MicroRNA-15a and -16-1 act via MYB to elevate fetal
hemoglobin expression in human trisomy 13
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Citation
Sankaran, V. G. et al. “MicroRNA-15a and -16-1 Act via MYB to
Elevate Fetal Hemoglobin Expression in Human Trisomy 13.”
Proceedings of the National Academy of Sciences 108.4 (2011) :
1519-1524. ©2011 by the National Academy of Sciences.
As Published
http://dx.doi.org/10.1073/pnas.1018384108
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National Academy of Sciences (U.S.)
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Final published version
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Wed May 25 18:28:42 EDT 2016
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Detailed Terms
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
a
Whitehead 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)
erythropoiesis
| globin gene regulation
H
uman syndromes that are attributable to chromosomal
imbalances or aneuploidy provide a unique opportunity to
understand the phenotypic consequences of altered gene dosage
(1, 2). Such observations also provide the prospect of gaining
insight into the mechanisms mediating normal human development and physiology. However, in the vast majority of
instances there is a limited understanding of how alterations in
specific genetic loci contribute to the consequent phenotypic
features seen in aneuploidy syndromes. Trisomy of chromosome
13 is one of the few viable human aneuploidies and is associated
with a number of unique features (1), including a delayed switch
from fetal to adult hemoglobin and persistently elevated levels of
fetal hemoglobin (HbF) (1, 3–5). This trait is of considerable
interest given that it is one of the few quantitative and objective
biochemical phenotypes described in such syndromes. Additionally, the regulation of HbF is of great interest given the wellcharacterized role of elevated HbF in ameliorating clinical severity in sickle cell disease and β-thalassemia (6, 7).
During human development a series of switches occurs involving the transcription of the globin genes residing within the
β-globin locus on human chromosome 11. A transient lineage of
red blood cells, the primitive erythroid lineage, is produced in
the first few weeks of human gestation (8). These cells produce
a unique embryonic β-like globin chain, ε-globin (9). Additionally
small amounts of other β-like globin genes are expressed in this
lineage (8, 9). Subsequently, definitive erythroid cells are produced from long-term self-renewing hematopoietic stem cells.
Initially these cells predominantly express the β-like fetal hemoglobin gene, γ-globin, and are produced in the fetal liver (10).
Around the time of birth, when production of erythroid and
www.pnas.org/cgi/doi/10.1073/pnas.1018384108
other hematopoietic cells shifts to the bone marrow, the predominant postnatal site for hematopoiesis, another switch
occurs, resulting in down-regulation of γ-globin and concomitant
up-regulation of the adult β-globin gene (6, 8, 10). There is
a limited understanding of the molecular control of these globin
gene switches that occur in human ontogeny, particularly with
regard to the fetal-to-adult hemoglobin switch within the definitive erythroid lineage. Recent insight into these mechanisms
has come from the field of human genetics (6) and resulted in the
identification of the transcription factor BCL11A as a major
regulator of this process (7). However, it is apparent that this
factor cannot solely be responsible for this switch in ontogeny.
Results
Mapping of partial trisomy cases provides an opportunity to
deduce genotype–phenotype relationships (1, 11). Given the
dramatic reduction in births with trisomy 13 following the
availability of prenatal diagnosis (12), mapping of such traits
must rely on cases that have previously been cytogenetically
mapped. Analysis of partial trisomy 13 cases has suggested that
specific regions on the proximal part of chromosome 13 may be
associated with elevations in HbF (1). Using eight well-annotated cases with detailed cytogenetic mapping data available
(11), chromosomal band 13q14 appears to be unambiguously
associated with elevated HbF levels (Fig. 1A). By accounting for
all 57 partial trisomy cases that have been reported with HbF
measurements (SI Appendix, Fig. S1) (13, 14), with varying
degrees of detail reported for cytogenetic mapping, a clear association with 13q14 is again deduced (Fig. 1B). This finding is
strongly supported by Bayesian chromosomal region association
models we developed (SI Appendix).
