G9a/GLP-dependent histone H3K9me2
patterning during human hematopoietic
stem cell lineage commitment
Xiaoji Chen,1,2Kyobi Skutt-Kakaria,2,3Jerry Davison,4Yang-Li Ou,2Edward Choi,5Punam Malik,6
Keith Loeb,5Brent Wood,7George Georges,5Beverly Torok-Storb,5and Patrick J. Paddison1,2,8
1Molecular and Cell Biology (MCB) Program,2Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle,
Washington 98109, USA;3The Evergreen State College, Olympia, Washington 98505, USA;4Public Health Sciences Division,
5Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA;
of Experimental Hematology, Cancer and Blood Diseases Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati,
Ohio 45229, USA;7Seattle Cancer Care Alliance, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA
G9a and GLP are conserved protein methyltransferases that play key roles during mammalian development
through mono- and dimethylation of histone H3 Lys 9 (H3K9me1/2), modifications associated with transcriptional
repression. During embryogenesis, large H3K9me2 chromatin territories arise that have been proposed to reinforce
lineage choice by affecting high-order chromatin structure. Here we report that in adult human hematopoietic
stem and progenitor cells (HSPCs), H3K9me2 chromatin territories are absent in primitive cells and are formed de
novo during lineage commitment. In committed HSPCs, G9a/GLP activity nucleates H3K9me2 marks at CpG
islands and other genomic sites within genic regions, which then spread across most genic regions during
differentiation. Immunofluorescence assays revealed the emergence of H3K9me2 nuclear speckles in committed
HSPCs, consistent with progressive marking. Moreover, gene expression analysis indicated that G9a/GLP activity
suppresses promiscuous transcription of lineage-affiliated genes and certain gene clusters, suggestive of regulation
of HSPC chromatin structure. Remarkably, HSPCs continuously treated with UNC0638, a G9a/GLP small
molecular inhibitor, better retain stem cell-like phenotypes and function during in vitro expansion. These results
suggest that G9a/GLP activity promotes progressive H3K9me2 patterning during HSPC lineage specification and
that its inhibition delays HSPC lineage commitment. They also inform clinical manipulation of donor-derived
[Keywords: G9a; GLP; H3K9me2; UNC0638; differentiation; hematopoietic stem and progenitor cell]
Supplemental material is available for this article.
Received July 5, 2012; revised version accepted September 28, 2012.
G9a/EHMT2 and GLP/EHMT1 are conserved protein
lysine methyltransferases that play key roles in regulat-
ing gene expression and chromosome structure during
mammalian development through de novo mono- and
dimethylation of histone H3 Lys 9 (H3K9me1/2) (for
review, see Collins and Cheng 2010), histone marks associ-
ated with transcriptional silencing (Litt et al. 2001; Noma
et al. 2001; Su et al. 2004; Wen et al. 2009). G9a and GLP
contain nearly identical Su(var)3-9 family SET methyl-
transferase domains, with which they bind and methylate
H3K9me0/1, and ankyrin repeat domains that create a
methyl-lysine-binding module that allows binding of
H3K9me1/2 marks separately from their catalytic domains
(Collins et al. 2008). Thus, G9a and GLP have separable
‘‘reading’’ and ‘‘writing’’ functions and can ‘‘read’’ their
own marks, which may allow nucleation and spreading
of H3K9me2 marks along chromatin, although to our
knowledge, this has not been demonstrated in mammals
(Collins and Cheng 2010).
G9a is essential for early mouse embryo development
and embryonic stem cell (ESC) differentiation (Tachibana
et al. 2002). Its loss abolishes methylated H3K9 in euchro-
matic regions (Tachibana et al. 2002; Rice et al. 2003).
However, H3K9 trimethylation (H3K9me3) (Peters et al.
2003), a transcriptional repressive mark found in hetero-
chromatic regions, is unaffected by G9a loss and is main-
tained by other methyltransferases; e.g., Suv39h or Setdb1
been associated with euchromatic gene silencing in several
cellular contexts: silencing of Oct4 gene in differentiating
mouse ESCs (Feldman et al. 2006), NRSF/REST-mediated
Article published online ahead of print. Article and publication date are
online at http://www.genesdev.org/cgi/doi/10.1101/gad.200329.112.
GENES & DEVELOPMENT 26:2499–2511 ? 2012 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/12; www.genesdev.org2499
silencing of neuronal genes in nonneuronal lineages
(Roopra et al. 2004), and PRDI-BF1-mediated silencing
during B-cell differentiation (Gyory et al. 2004). The
H3K9me2 mark can be found in isolated regions near
genes and also in large megabase chromatin blocks that
can be lineage-specific and/or lost in cancer cell lines,
which may be indicative of structural roles in maintain-
ing epigenetic memory during lineage formation (Wen
patterning in somatic cells or somatic stem cell self-
The mammalian hematopoietic system is hierarchi-
cally organized such that the developmental potential to
produce lineages and terminally differentiated cells is
progressively restricted (Supplemental Fig. S1; Doulatov
et al. 2012). However, our understanding of the molecular
events controlling hematopoietic stem cell (HSC) fate
decisions is only just emerging (Orkin and Zon 2008), and
methods to control stem cell fate remain elusive. This
has significantly limited the successful application of
HSC transplantation for patients with cancer, marrow
failure, hemoglobinopathies, autoimmune diseases, or
any other clinical condition that could benefit from an
infusion of HSCs or their progeny.
Here, we examined H3K9me2 patterning in normal
human hematopoietic stem and progenitor cells (HSPCs).
We show that G9a/GLPactivity drives progressive, genome-
wide H3K9me2 patterning in euchromatin during HSPC
lineage specification. Remarkably, HSPCs treated with
UNC0638, a G9a/GLP small molecular inhibitor (Vedadi
et al. 2011), altered H3K9me2 marks to better resemble
those observed in primitive CD34+CD90+CD38loCD45RA?
HSCs. UNC0638-treated HSPCs also better retain stem
cell-like phenotypes and function during in vitro expan-
sion. Moreover, cotreatment of HSPCs with UNC0638
and SR1, a small molecular inhibitor of the aryl hydro-
carbon receptor (AHR), recently shown to promote ex-
pansion of human HSPCs (Boitano et al. 2010), resulted in
further expansion of adult CD34+cells. Our findings
suggest that G9a/GLP-mediated H3K9me2 patterning is
involved in critical steps during HSPC lineage commit-
ment and that its inhibition leads to delayed differentia-
tion and retention of the primitive HSPCs.
G9a/GLP-mediated H3K9me2 patterning is progressive
during HSPC lineage commitment and reversed
by UNC0638 treatment
To investigate roles for G9a and GLP methyltransferase
function during human HSPC lineage specification, we
first examined global chromatin H3K9me2 patterning
using chromatin immunoprecipitation (ChIP) (O’Geen
et al. 2011). To this end, H3K9me2 ChIP sequencing
(ChIP-seq) analysis was performed on the following cell
cells (Majeti et al. 2007), unfractionated CD34+cells (which
contain mainly committed progenitors), CD41+CD61+
committed megakaryocytes (Megs) (Novershtern et al.
2011), CD3+T cells (Majeti et al. 2007), and the HS-5
human bone marrow stromal cell line (Fig. 1; Graf et al.
To ensure that H3K9me2 ChIP-seq peaks were specific
to H3K9me2 and G9a/GLP activity, in control ChIP-seq
experiments in unfractionated CD34+cells, we used
a recently developed chemical probe, UNC0638, which
potently and selectively inhibits both G9a and GLP
methyltransferase activity by blocking substrate access
to their SET methyltransferase domains (Vedadi et al.
