings with moderate MYB knockdown by miR-15a/16-1, these shRNAs were able to result terestingly, MYB knockdown in human cells and hypomorphic alleles in mice show similar phenotypes in megakaryocytes (19, in a marked slowing of cell cycle progression (Fig. 3I) and – marked up-regulation of γ-globin expression in erythroid cell 34 36), suggesting that miR-15a/16-1 overexpression and the lines (SI Appendix, Fig. S11). In support of the slowing of cell consequent reduction in MYB expression may alter other aspects cycle progression, we noted that the expression of several cell cy- of hematopoiesis in trisomy 13 patients. cle regulators were altered in our expression data from the MYB Discussion knockdown cells compared with controls (SI Appendix, Fig. S12). The altered cell cycle and differentiation kinetics of cells with We have demonstrated, using a combination of genetic and MYB knockdown are consistent with findings in erythroid cells functional approaches in human cells that the overexpression of using hypomorphic myb alleles in mice (33). Our findings suggest miR-15a/16-1 results in elevations in HbF gene expression and that MYB plays a critical role coordinating globin gene expres- this likely explains why patients with a trisomy of chromosome 13 sion, cell cycle regulation, and erythroid differentiation. have a delayed fetal-to-adult hemoglobin switch and persistence Alterations in γ-globin expression can occur in the context of of fetal hemoglobin. This effect is mediated, at least in part, stress erythropoiesis or other states where erythroid differenti- through down-modulation of the MYB transcription factor, ation is perturbed. Patients with trisomy 13 and elevated HbF which we have shown is a potent negative regulator of HbF ex- levels do not have anemia (4, 21), but the evaluation of in vivo pression and is a direct target of miR-15a and -16-1 (Fig. 5). It is erythropoiesis in these patients has not previously been reported. possible that other genes on chromosome 13 may also contribute To properly ascertain this, we examined autopsy specimens from to this phenotype, but miR-15a and -16-1 appear to have a major patients with trisomy 13 from the archives ranging over four decades at a single institution (SI Appendix). Initially all available trisomy 13 cases were selected for evaluation, of which 17 were used for our final analysis on the basis of confirmation of the MYB diagnosis of trisomy 13 and presence of appropriate histological

samples (SI Appendix, Table S4). In all of the samples examined, Euploid Erythroid MicroRNAs Progenitor Fetal erythropoiesis appeared appropriate for age, and full maturation 15a and 16-1 Hemoglobin of the erythroid lineage could be appreciated (Fig. 4 A, C, and D). This suggests that the elevated HbF levels in trisomy 13 can MYB occur without grossly perturbed erythropoiesis. In examining Midgestation Birth other aspects of hematopoiesis, we found a dramatic increase in Trisomy 13 megakaryocyte numbers in over 70% of the cases examined (Fig. Progenitor 4 B and C and SI Appendix, Table S4). In addition, the majority of the cases (64%) showed abnormal nuclear morphology of the Fig. 5. A model demonstrating how elevations of microRNAs 15a and 16-1 in trisomy 13 can result in elevated fetal hemoglobin expression. Normally, megakaryocytes, suggestive of decreased ploidy in these cells the basal level of these microRNAs can moderate expression of targets such (Fig. 4 B–D and SI Appendix, Fig. S13). These findings were fi as MYB during erythropoiesis. In the case of trisomy 13, elevated levels of speci c to patients with trisomy 13, as autopsy specimens from the these microRNAs results in additional down-regulation of MYB expression, same institution, in the same time period, and in patients of similar which in turn results in a delayed switch from fetal-to-adult hemoglobin and ages with other diagnoses lack these findings (SI Appendix). In- persistent expression of fetal hemoglobin.