We then used an integrative genomic approach to identify
candidates within the region implicated from the partial trisomy
cases, by analyzing a gene expression compendium to search for
genes with preferential expression in erythroid precursors
(CD71+) relative to other cell types (SI Appendix) (15). Of the 76
genes in the region, 14 (18%) passed this test (SI Appendix, Fig.
S2 and Table S1). We could further filter potential candidates
by examining whether the histone 3 lysine 4 trimethylation
(H3K4me3) modification, a well-characterized marker of active
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/
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
V.G.S., E.S.L., and H.F.L. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE25678).
1
To whom correspondence should be addressed. E-mail: lodish@wi.mit.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1018384108/-/DCSupplemental.
PNAS | January 25, 2011 | vol. 108 | no. 4 | 1519–1524
GENETICS
Many human aneuploidy syndromes have unique phenotypic
consequences, but in most instances it is unclear whether these
phenotypes are attributable to alterations in the dosage of specific
genes. In human trisomy 13, there is delayed switching and
persistence of fetal hemoglobin (HbF) and elevation of embryonic
hemoglobin in newborns. Using partial trisomy cases, we mapped
this trait to chromosomal band 13q14; by examining the genes in
this region, two microRNAs, miR-15a and -16-1, appear as top
candidates for the elevated HbF levels. Indeed, increased expression of these microRNAs in primary human erythroid progenitor
cells results in elevated fetal and embryonic hemoglobin gene
expression. Moreover, we show that a direct target of these
microRNAs, MYB, plays an important role in silencing the fetal and
embryonic hemoglobin genes. Thus we demonstrate how the developmental regulation of a clinically important human trait can
be better understood through the genetic and functional study of
aneuploidy syndromes and suggest that miR-15a, -16-1, and MYB
may be important therapeutic targets to increase HbF levels in
patients with sickle cell disease and β-thalassemia.
p
Chr.
13
q
B
Partial Trisomy Cases
13
12
11.2
11.1
11
12
13
1
0.9
Elevated HbF
Portion with Trisomy
A
Normal HbF
14
21
22
Elevated HbF
Normal HbF
0.8
0.7
0.6
0.5
0.4
0.3
31
0.2
32
33
34
0.1
0
pter−q11 q12
q13
q14
q21
q22
q31 q32−qter
Chr. 13 Band
Chromosome Band
LOC646982
LOC646982
LOC646982
FOXO1
MRPS31
SLC25A15
SUGT1L1
1.0
Relative Expression
D
miR-15a
Chromosome Bands Localized by FISH Mapping Clones
13q14.11
13q14.12
13q14.2
13q14.3
RefSeq Genes
KIAA0564
EPSTI1
SERP2
KCTD4
CPB2
ESD
SUCLA2
FNDC3A
KCNRG
FAM124A
NEK5
DGKH
ENOX1
NUFIP1
SIAH3
HTR2A
NUDT15
FNDC3A
KCNRG
SERPINE3
NEK3
DGKH
ENOX1
KIAA1704
ZC3H13
HTR2A
MED4
MLNR
DLEU1
INTS6
NEK3
AKAP11
CCDC122
GTF2F2
CPB2
ITM2B
CDADC1
DLEU7
DHRS12
SUGT1
TNFSF11
C13orf31
TPT1
LCP1
RB1
SETDB2
RNASEH2B
ATP7B
TNFSF11
C13orf31
SNORA31
LRCH1
LPAR6
SETDB2
RNASEH2B
ATP7B
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
0.8
miR-16
0.6
0.4
0.2
0.0
80
E
1
3
5
7
9
Days of Differentation
Relative Expression
C
60
40
20
0
1
3
5
7
9
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 chromosomal band is shown in this diagram. The association with chromosomal band 13q14 finding is supported using Bayesian probabilistic models (SI Appendix). (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 promoter of the genes
from chromosomal band 13q14 in erythroid cell lines (defined as
having a peak of H3K4me3 within 1.5 kb upstream from the
transcription start site). Using data derived from the ENCODE
project (16), we were able to find a number of unique peaks (SI
Appendix, Table S2) and of the gene list established from the
expression data, we could focus on 9 candidate genes (SI Appendix, Table S3). Of these, we noted that a top candidate in this
region was a precursor RNA (DLEU2) for two microRNAs, 15a
and 16-1, which have identical seed targeting sequences (Fig.