The ChIP-seq analysis revealed an unexpected series of
results (Fig. 1). The most primitive HSCs displayed small
and fewer H3K9me2 peaks. Unfractionated CD34+cells,
containing mainly committed progenitors, showed higher,
defined peaks that generally occur at CpG islands (CGIs).
In differentiated Megs and T cells, peaks arising in CD34+
cells were elaborated on and expanded to form nearly
identical H3K9me2 territories in genic regions. Figure 1A
and Supplemental Figure S2A show representative sam-
ples of 80 kb of chromosome 11 and chromosome 19 from
the ChP-seq data. Virtually all genic regions showed a
Evidence for this pattern arose from multiple analyses
of the ChIP-seq data. First, the frequency of sequence
reads per H3K9me2 peak (when examining either peak
height or width) showed progressive increases from more
primitive to differentiated cells, which were blocked and
reversed by UNC0638 treatment of CD34+HSPCs (Fig.
1B; Supplemental Fig. S2B).
Second, examination of the H3K9me2 peaks arising in
CD34+HSPCs revealed ;95% overlap peaks in Megs and
T cells (Fig. 1C–E). Figure 1D shows this result for the
entirety of chromosome 11 using a hive plot representa-
tion, where green lines show H3K9me2 marks shared
between HSPCs, Megs, and T cells, and red lines show
marks that have expanded in Megs and T cells. The
results show that almost all H3K9me2 marks found in
HSPCs are transmitted to Megs and Tcells. Hive plots for
other chromosomes showed identical results (Supple-
mental Fig. S2C; data not shown).
Third, another dramatic result revealed in the hive
plots was that there are no lineage-specific H3K9me2
patterns transmitted from HSPCs to Megs or from HSPCs
to T cells. These patterns would appear as purple lines
between HSPCs and Megs or HSPCs and Tcells. This was
true for all other chromosomes as well (Supplemental Fig.
S2C; data not shown). This notion is supported by corre-
lation of peak overlaps in different populations: Megs and
T cells share >90% of overlap (Fig. 1E). These analyses
support a model for H3K9me2 patterning in HSPC differ-
entiation that is progressive but not lineage-specific, at
least within the cell types we examined. However, further
analysis of other lineages needs to be done to confirm that
H3K9me2 patterning is not lineage-specific.
Fourth, H3K9me2 peaks formed in CD34+cells spread
to surrounding regions in chromatin. As shown in Figure
1A, H3K9me2 marks appear to be nucleated at CGIs and
then spread through genic regions in between. Spreading
is suggested by thefact thatthe differentiated populations
Chen et al.
2500GENES & DEVELOPMENT
are derived from CD34+HSPCs. In fact, for this exper-
iment, the Meg population was derived during in vitro
differentiation directly from the CD34+HSPCs used
for ChIP-seq. Moreover, these patterns were not due
to in vitro differentiation artifacts, since uncultured
Tcells (from two different donors) gave the same highly
reproducible pattern as the Megs. Thus, the results
indicate that H3K9me2 marks nucleated in CD34+
cells spread to surrounding regions to form larger
H3K9me2 nucleation sites in HSPCs are enriched
at H3K4me3 sites and CGIs
We next examined H3K9me2 HSPC peak overlap with
nine epigenetic marks and other genetic landmarks in
HSPCs and differentiated cells. For epigenetic marks, we
used the data from the work of Cui et al. (2009), who
examined multiple histone marks in CD133+human
HSPCs and also differentiated CD36+erythrocytes (Fig. 2A;
Supplemental Fig. S3A). The most frequent overlap
occurred with H3K4me3. This mark is found at tran-
scription start sites (TSSs) and is associated with active
transcription when present with histone H3K36me3,
which is found in gene bodies (Kolasinska-Zwierz et al.
2009), or epigenetic bivalency when found in combi-
nation with repressive marks (Attema et al. 2007;
Gaspar-Maia et al. 2011). Interestingly, almost 50% of
the H3K4me3 peaks in either CD133+or CD36+cells
overlapped H3K9me2 peaks in CD34+HSPCs. This
result is also consistent with H3K9me2 being enriched
at TSSs (Fig. 2B,C) and may suggest roles for G9a/GLP
in facilitating chromatin structure and bivalency at
promoters primed for expression in HSPCs.
Another interesting overlap was H4K20me1 (;21% of
peaks in CD36+cells) (Supplemental Fig. S3A). In contrast
to H3K4me3, this mark is found away from TSSs and has
been implicated in regulating DNA damage responses,
patterning during HSPC lineage commit-
ment. ChIP-seq was performed on cells
from two independent donors with antibody
against H3K9me2 in progressive stages of
the hematopoietic lineages or treated with
HSCs (denoted here as ‘‘CD90+’’) and CD41+
CD61+Megs were sorted from the same
donors as the CD34+HSPCs on day 4 and
day 10 of primary cell cultures, respectively.
CD3+T cells were sorted from the blood
of two different donors. ‘‘CD34+_UNC’’
UNC0638 for 48 h. (A) Representative
tracks from the Integrated Genome Viewer.
The Y-axis indicates the number of reads
(from 0 to 50) detected in 50-base-pair (bp)
windows. ‘‘_1’’ and ‘‘_2’’ indicate biological
replicates. (B) The number of reads for each
peak, limited by the sample with the fewest
called peaks. (C) Venn diagram showing
peak overlap between CD34+HSPCs and
share >100 bp. (D) Hive plot representing
chromosome 11. The peaks are displayed
with accurate genomic distances as blue
nodes along the length of the axes, while
peak overlaps are displayed as connected
lines. Green lines represent peaks shared
between CD34+HSPCs, Megs, and T cells.
Red lines represent peaks shared only be-
tween Megs and T cells. Unshared peaks by
the three populations would appear as pur-
ple lines. (E) Heat map representing the
fraction of overlaps between different sam-
ples. Overlaps are defined as read density
peaks sharing at least 100 bp. The number
of peaks overlapping is divided by the total
number of peaks in that sample and dis-
played as a value between 0 and 1. Com-
parisons are made with the samples in the
ChIP-seq analysis of H3K9me2
H3K9me2 patterning in HSPCs
GENES & DEVELOPMENT2501
mitotic condensation, and also gene expression (Beck
et al. 2012).
However, the most striking overlap was with CGIs
(Fig. 2D; Supplemental Fig. S3B), which are DNA regions
of high CpG density and are less likely to be methylated
(Gardiner-Garden and Frommer 1987; Hodges et al. 2011).
CGIs are generally near the gene promoters and are
associated with regulation of gene expression (Saxonov
et al. 2006). There are 28,691 predicted CGIs in the
human genome (Cocozza et al. 2011). Of these, 79% are
associated with an H3K9me2 peak in CD34+HSPCs
(Fig. 2D), which represents 47% of total H3K9me2. This
result strongly suggests that nucleation of H3K9me2
peaks is coordinated with CGIs in CD34+HSPCs.
patterning is progressive during HSPC lineage specifica-
tion, H3K9me2 nucleation frequently occurs at CGIs in
HSPCs, patterning events are dependent on G9a/GLP
methyltransferase activity, and UNC0638 treatment al-
ters H3K9me2 patterning to better resemble those ob-
served in primitive HSCs.
