Transcriptional activator DOT1L putatively regulates human embryonic stem cell differentiation into the cardiac lineage
Pursani et al. Stem Cell Research & Therapy
Transcriptional activator DOT1L putatively regulates human embryonic stem cell differentiation into the cardiac lineage
Varsha Pursani 0
Deepa Bhartiya 0 4
Vivek Tanavde 1 2
Mohsin Bashir 3
Prabha Sampath 3
0 Stem Cell Biology Department, ICMR-National Institute for Research in Reproductive Health , J.M. Street, Parel, Mumbai, Maharashtra 400 012 , India
1 Genome and Gene Expression Data Analysis Division , A
2 Division of Biological & Life Sciences, School of Arts & Sciences, Ahmedabad University , Ahmedabad 380009 , India
3 Division of Translational Control of Disease , A
4 Star-Institute of Medical Biology , Singapore 138648 , Singapore
Background: Commitment of pluripotent stem cells into differentiated cells and associated gene expression necessitate specific epigenetic mechanisms that modify the DNA and corresponding histone proteins to render the chromatin in an open or closed state. This in turn dictates the associated genetic machinery, including transcription factors, acknowledging the cellular signals provided. Activating histone methyltransferases represent crucial enzymes in the epigenetic machinery that cause transcription initiation by delivering the methyl mark on histone proteins. A number of studies have evidenced the vital role of one such histone modifier, DOT1L, in transcriptional regulation. Involvement of DOT1L in differentiating pluripotent human embryonic stem (hES) cells into the cardiac lineage has not yet been investigated. Methods: The study was conducted on in-house derived (KIND1) and commercially available (HES3) human embryonic stem cell lines. Chromatin immunoprecipitation (ChIP) was performed followed by sequencing to uncover the cardiac genes harboring the DOT1L specific mark H3K79me2. Following this, dual immunofluorescence was employed to show the DOT1L co-occupancy along with the cardiac progenitor specific marker. DOT1L was knocked down by siRNA to further confirm its role during cardiac differentiation. Results: ChIP sequencing revealed a significant number of peaks characterizing H3K79me2 occupancy in the proximity of the transcription start site. This included genes like MYOF, NR2F2, NKX2.5, and HAND1 in cardiac progenitors and cardiomyocytes, and POU5F1 and NANOG in pluripotent hES cells. Consistent with this observation, we also show that DOT1L co-localizes with the master cardiac transcription factor NKX2.5, suggesting its direct involvement during gene activation. Knockdown of DOT1L did not alter the pluripotency of hES cells, but it led to the disruption of cardiac differentiation observed morphologically as well as at transcript and protein levels. Conclusions: Collectively, our data suggests the crucial role of H3K79me2 methyltransferase DOT1L for activation of NKX2.5 during the cardiac differentiation of hES cells.
Human embryonic stem cells; Cardiac differentiation; DOT1L; Epigenetics; Gene expression; Histone methyltransferase
Pluripotent stem cells (PSCs) are blank cells with the
ability to differentiate into multiple cell types depending
upon the cues provided in vitro. They have open
euchromatin and complex epigenetic changes occur
when these PSCs become committed. Among these,
histone modifications take up the major role of opening
the chromatin structure for the subsequent transcription
activation. A number of studies have started unlocking
the molecular mechanisms of these epigenetic factors
that precisely orchestrate the development of specific
cell types from undifferentiated PSCs to aid in their wide
applications. Bivalency PSCs is a central discovery
involving an interesting interplay of histone methylations
H3K27me3 and H3K4me3. Deposited by EZH2 of the
polycomb group (PcG) and MLL2 of the trithorax group
(TrxG) of proteins respectively, bivalent domains are the
most widely studied mechanisms that render the gene
inactive and active while, on the other hand, the
presence of both marks keeps the gene poised for
subsequent activation or suppression upon differentiation
The relative distribution of these bivalent marks has
been extensively uncovered, assigning them a crucial
role in various mammalian developmental processes
including cardiogenesis. Our group recently reported a
vital role for EZH2 in the cardiac differentiation process
wherein EZH2 is recruited by NR2F2 (cardiac marker) at
the OCT4A promoter (pluripotency marker) for its
repression in early cardiac differentiation stages by
bringing about an H3K27me3 mark [
]. In addition to
MLL2, there are other histone active methyltransferases
recruited at the gene to activate transcription by
methylating the target locus.
