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HEMATOPOIESIS AND STEM CELLS
Undifferentiated hematopoietic cells are characterized by a genome-wide
undermethylation dip around the transcription start site and a hierarchical
epigenetic plasticity
*Yun Shin Chung,1 *Hye Joung Kim,1 Tae-Min Kim,1 Sung-Hyun Hong,1 Kyung-Rim Kwon,1 Sungwhan An,2
Jung-Hoon Park,1 Suman Lee,3 and Il-Hoan Oh1
1Catholic
Institute of Cell Therapy & Department of Cellular Medicine, College of Medicine, Catholic University of Korea, Seoul; 2Genomic Tree Inc, Daejeon; and
of Medical Life Science, CHA University, Pocheon, Republic of Korea
3Department
Evidence for the epigenetic regulation of
hematopoietic stem cells (HSCs) is growing, but the genome-wide epigenetic signature of HSCs and its functional significance remain unclear. In this study, from
a genome-wide comparison of CpG methylation in human CD34ⴙ and CD34ⴚ cells,
we identified a characteristic undermethylation dip around the transcription start
site of promoters and an overmethylation
of flanking regions in undifferentiated
CD34ⴙ cells. This “bivalent-like” CpG
methylation pattern around the transcrip-
tion start site was more prominent in
genes not associated with CpG islands
(CGIⴚ) than CGIⴙ genes. Undifferentiated
hematopoietic cells also exhibited dynamic chromatin associated with active
transcription and a higher turnover of
histone acetylation than terminally differentiated cells. Interestingly, inhibition of
chromatin condensation by chemical
treatment (5-azacytidine, trichostatin A)
enhanced the self-renewal of “stimulated” HSCs in reconstituting bone marrows but not “steady-state” HSCs in sta-
tionary phase bone marrows. In contrast,
similar treatments on more mature cells
caused partial phenotypic dedifferentiation and apoptosis at levels correlated
with their hematopoietic differentiation.
Taken together, our study reveals that the
undifferentiated state of hematopoietic
cells is characterized by a unique epigenetic signature, which includes dynamic
chromatin structures and an epigenetic
plasticity that correlates to level of undifferentiation. (Blood. 2009;114:4968-4978)
Introduction
Hematopoietic stem cells (HSCs) constitute a rare subpopulation in
hematopoietic tissues that can produce all lineages of blood cells
throughout life and reconstitute bone marrow when transplanted
into myeloablated hosts. The ability of HSCs to maintain or
reconstitute the hematopoietic system is dependent on their unique
ability to execute self-renewing divisions and multilineage differentiation potential.1 Numerous studies have identified key transcription factors involved in the self-renewal of HSCs2 and gene
expression patterns specific to primitive hematopoietic cells.3,4
However, it is still not clear how the dynamic regulation of gene
expression is coordinated to influence cell fate during the stochastic
process of cell fate decisions in HSCs.1
Recently, epigenetic modification of chromatin structures has
been implicated in the regulation of gene expression and development5 by influencing the accessibility of transcription factors to
DNA and altering the transcription profile of cells.6 The modification of chromatin structures is largely regulated by the acetylation
and methylation of histones (H3, H4), which act as switches
between permissive or repressive chromatin.7 In addition, DNA
methylation in promoter regions has been shown to cause transcriptional silencing of genes8 by promoting the binding of MeCP2, a
transcriptional repressor that recruits histone deacetylases (HDACs)
to methylated promoters.9
Accordingly, studies to characterize the epigenetic status of
pluripotent embryonic stem (ES) cells have revealed unique
features of ES cells, such as less condensed chromatin structures10,11 and the poised, that is “primed but held-in-check,”
expression of lineage-associated regulatory genes via a bivalent
mode of histone modification by Polycomb (PcG) group proteins.10,12 Accumulating evidence suggests that epigenetic modification also regulates hematopoietic differentiation.13-15 For example,
hematopoietic progenitor cells exhibit a promiscuous, low-level
expression of lineage-specific genes before commitment.16,17 Similarly, hematopoietic differentiation correlates to a stepwise decrease in the transcriptional accessibility of multilineage-affiliated
genes.16,18 In addition, changes in the expression of lineage-specifying
genes correlate with changes in chromatin structures in the promoter
regions during differentiation,19,20 and the epigenetic regulation of
defined sets of lineage-associated genes has been directly analyzed in
primary murine hematopoietic cells.21 However, the genome-wide
epigenetic signature of the undifferentiated state of hematopoietic cells
and its effect on HSC function in vivo still remain poorly understood.
In the current study, we analyzed the genome-wide DNA
methylation of undifferentiated human hematopoietic cells and
examined the effect of epigenetic modification on the hematopoietic function of HSCs in bone marrow under distinct physiologic
Submitted January 1, 2009; accepted August 9, 2009. Prepublished online as
Blood First Edition paper, September 14, 2009; DOI 10.1182/blood-2009-01197780.
The online version of this article contains a data supplement.
*Y.S.C. and H.J.K. contributed equally to this study.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
An Inside Blood analysis of this article appears at the front of this issue.
© 2009 by The American Society of Hematology
4968
BLOOD, 3 DECEMBER 2009 䡠 VOLUME 114, NUMBER 24
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BLOOD, 3 DECEMBER 2009 䡠 VOLUME 114, NUMBER 24
conditions. We show that undifferentiated hematopoietic cells can
be characterized by an undermethylation dip at the transcription
start site (TSS) of promoters and by chromatin structures in
dynamic equilibrium. We propose that this characteristic epigenetic
signature confers epigenetic plasticity and multilineage potential to
undifferentiated HSCs.
Methods
Animals
C57BL/6J-Ly 5.2 (BL6) mice or C57BL/6J-Pep3b-Ly5.1 (Pep3b) mice
were used as recipients or donors in congenic transplantations. Animals
were bred and maintained in sterile microisolator cages located in an
air-filtered room in the animal facility of the Catholic University of Korea.
Experiments were undertaken with the approval of the Animal Experiment
Board of the Catholic University of Korea.
Cells
Umbilical cord blood (UCB) cells were obtained from full-term normal
deliveries with informed consent from the parents and the approval of the Ethics
Committee for Medical Research of the Catholic University of Korea. CD34⫹
cells were purified by immunomagnetic selection (Dynal Biotech) from lowdensity cells, followed by flow-cytometric sorting for CD34⫹ in the FACSVantage (BD Biosciences). CD34⫺ cells were sorted from low-density cells. After
sorting, cells were reanalyzed by flow cytometry or xenotransplantation into
NOD/SCID mice to confirm depletion of HSCs (supplemental Figure 1,
available on the Blood website; see the Supplemental Materials link at the top of
the online article). To purify CD34⫹CD38⫹ or CD34⫹CD38⫺ cells, mononuclear cells were depleted of lineage marker⫹ cells by immunomagnetic column
(StemCell Technologies) and then stained with each antibody for further sorting.