G9a/GLP-H3K9me2 patterning is not required for
maintenance of global DNA methylation in HSPCs
The strong overlap of H3K9me2 nucleation sites in
CD34+cells with CGIs suggested the possibility that
H3K9me2 patterning may be coordinated with DNA
methylation. For example, G9a is shown to directly bind
to maintenance DNA methyltransferase DNMT1 during
S phase in Cos-7 cells (Este `ve et al. 2006), and G9a-
deficient mouse ESCs display DNA hypomethylation
(Ikegami et al. 2007). Therefore, we performed DNA
methylation array analysis probing 99% of RefSeq genes
and 96% of CGIs in CD34+cells with or without
UNC0638 treatment (Supplemental Fig. S4). However,
UNC0638 treatment did not lead to global changes in
DNA methylation. In fact, only ;0.02% of methylation
probes showed more than twofold difference compared
with the DMSO control. These results suggest that in-
hibition of G9a/GLP activity and H3K9me2 patterning
does not grossly perturb DNA methylation patterns in
HSPCs, consistent with previous observations in human
cancer cell lines (Vedadi et al. 2011).
Nuclear staining of H3K9me2 confirms progressive
patterning in committed HSPCs
To confirm that H3K9me2 patterning is progressive
during HSPC lineage commitment, we next performed
immunofluorescence (IF) staining of H3K9me2 marks in
primitive and committed HSPCs. This analysis revealed
that sorted CD34+CD90+CD38loCD45RA?HSCs showed
significantly less nuclear staining than the total CD34+
population (Fig. 3A,B). Cells stained with secondary
antibody only were used as a negative control (Fig. 3C).
Moreover, H3K9me2 staining in total CD34+cells re-
vealed the emergence of nuclear speckles or foci. As noted
above, UNC0638 treatment revealed that increases in
H3K9me2 staining and nuclear speckling arose as a result
CD34+HSPCs and nine other histone marks in CD133+HSPCs using data from the work of Cui et al. (2009). (B) Percentage of
H3K9me2 peak associations with gene bodies and TSSs. CD34+cells show enrichment at the TSS and deficiency in intergenic regions.
(C) H3K9me2 peak frequency relative to TSSs of genes. (D) Overlap between H3K9me2 peaks and University of California at Santa Cruz
(UCSC) CGIs in CD34+HSPCs.
Overlaps between H3K9me2 and other epigenetic marks and genetic landmarks. (A) Overlap between H3K9me2 peaks in
Chen et al.
2502 GENES & DEVELOPMENT
of G9a/GLP activity (Fig. 3A). The quantification of
staining did not examine foci per se but the entire nuclear
staining intensity (Fig. 3B); the difference in foci forma-
tion is likely to be more dramatic than total nuclear
staining. The formation of H3K9me2 foci in committed
HSPCs is consistent with progressive H3K9me2 pattern-
ing and development of H3K9me2-dependent higher-
order chromatin changes during lineage specification.
Nuclear staining of H3K9me2 in sorted stem and pro-
genitor cells during early commitment steps of HSC
differentiation also confirmed its progressive patterning
(Supplemental Fig. S5C,D).
Inhibition of G9a/GLP in HSPCs results
in promiscuous transcription of lineage-specific genes
and affects transcriptional regulation of certain
To evaluate the effect of G9a/GLP-dependent H3K9me2
on regulation of gene expression, we performed micro-
array gene expression analysis on unfractionated CD34+
cells with or without treatment with UNC0638 (Fig. 4;
Supplemental Fig. S6). Only 158 genes showed significant
alterations in expression (Supplemental Table S1). Inter-
estingly, among the 103 genes up-regulated by UNC0638
were those normally expressed in more mature hemato-
poietic cells as well as other tissues, including lung, liver,
and brain, as assessed using the UniProt tissue database
(Fig. 4A; Supplemental Table S2) and the Novartis normal
tissue compendium (Fig. 4B). Portions of these results
were confirmed by RT-qPCR analysis (Supplemental
Among the genes most significantly up-regulated by
UNC0638 were the embryonic and fetal hemoglobin
genes HBE1, HBG1, and HBG2 (Fig. 4C; Supplemental
Fig. S6A; Bauer and Orkin 2011). These genes are found in
a cluster of embryonic, fetal, and adult hemoglobin genes
on Chr11p15.5, which are progressively activated and
repressed during development by a DNA element up-
stream of the cluster called the locus control region (LCR)
(Chaturvedi et al. 2009). Consistent with our results,
G9a/GLP-H3K9me2 has been shown to facilitate silenc-
ing of HBE1, HBG1, and HBG2 during mammalian de-
velopment by altering the chromatin secondary structure
of LCR and the fetal hemoglobin genes (Chaturvedi et al.
HSCs (top panels), unfractionated CD34+cells (middle panels), and UNC0638-treated CD34+cells (bottom panels). Nuclei were
counterstained DAPI. (B,C) Mean intensity of H3K9me2 staining (B) or a secondary antibody-only staining (C); n > 380; (**) P < 10?15.
Nuclear staining of H3K9me2. (A) Deconvoluted Z-section pictures of H3K9me2 staining (green) in CD34+CD90+CD38loCD45RA?
H3K9me2 patterning in HSPCs
GENES & DEVELOPMENT 2503
In addition, we found evidence for G9a/GLP-dependent
regulation of other gene clusters, including Chr6p21
(CCL5/CCL23), and Chr19q13 (ZNF329/ZNF544) (Fig. 4D;
Supplemental Fig. S6B–D). For the latter zinc finger (ZNF)
cluster, UNC0638 treatment resulted in repression of gene
expression rather than derepression (Fig. 4D), suggesting
that G9a/GLP activity is required for the maintenance of
their expression in HSPCs. Previous studies have found
a protein motif called the Kruppel-associated box (KRAB)
domain in the majority of ZNF genes on chromosome 19,
which is critical for protein–protein interaction (Eichler
et al. 1998). KRAB-ZNF genes are largely involved in
transcriptional repression (Eichler et al. 1998).
These results suggest, first, that inhibition of G9a/GLP
by UNC0638 results in promiscuous transcription of
hematopoiesis-affiliated and nonhematopoiesis-affiliated
genes in HSPCs and, second, that G9a/GLP affects local
structure of chromatin at specific gene clusters in HSPCs.
Primitive HSCs have been hypothesized to have a more
‘‘open’’ chromatin structure that promotes promiscuous
transcription of both nonhematopoietic and hematopoi-
etic differentiation genes (Hu et al. 1997; Miyamoto et al.
2002; Ma ˚nsson et al. 2007). Our data indicate that G9a/
GLP and H3K9me2 patterning may help restrict tran-
scriptional promiscuity during HSPC differentiation.
Thus, one intriguing implication is that H3K9me2 helps
facilitate adoption of alternate chromatin structures re-
quired for lineage commitment and specification. If true,
inhibition of G9a/GLP and H3K9me2 patterning may
block or delay lineage commitment.
Inhibition of H3K9me2 patterning promotes primitive
cell phenotypes and expansion of CD34+cells, which
is further enhanced by SR1
To examine the possibility that G9a/GLP inhibition may
delay or block adoption of HSPC cell fates, we performed
a series of UNC0638 treatments on ex vivo cultures of
CD34+HSPCs followed by flow analysis of CD34 and
lineage cell surface markers (Fig. 5; Supplemental Fig. S7).
During a 2-wk time course, we observed that UNC0638
treatment increased the proportion of CD34+cells (23.6%
in UNC0638-treated vs. 9.2% in the cytokines alone)
while diminishing differentiated CD15+cells (Fig. 5A).