Histone methyltransferases have been shown to be
guided at the genomic locations in specific cell types by
directive roles of signaling pathways, histone variants,
nucleosome remodeling, and transcription factors [
although the mechanistic and specificity details are still
left to be uncovered. Cardiac differentiation has also been
shown as an integration of genomic (transcription factors)
and epigenetic (histone methyltransferases) information
that collectively activates and deactivates the cardiac
specific machinery. Epigenetic connection of cardiac
formation was first put forward in 2005 when the key
transcription factor GATA4 was shown to be coactivated
by an acetylation mark brought about by histone
acetyltransferase p300, thereby increasing its DNA binding
ability and stability in cardiac myocytes differentiated from ES
cells . Activated GATA4 further binds to NKX2.5,
another master cardiac transcription factor (TF) triggering
]. Similarly, essential roles for histone
demethylases like UTX and JMJD3 (H3K27me3
demethylases) have been reported to activate the cardiac genes
during ES cell transition from pluripotency to cardiomyocytes
]. NKX2.5 functions as an instrumental part of each
of the differentiation stages like chamber formation,
patterning of the conduction system, formation of the
interventricular septum, defined expression of critical downstream genes,
and terminal differentiation of the myocardium followed by
their maturation into adult equivalents [
Understanding the signals and the modifications for the expression of
cardiac transcription factors thus remains necessary to
expose the mechanistic details for stepwise depiction of cardiac
DOT1L, unlike all other histone methyltransferases,
represents the first crucial histone methyltransferase not
containing an evolutionarily conserved catalytic domain called
SET, referring to the Su(var)3-9, Enhancer of Zeste (E(Z)),
and Trithorax (trx) domain [
]. DOT1L represents the
only enzyme that activates its target by delivering
dimethylation at lysine 79 of histone H3 [
]. Although DOT1L
was initially identified for regulating heterochromatin
], accumulating literature now suggests its
role in the regulation of gene activation [
important area having DOT1L as an essential controller is
that of cell cycle and DNA damage repair [
Involvement of DOT1L in in-vivo cardiac development and
function has been shown by Nguyen and Zhang , wherein
the group noted severe dilated cardiomyopathy in DOT1L
knockout mice, which upon further study was rescued by
ectopic expression of DOT1L, and that DOT1L is the
possible target malfunctioning in dilated cardiomyopathy. The
contribution of DOT1L in cardiac formation from
undifferentiated mouse ES cells was reported recently [
study successfully proved DOT1L expression on cardiac
genes, which upon knocking down affects the expression
of these genes delaying the cardiac differentiation. To
conclude, DOT1L has an important role during cardiogenesis
both in vivo and in vitro, and demands much more
research efforts toward displaying its connection at the
molecular and genetic levels as its deletion results in cardiac
The present study was designed to understand whether
DOT1L is crucial for the cardiac progenitor differentiation
in vitro. Studies were carried out on the inhouse-derived
hES cell line KIND1 along with a well-studied HES3 hES
cell line. By performing chromatin immunoprecipitation
(ChIP) followed by sequencing (ChIP-seq), we
interrogated the hES cell-derived cardiac progenitors and beating
CMs for the occupancy of an H3K79me2 mark on the
specific cardiac genes. Dual immunofluorescence was
performed to investigate whether cardiac specific
transcription factor NKX2.5 is coexpressed with and activated by
H3K79me2 methyltransferase DOT1L.
Cell culture and differentiation
KIND1 is an in-house derived hES cell line derived at
our laboratory in Mumbai [
] and the HES3 cell line
(WiCell Research Institute Inc.) was available from Dr
Prabha Sampath’s laboratory for the present study.
Undifferentiated feeder-free KIND1 hES cells were
cultured in Stempro hES SFM medium (Invitrogen,
Carlsbad, CA, USA) supplemented with 8 ng of bFGF
(Peprotech, NJ, USA) as described earlier [
], while the
HES3 cell line was grown in mTeSR™1 medium
(STEMCELL Technologies Inc., Canada) at 37 °C and
5% CO2. For subjecting the confluent pluripotent KIND1
and HES3 hES cells to cardiac differentiation, they were
transitioned from growth medium into RPMI 1640
containing 5% B-27 and 1% glutamax (basal medium), and
the differentiation protocol was followed as reported by
our group earlier [
]. In brief, cells were first exposed to
basal medium supplemented with 100 ng/ml Activin A
(Peprotech) and 5 ng/ml of bFGF (R&D Systems, MN,
USA) for 24 h. This was followed by 15 ng/ml BMP4
(R&D Systems) and 5 ng/ml bFGF (R&D Systems) in basal
medium for another 4 days. Finally, the cells were treated
with WNT pathway blocker DKK1 (Peprotech) at 150 ng/ml
concentration for the next 4 days. From day 9 onward, the
cells were maintained in basal medium until day 20 wherein
the media were changed on every alternate day.