Sankaran et al. PNAS Early Edition | 5of6 Downloaded by guest on September 29, 2021 impact. Our study demonstrates a unique and previously un- that can teach us a great deal about normal human development appreciated pathway that regulates this intensively studied de- and physiology. velopmental process. The exact relevance of our findings to normal physiology is not clear, but it appears to be likely that Materials and Methods these pathways may play an important role in the normal fetal- Details of the cell culture approaches, constructs used, lentiviral preparation, to-adult hemoglobin switch (Fig. 5). Consistent with this notion, RNA analysis, flow cytometry, protein methods, and analysis can be found in + common genetic variants close to the MYB gene appear to be SI Appendix. CD34 cells were cultured and differentiated using a two-phase important regulators of HbF levels in adult humans (25). Further culture method in serum-free conditions, with appropriate cytokines added at the various stages of the culture (7). RNA extraction, quantitative RT-PCR, work will be needed to gain insight into the exact role of these and microarray expression analysis were performed in a manner similar to factors in the mechanisms mediating human hemoglobin what has previously been described (7, 38). Lentivirus production and in- switching. Nonetheless, it is apparent from our work that miR- fection was carried out using a modified spin-infection method for the 15a, -16-1, and MYB could be important therapeutic targets to erythroid progenitors that were grown in suspension (39). Flow cytometry elevate HbF expression to ameliorate the severity of sickle cell was performed using standard approaches (38) with data analysis occurring disease and β-thalassemia. in the FlowJo 7.5.5 software suite. Our findings demonstrate the power of using human genetic approaches to understand developmental processes, where the ACKNOWLEDGMENTS. We thank D. Nathan and S. Lux for their inspiration use of model organisms can be limited (37). Importantly, our and guidance; C. Epstein, D. Bartel, C. Walkley, C. Sieff, J. Menne, J. fi fi Hirschhorn, J. Flygare, M. Bousquet, D. Nguyen, B. Wong, Y. Jeong, H. B. ndings suggest that alterations of gene dosage at speci c ge- Larman, and G. Bell for valuable advice; T. DiCesare for valuable assistance in netic loci likely underlie the numerous phenotypes observed producing the model illustration; and C. Kitidis-Mitrokostas and N. Cohen for uniquely with specific aneuploidy syndromes (1). In relatively technical assistance. T.F.M. was supported by the Kay Kendall Leukaemia Fund. D.S. was supported by a Department of Energy Computational Science few cases has this been strongly supported by functional evi- Graduate Fellowship (Grant DE-FG02-97ER25308). This work was supported dence. Similar types of approaches may help to uncover other by National Institutes of Health Grants R01 DK068348 and P01 HL32262 genotype–phenotype correlations in these fascinating syndromes (to H.F.L.).

1. Epstein CJ (1986) The Consequences of Chromosome Imbalance: Principles, 21. Bard H (1972) Postnatal fetal and adult hemoglobin synthesis in D1 trisomy syndrome. Mechanisms, and Models (Cambridge Univ Press, Cambridge, New York). Blood 40:523–527. 2. Korbel JO, et al. (2009) The genetic architecture of Down syndrome phenotypes 22. Papayannopoulou T, Torrealba de Ron A, Veith R, Knitter G, Stamatoyannopoulos G revealed by high-resolution analysis of human segmental trisomies. Proc Natl Acad Sci (1984) Arabinosylcytosine induces fetal hemoglobin in baboons by perturbing USA 106:12031–12036. erythroid cell differentiation kinetics. Science 224:617–619. 3. Pinkerton PH, Cohen MM (1967) Persistence of hemoglobin F in D/D translocation 23. Letvin NL, et al. (1985) Influence of cell cycle phase-specific agents on simian fetal with trisomy 13-15 (D1). JAMA 200:647–649. hemoglobin synthesis. J Clin Invest 75:1999–2005. 4. Huehns ER, Hecht F, Keil JV, Motulsky AG (1964) Developmental hemoglobin 24. Friedman RC, Farh KK, Burge CB, Bartel DP (2009) Most mammalian mRNAs are anomalies in a chromosomal triplication: D1 trisomy syndrome. Proc Natl Acad Sci USA conserved targets of microRNAs. Res 19:92–105. 51:89–97. 