UNC0638 treatment also led to increases in the number
of total nucleated cells and CD34+cells, with 1 mM
UNC0638 having the best expansion effect (Fig. 5B).
transcriptional regulation of certain gene clusters. (A) Tissue classifications of genes that were significantly changed in expression
by UNC0638 with the UniProt tissue database. See also Supplemental Table S2. (B) Gene expression profiling performed using the
Broad Institute’s Molecular Signatures database. Genes that were considered differentially expressed were compared with the Novartis
normal tissue compendium. Multilineage genes up-regulated by UNC0638 are shown. (C,D) Clusters of genes that were significantly
changed in expression by UNC0638. Looking for genes in close proximity in the differential expression set identified these clusters. The
fold change represents the gene expression change between CD34+HSPCs treated with UNC0638 compared with the DMSO control.
Arrows indicate transcription directions.
Inhibition of H3K9me2 patterning in HSPCs results in up-regulation of multilineage gene expressions and affects
Chen et al.
2504GENES & DEVELOPMENT
Moreover, UNC0638-treated HSCs also better retained
CD49f, a marker associated with long-term repopulating
HSCs (Supplemental Fig. S7B; Notta et al. 2011). These
experiments were repeated multiple times with CD34+
cells derived from bone marrow of normal donors or
peripheral blood of G-CSF-mobilized donors with similar
effects. Molecular studies revealed that G9a and GLP had
similar expression in CD34+primitive cells and CD34?
differentiated cells (Supplemental Fig. S9A) and that
UNC0638 treatment led to global loss of H3K9me2
(about twofold to fourfold) in HSPCs and a lesser decrease
in H3K9me1 (;1.4-fold) without affecting H3K9me3
levels or the expression of G9a, consistent with direct
inhibition of its catalytic activity (Supplemental Fig. S9).
To further evaluate the effect of UNC0638 in pro-
moting primitive HSPCs, we compared and combined it
with treatments of SR1, a small molecule inhibitor of
AHR, which was recently shown to promote expansion
of human HSPCs in ex vivo cultures (Boitano et al.
2010). Flow analysis revealed that single treatments
with UNC0638 or SR1 enhanced the proportion of more
primitive HSPCs (Fig. 6A)—indicated by CD34+, CD38lo,
CD90+, and CD45RA?(Manz et al. 2002; Majeti et al.
2007)—compared with the no-drug control on day14. SR1
treatments reproduced previously published results.
Remarkably, cotreatment with SR1 and UNC0638 ap-
proximately doubled the effect of either drug alone for
retention of CD34+CD38loand CD34+CD90+cells, result-
ing in CD34+retention as high as 84% after14 d of culture,
compared with only 12% in untreated controls. Similar
results were obtained from CD34+cells from G-CSF-
mobilized and bone marrow cells from multiple donors
(Fig. 6A; Supplemental Fig. S10A).
To determine the cause of increase in CD34+cells, we
performed cumulative cell counts and viability assays
for the total nucleated cells produced by HSPC cultures
for 21 or 31 d as well as total CD34+cells produced
during the same time period. The results demonstrated
that the increase in proportion of CD34+cells was due to
increased expansion of CD34+cells. Notably, double
treatment increased G-CSF-mobilized CD34+expansion
>120-fold by day 17 and increased bone marrow CD34+
cells nearly 400-fold by day 31, while individual treat-
ments and mock controls were considerably less potent
(Fig. 6C; Supplemental Fig. S10C). Importantly, the live–
dead cell ratio did not change significantly between mock
and treatments (Supplemental Fig. S12A), demonstrating
that the increased cell counts were not due to increase in
cell survival. Moreover, carboxyfluorescein succinimidyl
ester (CFSE) dye retention assays revealed that by day 7,
all cells in culture had undergone at least four cell di-
visions, including CD34+cells (Supplemental Fig. S12B).
Thus, SR1/UNC0638 treatments did not result in main-
tenance of large numbers of quiescent cells. However,
treated cultures did exhibit better dye retention, sugges-
tive of expansion of slower-dividing primitive cells (Cheng
et al. 2000; Zhang et al. 2006).
Similar results were obtained using different medium
formulations, with or without serum and altered cyto-
kine conditions (e.g., using EPO instead of TPO or EPO
plus TPO) (Supplemental Fig. S13A,B; Birkmann et al.
1997), albeit overallCD34+retention variedbytreatment.
Moreover, another substrate-competitive inhibitor of
G9a/GLP, BIX01294 (Kubicek et al. 2007), phenocopied
UNC0638 treatments for CD34+retention (Supplemen-
tal Fig. S13C), while the use of an N-methyl analog of
UNC0638, UNC0737, which is highly similar in struc-
tural but >300-fold less potent against G9a/GLP than
UNC0638 (Vedadi et al. 2011), had no effect on HSPC
expansion (data not shown). Both results further sug-
gest that UNC0638 inhibition is specific to G9a/GLP
SR1 and UNC0638 have divergent effects on HSPC
expansion and gene expression
Given the observed differences in HSPC responses to SR1
and UNC0638 single treatments, we further investigated
motes primitive cell phenotypes and expansion of
CD34+cells. (A) Flow analysis of day 1 versus day 11
bone marrow (BM) CD34+CD38locells. CD34+CD38lo
cells were cultured with or without UNC0638 for
11 d. Multicolor flow analysis was performed on
CD34 (HSPC marker) and CD15 (differentiated
granulocyte marker). Lineage markers include CD3
(T cell), CD11b (monocyte/granulocyte), CD14
(monocyte), CD15, CD19 (B cell), CD56 (natural
killer cell), and CD235a (erythrocyte) (Baum et al.
1992; Manz et al. 2002). The colors shown are red
(CD34)+, magenta (CD15+), blue (HLA-DRhi), cyan
(HLA-DRlo), and green (CD13hi). Data are represen-
tative of multiple independent experiments using
G-CSF-mobilized peripheral blood mononuclear
cells (PBMC) or bone marrow-derived CD34+cells.
(B) UNC0638 from 0.5 mM to 2 mM increased the
number of both total nucleated cells (TNCs) and
CD34+cells, with 1 mM having the greatest expan-
sion effect on CD34+cells.
Inhibition of H3K9me2 patterning pro-
H3K9me2 patterning in HSPCs
GENES & DEVELOPMENT 2505
(pretreatment) or day 14 cultured with control (0.01% DMSO), UNC0638, SR1, or UNC0638/SR1 dual treatment. Primitive HSPCs are
detected by CD34+CD38lo(top panels), CD34+CD90+(middle panels), and CD34+CD45RA?(bottom panels). See also Supplemental
Figure S10 for flow analysis of bone marrow CD34+cells. (B,C) Fold expansion of total nucleated cells (TNCs) (B) and CD34+cells (C)
from control (blue diamonds), UNC0638-treated (green triangles), SR1-treated (red squares), or UNC0638 plus SR1-treated (purple
crosses) conditions (n = 3). (*) Student’s t-test, P < 0.01. (D) Engraftments of human day 14 expanded HSPCs in immunodeficient mice.
The frequency of SCID-repopulating cells (SRCs) was calculated by Poisson statistics (n = 5). The number of SRCs in each group was
calculated by multiplying its frequency by the total cell number at day 14. (E) Engraftment kinetics of absolute ANC in dog H501 that
received 9.2 Gy of TBI and 1.7 3 107expanded autologous CD34+cells per kilogram, which were cultured for 14 d in UNC0638 and SR1
(red line). As a positive control (gray lines), shown is ANC recovery after major histocompatibility complex-matched littermate or
unrelated cord blood progenitor cell transplantation with unmanipulated cells in 13 dogs after 9.2 Gy of TBI infused with cell doses
comparable with H501. Dotted line indicates ANC = 500 cells per microliter.