Quantitative polymerase chain reaction
Total RNA was extracted using an RNeasy Mini Kit
(Qiagen, Germany) and quantified using an ND1000
Spectrophotometer (NanoDrop Technologies, Inc., DE,
USA). Then 500 ng was subjected to reverse
transcription for cDNA synthesis using SuperScript® III Reverse
Transcriptase (Thermo Fisher Scientific, MA, USA) as per
the manufacturer’s instructions using a 7900HT Fast
RealTime PCR System (Thermo Fisher Scientific). Quantitative
polymerase chain reaction (qPCR) was performed using
SYBR Green chemistry (Thermo Fisher Scientific) on a
7900HT Fast Real-Time PCR System. The program applied
for amplification included 25 °C for 10 min, 50 °C for 50
min, and 85 °C for 5 min. The fold change was determined
by the 2–ΔΔCt method and was expressed relative to that of
an internal control, RPLPO. The expression level of each
gene transcript is normalized to a value of 1.0 for
undifferentiated cells. The error bars represent ±standard error of
the mean (SEM). All results are an average of at least three
biological replicates. The primers used are presented in
ChIP was performed as per our recent report [
about 1–2 million KIND1 and HES3 hES cells each
harvested at days 0, 12, and 20 were subjected to
formaldehyde crosslinking and sonication (Bioruptor;
Cosmo Bio Co. Ltd, Japan). Sonicated protein–DNA
complexes (200–500 bp) were precipitated with 10 μg of
anti-H3K79me2 antibody (Cell Signaling Technology, MA,
USA) overnight at 4 °C. Post thorough washing and
elution, the ChIPped samples were subjected to standard
DNA extraction protocol employing
phenol:chloroform:isoamyl alcohol as per the manufacturer’s instructions
(Thermo Fisher Scientific). Extracted DNA samples were
sent to the Genome Institute of Singapore (GIS), Singapore
for sequencing on Illumina HiSeq2500 sequencer. The
analysis of sequencing results obtained was performed
at Sandor Life Sciences Pvt. Ltd (Hyderabad, India).
The raw sequencing reads mapped with Humanhg19
were aligned using Bowtie (Galaxy tool) while peak
calling was performed using MACS (Galaxy tool).
Integrative Genome Viewer [
] was used for visualization of
the resulting peaks.
The standard immunofluorescence protocol was
followed to study expression of DOT1L and NKX2.5.
Cells were grown in chamber slides followed by their
differentiation and fixation with 4% paraformaldehyde
(PFA) (Sigma-Aldrich, MO, USA) at days 0, 12, and 20
for 15 min followed by permeabilization with 0.3% triton
X-100 (Sigma-Aldrich). Blocking was performed using
phosphate buffer saline (PBS) containing 5% BSA (Sigma
Aldrich) plus 1% normal goat serum (Bangalore Genei,
Bangalore, India) for 60 min at room temperature. Cells
were then incubated at 4 °C overnight with primary
antibodies against DOT1L (1:200) (Abcam) and NKX2.5
(1:200) (R&D Systems) diluted in blocking buffer. Later
the cells were incubated in appropriate secondary
antibodies (Thermo Fisher Scientific) diluted in blocking buffer
for 2 h at room temperature. Representatives images were
captured using a confocal microscope (Olympus FV1000).
A small interfering RNA (siRNA)-based transfection
technique for knocking down was employed to study the
expression of DOT1L in both KIND1 and HES3 cells at days 0, 12,
and 20. siRNAs for DOT1L along with a nontarget siRNA
pool (control), with the following target sequences, were
procured from GE Dharmacon™. DOT1L siRNAs
(LQ014900-01-0010), (1) UCACUAUGGCGUCGAGAAA, (2)
GCUAUGGAGAAUUACGUUU, (3) GCAGAAUCGUGU
CCUCGAA, (4) AAGAGUGGAGGGAGCGAAU; and
nontarget siRNA pool (D-001810-10-20), (1)
UGGUUUACAUGUCGACUAA, (2) UGGUUUACAUGUUGUGUGA, (3)
UGGUUUACAUGUUUUCCUA. Lipofectamine™ RNAiMAX Transfection
Reagent (Thermo Fisher Scientific) was used to dilute the
siRNAs for transfection as per the manufacturer’s
instructions. The pool of DOT1L siRNAs 1 and 3 gave the
maximum knockdown at a concentration of 25 nM. Post
incubation for 5 min, siRNA DOT1L–lipid complex diluted in
Opti-MEM™ Reduced Serum Medium (Thermo Fisher
Scientific) was added to the cells. The medium was changed
with Stempro hES SFM growth medium after 24 h. Cells
were harvested 72 h post knock down for subsequent
analysis. For knocking down DOT1L in cardiac progenitors
(day 12), transfection was performed at day 9 of
differentiation protocol (described earlier), following which the
progenitor cells were collected after 72 h (i.e., at day 12).