1C). This was of particular interest, given the role that micro1520 | www.pnas.org/cgi/doi/10.1073/pnas.1018384108
RNAs play in modulating various aspects of hematopoiesis and
erythropoiesis specifically (17, 18). MiR-15a and -16 are expressed throughout human erythroid maturation in adult bone marrow cells and increase modestly with terminal differentiation
(Fig. 1 D and E), consistent with prior observations in cord blood
and erythroid cell lines (19, 20).
These observations suggest that increased expression of miR15a/16-1 in trisomy 13 could potentially result in elevated levels
of HbF expression. To directly test this hypothesis, we used
a lentiviral vector to increase expression of these microRNAs in
adult bone marrow-derived hematopoietic progenitors that subSankaran et al.
equivalent to controls at the later stages that represent more mature (basophilic) erythroblasts (Fig. 2 E and F) (7). Such findings
are reminiscent of the observations made using S-phase inhibitors
for HbF induction in primate models, where the most responsive
stages appeared to be the CFU-Es and proerythroblasts (22, 23).
To gain further insight into the molecular etiology for these
observations, we examined whether specific miR-15a/16-1 targets
may be potential mediators of the increased HbF expression.
Because evolutionary conservation of seed-matched targets shows
great utility in identifying bona fide microRNA targets (24), we
used a metric of target context and conservation (aggregate PCT,
which is a Bayesian estimate of the probability that a site is conserved due to selective maintenance of miRNA targeting rather
than by chance) and compared this with relative expression of
mRNAs in the erythroid target tissues of interest relative to a large
number of other tissues (Fig. 3A and SI Appendix, Fig. S6). Using
this approach, MYB was one miR-15a/16-1 target that appeared to
be of great interest, given that it was highly expressed in erythroid
progenitors and had two conserved 8mer miR-15a/16-1 targeting
sites (Fig. 3B). This was particularly notable, because common
genetic variants from genomewide association studies have suggested that polymorphisms in the MYB locus are important mediators of variation in HbF levels in humans (25), which is further supported by the finding that overexpression of MYB in cell
lines causes a decrease in γ-globin expression (26). We found that
MYB protein levels were reduced with even a modest (two- to
threefold) increase in miR-15a/16-1 expression in erythroid cell
lines (Fig. 3C) and a luciferase reporter assay confirmed that
sequently underwent synchronous differentiation toward the erythroid lineage (7). By increasing expression of miR-15a and -16
by an average of 1.5-fold (SI Appendix, Fig. S3), similar to what
would be expected in the context of a trisomy, we found that the
levels of γ-globin gene expression were robustly increased by an
average of 2.4-fold (Fig. 2A). Because trisomy 13 can result in
elevated γ-globin synthesis even in newborns with elevated HbF
levels at baseline (21), we increased expression of these microRNAs by 2- to 3-fold in human erythroleukemia K562 cells (SI
Appendix, Fig. S4), which endogenously express high levels of
γ-globin, and found that the γ-globin levels could be further increased by 50% (Fig. 2B). Newborns with trisomy 13 also show
elevated expression of the embryonically expressed ε-globin, as
illustrated by the persistence of low level hemoglobin Gower 2
(α2ε2) expression (4). This increase was recapitulated in the primary bone marrow-derived cells with increased miR-15a/16-1
expression (Fig. 2C). Since alterations in γ-globin expression may
accompany altered differentiation of cells, we examined the lentivirally transduced primary adult erythroid progenitors and found
no major differences in the morphology or phenotype of cells with
increased miR-15a/16-1 expression compared with controls (Fig.