25. Thein SL, Menzel S, Lathrop M, Garner C (2009) Control of fetal hemoglobin: New 5. Powars D, Rohde R, Graves D (1964) Foetal haemoglobin and neutrophil anomaly in the D1-trisomy syndrome. Lancet 1:1363–1364. insights emerging from genomics and clinical implications. Hum Mol Genet 18(R2): 6. Orkin SH, Higgs DR (2010) Medicine. Sickle cell disease at 100 years. Science 329: R216–R223. 291–292. 26. Jiang J, et al. (2006) cMYB is involved in the regulation of fetal hemoglobin 7. Sankaran VG, et al. (2008) Human fetal hemoglobin expression is regulated by the production in adults. Blood 108:1077–1083. developmental stage-specific repressor BCL11A. Science 322:1839–1842. 27. Klein U, et al. (2010) The DLEU2/miR-15a/16-1 cluster controls B cell proliferation and 8. McGrath K, Palis J (2008) Ontogeny of erythropoiesis in the mammalian embryo. Curr its deletion leads to chronic lymphocytic leukemia. Cancer Cell 17:28–40. Top Dev Biol 82:1–22. 28. Chung EY, et al. (2008) c-Myb oncoprotein is an essential target of the dleu2 tumor 9. Peschle C, et al. (1985) Haemoglobin switching in human embryos: Asynchrony of suppressor microRNA cluster. Cancer Biol Ther 7:1758–1764. — — fi zeta alpha and epsilon gamma-globin switches in primitive and de nite 29. Bianchi E, et al. (2010) c-myb supports erythropoiesis through the transactivation of erythropoietic lineage. Nature 313:235–238. KLF1 and LMO2 expression. Blood 116:e99–e110. 10. Sankaran VG, Xu J, Orkin SH (2010) Advances in the understanding of haemoglobin 30. Subramanian A, et al. (2005) Gene set enrichment analysis: A knowledge-based switching. Br J Haematol 149:181–194. approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 11. Yunis JJ (1977) New Chromosomal Syndromes (Academic, New York). 102:15545–15550. 12. Parker MJ, Budd JL, Draper ES, Young ID (2003) Trisomy 13 and trisomy 18 in adefined population: Epidemiological, genetic and prenatal observations. Prenat 31. Watkins NA, et al. (2009) A HaemAtlas: Characterizing gene expression in – Diagn 23:856–860. differentiated human blood cells. Blood 113:e1 e9. 13. Tharapel SA, Lewandowski RC, Tharapel AT, Wilroy RS, Jr. (1986) Phenotype- 32. Welch JJ, et al. (2004) Global regulation of erythroid gene expression by transcription karyotype correlation in patients trisomic for various segments of chromosome 13. factor GATA-1. Blood 104:3136–3147. J Med Genet 23:310–315. 33. Vegiopoulos A, García P, Emambokus N, Frampton J (2006) Coordination of 14. Rogers JF (1984) Clinical delineation of proximal and distal partial 13q trisomy. Clin erythropoiesis by the transcription factor c-Myb. Blood 107:4703–4710. Genet 25:221–229. 34. Kasper LH, et al. (2002) A transcription-factor-binding surface of coactivator p300 is 15. Su AI, et al. (2004) A gene atlas of the and human protein-encoding required for haematopoiesis. Nature 419:738–743. transcriptomes. Proc Natl Acad Sci USA 101:6062–6067. 35. Emambokus N, et al. (2003) Progression through key stages of haemopoiesis is 16. Rosenbloom KR, et al. (2010) ENCODE whole-genome data in the UCSC Genome dependent on distinct threshold levels of c-Myb. EMBO J 22:4478–4488. – Browser. Nucleic Acids Res 38(Database issue):D620 D625. 36. Carpinelli MR, et al. (2004) Suppressor screen in Mpl-/- mice: c-Myb mutation causes 17. Chen CZ, Li L, Lodish HF, Bartel DP (2004) MicroRNAs modulate hematopoietic lineage supraphysiological production of platelets in the absence of thrombopoietin differentiation. Science 303:83–86. signaling. Proc Natl Acad Sci USA 101:6553–6558. 18. Zhao G, Yu D, Weiss MJ (2010) MicroRNAs in erythropoiesis. Curr Opin Hematol 17: 37. Sankaran VG, et al. (2009) Developmental and -divergent globin switching are 155–162. driven by BCL11A. Nature 460:1093–1097. 19. Lu J, et al. (2008) MicroRNA-mediated control of cell fate in megakaryocyte- 38. Sankaran VG, Orkin SH, Walkley CR (2008) Rb intrinsically promotes erythropoiesis by erythrocyte progenitors. Dev Cell 14:843–853. – 20. Zhao H, Kalota A, Jin S, Gewirtz AM (2009) The c-myb proto-oncogene and microRNA- coupling cell cycle exit with mitochondrial biogenesis. Genes Dev 22:463 475. 15a comprise an active autoregulatory feedback loop in human hematopoietic cells. 39. Moffat J, et al. (2006) A lentiviral RNAi library for human and mouse genes applied to Blood 113:505–516. an arrayed viral high-content screen. Cell 124:1283–1298.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1018384108 Sankaran et al. Downloaded by guest on September 29, 2021