Similarly, for knocking down the DOT1L at day 20
(cardiomyocyte stage), siRNA transfections were performed at day
17 of the cardiac-directed differentiation protocol.
Differentiation of hES cells: KIND1 and HES3 cells display an identical cardiac differentiation pattern
Sequential addition of growth factors to undifferentiated
KIND1 and HES3 cells led to a stepwise differentiation
pattern into the cardiac lineage. Directed differentiation
of pluripotent HES3 cells into the cardiac lineage was
associated with distinct morphological changes ( see
Additional file 1 for images taken at different time points of
differentiation). Similar changes were observed when
KIND1 cells were subjected to a similar differentiation
protocol recently reported by our group [
of the BMP and TGF-β pathways with a high
concentration of growth factor ligands like BMP4 and Activin
A resulted in upregulation of transcripts like GATA4
and KDR2 that represent the formation of early cardiac
mesoderm. Following this, addition of DKK1 for inhibition
of the WNT pathway further drove the differentiation
toward the cardiac fate evident by expression of MESP1,
MEF2C, ISL1, NKX2.5, and CTNT transcripts specific for
cardiac mesoderm, cardiac progenitors, and beating
cardiomyocytes (observed only in KIND1 cells). Depending upon
the gene expression pattern, we harvested the cells at days 0,
12, and 20 during differentiation of both KIND1 and HES3
cells, which depict undifferentiated pluripotent hES cells,
cardiac progenitors, and beating cardiomyocytes for carrying
out further studies. The corresponding changes in specific
transcripts that were expected to change during
differentiation and protein expression of CTNT and NKX2.5 in
differentiating HES3 cells are presented in Additional files 2 and
3. These results are also similar to earlier published data
using KIND1 cells [
Interestingly with these results, we for the first time
report the typical cardiac differentiation pattern following a
20-day directed differentiation protocol in the HES3 cell
line. In addition, a similar morphology observed in both cell
lines at each stage during cardiac differentiation, further
validating our protocol as well as the pattern of cardiac
differentiation from hES cells. Beating cardiomyocytes were
observed only in KIND1 cells perhaps because of intrinsic
differences between the two cell lines. KIND1 cells were
derived on human feeder fibroblasts whereas HES3 cells were
derived initially on mouse embryonic fibroblast support.
ChIP-seq: H3K79me2 targets cardiac lineage genes during differentiation of KIND1 and HES3 cells
We first sought to understand the occupancy of
H3K79me2 modification onto the genomic regions in
undifferentiated as well as differentiating hES cells
toward the cardiac lineage. Chromatin
immunoprecipitation followed by sequencing (ChIP-seq) visualized an
interesting expression pattern of an H3K79me2 methyl
mark onto the expressed genes. The enrichment of
H3K79me2 mark appeared upstream of exons including
the promoters and the 5′UTR regions of genes, with a
significant proportion of peaks occupying downstream
regions of the transcription start site (TSS) as per the
known localization pattern of this mark (Fig. 1). Analyzing
the status of this dimethyl mark at day 0, transcripts like
POU5F1, NANOG, and SOX2 representing the pluripotent
hES cells accumulated H3K79me2 peaks toward the
downstream of TSS and disappeared at both the day 12
and day 20 stage of cardiac differentiation (Fig. 2). This
highlights the possible role of an H3K79me2 methyl mark
in their activation in an undifferentiated state. Further
analyzing the cells at days 12 and 20, the active genes
comprised key transcripts representing the cardiac lineage
like BMP4, GATA4, KDR2, ISL1, MEF2C, MSX1, MYH6,
MYL2, NKX2.5, NR2F2, T, TNNT2, WNT5A, HAND1, and
MYOF (Fig. 2a, b). On extending our analysis, significant
signals of H3K79me2 peaks were noted at the intron, 3′
UTR, and splice site regions of genes including HNF4A,
LEFTY1, NOGGIN, NQO1, OTX2, SOX7, and NPTX2
(results shown for both KIND-1 and HES3 cells in
Additional file 4). Expression patterns in both hES cell lines
employed in the present study displayed high similarity,
implying the crucial collaboration of H3K79me2
modification with the cardiac gene expression machinery. With
respect to the various findings describing its roles in
transcriptional activation or for the derepression of
heterochromatin locally [
31, 33, 43–45
], DOT1L is also
known as the critical regulator of the cell cycle process
wherein enriched expression of DOT1L is found during
the G2/M phase [
On the other hand, deficiency of DOT1L results in cell
cycle progression defects and aneuploidy in
differentiating ES cells [
]. In support of this, we in our
present analysis also noted the significant peaks at
PCNA, the gene required for DNA replication known as
the marker of cell proliferation of cardiac muscle cells
. Additional cell cycle regulators containing an
H3K79me2 mark include BUB1 and BRCA1 along with
E2F5, an antiapoptotic factor in cardiomyocytes. These
analyses, besides confirming the differentiation of
KIND1 and HES3 hES cells into the cardiac lineage, also
reveal the necessary involvement of dimethylation of
H3K79 as a putative activation mark for induction of
gene activation during cardiogenesis in vitro.