Murine bone marrow cells enriched with progenitors were obtained after a 4-day
treatment with 5-fluorouracil (Sigma).22 For subsets of hematopoietic populations, bone marrow cells were first depleted of lineage (CD5, CD45R, CD11b,
TER119, Gr-1, 7-4)–positive cells using an immunomagnetic column (StemSep,
StemCell Technologies; Lin⫺) and then stained with Sca-1, c-Kit, or CD34 for
sorting for each specific population. Lineage-positive (Lin⫹) cells or their
subpopulations (B220⫹Sca-1⫺ or Mac-1⫹/Gr-1⫹Sca-1⫺) were also obtained by
sorting. Antibodies for fluorescence-activated cell sorting (FACS) were purchased from BD Biosciences PharMingen. Gates were set to exclude greater than
99.99% of nonspecifically stained propidium iodide (PI)⫺ cells that were
incubated with isotype-matched control antibodies labeled with corresponding
fluorochromes.
Genome-wide CpG methylation analysis
Genomic DNAobtained from CD34⫹ and CD34⫺ cells was fragmented by either
sonication or digestion with MseI. Recombinant methyl-binding domain (Genomic
Tree) was incubated with sonicated genomic DNA in a binding reaction mixture
(10mM Tris-HCl, pH 7.5, 50mM NaCl, 1mM ethylenediaminetetraacetic acid,
1mM dithiothreitol, 3mM MgCl2, 0.1% Triton X-100, 5% glycerol, 25 mg/mL
bovine serum albumin, and 1.25 ␮g/mL sonicated JM110 bacterial DNA) for
4 hours at 4°C on a rocking platform. This mixture was bound to Ni-NTA agarose
beads, and the pelleted beads were washed 3 times with binding buffer containing
700mM NaCl. The methyl-enriched DNA fraction was purified using Qiaquick
PCR purification kits (QIAGEN) and amplified using a whole genome amplification kit as recommended by the manufacturer (Agilent Technologies). Amplified
DNA products from CD34⫹ and CD34⫺ cells were labeled with Cy3-dUTP and
Cy5-dUTP, respectively, by random priming and hybridized onto Agilent human
CpG island (CGI) microarrays using conditions specified by the manufacturer
(Agilent Technologies). After washing, the slides were scanned using an Agilent
scanner and images were quantified using the Feature Extraction Software,
Version 9.3 (Agilent Technologies). Genes exhibiting significant (P ⬍ .01)
undermethylation around TSS or flanking overmethylation were identified using
modified algorithms previously reported.23 Briefly, 3-probe moving average of
log2 ratios were plotted in normal distribution, and the significance of relative
EPIGENETIC REGULATION OF HEMATOPOIETIC STEM CELLS
4969
undermethylation or overmethylation was calculated using z-statistics. Enrichment of gene function was analyzed using Database for Annotation, Visualization, and Integrated Discovery (http://david.niaid.nih.gov) as described.24 All
microarray data have been deposited in the GEO public database under accession
number GSE17833.
Analysis of methylation in repetitive DNA elements by
pyrosequencing
For methylation in repetitive elements, 500 ng of genomic DNA was treated
with sodium bisulfite using the EZ DNA methylation gold kit according to
the manufacturer’s protocol (Zymo Research). The bisulfite-treated DNA
(50 ng) was amplified by PCR. Pyrosequencing reactions were performed
automatically using a PSQ 96MA system (Pyrosequencing AB) according
to the manufacturer’s instructions. Methylation was quantified using the
PyroQ-CpG Software. The sequence of each primer is shown in supplemental Table 3.
Ex vivo culture
Stroma-free murine hematopoietic cells were cultured for 5 days in
serum-free medium containing Iscove modified Dulbecco medium plus BIT
(StemCell Technologies), supplemented with 10⫺4M 2-mercaptoethanol plus
40 ␮g/mL low-density lipoprotein (Sigma), 100 ng/mL murine steel factor
(R&D Systems), 100 ng/mL human Flt3 ligand (R&D Systems), and
50 ng/mL human thrombopoietin (CytoLab/PeproTech).22 Human hematopoietic cells were similarly cultured ex vivo in serum-free substitution
media containing 20% BIT as described.25 Each culture was supplemented
with growth factor cocktails containing 100 ng/mL human Flt3 ligand
(R&D Systems), 100 ng/mL human stem cell factor (R&D Systems), and
20 ng/mL human interleukin-3, granulocyte colony-stimulating factor, and
human interleukin-6 (R&D Systems).
In vivo and in vitro treatment with epigenetic inhibitors
For in vivo treatment during the regenerative phase, 1 ␮g/g 5-azacytidine (AZA)
or 0.5 ␮g/g trichostatin A (TSA) was intraperitoneally injected into recipient mice
for 2 weeks starting 3 days after transplantation. For treatment during the
stationary phase, the same dose of AZA or TSA was injected daily into donor
mice for 2 weeks. For treatment with AZA and TSA in an ex vivo culture, sorted
murine or human hematopoietic cells were precultured for 18 hours in serum-free
media supplemented with growth factors, and AZA (25 ng/mL; Sigma-Aldrich)
and TSA (25 ng/mL; Sigma-Aldrich) were added for 24 to 48 hours. The treated
cells were washed and injected into irradiated recipient mice or restained and
analyzed for phenotypes.
LTC-IC assays
Sorted CD34⫹CD38⫺ cells and CD34⫺ cells from human UCB were
serum-free cultured for 18 hours and treated with 5-AZA (25 ng/mL) and
TSA (25 ng/mL) for 48 hours ex vivo and then washed and subjected to a
long-term culture assay as described.26 Briefly, cells were transferred to a
35-mm dish containing a feeder layer of irradiated (8000 cGy) mouse
stromal cells, M210B4 (ATCC), and cultured in Myelocult medium
(StemCell Technologies) containing 10⫺6M hydrocortisone (Sigma) and
10⫺4M 2-mercaptoethanol (Sigma) for 6 weeks with half-medium changes.
For colony formation, cells in a long-term culture were harvested and plated
on semisolid methylcellulose media supplemented with cytokines.
In vivo repopulation and CRU assays
Transplantation of HSCs from primary recipient mice into congenic
secondary recipient mice was performed as previously described.27 To
calculate HSC numbers quantitatively, competitive repopulating unit (CRU)
assays were performed as previously described.28 Briefly, serially diluted
cells were transplanted into lethally irradiated (900 cGy) mice with
105 helper cells, and lymphoid and myeloid engraftment levels in the
recipient mice blood were determined after 16 weeks. Lineages of repopulated hematopoietic cells were analyzed by staining with anti-Mac-1/Gr-1
(BD Biosciences PharMingen) for myeloid cells and with anti-TB104 or
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4970
BLOOD, 3 DECEMBER 2009 䡠 VOLUME 114, NUMBER 24
CHUNG et al
anti-B220 antibodies (BD Biosciences PharMingen) for lymphoid cells.27
Recipient mice whose white blood cells contained 1% or more donor-derived
lymphoid and myeloid cells were scored as positive; 1 CRU was defined as the
cell dose at which 37% of the mice tested were negative for donor-derived
lymphoid and myeloid cells (negative mice).28 CRU frequencies and 95%
confidence intervals (CIs) were calculated by applying Poisson statistics to the
proportion of negative mice in groups of recipients given different numbers of
cells using L-Calc software (StemCell Technologies).
sulfate (SDS) loading buffer was added to the beads and samples were
electrophoresed for autoradiographic visualization.
