The histone H3K79 methyltransferase Dot1L is essential for mammalian development and heterochromatin structure.

Abstract

Dot1 is an evolutionarily conserved histone methyltransferase specific for lysine 79 of histone H3 (H3K79). In Saccharomyces cerevisiae, Dot1-mediated H3K79 methylation is associated with telomere silencing, meiotic checkpoint control, and DNA damage response. The biological function of H3K79 methylation in mammals, however, remains poorly understood. Using gene targeting, we generated mice deficient for Dot1L, the murine Dot1 homologue. Dot1L-deficient embryos show multiple developmental abnormalities, including growth impairment, angiogenesis defects in the yolk sac, and cardiac dilation, and die between 9.5 and 10.5 days post coitum. To gain insights into the cellular function of Dot1L, we derived embryonic stem (ES) cells from Dot1L mutant blastocysts. Dot1L-deficient ES cells show global loss of H3K79 methylation as well as reduced levels of heterochromatic marks (H3K9 di-methylation and H4K20 tri-methylation) at centromeres and telomeres. These changes are accompanied by aneuploidy, telomere elongation, and proliferation defects. Taken together, these results indicate that Dot1L and H3K79 methylation play important roles in heterochromatin formation and in embryonic development.

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The Histone H3K79 Methyltransferase Dot1L Is Essential
for Mammalian Development and Heterochromatin
Structure
Brendan Jones1, Hui Su1, Audesh Bhat2, Hong Lei3, Jeffrey Bajko1, Sarah Hevi1, Gretchen A. Baltus1,
Shilpa Kadam1, Huili Zhai4, Reginald Valdez3, Susana Gonzalo2, Yi Zhang5,6, En Li1, Taiping Chen1*
1Epigenetics Program, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, United States of America, 2Department of Radiation Oncology, Radiation
and Cancer Biology Division, Washington University School of Medicine, St. Louis, Missouri, United States of America, 3Developmental and Molecular Pathways, Novartis
Institutes for Biomedical Research, Cambridge, Massachusetts, United States of America, 4Analytical Sciences, Novartis Institutes for Biomedical Research, Cambridge,
Massachusetts, United States of America, 5Howard Hughes Medical Institute, Chevy Chase, Maryland, United States of America, 6Department of Biochemistry and
Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
Abstract
Dot1 is an evolutionarily conserved histone methyltransferase specific for lysine 79 of histone H3 (H3K79). In Saccharomyces
cerevisiae, Dot1-mediated H3K79 methylation is associated with telomere silencing, meiotic checkpoint control, and DNA
damageresponse.Thebiologicalfunctionof H3K79methylation inmammals,however, remains poorlyunderstood.Usinggene
targeting, we generated mice deficient for Dot1L, the murine Dot1 homologue. Dot1L-deficient embryos show multiple
developmental abnormalities, including growth impairment, angiogenesis defects in the yolk sac, and cardiac dilation, and die
between 9.5 and 10.5 days post coitum. To gain insights into the cellular function of Dot1L, we derived embryonic stem (ES)
cells from Dot1L mutant blastocysts. Dot1L-deficient ES cells show global loss of H3K79 methylation as well as reduced levels of
heterochromatic marks (H3K9 di-methylation and H4K20 tri-methylation) at centromeres and telomeres. These changes are
accompanied by aneuploidy, telomere elongation, and proliferation defects. Taken together, these results indicate that Dot1L
and H3K79 methylation play important roles in heterochromatin formation and in embryonic development.
Citation: Jones B, Su H, Bhat A, Lei H, Bajko J, et al. (2008) The Histone H3K79 Methyltransferase Dot1L Is Essential for Mammalian Development and
Heterochromatin Structure. PLoS Genet 4(9): e1000190. doi:10.1371/journal.pgen.1000190
Editor: Wendy A. Bickmore, Medical Research Council Human Genetics Unit, United Kingdom
Received February 27, 2008; Accepted August 5, 2008; Published September 12, 2008
Copyright: ? 2008 Jones et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Novartis Institutes for Biomedical Research.
Competing Interests: All authors except AB, SG, and YZ are employees of Novartis Institutes for Biomedical Research.
* E-mail: taiping.chen@novartis.com
Introduction
Histones are subject to a variety of post-translational modifica-
tions, including acetylation, phosphorylation, ubiquitination, and
methylation. These modifications dictate chromatin structure by
affecting the recruitment of nonhistone proteins and/or the
interactions between nucleosomes [1,2]. Heterochromatin is
associated with high levels of methylation at H3K9, H3K27,
and H4K20 and low levels of acetylation, whereas actively
transcribed euchromatin is typically enriched with acetylation and
methylated H3K4, H3K36, and H3K79.
Most histone H3 modifications occur on residues within the N-
terminal tail. In contrast, H3K79 is located in a loop within the
globular domain, exposed on the nucleosome surface. The yeast
Dot1 and its homologues in other species are the only known
H3K79 methyltransferases [3–5]. Unlike other histone lysine
methyltransferases, Dot1 family members do not have a SET
domain [3–5]. Instead, their catalytic domain contains conserved
sequence motifs characteristic of class I methyltransferases such as
DNA methyltransferases (DNMTs) and the protein arginine
methyltransferase PRMT1 [6].