Dual immunofluorescence: coexpression of DOT1L and
NKX2.5 in cardiac progenitors
NKX2.5 is an essential marker for cardiac progenitors
during in-vivo embryonic development as well as in-vitro
differentiation of pluripotent cells. In continuation of the
observation that an H3K79me2 mark occurs on NKX2.5,
we were then interested to see whether H3K79me2
methyltransferase DOT1L is involved in activation of the
NKX2.5 gene by delivering an H3K79me2 mark leading to
its expression that is essential for the downstream
cardiogenesis. This coappearance of DOT1L and NKX2.5 was
investigated by dual immunofluorescence performed at
days 0, 12, and 20. Figure 3 clearly reveals the significant
number of areas coexpressing DOT1L and cardiac marker
NKX2.5 at the progenitor (day 12) and the beating
cardiomyocyte (day 20) stages obtained from KIND1 and HES3
cells. On the other hand, no colocalization was observed
for DOT1L and NKX2.5 at the pluripotent (day 0) stage
of the cells. These obvious results further indicate that
DOT1L might be coactivating the key cardiac transcript
NKX2.5 by bringing about an H3K79me2 activation mark.
In support of these results, Dystrophin (DMD)
expression was upregulated in cardiac progenitors and beating
cardiomyocytes compared to undifferentiated KIND1 cells
(Additional file 5). ChIP sequencing analysis also revealed
significant peaks representing the H3K79me2 occupancy
at the DMD gene in differentiated cardiac cells obtained
from both KIND1 and HES3 cells (see Additional file 6).
In their mechanistic study, Nguyen and Zhang [
revealed that DOT1L functions in cardiomyocytes through
regulating DMD transcription and that DMD is a direct
target of DOT1L. The present study clearly reports a
correlated expression of DMD and DOT1L-mediated
H3K79me2 methylation in cardiac cells. The results are in
compliance with published studies and report the
expression of DMD in cardiac cells differentiated from hES cells
for the first time, further implying the active involvement
of DOT1L during cardiac differentiation.
Knockdown studies: DOT1L deficiency leads to compromised cardiogenesis from KIND1 and HES3 cells
Since DOT1L and its methylation mark H3K79me2 were
found to be expressed along with NKX2.5, we then
planned to explore whether DOT1L is crucially required
in both regulating the pluripotent state as well as for
obtaining cardiac progenitors. For this, we moved ahead
with knocking down the DOT1L expression in both
KIND1 and HES3 cells followed by their inspection both
at pluripotent as well as during cardiac differentiation
stages. In addition to comparing the gene expression
status in DOT1L knocked down hES cells with that of
wild-type hES cells, both cell lines were simultaneously
transfected with nontarget siRNA control. With the
achievement of about 70% and 75% of DOT1L
knockdown in KIND1 and HES3 cells (Fig. 4), we first
monitored its effect upon proliferation and maintenance of
hES cells in their pluripotent state. Interestingly, post
knock down both KIND1 and HES3 cells were
morphologically indistinguishable when compared to
their wild-type or control counterparts (Fig. 5).