UNC0638 and SR1 additively enhanced retention of primitive HSPCs. (A) Flow analysis of CD34+PBMCs on day 1
the mechanism of SR1- and UNC0638-dependent HSPC
expansion on different HSPCs subpopulations. We began
by analyzing CD45RA-positive and CD45RA-negative
progenitor pools, since UNC0638 increased the propor-
tion of CD34+CD45RA?cells, while SR1 increased the
proportion of CD34+CD45RA+cells (Fig. 6A). To this end,
we purified CD34+CD45RA?and CD34+CD45RA+pop-
ulations from day 4 HSPC cultures, which had not
previously been treated with SR1 or UNC0638, and per-
formed expansion assays on the isolated cells (Supplemen-
tal Fig. S14A). The results revealed that UNC0638 had
no effect on the CD34+CD45RA+subpopulation, while
SR1 preferentially stimulates its expansion. In contrast,
both drugs had similar effects on CD34+CD45RA?cells
(about eightfold peak expansion), and combined treat-
ment resulted in 13-fold peak expansion (Supplemental
To further refine this experiment, expansion assays
were performed on five pools of myeloid progenitors
available in the HSPC culture system: HSCs, MPPs,
CMPs, MEPs, and GMPs (Supplemental Fig. S14B). Pro-
genitor pools were isolated by FACS and then expanded
with or without SR1 and UNC0638. In these assays, SR1/
UNC0638 cotreatment most dramatically affected the
primitive HSCs (i.e., CD34+CD90+CD38loCD45RA?)—the
CD34+cells expanded ;50-fold after 14 d in SR1/
UNC0638, compared with about sixfold expansion for
untreated controls. Moreover, single treatments were less
effective than cotreatment, except for the CD45RA+
GMP pool, where UNC0638 had no effect (Supplemental
We next examined SR1- and UNCC0638-dependent
changes in HSPC gene expressions to help determine the
degree of similarity in their molecular mechanisms of
action. We performed gene expression analysis for single-
treated and cotreated CD34+HSPCs (Supplemental
Tables S1, S3). The results revealed a degree of divergence
for singly treated HSPCs by cluster and multidimensional
scaling analysis (Supplemental Fig. S15A,B). In fact, there
was little overlap between single treatments and few
overall genes that showed significant changes (Supple-
mental Fig. S15C). These results suggest that UNC0638
and SR1 act through different mechanisms.
UNC0638- and SR1-treated HSPCs better retain
the ability to engraft and repopulate in vivo in mice
These results suggest that both UNC0638 and SR1
treatments are capable of enhancing the ex vivo expan-
sion of CD34+populations, and combining the two
compounds multiplies the effect of either one alone. To
demonstrate the retention of stem cell activity in vivo,
we performed engraftment and repopulation experiments
using SR1 and UNC0638 expanded HSPCs in small and
large animal models of HSC transplantation.
We first measured SCID-repopulating cells (SRCs) in
expanded human HSPC cultures using limiting dilution
assays (LDAs) in immune-compromised mice (Szilvassy
et al. 1990). In these assays, day 14 expanded HSPCs in
mock-, single-, or cotreated conditions were injected into
sublethally irradiated NOD/Scid/IL-2 receptor-g-null
(NSG) mice (n = 5). Eight weeks post-injection, percent-
ages of human CD45+cells in mouse bone marrow were
examined to determine SRC frequency. By this assay,
single-drug treatments resulted in an approximately two-
fold increase in the number of SRCs over mock treatment,
while cotreatment resulted in an approximately fivefold
increase (Fig. 6D). Importantly, human CD45+cells con-
tained both myeloid (CD33+) and lymphoid (CD19+) cells,
indicating that the expanded cells retained multilineage
reconstitution potential in vivo (Supplemental Fig. S16).
However, it should benoted thatincontrol experimentsin
which freshly isolated G-mobilized CD34+cells were
injected into NSG mice, as few as 200,000 CD34+cells
displayed engraftment and multilineage differentiation
in mice (n = 5), whereas no mouse was engrafted with
200,000 UNC0638/SR1 expanded day 14 CD34+cells (n =
5). This suggests that while SR1/UNC0638 treatmentdoes
enhance retentionofprimitivestem cellmarkersandstem
cell activity compared with the no-drug control expanded
cells, the overall stem cell activity as measured in this
surrogate assay still diminishes when compared with
We next examined the effects of SR1/UNC0638 treat-
ment on HSC activity during expansion of canine CD34+
cells, as previous studies conducted over four decades
show that the outcomes of HSC transplantation in dogs
accurately predict the outcomes in human patients
(Ostrander and Giniger 1997; Thomas and Storb 1999). To
this end, a recipient dog was given 9.2 Gy of total body
irradiation (TBI), a myeloablative dose, and then infused
with autologous day 14 SR1/UNC0638 expanded HSPCs,
1.7 3 107total nucleated cells per kilogram. To evaluate
reconstitution, absolute neutrophil counts (ANCs) were
monitored daily until complete hematopoietic recovery
for 84 d post-transplantation. Remarkably, transplanta-
tion of SR1/UNC0638 expanded cells led to full recovery
of the recipient (Fig. 6E; Georges et al. 2010). These results
are consistent with the notion that SR1/UNC0638 expan-
sion at the very least sustains canine HSC activity during
14-d expansion of HSPCs. This is in contrast to an
unpublished study (n = 4) in which canine CD34+HSPCs
cultured for 7–10 d in cytokines only failed to engraft in
dogs conditioned with 9.2 Gy of TBI (M Mielcarek, pers.
These results strongly suggested that SR1/UNC0638
cotreatment allows retention of primitive HSCs during in
vitro expansion. It holds promise for expanding HSPCs for
transplantation purposes (Dahlberg et al. 2011).
Here, we examined roles for G9a/GLP activity in normal
human HSPCs using an in vitro culture and differentia-
tion system and a newly developed chemical probe
targeting G9a/GLP, UNC0638 (Vedadi et al. 2011). Our
studies led to several unexpected findings. First, they
revealed that G9a/GLP-dependent H3K9me2 patterning
H3K9me2 patterning in HSPCs
GENES & DEVELOPMENT 2507
marks are nucleated at 79% of CGIs in CD34+HSPCs and
then spread to surrounding regions during differentiation
to form characteristic H3K9me2 territories in euchro-
matic regions of all chromosomes. Second, they sug-
gested that G9a/GLP and H3K9me2 patterning may help
restrict transcription of multilineage genes during HSPC
differentiation. Third, they showed that UNC0638 treat-
ment in G-CSF mobilized peripheral blood and bone
marrow-derived CD34+HSPCs promotes retention of
primitive HSCs in vitro and that this effect is enhanced
by cotreatment with the AHR inhibitor SR1 (Boitano
et al. 2010). Fourth, they demonstrated that UNC0638
and SR1 target primitive HSCs but through different
mechanisms, as judged by differences in expansion effects
on committed progenitors and gene expression profiles
after treatments. Taken together, these results suggest
that G9a/GLP-dependent H3K9me2 patterning plays key
roles in early lineage commitment of adult HSPCs.