2D and SI Appendix, Fig. S5). To gain further insight into the
mechanism by which these microRNAs may be acting to elevate
γ-globin expression, we assessed cell cycle progression in the synchronously differentiating primary erythroid cells (7). Interestingly, we noted that cell cycle progression was slowed by miR-15a/
16-1 overexpression at the early stages of differentiation that
represent colony-forming unit erythroid cells (CFU-Es) or proerythroblasts (G1- and S-phase difference, P < 0.001), but was
C
B
Percentage of ε -Globin
40
-1
5a
-1
6
80
G1
S
G2/M
40
iR
-1
5
on
tr
ol
m
m
iR
-1
5
on
tr
ol
a16
0
C
0
GENETICS
80
C
-1
iR
m
F 120
Percentage of Cells
m
E 120
Percentage of Cells
miR
15a-16
C
iR
iR
m
Control
on
tr
ol
on
tr
ol
C
-1
5
a16
on
tr
ol
C
D
0.00005
0.00000
0.0
-1
0
0.5
-1
10
1.0
**
0.00010
a16
20
1.5
0.00015
-1
5
30
2.0
a16
40
Relative γ -Globin Expression
Percentage of γ-Globin
A
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 | January 25, 2011 | vol. 108 | no. 4 | 1521
MYB
5
MYB
0
1.1
-5
GAPDH
Aggregate PCT
D
B
0.8
***
0.4
0.0
40
20
a16
iR
m
Percentage of ε -Globin
60
0.0008
***
0.0006
***
0.0004
0.0002
0.0000
on
tr
ol
sh
M
YB
1
sh
M
YB
2
0
C
0.0
Percentage of γ -Globin
*** ***
*** ***
on
tr
ol
sh
M
YB
1
sh
M
YB
2
0.5
80
C
1.0
C
I
0.8
100
% of Maximum
Enrichment Score
H
G
F
on
tr
ol
sh
M
YB
1
sh
M
YB
2
Relative MYB Expression
E
-1
5
C
on
tr
ol
MYB 3’UTR 654-661 5’...AUGAAAAACGUUUUUUGCUGCUA...3’
MYB 3’UTR 681-688 5’...CUUAGCCUGUAGACAUGCUGCUA...3’
|||||||
hsa-miR-15a
GUGUUUGGUAAUACACGACGAU-5’
hsa-miR-16-1
GCGGUGAUAAAUGCACGACGAU-5’
1.2
-1
Log2 Expression Ratio
Co
nt
ro
m l
iR
-1
5a
-1
6
C
10
Relative Luciferase Activity
A
0.6
0.4
0.2
0.0
80
60
Cells in S-phase
Control - 68.65%
shMYB 1 - 39.94 %
shMYB 2 - 36.43 %
40
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 log 2 ratio. (B) Two 8mer target sites for miR-15a and -16-1 are located in the 3′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) Percentage of γ-globin (F ) and ε-globin (G) gene expression as a percentage of all
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),
consistent with previous studies (20, 27, 28).
To test whether MYB may be a critical mediator of γ-globin
expression, we reduced MYB expression with two shRNAs in
synchronously differentiating adult erythroid progenitors (Fig.
3E). This knockdown robustly increased γ-globin expression, as
1522 | www.pnas.org/cgi/doi/10.1073/pnas.1018384108
occurs to a lesser extent with miR-15a/16-1 elevation (Figs. 2A
and 3F). Concomitantly, we found that expression of the embryonic globin chain, ε-globin, was also dramatically increased by
MYB knockdown (Fig. 3G). Our examination of expression data
from a recent study of MYB siRNA treatment of umbilical cord
blood erythroid progenitors (29) demonstrated robust elevations
Sankaran et al.
Sankaran et al.