Statistical analysis
The differences between the groups were evaluated using the Student t test
(P ⬍ .05). CRU frequencies and 95% CIs were calculated according to
Poisson statistics. The data represent the mean plus or minus SEM.
Western blot and pulse-chase labeling for histone acetylation
Sorted murine and human hematopoietic cells were lysed in 2⫻ Laemmli buffer
and subjected to electrophoresis and analyzed using antibodies against histone
acetyl transferase 1 (HAT1), HDAC1, HDAC2, DNMT1, DNMT3a, DNMT3b
(Santa Cruz Biotechnology), Me-H3K4, Di-MeH3K9, Ac-H4, Ac-H3K9/14,
MeCP2 (Upstate Biotechnology), or actin (Chemicon International). For pulsechase labeling of histone acetylation, sorted cell fractions were first cultured for
18 hours in serum-free media containing growth factors and then supplemented
with 3H-acetic acid (1 mCi/mL final concentration; GE Healthcare). After
4 hours, labeled cells were harvested and washed with cold PBS twice, then lysed
in 1⫻ radioimmunoprecipitation assay (RIPA) buffer supplemented with a
protease inhibitor cocktail (Roche Diagnostics), and phenylmethylsulfonyl
fluoride (Pierce). Lysates from 106 cells were precleared with protein A–Sepharose for 3 hours, and supernatants were incubated with Ac-H4 antibody or rabbit
IgG (Santa Cruz Biotechnology). Protein A–Sepharose beads were then added,
and the reactions were rotated for 3 hours at 4°C. After 3 washes, sodium dodecyl
A
Results
Undifferentiated hematopoietic cells display a genome-wide
undermethylation dip in the TSS of promoters
As a first approach to characterize the epigenetic signature of
undifferentiated hematopoietic cells, we compared the genomewide DNA methylation of undifferentiated (CD34⫹) and differentiated (CD34⫺) human hematopoietic cells. Methylated CpGenriched DNA fractions were collected from purified CD34⫹ and
CD34⫺ cells using methyl-CpG binding proteins29 and hybridized
to high-resolution CpG microarrays. The relative overmethylation
and undermethylation in CD34⫹ cells was determined by comparison with methylation levels in CD34⫺ cells. We first plotted mean
log2 intensity ratios (CD34⫹/CD34⫺) with respect to the distance
B
1500
Number of genes
Log2 ratio (CD34+/CD34-)
0.2
0.1
0
-150~+300bp
1000
+300~+1500bp
-1500~-150bp
500
-0.1
-0.2
-3000
0
-2000
0
1000
-1000
Distance (bp) from TSS
2000
3000
C
-2
D
0.4
-1
1
1000
-150~+300bp
CGI(+)
CGI(-)
Number of genes
0.3
Log2 ratio (CD34+/CD34-)
0
Mean Log2 ratio
0.2
0.1
+300~+1500bp
-1500~-150bp
CGI(+)
CGI(-)
CGI(+)
CGI(-)
CGI(+)
CGI(-)
500
0
-0.1
-0.2
-3000
-2000
-1000
0
1000
Distance (bp) from TSS
2000
3000
0
-2
-1
0
Mean Log2 ratio
1
2
Figure 1. Genome-wide analysis of CpG methylation in human CD34ⴙ cells compared with CD34ⴚ cells. The log2 intensity ratio of CpG methylation (CD34⫹/CD34⫺) was
averaged from 4 experiments. (A) The mean intensity ratio of probes is shown in 100-bp–sized windows up to 3 kb upstream and downstream of the TSS of known genes; error
bar represents 95% CI. Negative log2 ratio values are indicative of a relative undermethylation around the TSS (⫺150 bp to 300 bp of the TSS) in CD34⫹ cells compared with
CD34⫺ cells. In flanking regions, positive log2 ratio values are indicative of a relative overmethylation in this region, up to 3 kb of upstream and downstream of the TSS, in
CD34⫹ cells. (B) Genome-wide plot of the number of genes with respect to mean log2 ratios for 3 regions around the TSS (⫺150 bp to 300 bp, ⫺1.5 kb to ⫺150 bp, and 300 bp
to 1.5 kb of the TSS). The log2 distribution encompassing the TSS (⫺150 bp to 300 bp) exhibits a normal-shaped curve with a peak markedly shifted toward negative direction,
indicating an undermethylation in this region in CD34⫹ cells compared with CD34⫺ cells. (C) Separate analysis of log2 intensity ratios for CGI⫹ and CGI⫺ gene clusters. Log2
ratios for probes for CpG islands (CGI⫹) and non-CpG islands (CGI⫺) were separately plotted with respect to distance from the TSS. Both CGI⫹ and CGI⫺ genes exhibit an
undermethylation dip around the TSS in CD34⫹ cells compared with CD34⫺ cells and CGI⫺ genes are hypermethylated in the flanking regions. (D) A log2 distribution plot of
CGI⫹ and CGI⫺ genes shows the TSS flanking regions of CGI⫺ genes are skewed toward positive values, indicating hypermethylation.
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BLOOD, 3 DECEMBER 2009 䡠 VOLUME 114, NUMBER 24
EPIGENETIC REGULATION OF HEMATOPOIETIC STEM CELLS
Table 1. The significance of enrichment of stemness-related
gene sets
Gene set ID*
-1.5 kb ~ -150 bp
-150 bp ~ +300 bp +300 bp ~ +1.5 kb
NANOG targets
0.201 (OM)
0.00652 (OM)
0.703 (OM)
OCT4 targets
0.984 (OM)
0.013 (OM)
0.272 (UM)
SOX2 targets
0.220 (OM)
0.006 (OM)
0.751 (UM)
NOS targets†
0.906 (UM)
0.240 (OM)
0.204 (UM)
NOS TFs
0.788 (UM)
0.394 (OM)
0.770 (UM)
SUZ12 targets
0.968 (OM)
0.130 (OM)
1.9E-06 (UM)
EED targets
0.244 (UM)
0.344 (OM)
6.2E-06 (UM)
H3K27 bound
0.423 (UM)
0.238 (OM)
8.5E-05 (UM)
PRC2 targets‡
0.919 (OM)
0.027 (OM)
2.5E-04 (UM)
MYC targets 1
0.403 (UM)
0.678 (OM)
0.273 (OM)
MYC targets 2
0.012 (OM)
0.123 (OM)
0.050 (OM)
For each gene set, 1.5 kb upstream and downstream of TSS regions were
subdivided into ⫺1.5 kb ⬃ ⫺150 bp, ⫺150 bp ⬃ ⫹300 bp, and ⫹300 bp ⬃ ⫹1.5 kb.