However, these results also raise several questions re-
garding G9a/GLP function and H3K9me2 marks in
Key among these is: What specific roles do H3K9me2
marks play during HSPC lineage specification? During
mammalian development, G9a/GLP activity gives rise to
large organized chromatin K9me2 modification (LOCK)
regions of up to 4.9 Mb (Wen et al. 2009), which have been
proposed to facilitate retention of higher-order chromatin
structure and epigenetic memory. LOCKs show apparent
marks in the mouse brain and liver and human placenta
(Wen et al. 2009). Our results are consistent with forma-
tion of LOCK-like H3K9me2 territories during human
hematopoiesis and, moreover, support roles for H3K9me2
in the development of higher-order chromatin structures
during HSPC lineage specification. G9a/GLP-dependent
H3K9me2 marks arise in HSPC nuclei as ‘‘speckles,’’
which likely indicates the formation of organized chro-
matin structures during lineage commitment. Further-
more, UNC0638 treatment affects expression of multiple
genes appearing in chromosome gene clusters in HSPCs
(e.g., 6p21, 11p15, and 17q11), suggesting that H3K9me2
facilitates formation of local chromosome structures at
these loci. For example, G9a/GLP-H3K9me2 has been
shown to facilitate silencing of HBE1, HBG1, and HBG2
during mammalian development by altering the chroma-
tin secondary structure of LCR and the fetal hemoglobin
genes (Chaturvedi, et al. 2009). Expression of these genes
is derepressed in UNC0638-treated HSPCs. Thus, the
results are consistent with the notion that UNC0638
treatment partially blocks formation of higher-order
chromatin structure in HSPCs.
Another question is how H3K9me2 marks arise in
committed hematopoietic cells. Since G9a/GLP-depen-
dent H3K9 methylation can occur de novo, pre-existing
epigenetic marks are not required (Collins and Cheng
2010). Our results show that H3K9me2 nucleation sites
in HSPCs most strongly overlap with CGIs, perhaps
suggesting a functional link between H3K9me2 and
DNA methylation. Since CGIs are generally hypomethyl-
ated in HSPCs (Hodges et al. 2011), one possibility is that
G9a/GLP and DNMT activity are coordinated such that
H3K9me2 marks are laid down by default only where
CpGs are unmethylated (i.e., in CGIs). Alternatively,
specificity factors might target G9a/GLP to CGIs. For
example, in ESCs, G9a/GLP bind to UHRF1, which in
turn binds to hemimethylated CpG sites (Kim et al.
One intriguing implication of our results is that the
absence of H3K9me2 marks in HSCs may facilitate
adoption of alternate chromatin and chromosomal struc-
tures required for lineage commitment and specification.
This would be consistent with the concept that HSCs
harbor an open chromatin structure that results in pro-
miscuous transcription (Akashi et al. 2003), which is
incompatible with the presence of H3K9me2 chromatin
This study also has important implications for clinical
uses of human HSCs. One of the long-standing road-
blocks limiting application of HSCs has been ourinability
to effectively expand and/or immortalize HSCs ex vivo
(Dahlberg et al. 2011). Initial attempts at ex vivo expan-
sion of HSPCs focused on cytokine stimulation to sup-
port survival and proliferation of lineage-committed
progeny in the hope of expanding true HSCs as well
(Sauvageau et al. 2004; Dahlberg et al. 2011). However,
these attempts have largely failed to enhance in vivo
engraftment in patients.
One notable exception is stimulation of Notch signal-
ing in cord blood units (Ohishi et al. 2002; Delaney et al.
2010), which allows more rapid myeloid reconstitution in
patients with post-transplantation cytopenias. However,
it appears that Notch-expanded cord blood units may be
depleted of long-term repopulating HSCs (Dahlberg et al.
2011) and,as a result, are givenin combination with naive
cord blood units to provide stem cells to improve long-
term engraftment. Moreover, Notch-driven expansion
only affects fetal cord blood stem cells but has no effect
on adult human HSC expansion (Dahlberg et al. 2011).
However, Cooke and colleagues (Boitano et al. 2010)
recently discovered that SR1, a small molecular inhibitor
of the AHR, promotes expansion of CD34+human HSPCs
in ex vivo cultures. Our SR1 trials similarly support these
findings in adult stem cells, although SR1 treatment did
not dramatically affect expression of AHR pathway tar-
gets, as previously reported (Boitano et al. 2010).
Our studies with UNC0638 revealed that this drug on
its own had effects similar to SR1 with respect to re-
tention of CD34+HSPCs and also HSPC engraftment
activity in immunocompromised mice. Moreover, exam-
ination of SR1 and UNC0638 treatment revealed that
each affects both common and distinct populations of
HSPCs, with most dramatic effects observed on primitive
HSCs. The mechanisms giving rise to their expansion
effects were clearly divergent based on transcriptional
profiling, and there was no evidence of cross regulation of
AHR pathway and/or G9a/GLP gene expression.
We envision several clinical applications of SR1/
UNC0638 treatments. First is in the expansion of HSCs
for transplantation. There are many cases in which
transplantation products are critically limited (e.g., young
Chen et al.
2508 GENES & DEVELOPMENT
or small donors, prior treatment of the donor, or failure to
mobilize). Second is in accelerating transplantation re-
covery. Post-transplantation cytopenias, including neu-
tropenias and thrombocytopenias, are commonplace and
lead to life-threatening infections or bleeding and result
in costly, extended hospitalization (Dahlberg et al. 2011).
Our results from modeling transplantation of SR1/
UNC0638 cultures in a canine model suggest that com-
bining day 14 expanded cultures with nonmanipulated
HSPCs may help bridge post-transplantation neutropenia
in addition to providing long-term engraftment. Third, it
is conceivable that UNC0638 has a potential benefit to
patients with b-hemoglobinopathies by reactivating the
embryonic and fetal hemoglobin, whose activation is
associated with milder symptoms (Akinsheye et al.
2011). Last, we envision that SR1 and UNC0638 may be
combined with additional experimental manipulations to
practically immortalize single HSCs for unlimited expan-
sion while retaining developmental potential, similar to
ESCs or induced pluripotent stem cells.
In conclusion, our data strongly suggest that G9a/GLP-
mediated H3K9me2 patterning is required for HSPC
lineage specification and that its inhibition leads to
delayed differentiation and retention of the HSPCs. These
findings should prove useful for clinical and experimental
applications limited by current techniques to maintain
HSPCs in vitro.
Materials and methods
Human CD34+cells from G-CSF-mobilized peripheral blood or
bone marrow of healthy adults were purchased from the Fred
Hutchinson Cancer Research Center Cell Processing Shared
Resource. Cells were maintained in either serum-containing
medium (IMDM with 10% fetal calf serum, supplemented with
IL-3) or the serum-free medium (SFEM; StemCell Technologies),
supplemented with 13 antibiotics and 100 ng/mL SCF, IL-6,
Flt3L, and TPO. UNC0638 (Sigma) and SR1 (AMRI) were resus-
pended in DMSO and used at the indicated concentrations. Cells
were cultured at 37°C in 5% CO2/95% air at a density between
0.5 million and 1.5 million cells per milliliter.
Cells were cross-linked with 1% formaldehyde and sonicated to
achieve chromosome fragments of 200–400 bp. ChIPs were
performed using antibodies directed against H3K9me2 (Abcam).
Samples are prepared for Illumina-based sequencing using the
Encore NGS Library System I (NuGEN). High-throughput se-
quencing by synthesis (HT-SBS) was performed on an Illumina
HiSeq 2000 sequencer.