A
C
B
D
Fig. 4. Pathology from trisomy 13 autopsy cases reveals normal erythropoiesis and perturbed megakaryopoiesis. (A) Bone marrow (BM) section
from a trisomy 13 case reveals normal erythroid maturation with a normal
immature:mature progenitor ratio. (B) Abnormal and increased numbers of
megakaryocytes that have hypolobulated nuclei with a “staghorn” appearance are seen in a BM section. (C) Normal erythropoiesis and increased
megakaryocytes with hypolobulated nuclei seen in a BM section. (D) Normal
erythropoietic maturation and a megakaryocyte with a staghorn appearance are seen on this BM section. Examples of some dysplastic megakaryocytes with staghorn and hypolobulated nuclei are highlighted in the
images with cyan arrows. All images are shown at 400× magnification and
slides were stained with hematoxylin and eosin.
terestingly, MYB knockdown in human cells and hypomorphic
alleles in mice show similar phenotypes in megakaryocytes (19,
34–36), suggesting that miR-15a/16-1 overexpression and the
consequent reduction in MYB expression may alter other aspects
of hematopoiesis in trisomy 13 patients.
Discussion
We have demonstrated, using a combination of genetic and
functional approaches in human cells that the overexpression of
miR-15a/16-1 results in elevations in HbF gene expression and
this likely explains why patients with a trisomy of chromosome 13
have a delayed fetal-to-adult hemoglobin switch and persistence
of fetal hemoglobin. This effect is mediated, at least in part,
through down-modulation of the MYB transcription factor,
which we have shown is a potent negative regulator of HbF expression and is a direct target of miR-15a and -16-1 (Fig. 5). It is
possible that other genes on chromosome 13 may also contribute
to this phenotype, but miR-15a and -16-1 appear to have a major
MYB
Euploid Erythroid
Progenitor
MicroRNAs
15a and 16-1
Fetal
Hemoglobin
MYB
Midgestation
Birth
Trisomy 13
Progenitor
Fig. 5. A model demonstrating how elevations of microRNAs 15a and 16-1
in trisomy 13 can result in elevated fetal hemoglobin expression. Normally,
the basal level of these microRNAs can moderate expression of targets such
as MYB during erythropoiesis. In the case of trisomy 13, elevated levels of
these microRNAs results in additional down-regulation of MYB expression,
which in turn results in a delayed switch from fetal-to-adult hemoglobin and
persistent expression of fetal hemoglobin.
PNAS | January 25, 2011 | vol. 108 | no. 4 | 1523
GENETICS
of both γ-globin and ε-globin gene expression, even with higher
baseline levels of these globin genes at this stage of human ontogeny (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 performed with cells from one of the MYB knockdowns along with
a set of controls. We did not observe any significant difference in
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,
a similar type of knockdown can also result in increased production of megakaryocytes (19). Moreover, when gene sets were
created from the MYB knockdown expression sets compared
with controls, the up-regulated genes were found to be significantly enriched in the later stages of erythroid differentiation,
supporting the notion that precocious erythroid differentiation
was occurring in this context (SI Appendix, Fig. S10) (32). Together, these findings suggest that MYB is necessary for the
normal differentiation kinetics of adult erythroid cells and reduction in the level of this gene results in altered erythroid differentiation 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
in a marked slowing of cell cycle progression (Fig. 3I) and
marked up-regulation of γ-globin expression in erythroid cell
lines (SI Appendix, Fig. S11). In support of the slowing of cell
cycle progression, we noted that the expression of several cell cycle regulators were altered in our expression data from the MYB
knockdown cells compared with controls (SI Appendix, Fig. S12).
The altered cell cycle and differentiation kinetics of cells with
MYB knockdown are consistent with findings in erythroid cells
using hypomorphic myb alleles in mice (33). Our findings suggest
that MYB plays a critical role coordinating globin gene expression, cell cycle regulation, and erythroid differentiation.