Confirming this at the transcript by qPCR, the expression of key
pluripotent marker OCT4 was seen to be unchanged in
wild-type, negative control, and post-DOT1L
knockdown hES cells at day 0 (undifferentiated) (Fig. 6). This is
consistent with a couple of earlier reports stating that
DOT1L does not inhibit the pluripotency of ES cells and
that they retain the expression of pluripotent markers as
evident at both transcript and protein levels [
Extending ahead from the pluripotent state, we then worked
further to examine the potential of both cell lines post
knock down to differentiate and give rise to cardiac
progenitors using the same differentiation protocol as
described earlier. We first looked for the shift from the
typical morphology that we observed during cardiac
differentiation from KIND1 and HES3 cells as described.
DOT1L knockdown severely impaired the normal
differentiation of hES cell lines into cardiac progenitors,
as evidenced by the depleted morphological pattern of cells
at day 12 obtained post DOT1L knock down; as compared
to that seen in cells containing no target siRNA pool or
the wild-type KIND1 and HES3 cells (Fig. 5).
Further qPCR analysis was carried out to understand
the status of NKX2.5 expression at the transcript level
(Fig. 6). While the expression of NKX2.5 in progenitor
cells containing no target siRNA was comparable to that
obtained from wild-type cells, the levels of NKX2.5
expression in progenitors derived from cells deficient for
DOT1L were drastically affected at both progenitor (day
12) and cardiomyocyte (day 20) stages when compared
with control or wild-type cells at the respective stages.
About 60% and 80% of reduced NKX2.5 levels were
denoted at day 12 and day 20 respectively, differentiated
from both KIND1 and HES3 cells. This indicates that
depletion of DOT1L in hES cells resulted in significantly
reduced NKX2.5 expression and thus cardiac progenitor
formation. To further investigate whether DOT1L is
crucial for the downstream targets of NKX2.5, we
checked for the expression of other cardiac representative
genes like MESP1, MEF2C, ISL1, and CTNT in
progenitors and cardiomyocytes obtained from KIND1 and HES3
cells. An intensely diminished gene expression level of
these markers was noted at day 12 that was further
reduced at day 20. These observations extend the effects
of depletion of DOT1L according to which not only
NKX2.5 but also the downstream transcriptional targets of
NKX2.5 are influenced leading to much less or no
cardiomyocyte formation in vitro. To further confirm these
results, we performed dual immunofluorescence to study
the coexpression of NKX2.5 and DOT1L in cardiac
progenitors deficient for DOT1L (Figs. 7 and 8). While we
observed considerable areas, displaying their colocalization in
control and wild-type hES cells, there was very little or no
expression of either markers in the cardiac progenitors
containing siDOT1L. These results further help establish the
fact that DOT1L has a decisive participation in early
incidents of cardiac differentiation from hES cells.
In the present study, we aimed to understand the
importance of active histone modifier DOT1L during
cardiac differentiation in vitro on hES cells.
In-housederived KIND1 and HES3 cell lines were used for the
study. The ability of KIND1 cells to differentiate into
cardiac progenitors and beating cardiomyocytes has been
published previously [
]; however, the present study
provides the first results on cardiac differentiation of
HES3 cells using a directed differentiation protocol.
ChIP sequencing was performed to look for the DOT1L
specific mark H3K79me2 in differentiating KIND1 and
HES3 cells. As per the known localization of H3K79me2
modification, significant peaks were noted at the
downstream regions of pluripotent genes like OCT4, SOX2
and NANOG, whereas genes like GATA4, HAND1,
NR2F2, NKX2.5, MESP1, ISL1, and WNT5A harbored
the H3K79me2 peaks as the cells underwent
differentiation into cardiac progenitor and cardiomyocyte stages.
ChIP sequencing analysis also revealed the significant
peaks of H3K79me2 on the DMD gene at days 12 and
20 in both KIND1 and HES3 cells, suggesting its direct
upregulation by DOT1L during cardiac differentiation.
Employing dual immunofluorescence, colocalization of
DOT1L was studied with the master cardiac
transcription factor NKX2.5. While NKX2.5 was not expressed in
the pluripotent stage, substantial areas showing
coexpression of DOT1L and NKX2.5 were located in cardiac
progenitors upon differentiation from both KIND1 and
HES3 cells. Moreover, expression of DMD was also
increased as undifferentiated KIND1 hES cells
differentiated into cardiac progenitors and cardiomyocytes, and
correlated with the DOT1L expression and H3K79me2
methylation. Further studies were undertaken to study
the effects of loss of DOT1L on differentiating KIND1
and HES3 cells. A 70–75% knock down of DOT1L was
obtained in hES cells using siRNA technology.