Mean log2 ratios of the 3 promoter regions were calculated, and the significance of
enrichment was calculated using the PAGE algorithm, while discriminating between
relative overmethylation (OM) and undermethylation (UM) of the gene sets in CD34⫹
cells relative to CD34⫺ cells. Significant (P ⬍ .05) enrichment is indicated by the
shaded boxes.
*Eleven stemness-related gene sets were selected from previously published
literature.
†NOS targets represent the overlap of the NANOG, OCT4, and SOX2 gene sets.
‡PRC2 targets represent the overlap of SUZ, EED, and H3K27 target genes.
from the TSS of known genes (Figure 1A). In a promoter-wide log2
intensity ratio plot, a dip in the log2 ratio was observed in the region
in close vicinity to the TSS (⫺150 bp to 300 bp), revealing a
relative undermethylation of this area in CD34⫹ cells. In contrast,
the flanking regions up to 1.5 kb of upstream and downstream of
the TSS were overmethylated in CD34⫹ cells. These results show
that, in CD34⫹ cells, CpG methylation around the TSS displays a
bivalent-like pattern. To delineate the relative undermethylation
and overmethylation around the TSS in CD34⫹ cells, we plotted the
number of genes with respect to the mean log2 ratios for 3 regions
around the TSS (⫺150 bp to 300 bp, ⫺1.5 kb to ⫺150 bp, and
300 bp to 1.5 kb from the TSS; Figure 1B). The log2 distribution
encompassing TSS (⫺150 bp to 300 bp) generated a normalshaped curve with a peak markedly shifted toward negative log2
values, indicating that undermethylation in this region is global and
not limited to a gene subset. Plots for the flanking regions upstream
A
4971
(⫺1.5 kb to ⫺150 bp) and downstream (300 bp to 1.5 kb) of the
TSS were skewed toward positive log2 values, indicating a
generalized overmethylation of genes in these regions.
Next, to analyze CpG methylation with respect to CGIs, probes
specific for CGI (CGI⫹) and non-CGI (CGI⫺) genes were analyzed. As
shown in Figure 1C, the mean log2 ratios reveal that the TSS regions of
both CGI⫹ and CGI⫺ genes had undermethylation/overmethylation
patterns but that, overall, CGI⫹ genes had a more prominent undermethylation dip and CGI⫺ gene were characterized by more prominent
overmethylation in the flanking regions of the TSS. In addition, the
mean log2 distributions show more prominent skewing toward positive
mean log2 values in the flanking regions of CGI⫺ genes (Figure 1D).
These results show that CGI⫹ and CGI⫺ genes are distinctively
regulated by CpG methylation.
Having observed global differences in CpG methylation in
CD34⫹ and CD34⫺ cells, we next examined CpG methylation
around 11 known stemness-related genes30 (Table 1), including
transcriptional targets of Nanog, OCT4, SOX2, Myc, and previously identified PcG-regulated genes. Mean log2 ratios were
calculated for 3 regions around the TSS of these 11 genes, and the
significance of enrichment for individual gene sets was determined
using the PAGE algorithm.31 As shown, the log2 intensity ratio of
the transcriptional targets of known pluripotency-related factors,
NANOG, OCT4, and SOX2,23 showed significant deviation toward
positive log2 ratio in the TSS region, indicating a reduced extent of
undermethylation around the TSS (Figure 2A; Table 1). In contrast,
log2 intensity ratio plots of PcG-regulated genes, including the identified
targets of SUZ, EED, and H3-lysine 27,32 revealed significant undermethylation in downstream flanking regions of the TSS (Figure 2B;
Table 1). Taken together, these results show that pluripotent genes
in CD34⫹ cells are relatively overmethylated in their TSS and
genes involved in “poised” gene expression12 are relatively undermethylated downstream of their TSS. However, we detected no
significant shift in log2 plots of specific genes that are highly
up-regulated in embryonic stem cells30 or in log2 plots of hematopoiesis-related lineage specific regulators33 (data not shown).
Next, we examined repetitive elements of the genome for CpG
methylation, as demethylation has been shown to be regulated within
these elements during early development.34,35 Methylation in CpG
islands of Alu-1, LINE-1,36 and Satellite-234 in human hematopoietic
cells at various differentiation stages was examined by pyrosequencing.
B
0.25
0.25
Mean
0.2
NANOG
OCT4
SOX2
0.15
0.15
0.1
0.1
0.05
0.05
0
0
-0.05
-0.05
-0.1
-0.1
-0.15
Mean
0.2
SUZ12
EED
H3K27
-0.15
-1.5
-1
-0.5
0
0.5
1
1.5
-1.5
-1
-0.5
CD34⫹/CD34⫺
0
0.5
1
1.5
Figure 2. Analysis of CpG methylation for selected stemness-related genes. Mean log2 ratios of plots for
of 11 stemness-related genes (Table 1). (A) Log2
ratios with respect to the distance from the TSS for transcription targets of known pluripotency-related factors, NANOG, OCT4, and SOX2 (average of 4 experiments). Positive
log2 ratios around the TSS indicate that this region is less undermethylated. (B) Mean log2 ratio plots for PcG-regulated genes, including the genes identified as targets of Suz,
Eed, and H3-lysine 27, show significant enrichment of negative log2 ratios in downstream flanking regions of the TSS. A characteristic right shift indicates an undermethylation
dip in the downstream flanking region of TSS for PcG-regulated genes.
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BLOOD, 3 DECEMBER 2009 䡠 VOLUME 114, NUMBER 24
CHUNG et al
A
CD34+
CD38101
102
103
c-Kit-PE
CD38-PE
CD34+
CD38+
100
100
104
40
102
103
104
6
30
ALU
element
101
CD34-FITC
CD34-FITC
B
Figure 3. Comparison of CpG methylation in repetitive elements.
(A) Representative FACS profiles for human and murine hematopoietic
subpopulations. (B) Methylation levels of CpG dinucleotides in each
repetitive element as indicated. Shown are the mean percentage of
methylated CpGs for 3 independent locus (denoted as CpG 1, 2, and 3) in
each region as determined by pyrosequencing analysis (n ⫽ 3).
Lin- c-Kit+ Lin-c-kit+
CD34+
CD34-
CD34-
4
B1
element
20
2
10
0
100
0
30
80
20
60
LINE1 (1.3)
40
ID
element
10
20
Satellite 2
0
100
0
100
80
80
60
60
40
40
20
20
0
Microcentric
Satellite
0
CD34+ CD34+ CD34CD38- CD38+
CpG1
LinLinckit+ ckit+
CD34- CD34+
CpG2
Lin+
CpG3
Comparable levels of CpG methylation in these regions were found in
primitive CD34⫹CD38⫺, undifferentiated but committed CD34⫹CD38⫹,
and differentiated CD34⫺ cells (Figure 3). Similar results were obtained
for repetitive elements (B1, ID, and microcentric satellite) of murine
hematopoietic cells (Figure 3). These results show that undermethylation and overmethylation patterns in undifferentiated hematopoietic cells are limited to nonrepetitive DNA elements during
post-developmental stages.