Dot1 was initially identified as a disruptor of telomeric silencing in
Saccharomyces cerevisiae [3]. Subsequent studies showed that both
overexpression and inactivation of Dot1 as well as mutations at
H3K79 all lead to loss of telomeric silencing [7–9]. Although the
mechanisms by which Dot1 affects telomere structure and function
are not fully understood, it is believed that H3K79 methylation plays
an important role in restricting the Sir proteins at heterochromatic
regions [7,8,10]. Dot1-dependent H3K79 methylation has also been
shown to be involved in meiotic checkpoint control and in G1 and S
phase DNA damage checkpoint functions of Rad9 in yeast [11,12].
H3K79 methylation is also a widespread histone modification in
mammalian cells [4]. Abnormal H3K79 methylation has been
linked to leukemogenesis in humans [13,14]. However, the
biological function of H3K79 methylation in mammals remains
largely unknown. Here we generated a mouse line containing a
null mutation in Dot1L, the murine Dot1 homologue, and
investigated the role of Dot1L and H3K79 methylation in
embryonic development and cellular function. We provide
evidence that Dot1L is required for embryogenesis and for the
integrity of constitutive heterochromatin at the cellular level.
Results
Generation of Dot1L Conditional and Null Alleles in Mice
To target the Dot1L gene, we constructed a targeting vector in
which a 2.3-kb genomic region containing exons 5 and 6 and a
promoterless b-geo selection cassette were flanked, respectively, by
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three loxP sites (Figure 1A). Exons 5 and 6 encode 108 amino acids
that form several conserved motifs in the Dot1L catalytic domain,
including the SAM-binding motif and motifs X, I, and II [6]. Since
mutations of conserved residues within motif I abolish the
methyltransferase activity of Dot1L [4], we predicted that deletion
of exons 5 and 6 would inactivate Dot1L.
ES cells were transfected with the targeting vector and selected
in G418-containing medium. Clones with homologous recombi-
nation were identified by Southern blot analysis with a 59 external
probe (Figure 1B). Three of these clones, referred to as Dot1L3lox/+,
were injected into blastocysts to generate chimeric mice, which
subsequently transmitted the mutant allele to their offspring.
Deletion of the b-geo cassette plus exons 5 and 6 was achieved by
breeding the Dot1L3loxallele into mice expressing Cre recombinase
in the germ line. The resulting null allele is referred to as Dot1L1lox
(Figure 1A). Genotypes were determined using PCR (Figure 1C).
Dot1L Is Essential for Embryonic Development
We first determined the expression of Dot1L during embryonic
development, taking advantage of the fact that cells containing the
Dot1L3loxallele express lacZ under the control of the endogenous
Dot1L promoter. We conducted X-gal staining on Dot1L3lox/+
heterozygous embryos and wild-type littermates at different stages
of development. Dot1L expression is ubiquitous as early as 7.5-dpc
(the earliest time point tested, Figure 2A). At 9.5-dpc, Dot1L
expression remains ubiquitous and areas of elevated expression are
Author Summary
Histone methylation plays a critical role in the regulation of
gene expression and chromatin structure. Among the sites
of histone methylation, lysine 79 of histone H3 (H3K79) is
unique in that it is not located within the H3 N-terminal tail
but in the globular domain. Our knowledge about H3K79
methylation comes primarily from studies in yeast. This
study focuses on the role of H3K79 methylation in
mammalian development and cellular function. We show
that genetic disruption of Dot1L, the only known H3K79
methyltransferase gene in mouse, results in embryonic
lethality. At the cellular level, Dot1L deficiency leads to
alterations in constitutive heterochromatin, accompanied
by telomere elongation, aneuploidy, and proliferation
defects. Our work represents a key step toward under-
standing the function of H3K79 methylation in mammals.
Figure 1. Generation of mutant Dot1L alleles in mice. (A) Schematic depiction of the strategy used to generate the Dot1L3loxand Dot1L1lox
alleles. The exons are numbered. The locations of the Southern probe and PCR primers (DF1, DR1, and DR2) used for genotyping, as well as the sizes
of the diagnostic fragments recognized by the Southern probe, are indicated (E, EcoRI). loxP sites are shown as triangles. (B) Southern blot analysis of
EcoRI-digested genomic DNA probed with an 860-bp 59 probe external to the targeting vector. The presence of the 8.3-kb band confirms
homologous recombination. (C) PCR genotyping of DNA from ES cells. WT, 485 bp; 1lox, 233 bp.
doi:10.1371/journal.pgen.1000190.g001
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apparent. Tissues that demonstrate high levels of lacZ staining
include the optic vesicle, the first branchial arch, the limb buds, the
heart, the otic pit, and the neural ectoderm (Figure 2B). Dot1L is
also expressed at high levels in extra-embryonic tissues, including
the visceral endoderm and visceral mesoderm of the yolk sac, and
in primitive erythrocytes (Figure 2C). Similar lacZ staining patterns
are observed in embryos harvested at 10.5-dpc, 11.5-dpc, and
12.5-dpc (data not shown), suggesting that Dot1L is broadly
expressed during embryonic development.