Alterations in γ-globin expression can occur in the context of
stress erythropoiesis or other states where erythroid differentiation is perturbed. Patients with trisomy 13 and elevated HbF
levels do not have anemia (4, 21), but the evaluation of in vivo
erythropoiesis in these patients has not previously been reported.
To properly ascertain this, we examined autopsy specimens from
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
diagnosis of trisomy 13 and presence of appropriate histological
samples (SI Appendix, Table S4). In all of the samples examined,
erythropoiesis appeared appropriate for age, and full maturation
of the erythroid lineage could be appreciated (Fig. 4 A, C, and
D). This suggests that the elevated HbF levels in trisomy 13 can
occur without grossly perturbed erythropoiesis. In examining
other aspects of hematopoiesis, we found a dramatic increase in
megakaryocyte numbers in over 70% of the cases examined (Fig.
4 B and C and SI Appendix, Table S4). In addition, the majority
of the cases (64%) showed abnormal nuclear morphology of the
megakaryocytes, suggestive of decreased ploidy in these cells
(Fig. 4 B–D and SI Appendix, Fig. S13). These findings were
specific to patients with trisomy 13, as autopsy specimens from the
same institution, in the same time period, and in patients of similar
ages with other diagnoses lack these findings (SI Appendix). In-
impact. Our study demonstrates a unique and previously unappreciated pathway that regulates this intensively studied developmental process. The exact relevance of our findings to
normal physiology is not clear, but it appears to be likely that
these pathways may play an important role in the normal fetalto-adult hemoglobin switch (Fig. 5). Consistent with this notion,
common genetic variants close to the MYB gene appear to be
important regulators of HbF levels in adult humans (25). Further
work will be needed to gain insight into the exact role of these
factors in the mechanisms mediating human hemoglobin
switching. Nonetheless, it is apparent from our work that miR15a, -16-1, and MYB could be important therapeutic targets to
elevate HbF expression to ameliorate the severity of sickle cell
disease and β-thalassemia.
Our findings demonstrate the power of using human genetic
approaches to understand developmental processes, where the
use of model organisms can be limited (37). Importantly, our
findings suggest that alterations of gene dosage at specific genetic loci likely underlie the numerous phenotypes observed
uniquely with specific aneuploidy syndromes (1). In relatively
few cases has this been strongly supported by functional evidence. Similar types of approaches may help to uncover other
genotype–phenotype correlations in these fascinating syndromes
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1524 | www.pnas.org/cgi/doi/10.1073/pnas.1018384108
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Materials and Methods
Details of the cell culture approaches, constructs used, lentiviral preparation,
RNA analysis, flow cytometry, protein methods, and analysis can be found in
SI Appendix. CD34+ cells were cultured and differentiated using a two-phase
culture method in serum-free conditions, with appropriate cytokines added
at the various stages of the culture (7). RNA extraction, quantitative RT-PCR,
and microarray expression analysis were performed in a manner similar to
what has previously been described (7, 38). Lentivirus production and infection was carried out using a modified spin-infection method for the
erythroid progenitors that were grown in suspension (39). Flow cytometry
was performed using standard approaches (38) with data analysis occurring
in the FlowJo 7.5.5 software suite.
ACKNOWLEDGMENTS. We thank D. Nathan and S. Lux for their inspiration
and guidance; C. Epstein, D. Bartel, C. Walkley, C. Sieff, J. Menne, J.
Hirschhorn, J. Flygare, M. Bousquet, D. Nguyen, B. Wong, Y. Jeong, H. B.
Larman, and G. Bell for valuable advice; T. DiCesare for valuable assistance in
producing the model illustration; and C. Kitidis-Mitrokostas and N. Cohen for
technical assistance. T.F.M. was supported by the Kay Kendall Leukaemia
Fund. D.S. was supported by a Department of Energy Computational Science
Graduate Fellowship (Grant DE-FG02-97ER25308). This work was supported
by National Institutes of Health Grants R01 DK068348 and P01 HL32262
(to H.F.L.).
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