Undifferentiated hematopoietic cells undergo dynamic
epigenetic modifications
Next, we examined nucleosomal histone modifications at different
stages of hematopoietic differentiation. We compared the acetylation of histone (H4 and H3) in 3 human hematopoietic subpopulations37: CD34⫹CD38⫺, CD34⫹CD38⫹ cells, and CD34⫺ cells. As
shown in Figure 4A, human CD34⫹CD38⫺ and CD34⫹CD38⫹
cells exhibited higher levels of H4 and H3 acetylation than
terminally differentiated CD34⫺ cells. Moreover, the level of
H3K4 methylation, associated with active chromatin regions,38 was
highly enriched in undifferentiated (CD34⫹) cells, whereas the
level of H3K9 methylation, associated with repressed chromatin
regions,39 was enriched in terminally differentiated (CD34⫺)
hematopoietic cells (Figure 4A). Similarly, undifferentiated Lin⫺ckit⫹CD34⫺ and Lin⫺c-kit⫹CD34⫹ murine bone marrow cells also
exhibited higher levels of H4/H3 acetylation and H3K4 methylation, the histone modification associated with active transcription,
but lower levels of repressive H3K9 methylation compared with
differentiated (Lin⫹) cells (Figure 4A). Interestingly, undifferentiated cells of both human (CD34⫹) and murine (Lin⫺) origin
expressed higher levels of HDAC 1 and 2 and HAT1 than
differentiated cells (human CD34⫺ and murine Lin⫹ cells; Figure
4B), suggesting that histone acetylation turnover may be higher in
undifferentiated cells. To examine the rate of histone acetylation
turnover in these cells, we performed pulse-chase experiments
using hematopoietic cells exposed to 3H-acetic acid in a short-term
culture and compared the rate of de novo histone acetylation. As
shown in Figure 4C-D, incorporation of 3H-acetyl residues and H4
acetylation was higher in undifferentiated human and murine
hematopoietic cells (CD34⫹ and Lin⫺, respectively) compared
with differentiated cells (human CD34⫺ and murine Lin⫹ cells),
indicating that histone modification is more dynamic in these cells.
Taken together, these results show that undifferentiated hematopoietic cells are characterized by more open chromatin structures and
undergo more dynamic chromatin modifications than differentiated
cells, which have more condensed chromatin structures and more
stable epigenetic modifications.
Epigenetic modifications enhance self-renewal of HSCs in
“stimulated” but not “steady-state” bone marrows
Having observed epigenetic modifications characteristic of undifferentiated cells, we next addressed whether the epigenetic status of
HSCs has a functional impact on hematopoietic functions in vivo
by examining if epigenetic changes induce changes in the selfrenewal of HSCs during various physiologic conditions of bone
marrow. To this end, we first examined the effects of chemically
inhibiting DNA methylation (with AZA) and histone deacetylation
(TSA) on the self-renewal of CRUs28 during regenerating bone
marrow condition. HSCs were transplanted into recipients myeloablated by irradiation, and AZA or TSA was injected during the
initial 2 weeks of reconstitution, the time period when transplanted
HSCs undergo active self-renewal in the bone marrow.27 Sixteen
weeks after transplantation, the number of donor-derived CRU
regenerated in each recipient mice marrows was determined by
limiting dilution transplantations into secondary recipients (Figure
5A top panel). As shown in Figure 5Bi, a high number of CRUs
were regenerated from the TSA-treated group and a moderate
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BLOOD, 3 DECEMBER 2009 䡠 VOLUME 114, NUMBER 24
A
CD34+ CD34+
CD38- CD38+ CD34-
EPIGENETIC REGULATION OF HEMATOPOIETIC STEM CELLS
LinLinc-kit+ c-kit+
CD34- CD34+ Lin+
C
Lin-
4973
Lin+
Inp. IgG Ac-H4 Inp. IgG Ac-H4
Ac-H3K9/14
3
Ac-H4
LinIgG
Me-H3K4
Lin+
H4 IgG
H4
anti-H4
Di-MeH3 K9
H-Ac-H4
H4
β-actin
B
D
HDAC1
HDAC2
CD34+
CD34-
Inp. IgG Ac-H4 Inp. IgG Ac-H4
3
H-Ac-H4
HAT1
CD34+
β-actin
IgG
anti-H4
H4
CD34IgG
H4
H4
Figure 4. Undifferentiated hematopoietic cells have more open and dynamic chromatin than differentiated cells. (A) Levels of active (Me-H3K4) and repressive
(Di-Me-H3K9) histone modifications and acetylation of histones (Ac-H3K9/14, Ac-H4) were compared at different stages in human (left row) and murine (right row)
hematopoietic cells. (B) Protein expression levels of each indicated histone-modifying enzymes from human (left) and murine (right) hematopoietic cells.
(C-D) Pulse-chase labeling of histone acetylation in lineage-negative (Lin⫺) and lineage-positive (Lin⫹) murine bone marrow cells (C) and in human CD34⫹ and CD34⫺
cells (D). (Top panels) Autoradiography of immunoprecipitations using the antibodies as indicated from 106 input cells. Inp indicates input cells; IgG, immunoprecipitation with
isotype IgG; Ac-H4, immunoprecipitation with antibody against acetylated H4. (Bottom panels) Immunoblots using antibody against total form of H4 from 106 input cells.
number from the AZA-treated group compared with the number of
donor-derived CRUs regenerated from control mice (751 CRUs for
TSA-treated, 256 CRU for AZA-treated, and 27 CRUs for control
mice). These results suggest that open chromatin structures influence the self-renewal properties of HSCs in vivo during the
regenerative process of bone marrow.
We next examined whether TSA and AZA treatment has similar
effects on steady-state HSCs in nonstimulated bone marrow.
Nonirradiated mice were injected with TSA or AZA for 2 weeks,
and the bone marrow CRU content of the treated mice was
determined by a limiting dilution assay (Figure 5A bottom panel of
schematic). No significant difference in CRU frequency among
each mice group was found (10 000 CRUs vs 9220 and 10 000 CRUs
for control vs TSA- and AZA-treated groups, respectively; Figure
5Bii). Thus, our data reveal that increased HSC self-renewal in
response to epigenetic modifications occurs only in “stimulated”
but not “steady-state” marrows. These results suggest that
opening chromatin structure maintains HSCs in an undifferentiated state and that extrinsic signals are required to execute their
self-renewal. In support of this, primitive hematopoietic cells
(LSK) cultured in vitro with AZA and TSA maintained their
level of undifferentiation to a much higher level than cells
cultured with cytokine alone (45% in treated vs 3.3% in control
group, respectively, P ⬍ .05; Figure 6B).
Taken together, these results show that epigenetic modifications
indeed influence HSC function in vivo and that open chromatin
structures are associated with a higher maintenance of the undifferentiated state and a corresponding higher probability of selfrenewal after extrinsic stimulation.