Additional methods and data analysis can be found in the
We thank Shelly Heimfeld, Irv Bernstein, Andrew Emili,
Matthew Ferro, David Emery, Tony Blau, and members of the
Paddison and Torok-Storb laboratories for helpful discussions.
We thank Shelly Heimfeld for providing human HSPCs; Melissa
Comstock, LaKeisha Perkins, and Cynthia Nourigat for their
help with xenograft studies; Robert Jordan for technical assis-
tance with canine transplants; Alyssa Dawson and Andy Marty
for technical assistance with microarray and next-generation
sequencing; the Cincinnati Cell Characterization Core (C4) and
its Molecular (Elke Grassman), Genomic (Mehdi Kedachhe), and
Bioinformatics (Bruce Aronow and Philip Dexheimer) subcores
for help with DNA methylation array and analysis; Julio Vazquez
Lopez for technical assistance with the fluorescence microscope;
Gretchen Johnson and Megan Wilson for help with flow analysis;
and Pam Lindberg for assistance in manuscript preparation. This
work was supported by FHCRC institutional funds (P.J.P); grants
from the Pew scholar program (to P.J.P.), NIH/NHLBI (U01
HL099993 to B.T.S., P.J.P., and X.C), and NIDDK/NIH (P30
DK56465 to B.T.S. and P.J.P.); the P30DK056465 740 CCEH Pilot
awards (to P.J.P. and X.C.); NHLBI U01-HL099997 Pilot award (to
P.J.P. and X.C.); and the HHMI/UW Molecular Medicine Scholar
award (to X.C.).
Akashi K, He X, Chen J, Iwasaki H, Niu C, Steenhard B, Zhang J,
Haug J, Li L. 2003. Transcriptional accessibility for genes of
multiple tissues and hematopoietic lineages is hierarchically
controlled during early hematopoiesis. Blood 101: 383–389.
Akinsheye I, Alsultan A, Solovieff N, Ngo D, Baldwin CT,
Sebastiani P, Chui DH, Steinberg MH. 2011. Fetal hemoglo-
bin in sickle cell anemia. Blood 118: 19–27.
Attema JL, Papathanasiou P, Forsberg EC, Xu J, Smale ST,
Weissman IL. 2007. Epigenetic characterization of hemato-
poietic stem cell differentiation using miniChIP and bisulfite
sequencing analysis. Proc Natl Acad Sci 104: 12371–12376.
Bauer DE, Orkin SH. 2011. Update on fetal hemoglobin gene
regulation in hemoglobinopathies. Curr Opin Pediatr 23:
Baum CM, Weissman IL, Tsukamoto AS, Buckle AM, Peault B.
1992. Isolation of a candidate human hematopoietic stem-
cell population. Proc Natl Acad Sci 89: 2804–2808.
Beck DB, Oda H, Shen SS, Reinberg D. 2012. PR-Set7 and
H4K20me1: At the crossroads of genome integrity, cell cycle,
chromosome condensation, and transcription. Genes Dev 26:
Birkmann J, Oez S, Smetak M, Kaiser G, Kappauf H, Gallmeier
WM. 1997. Effects of recombinant human thrombopoietin
alone and in combination with erythropoietin and early-
acting cytokines on human mobilized purified CD34+pro-
genitor cells cultured in serum-depleted medium. Stem Cells
Boitano AE, Wang J, Romeo R, Bouchez LC, Parker AE, Sutton
SE, Walker JR, Flaveny CA, Perdew GH, Denison MS, et al.
2010. Aryl hydrocarbon receptor antagonists promote the
expansion of human hematopoietic stem cells. Science 329:
Chaturvedi CP, Hosey AM, Palii C, Perez-Iratxeta C, Nakatani
Y, Ranish JA, Dilworth FJ, Brand M. 2009. Dual role for the
methyltransferase G9a in the maintenance of b-globin gene
transcription in adult erythroid cells. Proc Natl Acad Sci
Cheng T, Rodrigues N, Shen H, Yang Y, Dombkowski D, Sykes
M, Scadden DT. 2000. Hematopoietic stem cell quiescence
maintained by p21cip1/waf1. Science 287: 1804–1808.
Cocozza S, Akhtar MM, Miele G, Monticelli A. 2011. CpG
islands undermethylation in human genomic regions under
selective pressure. PLoS ONE 6: e23156. doi: 10.1371/journal.
H3K9me2 patterning in HSPCs
GENES & DEVELOPMENT2509
Collins R, Cheng X. 2010. A case study in cross-talk: The
histone lysine methyltransferases G9a and GLP. Nucleic
Acids Res 38: 3503–3511.
Collins RE, Northrop JP, Horton JR, Lee DY, Zhang X, Stallcup
MR, Cheng X. 2008. The ankyrin repeats of G9a and GLP
histone methyltransferases are mono- and dimethyllysine
binding modules. Nat Struct Mol Biol 15: 245–250.
Cui K, Zang C, Roh TY, Schones DE, Childs RW, Peng W,
Zhao K. 2009. Chromatin signatures in multipotent hu-
man hematopoietic stem cells indicate the fate of bivalent
genes during differentiation. Cell Stem Cell 4: 80–93.
Dahlberg A, Delaney C, Bernstein ID. 2011. Ex vivo expansion
of human hematopoietic stem and progenitor cells. Blood
Delaney C, Heimfeld S, Brashem-Stein C, Voorhies H, Manger RL,
Bernstein ID. 2010. Notch-mediated expansion of human cord
blood progenitor cells capable of rapid myeloid reconstitution.
Nat Med 16: 232–236.
Doulatov S, Notta F, Laurenti E, Dick JE. 2012. Hematopoiesis:
A human perspective. Cell Stem Cell 10: 120–136.
Eichler EE, Hoffman SM, Adamson AA, Gordon LA, McCready P,
Lamerdin JE, Mohrenweiser HW. 1998. Complex b-satellite
repeat structures and the expansion of the zinc finger gene
cluster in 19p12. Genome Res 8: 791–808.
Este `ve PO, Chin HG, Smallwood A, Feehery GR, Gangisetty
O, Karpf AR, Carey MF, Pradhan S. 2006. Direct interac-
tion between DNMT1 and G9a coordinates DNA and
histone methylation during replication. Genes Dev 20:
Feldman N, Gerson A, Fang J, Li E, Zhang Y, Shinkai Y, Cedar H,
Bergman Y. 2006. G9a-mediated irreversible epigenetic in-
activation of Oct-3/4 during early embryogenesis. Nat Cell
Biol 8: 188–194.
Gardiner-Garden M, Frommer M. 1987. CpG islands in verte-
brate genomes. J Mol Biol 196: 261–282.
Gaspar-Maia A, Alajem A, Meshorer E, Ramalho-Santos M.
2011. Open chromatin in pluripotency and reprogramming.
Nat Rev Mol Cell Biol 12: 36–47.
Georges GE, Lesnikov V, Baran SW, Aragon A, Lesnikova M,
Jordan R, Laura Yang YJ, Yunusov MY, Zellmer E, Heimfeld
S, et al. 2010. A preclinical model of double- versus single-
unit unrelated cord blood transplantation. Biol Blood Mar-
row Transplant 16: 1090–1098.
Graf L, Iwata M, Torok-Storb B. 2002. Gene expression profiling
of the functionally distinct human bone marrow stromal cell
lines HS-5 and HS-27a. Blood 100: 1509–1511.
Gyory I, Wu J, Feje ´r G, Seto E, Wright KL. 2004. PRDI-BF1
recruits the histone H3 methyltransferase G9a in transcrip-
tional silencing. Nat Immunol 5: 299–308.