Remarkably, DOT1L knockdown did not show any deleterious
effects on the pluripotency of hES cells maintaining their
typical morphology as well as the expression of
pluripotency gene OCT4; however, deficiency of DOT1L
severely attenuated the cardiac differentiation pattern in
KIND1 cells as well as HES3 cells. Furthermore,
transcription factor NKX2.5 and its downstream targets like
GATA4, TBX5, and ISL1 were found to be critically
downregulated at cardiac progenitor and cardiomyocyte
stages when DOT1L was knocked down. This was
further confirmed when cells lacking DOT1L did not
show coexpression of NKX2.5 and DOT1L by
immunofluorescence. We report the possible involvement of
histone activating methyltransferase DOT1L during cardiac
differentiation of hES cells. Such studies, besides helping to
improve the efficiency of hES cell differentiation, would
further aid in better understanding the early events underlying
cardiac differentiation in vitro.
High levels of active methylation marks occur upon
the euchromatin or the open chromatin that is accessible
to the transcription machinery. Cardiac cell fate also
depends upon its specific and timely gene expression
mechanisms that in turn are largely regulated by active
epigenetic modifications like H3K4me3, H3K36me3, and
H3K79me2. H3K4me3 is indispensable in cardiac
developmental genes, evidenced by exome sequencing to
identify the underlying mutations in CHD patients. This
includes genes like GATA4, NKX2.5, and TBX5 read
with mutated H3K4 methylation [
involvement of MLL2 is also visualized in differentiating mES
cells that directly controls cardiac specific genes by
promoting H3K4me3 deposition [
represents a second crucial active methylation mark found to
be enriched upon NKX2.5. Interaction of H3K36me3
methyltransferase WHSC1 and NKX2.5 has remarkably
shown the regulation of another set of genes like PDGFRA
and NPPA. In support of this, WHSC1 mutant hearts
resulted in cardiac developmental defects consistent with
NKX2.5 heterozygous mutants, further confirming their
functional link [
]. Results of the present study show the
key role of the transcription activating mark represented
by H3K79me2 deposited onto the actively transcribing
genes by DOT1L. These findings suggest the involvement
of multiple histone activating methyltransferases for the
activation of a gene in cardiac differentiation. However,
what further remains to be revealed is whether there exists
a collaborative effort by these epigenetic modifiers and
also the effects of loss of one enzyme on the functions of
another epigenetic modifier.
NKX2.5 represents a critical cardiac developmental
factor that essentially directs the multiple downstream
genes required for cardiac morphogenesis and maturation.
This is supported by the conduction and contraction
defects leading to premature death upon its deletion in
]. NKX2.5 mutations also predispose the
patients to cardiac developmental disorder termed dilated
cardiomyopathy (DCM). Involvement of NKX2.5 in
processes like cardiomyocyte specification and their
homeostasis, development of the conduction system and
cardiac muscle cells, as well as septation and nodal
formation makes it a central participant in the genetic
model for DCM [
]. Several reports also implicate
epigenetic factors as causal events of DCM. Nguyen and
Zhang  showed the significant role of DOT1L in the
pathogenesis of DCM and that cardiac-specific conditional
knockout for DOT1L in mice was lethal in nature. This
study also reported mechanistic details showing DMD as
the key target mediating DOT1L function in cardiac cells.
Our results are in agreement with these published reports
and provide a possible mechanism involving DOT1L
during cardiogenesis leading to various pathologies. This
further opens up the question of whether the genetic cause
of DCM with respect to NKX2.5 is due to failure of its
activation by DOT1L and hence its loss of function.
Our study opens up a number of areas that further
need to be explored in order to design a
DOT1Lcentered gene expression model. Similarly, the other
gene activating factors might also be involved along with
DOT1L since DOT1L is known to function in
coordination with MLL2 for maintenance of gene expression in
]. Crosstalk among histone methyltransferases
also requires further investigation. On the other hand,
understanding the effects of overexpression of DOT1L
during in-vitro cardiac differentiation might also uncover
newer layers of regulation of cardiac gene expression.
The present study, besides uncovering the contribution
of DOT1L in cardiac differentiation from hES cells, puts
forward a wide range of exciting possibilities that would
aid in enhancing the efficiency of cardiac differentiation
from hES cells as well as their clinical applications.
However, further studies showing altered occupancy of
H3K79me2 mark post DOT1L knock down as well as
demonstrating direct binding of DOT1L to NKX2.5 in a
pure population of cardiomyocytes need to be studied in
order to further substantiate our findings.