Hematopoietic cells display a hierarchical flexibility to
epigenetic alteration and reprogramming
Based on the observations in HSCs, we next examined the effects
of AZA and TSA on more mature cell populations to determine
whether dedifferentiation can be induced in these cells. Terminally
differentiated myeloid (Mac-1/Gr-1⫹Sca-1⫺) or B-lymphoid
(B220⫹Sca-1⫺) cells, or cells at an intermediate level of differentiation (Lin⫺Sca-1⫺c-kit⫺; L⫺S⫺K⫺) were purified, cultured in vitro
with TSA and AZA, and compared with chemically treated
primitive Lin⫺Sca-1⫹c-kit⫹ (L⫺S⫹K⫹) cells.
As shown, L⫺S⫺K⫺ and Lin⫹ (both myeloid and Blymphoid) cells exhibited extensive apoptosis in response to
AZA and TSA treatment compared with L⫺S⫹K⫹ cells (80% for
L⫺S⫺K⫺, 97% for Lin⫹, and 20% for L⫺S⫹K⫹; Figure 6A). The
extent of apoptosis thus correlated with the degree of terminal
differentiation of these cells. Interestingly, of the surviving
L⫺S⫺K⫺ cells, 11% were now L⫺S⫹K⫹, a more primitive
phenotype indicating that a phenotypic dedifferentiation had
occurred in these cells (Figure 6B). Similarly, although terminally differentiated (myeloid and B-lymphoid) cells treated with
AZA and TSA underwent extensive apoptosis, the phenotype of
some of the surviving cells was Lin⫺, a more immature cell
phenotype (54% and 30% of the surviving myeloid-treated and
B lymphoid–treated cells, respectively; Figure 6C-D), but no
cells with a L⫺S⫹K⫹ phenotype were detected in either cell
population (data not shown). Moreover, myeloid and B-lymphoid
cells that had been treated with TSA and AZA in vitro had no
repopulating activity when transplanted into mice (Figure 6E),
indicating that the extent of their dedifferentiation is limited.
Similarly, treatment of primitive human CD34⫹38⫺ cells with
AZA/TSA caused low levels of apoptosis (1.3%; Figure 6F) and a
high maintenance of undifferentiated (CD34⫹) cells during ex vivo
culture compared with untreated control cells (90% vs 68% of
cultured cells for the treated and control group, respectively,
P ⬍ .05; Figure 6G), which correlated to a concomitant increase in
the number of long-term culture colonies (986 vs 312 from treated
and control group, respectively, P ⬍ .05; Figure 6H left). In
contrast, treatment of differentiated CD34⫺ cells with AZA/TSA
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4974
BLOOD, 3 DECEMBER 2009 䡠 VOLUME 114, NUMBER 24
CHUNG et al
A
B
Tx. during regenerative phase
Ly5.2 2 wk
Donor
cell
Ly5.1
1º Recipients
16 wk
i
Regenerated
HSCs
DMSO
AZA
TSA
Donor-derived
CRUs
regenerated
in 1¶ mice
(95% C.I)
1/6
0/6
0/6
1/692,600
(1/98,700
~1/4,861,300
)
27
(4~192)
191000
47800
12000
6/6
2/5
2/5
1/50,900
(1/22,700
~1/114,100)
751
(335~1682)
304000
76000
19000
4/5
1/5
0/5
1/244,900
(1/100,400
~1/597,500)
256
(105~624)
No. donor
cells
regeneration
DMSO
5.3x107
4.74x106
93800
23700
5930
TSA
4.94x107
9.56x106
5-AZA
5.07x107
1.57x107
Limiting
dilution
No. CRUs
regenerated in
1º Recipients
CRU
Freq.
Total
BM cell
No. in
1¶ mice
2º Recipients
Donor
Reconst.
cells
in 2¶
into
mice
2¶ mice
ii
Tx. during stationary phase
Ly5.1
DMSO
AZA
TSA
Treated
donor
No. of
CRUs
in
donor
mice
Total
BM cell No.
in 1¶ mice
In put
cell
dose
Freq. of
reconstitution
CRU
frequency
CRU/
mouse
(95% C.I)
DMSO
5.73X107
2000
10000
50000
0/5
1/5
5/5
1/22,900
(1/9,700
~1/54,200)
10010
(4230~23670)
TSA
5.19X107
2000
10000
50000
0/5
3/5
4/5
1/22,900
(1/9,700
~1/54,100)
9080
(3840~21470)
5-AZA
3.33X107
2000
10000
50000
1/5
2/5
5/5
1/13,700
(1/5,700
~1/33,100)
9720
(4020~23500)
Figure 5. Comparison of the effects of AZA or TSA treatment on HSC self-renewal in distinct bone marrow conditions. (A) Schematic illustration of the experimental
design comparing the effects of AZA and TSA treatment on HSCs during the regenerative or stationary phase in bone marrow. (Top panel) Epigenetic treatment on HSCs during
the regenerative phase of bone marrow. Donor bone marrow cells (105/mouse; Pep 3b, Ly5.1) were transplanted into lethally irradiated recipient mice (BL6, Ly5.2; n ⫽ 3).
Recipient mice were then injected daily with AZA or TSA for 2 weeks starting 3 days after transplantation, the time period when transplanted HSCs undergo active
self-renewal.27 After 16 weeks, the total number of donor-derived CRU regenerated in the primary recipients was determined by a limiting dilution analysis into secondary
recipient mice. (Bottom panel) Epigenetic treatment on HSCs during the stationary phase of bone marrow. Mice (Pep3b, Ly5.1) in the homeostatic phase were injected daily
with TSA or AZA for 2 weeks, and CRU frequencies and total CRU numbers in the bone marrows of treated mice were determined by a limiting dilution analysis. (B) Effects of
epigenetic treatment on HSC self-renewal during the regenerative (i) and stationary (ii) phases of bone marrow. Shown are the CRU frequencies of donor-derived cells
determined by Poisson statistics. CRU frequencies and 95% CIs were calculated by applying Poisson statistics. Total number of CRU in the mice was calculated assuming that
2 femurs and tibias represent 25% of the total marrow.
caused high levels of apoptosis (27%; Figure 6F) and limited levels
of phenotypic dedifferentiation into CD34⫹ cells (14% in treated
CD34⫺ cells; Figure 6G), which was associated with a limited
acquisition of long-term culture cells from treated CD34⫺ cells
(5 colonies in the treated group only; Figure 6H right).
Taken together, these results demonstrate that hematopoietic
cells at different stages of differentiation have distinct responses
to epigenetic alterations; undifferentiated cells are permissive to
changes in their chromatin structures, whereas differentiated
cells are nonpermissive to epigenetic changes and exhibit high
levels of apoptosis. This suggests that hematopoietic cells have
an epigenetic flexibility that correlates to their degree of
undifferentiation.
Discussion
Numerous studies on ES cells10-12 and hematopoietic progenitors16-18,21
have provided evidence for epigenetic regulation of stem cell fates
during HSC maintenance. However, the effects of a particular
epigenetic status on HSC cell fate decisions or on hematopoietic
functions in vivo are key questions that still remain unresolved.
In the current study, we have attempted to identify the global
epigenetic signature for the undifferentiated state of hematopoietic
cells and determine its effect on hematopoietic functions in vivo.
We first compared global CpG methylation in nonrepetitive
elements of genes in human CD34⫹ cells to that in CD34⫺ cells.