Hodges E, Molaro A, Dos Santos CO, Thekkat P, Song Q, Uren
PJ, Park J, Butler J, Rafii S, McCombie WR, et al. 2011.
Directional DNA methylation changes and complex inter-
mediate states accompany lineage specificity in the adult
hematopoietic compartment. Mol Cell 44: 17–28.
Hu M, Krause D, Greaves M, Sharkis S, Dexter M, Heyworth C,
Enver T. 1997. Multilineage gene expression precedes com-
mitment in the hemopoietic system. Genes Dev 11: 774–
Ikegami K, Iwatani M, Suzuki M, Tachibana M, Shinkai Y,
Tanaka S, Greally JM, Yagi S, Hattori N, Shiota K. 2007.
Genome-wide and locus-specific DNA hypomethylation in
G9a deficient mouse embryonic stem cells. Genes Cells 12:
Kim JK, Este `ve PO, Jacobsen SE, Pradhan S. 2009. UHRF1 binds
G9a and participates in p21 transcriptional regulation in
mammalian cells. Nucleic Acids Res 37: 493–505.
Kolasinska-Zwierz P, Down T, Latorre I, Liu T, Liu XS, Ahringer
J. 2009. Differential chromatin marking of introns and
expressed exons by H3K36me3. Nat Genet 41: 376–381.
Kubicek S, O’Sullivan RJ, August EM, Hickey ER, Zhang Q,
Teodoro ML, Rea S, Mechtler K, Kowalski JA, Homon CA,
et al. 2007. Reversal of H3K9me2 by a small-molecule
inhibitor for the G9a histone methyltransferase. Mol Cell
Litt MD, Simpson M, Gaszner M, Allis CD, Felsenfeld G. 2001.
Correlation between histone lysine methylation and devel-
opmental changes at the chicken b-globin locus. Science
Majeti R, Park CY, Weissman IL. 2007. Identification of a hier-
archy of multipotent hematopoietic progenitors in human
cord blood. Cell Stem Cell 1: 635–645.
Ma ˚nsson R, Hultquist A, Luc S, Yang L, Anderson K, Kharazi S,
Al-Hashmi S, Liuba K, Thore ´n L, Adolfsson J, et al. 2007.
Molecular evidence for hierarchical transcriptional lineage
priming in fetal and adult stem cells and multipotent pro-
genitors. Immunity 26: 407–419.
Manz MG, Miyamoto T, Akashi K, Weissman IL. 2002. Pro-
spective isolation of human clonogenic common myeloid
progenitors. Proc Natl Acad Sci 99: 11872–11877.
Miyamoto T, Iwasaki H, Reizis B, Ye M, Graf T, Weissman IL,
Akashi K. 2002. Myeloid or lymphoid promiscuity as a crit-
ical step in hematopoietic lineage commitment. Dev Cell 3:
Noma K, Allis CD, Grewal SI. 2001. Transitions in distinct
histone H3 methylation patterns at the heterochromatin
domain boundaries. Science 293: 1150–1155.
Notta F, Doulatov S, Laurenti E, Poeppl A, Jurisica I, Dick JE.
2011. Isolation of single human hematopoietic stem cells
capable of long-term multilineage engraftment. Science 333:
Novershtern N, Subramanian A, Lawton LN, Mak RH, Haining
WN, McConkey ME, Habib N, Yosef N, Chang CY, Shay T,
et al. 2011. Densely interconnected transcriptional circuits
control cell states in human hematopoiesis. Cell 144: 296–
O’Geen H, Echipare L, Farnham PJ. 2011. Using ChIP-seq
technology to generate high-resolution profiles of histone
modifications. Methods Mol Biol 791: 265–286.
Ohishi K, Varnum-Finney B, Bernstein ID. 2002. Delta-1
enhances marrow and thymus repopulating ability of hu-
man CD34+CD38?cord blood cells. J Clin Invest 110: 1165–
Orkin SH, Zon LI. 2008. Hematopoiesis: An evolving paradigm
for stem cell biology. Cell 132: 631–644.
Ostrander EA, Giniger E. 1997. Semper fidelis: What man’s best
friend can teach us about human biology and disease. Am J
Hum Genet 61: 475–480.
Peters AH, Kubicek S, Mechtler K, O’Sullivan RJ, Derijck AA,
Perez-Burgos L, Kohlmaier A, Opravil S, Tachibana M,
Shinkai Y, et al. 2003. Partitioning and plasticity of re-
pressive histone methylation states in mammalian chroma-
tin. Mol Cell 12: 1577–1589.
Rice JC, Briggs SD, Ueberheide B, Barber CM, Shabanowitz J,
Hunt DF, Shinkai Y, Allis CD. 2003. Histone methyltrans-
ferases direct different degrees of methylation to define
distinct chromatin domains. Mol Cell 12: 1591–1598.
Roopra A, Qazi R, Schoenike B, Daley TJ, Morrison JF. 2004.
Localized domains of G9a-mediated histone methylation are
required for silencing of neuronal genes. Mol Cell 14: 727–738.
Sauvageau G, Iscove NN, Humphries RK. 2004. In vitro and in
vivo expansion of hematopoietic stem cells. Oncogene 23:
Chen et al.
2510GENES & DEVELOPMENT
Saxonov S, Berg P, Brutlag DL. 2006. A genome-wide analysis of
CpG dinucleotides in the human genome distinguishes two
distinct classes of promoters. Proc Natl Acad Sci 103: 1412–
Su RC, Brown KE, Saaber S, Fisher AG, Merkenschlager M,
Smale ST. 2004. Dynamic assembly of silent chromatin
during thymocyte maturation. Nat Genet 36: 502–506.
Szilvassy SJ, Humphries RK, Lansdorp PM, Eaves AC, Eaves CJ.
1990. Quantitative assay for totipotent reconstituting hema-
topoietic stem cells by a competitive repopulation strategy.
Proc Natl Acad Sci 87: 8736–8740.
Tachibana M, Sugimoto K, Nozaki M, Ueda J, Ohta T, Ohki M,
Fukuda M, Takeda N, Niida H, Kato H, et al. 2002. G9a
histone methyltransferase plays a dominant role in euchro-
matic histone H3 lysine 9 methylation and is essential for
early embryogenesis. Genes Dev 16: 1779–1791.
Thomas ED, Storb R. 1999. The development of the scientific
foundation of hematopoietic cell transplantation based on
animal and human studies. In Hematopoietic cell trans-
plantation (ed. ED Thomas et al.), pp. 1–11. Blackwell
Science, Malden, MA.
Vedadi M, Barsyte-Lovejoy D, Liu F, Rival-Gervier S, Allali-
Hassani A, Labrie V, Wigle TJ, Dimaggio PA, Wasney GA,
Siarheyeva A, et al. 2011. A chemical probe selectively
inhibits G9a and GLP methyltransferase activity in cells.
Nat Chem Biol 7: 566–574.
Wen B, Wu H, Shinkai Y, Irizarry RA, Feinberg AP. 2009. Large
histone H3 lysine 9 dimethylated chromatin blocks distin-
guish differentiated from embryonic stem cells. Nat Genet
Zhang J, Grindley JC, Yin T, Jayasinghe S, He XC, Ross JT, Haug
JS, Rupp D, Porter-Westpfahl KS, Wiedemann LM, et al.
2006. PTEN maintains haematopoietic stem cells and acts in
lineage choice and leukaemia prevention. Nature 441: 518–
H3K9me2 patterning in HSPCs
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