Additional file 1: Brightfield images of HES3 hES cells during directed
differentiation into cardiac lineage. Differentiation results in distinct
morphological changes leading to increased compaction among the
cells as differentiation proceeds from day 0 to day 20. Similar changes
observed when KIND1 cells were differentiated into cardiac cells as
described earlier [
]. Magnification 10×. (PDF 554 kb)
Additional file 2: Characterization of cardiac differentiation of HES3 cells
by quantitative real-time PCR (qRT-PCR). Expression of transcripts representing
pluripotency (OCT4), cardiac mesoderm (MESP1), cardiac progenitors (NKX2.5,
MEF2C), and cardiomyocytes (CTNT) at days 0, 12, and 20 during 20 days of
cardiac differentiation. Note OCT-4 expression in undifferentiated cells is
downregulated as the cells initiate differentiation. Early cardiac markers
detected on day 12 and mature markers upregulated on day 20. Similar
changes in transcripts expression observed when KIND1 cells were
differentiated into cardiac cells as described earlier [
]. Error bars represent
±SEM. (PDF 410 kb)
Additional file 3: Characterization of cardiac differentiation of HES3 cells
by immunofluorescence studies. Expression of NKX2.5 (A) and CTNT (B)
on days 12 and 20 observed by immunofluorescence. (A) Distinct nuclear
expression of NKX2.5 observed and (B) CTNT cell surface expression.
Similar changes observed when KIND1 cells were differentiated into
cardiac cells as described earlier [
]. Counterstaining using DAPI.
Magnifications 20×. (PDF 450 kb)
Additional file 4: ChIP sequencing in KIND1 and HES3 cells during
cardiac differentiation visualized by Integrated Genome Viewer shows
binding profile of H3K79me2 modification across genes HNF4A, LEFTY1,
NOGGIN, NQO1, OTX2, and NPTX2 in KIND1 (green) and HES3 (red) cells at
days 0, 12, and 20 of cardiac differentiation. (PDF 548 kb)
Additional file 5: Dystrophin gene expression during cardiac differentiation
of KIND1 hES cells on days 0, 12, and 20 during cardiac differentiation of
KIND1 hES cell line. Expression of Dystrophin increased in cardiac progenitors
and cardiomyocytes compared to undifferentiated KIND1 cells. Results in
agreement with earlier reports in DOT1L conditional knockout mice heart
concluding Dystrophin as a direct target of DOT1L [
]. Error bars represent
±SEM. (PDF 329 kb)
Additional file 6: ChIP sequencing of occupancy of H3K79me2 on DMD
gene during cardiac differentiation of KIND1 and HES3 cells showing
occupancy of H3K79me2 methylation mark brought about by DOT1L on
DMD gene during cardiac differentiation. Results clearly show significant
peaks representing the DOT1L specific methylation mark on days 12 and
20 as compared to day 0 suggestive of its activation by DOT1L during
cardiac differentiation in vitro. (PDF 614 kb)
bFGF: Basic fibroblast growth factor; CHD: Chronic heart disease;
ChIP: Chromatin immunoprecipitation; hES: Human embryonic stem;
PBS: Phosphate buffered saline; PcG: Polycomb group; PCR: Polymerase chain
reaction; PSC: Pluripotent stem cell; TrxG: Trithorax group
The authors thank the sequencing facility of the Genome Institute of
Singapore for sequencing services, Sandor Life Sciences for sequencing data
analysis, the Institute of Medical Biology (IMB) Microscopy Unit for confocal
studies, Paul for assistance with providing HES3 cell cultures, Colin Stewart
for cell culture facility, and Wai Kay Kok and Rafidah for their technical and
experimental inputs. Special thanks to Gopinath Sundaram for guidance in
confocal and knockdown studies.
The Council of Scientific & Industrial Research (CSIR), Government of India,
New Delhi and core funding by the Indian Council of Medical Research,
Government of India, New Delhi supported this study. The authors also
acknowledge the Department of Science and Technology—INSPIRE
(DSTINSPIRE), Government of India for providing an INSPIRE fellowship to VP.
Availability of data and materials
The ChIP sequencing raw datasets generated during the current study are
available in the NCBI Sequence Read Archive (SRA) repository under
accession number SRP115341.
VP was involved in study design, carrying out experiments, performing data
analysis and interpretation, and manuscript preparation. DB was involved in
obtaining funds, data interpretation, and manuscript preparation. VT was
involved in experimental design and discussions. MB was involved in
carrying out experiments. PS was involved in experimental and scientific
inputs. All authors read and approved the submitted version of the
Ethics approval and consent to participate
Consent for publication
All authors read and approved the final version of the manuscript for
submission. ICMR-NIRRH Accession number RA/502/07-2017.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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