We found a striking genome-wide undermethylation dip around the
TSS and an overmethylation of flanking regions in undifferentiated
CD34⫹ cells compared with CD34⫺ cells.
To have further insight on this methylation pattern, genes
exhibiting significant (P ⬍ .01) undermethylation dip around
TSS region was identified (supplemental Table 1). Interestingly,
89 genes thus identified were significantly enriched with nuclear
proteins for chromatin modeling (supplemental Table 2). When
gene expression study was performed for selected genes in the
group using real-time PCR, 10 of 11 genes examined showed
higher level transcripts in CD34⫹ cells than in CD34⫺ cells
(supplemental Figure 2). These results suggest undermethylation near TSS is an epigenetic structure related to active
transcription of genes, and that, at least, one function of such
patterns in CD34⫹ cells should include active modeling of
chromatin structures in undifferentiated state of the cells.
Consistent with this view, we found that undifferentiated
hematopoietic cells of both human and murine origins exhibited
rather higher-level expression of the DNMT3a, DNMT3b, an
enzymes for de novo DNA methylation being essential for
self-renewal of hematopoietic cells,40 as well as higher levels of
MeCP2 (supplemental Figure 3). Thus, undifferentiated hematopoietic cells, despite cellular conditions for higher DNA methylation, are under active maintenance of undermethylation dip
around TSS, which allows higher-level transcription of
genes, including those for chromatin remodeling. However,
variations were also observed in the extent of undermethylation
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BLOOD, 3 DECEMBER 2009 䡠 VOLUME 114, NUMBER 24
4975
B
A
: Control
: AT
80
60
40
20
40
30
20
10
B
L-S-K-
Mac-1/Gr-1+Sca-1-
Myeloid
L-S+K+
D
B220+Sca-1-
B220
Mac-1/Gr-1
Control
: Control
: AT
40
0
E
L-S+K+
60
B220
B220+Sca-1-
*
L-S-K-
20
AT
Mac-1/Gr-1
B
80
% of Lin- cells
Myeloid
*
*
0
0
C
: Control
: AT
50
% of L-S+K+ cells
% of PI positive cells
100
% of engraftment (Ly5.1)
Figure 6. Effects of epigenetic treatment on mature hematopoietic populations. (A-D) Murine bone marrow cells were
sort-purified into primitive (Lin⫺Sca-1⫹c-Kit⫹; LSK), intermediate (Lin⫺Sca-1⫺c-Kit⫺; L⫺S⫺K⫺), and terminally differentiated
B-lymphoid (B220⫹Sca-1⫺) or myeloid (Mac-1⫹/Gr-1⫹Sca-1⫺)
cell populations. Each cell population was treated with AZA
(25 ng/mL) and TSA (25 ng/mL) (denoted as AT) for 24 hours.
(A) The extent of apoptosis after treatment was measured as
the percentage of PI⫹ cells. (B) The percentage of L⫺S⫹K⫹
cells was determined by restaining the cells with the indicated
antibodies. *Less than 0.1%. (C-D) Effects of epigenetic
treatment on B-lymphoid (B220⫹Sca-1⫺) and myeloid (Mac-1⫹/
Gr-1⫹Sca-1⫺) cells were examined after restaining cultured
cells with the indicated antibodies. Representative FACS
profile (C) and mean ⫾ SD% Lin⫺ cells (D) after culture are
shown (5 independent experiments). AT indicates treatment
with both AZA and TSA. (E) Effects of epigenetic treatment on
the repopulating activity of mature hematopoietic cells.
B220⫹Sca-1⫺ and Mac-1⫹/Gr-1⫹Sca-1⫺ cells (each 105 cells)
were treated with epigenetic modifiers (AT) for 24 hours and
transplanted with helper cells (105 cells each) into irradiated
recipient mice together. Shown is the percentage of donorderived cells in the peripheral blood of individual recipient
mice 16 weeks after transplantation (n ⫽ 5 for control and
8 for treated group). (F-G) Effects of epigenetic treatment on
human UCB-derived hematopoietic cells. Sorted CD34⫺ and
CD34⫹CD38⫺ cells (106 and 105, respectively) were treated
with AZA (25 ng/mL) and TSA (25 ng/mL) for 48 hours and
analyzed for apoptosis with PI⫹ cells (F) and percentage
CD34⫹ cells after culture (G). Shown are the mean ⫾ SD from
3 experiments. (H) Effects of epigenetic treatment on hematopoietic cells in a long-term culture. CD34⫹38⫺ and CD34⫺
cells (104 and 106 cells, respectively) were treated with AZA
and TSA for 48 hours and subjected to a long-term culture as
described in “LTC-IC assays” in “Methods.” Shown is the total
number of colonies and 12-day colony assays obtained after a
6-week long-term culture from 3 independent experiments.
*No colonies were obtained.
EPIGENETIC REGULATION OF HEMATOPOIETIC STEM CELLS
*Myeloid
B
Mc-1+Gr-1+Sca-1-
3
2.3
2
1.9
1.8
1.8
1
0
Control
AT
Control
AT
G
F
100
40
: Control
: AT
20
10
0
: AT
80
30
% of CD34+ cells
% of PI positive cells
: Control
60
40
20
CD34+CD38-
0
CD34-
CD34+CD38-
CD34-
1200
No. of colonies from1x106 cells
No. of total colonies from 1x104 cells
H
CD34+CD38-
1000
800
600
400
200
0
control
near TSS when 6 of the literature-based genes being overexpressed in primitive hematopoietic cells41 were randomly selected and subjected to pyrosequencing analysis (supplemental
Figure 4). Further studies in larger-scale analysis would be
necessary to elucidate genome-wide significance of undermethylation dip.
Interestingly, the genes with (CGI⫹) and without (CGI⫺)
CpG islands were distinctly regulated by CpG methylation;
CGI⫹ genes had a prominent undermethylation dip near TSS,
whereas non-CGI genes were characterized with a TSSlocalized undermethylation and a prominent overmethylation of
AT
10
CD348
6
4
2
0
Q
control
AT
the flanking regions. It has been previously shown that CGI⫺
genes are highly enriched for lineage-specific genes, whereas
CGI⫹ genes are largely composed of either developmental
regulators or housekeeping genes.33,42,43 Consistent with this
notion, our enrichment analysis revealed that the genes with
significant (P ⬍ .01) overmethylation in the flanking regions are
enriched with negative regulators of transcriptions or lineagespecific genes (supplemental Table 2). Thus, it appears that
overmethylation in flanking regions is related to restriction for
lineage-specific genes but derepression for transcription factors,
whereas undermethylation near TSS is related to permissive
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4976
BLOOD, 3 DECEMBER 2009 䡠 VOLUME 114, NUMBER 24
CHUNG et al
Epigenetic
Plasticity
Multi-lineage
potential
Figure 7. Schematic illustration of the proposed
model for the epigenetic plasticity of hematopoietic
cells. Undifferentiated hematopoietic cells (A) have dynamic chromatin and display permissiveness to changes
in chromatin structures and multilineage differentiation
potential. In the undifferentiated state, epigenetic treatment (B) that opens chromatin structures (AZA/TSA)
enhances HSC self-renewal when other extrinsic signals
are present. In contrast, differentiated cells (C) have
stable chromatin and are resistant to epigenetic changes.
Epigenetic treatment with AZA/TSA leads to a partial
dedifferentiation toward more immature cell phenotypes
(D), although most of differentiated cells undergo extensive apoptosis. Thus, epigenetic plasticity correlates to
the level of undifferentiation of hematopoietic cells.
Stem cell state
undifferentiated
TSA/AZA
Self-renewal
A
dynamic
chromatin
B
stable
chromatin
D
Non-Stem cell
state
TSA/AZA
Partial
de-differentiation
C
apoptosis
Stability of
Epigenome
& cell type
Differentiation
expression of multiple transcriptional regulators. It is therefore
probable that both undermethylation around TSS and flanking
overmethylation may independently contribute to the low-level
“poised” expression of lineage-specific genes, which is a
characteristic of undifferentiated hematopoietic cells.16,17 A
similar difference in the regulation of CGI⫹ and CGI⫺ genes was
also recently reported in a study using a murine hematopoietic
progenitor cell line, where most poised H3K4Me2⫹/Me3⫺ genes
were CGI⫺ genes, whereas unpoised genes were GGI⫹ genes.33
However, it is yet to be explored to what extent the “bivalentlike” DNA methylation patterns contribute to the chromatin
structures in undifferentiated cells.
Importantly, in addition to dynamic maintenance of DNA
methylation during undifferentiation, we also show that the
dynamics of chromatin structures vary with hematopoietic
differentiation. Previous studies have shown that chromatin
exists in a dynamic equilibrium between “open” and “closed”
states, the so-called “fluidity” of chromatin,44 and that these
dynamic changes in the chromatin states may be mediated by
nucleosome remodeling and histone acetylation.45 We found that
undifferentiated hematopoietic cells displayed the characteristics of less condensed chromatin structures; they had higher
levels of the active forms of histone modification and histone
acetylation, similar to those seen in pluripotent ES cells.10,11 In
addition, undifferentiated hematopoietic cells of both murine
and human origin were enriched with both HATs and HDACs
and exhibited a higher incorporation rate of histone acetylation
compared with mature cells. These results suggest that the
undifferentiated state of hematopoietic cells can be also characterized by a higher turnover rate of chromatin modifications, or
chromatin fluidity, than differentiated cells.
However, one key question is whether such epigenetic
signatures are predictive of and therefore determine a particular
property of HSCs, particularly during hematopoietic processes
in vivo. In previous studies using ex vivo culture models,
chemically inhibiting epigenetic modifications resulted in
changes in HSC cell fate and increased HSC self-renewal.46-48
However, it was shown that these in vitro cultures were prone to
epigenetic changes,49 and the effect of epigenetic modification
on in vivo hematopoietic functions remained, until now, unclear.
Here, we found that treatment with TSA and AZA enhanced
HSC self-renewal during bone marrow reconstitution as demon-
strated by increased donor-derived CRUs. Interestingly, TSA
preferentially promoted HSC self-renewal in vivo compared
with AZA (Figure 5B top panel), whereas AZA rather than TSA
has been shown to preferentially promote self-renewal in vitro,48
revealing that DNA methylation and histone acetylation have
distinct effects on HSC properties in vitro and in vivo. In
contrast, the self-renewal of HSCs in stationary phase bone
marrow was not enhanced after chemically induced epigenetic
modifications (Figure 5B bottom panel). The differences in HSC
self-renewal observed for chemically treated “stimulated” and
“steady-state” bone marrows suggest that priming and/or maintenance of undifferentiated state, rather than the direct induction
of self-renewing divisions, is induced by the open chromatin
structures after epigenetic modification.
In support of this, undifferentiated murine and hematopoietic
cells treated with AZA and TSA had a higher maintenance of
undifferentiated cells. Moreover, mature (L⫺S⫺K⫺ or Lin⫹) cell
populations exhibited dedifferentiation after similar treatments,
indicating that the open chromatin structures of hematopoietic
cells induce maintenance or acquisition of an undifferentiated
state. In contrast to the response observed in undifferentiated
hematopoietic cells, mature hematopoietic cells responded to
the epigenetic treatments with rapid and extensive levels of
apoptosis, which correlated to the degree of terminal differentiation of the cells. Thus, our findings reveal that the chromatin
structures of undifferentiated hematopoietic cells are more
dynamic than those of differentiated cells and that observed
differences in permissiveness to epigenetic changes reflect
differences in the epigenetic plasticity of the chromatins in
hematopoietic cells. Consistent with our observations, ES cells
were recently shown to have hyperdynamic chromatin proteins
when the cells are in a pluripotent phase but proteins immobilized on chromatin on differentiation.50
Therefore, as illustrated in Figure 7, we propose that the
undifferentiated state of hematopoietic cells correlates to the level
of epigenetic flexibility and, therefore, that high epigenetic plasticity correlates with a high multilineage differentiation potential.
Further studies are required to better elucidate the functional
significance of epigenetic regulation and chromatin plasticity on
the multilineage potential and hematopoietic activities of HSCs.
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BLOOD, 3 DECEMBER 2009 䡠 VOLUME 114, NUMBER 24
Nevertheless, in the current study, we show that the undifferentiated state of hematopoietic cells can be epigenetically characterized by a TSS-localized undermethylation dip and dynamic chromatin structures with high epigenetic plasticity.
Acknowledgments
This work was supported by the Korea Science and Engineering Foundation (grant 2008-05981) and in part by the Ministry of Health, Welfare & Family of the Republic of Korea,
High-Performance Cell Therapy R&D Project (0405-DB010104-0006).
EPIGENETIC REGULATION OF HEMATOPOIETIC STEM CELLS
4977
Authorship
Contribution: Y.S.C., H.J.K., S.-H.H., K.-R.K., S.A., J.-H.P., and
S.L. performed experiments; T.-M.K. performed bioinformatics
analysis and interpretation; and I.-H.O. designed experiments,
interpreted data, supported grant, and wrote the manuscript.
Conflict-of-interest disclosure: S.A. is the founder of Genomic
Tree Inc. The remaining authors declare no competing financial
interests.
Correspondence: Il-Hoan Oh, Catholic Institute of Cell Therapy
& Department of Cellular Medicine, Catholic University of Korea,
School of Medicine, 505 Banpo-Dong, Seocho-Ku, Seoul, Korea;
e-mail: [email protected].
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2009 114: 4968-4978
doi:10.1182/blood-2009-01-197780 originally published
online September 14, 2009
Undifferentiated hematopoietic cells are characterized by a
genome-wide undermethylation dip around the transcription start site
and a hierarchical epigenetic plasticity
Yun Shin Chung, Hye Joung Kim, Tae-Min Kim, Sung-Hyun Hong, Kyung-Rim Kwon, Sungwhan An,
Jung-Hoon Park, Suman Lee and Il-Hoan Oh
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