Dot1L1lox/+mice were grossly normal and fertile. However,
intercrosses of Dot1L1lox/+mice produced no viable Dot1L1lox/1lox
homozygous offspring, suggesting embryonic lethality (Table 1).
Dot1L1lox/1loxembryos harvested at 8.5-dpc were indistinguishable
from wild-type and Dot1L1lox/+littermates (data not shown). At 9.5-
dpc, Dot1L1lox/1loxembryos were smaller than littermates, had
enlarged hearts and stunted tails on gross observation when viewed
Figure 2. Essential role for Dot1L in mouse embryonic development. (A) A representative X-gal stained 7.5-dpc Dot1L3lox/+embryo
demonstrating ubiquitous Dot1L transcription throughout the embryo. (B) A representative X-gal stained 9.5-dpc Dot1L3lox/+embryo demonstrating
ubiquitous Dot1L transcription throughout the embryo with elevated Dot1L expression in the indicated regions. (C) A representative X-gal stained 9.5-
dpc Dot1L3lox/+yolk sac demonstrating Dot1L transcription in visceral endoderm, visceral mesoderm, and primitive erythrocytes. (D) Representative
pictures of 9.5-dpc Dot1L1lox/+and Dot1L1lox/1loxembryos. Dot1L1lox/+embryos (left) were indistinguishable from wild-type embryos. Most Dot1L1lox/1lox
embryos were undersized, had an enlarged heart (cardiac dilation) and stunted tail (center), while approximately 15% exhibited developmental arrest
at E8.5 (right). (E) Representative pictures of a 10.5-dpc Dot1L1lox/1loxembryo (right) and a heterozygous littermate (left). (F) Representative pictures
showing the yolk sac vasculature of 9.5-dpc Dot1L1lox/+(left) and Dot1L1lox/1lox(right) embryos. The vasculature of the Dot1L1lox/1loxyolk sac is thinner
and less organized than that of the heterozygous littermate.
doi:10.1371/journal.pgen.1000190.g002
Table 1. Dot1L deficiency results in embryonic lethality.
8.5-dpc 9.5-dpc 10.5-dpc 11.5-dpc 12.5-dpcBirth
Litters8125326
Embryos
Dot1L+/+
Dot1L1lox/+
67105 372816 58
14 (21%)26 (25%) 11 (30%)8 (29%) 3 (19%)20 (34%)
34 (51%)50 (48%)17 (46%)13 (46%) 5 (31%)38 (66%)
Dot1L1lox/1lox
14 (21%) 20 (19%)3 (8%) 0 (0%)0 (0%)0 (0%)
resorbed5 (7%)9 (8%) 6 (16%)7 (25%)8 (50%)NA
The numbers and percentages of Dot1L+/+, Dot1L1lox/+, Dot1L1lox/1lox, and
resorbed embryos harvested at the indicated time points are shown. NA: not
applicable.
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under a dissecting microscope (Figure 2D, center). Approximately
15% of the Dot1L1lox/1loxembryos demonstrated a severe phenotype,
exhibiting developmental arrest at E8.5 (Figure 2D, right).
Histological examination of 9.5-dpc Dot1L1lox/1loxembryo sections
revealed focal areas of extensive apoptosis, but no obvious structural
defects (Figure S1). At 10.5-dpc, the percentage of viable Dot1L1lox/
1loxembryos was substantially below the expected Mendelian ratio
(Table 1), suggesting that many of the Dot1L1lox/1loxembryos die
duringthistimeinterval.Thefewthatsurvivedtothisstageexhibited
developmental arrest at E9.5 and severe cardiac dilation (Figure 2E).
No Dot1L1lox/1loxembryos survived beyond 10.5-dpc (Table 1).
As stunted growth and enlarged heart are phenotypes that often
occur as a result of defects in extraembryonic tissues, we examined
the yolk sac and placenta of 9.5-dpc Dot1L1lox/1loxembryos. While
the placenta showed no obvious defects, the yolk sac exhibited
abnormal vascular morphology. The yolk sac vasculature was
present and contained primitive erythrocytes, but was frequently
underdeveloped and disorganized when compared to control
littermates (Figure 2F). These observations indicate that, in the
absence of Dot1L, vasculogenesis took place in the yolk sac but
angiogenesis was defective.
Dot1L Deficiency in ES Cells Results in Growth Defects
To investigate the cellular function of Dot1L, we derived Dot1L
mutant ES cells from blastocysts produced from intercrosses of
Dot1L1lox/+mice. Two Dot1L1lox/1loxand multiple Dot1L1lox/+and
Dot1L+/+lines were established. As expected, H3K79 di- and tri-
methylation was greatly reduced in Dot1L1lox/1loxcells compared to
Dot1L+/+cells (Figure 3A). Dot1L1lox/+cells had intermediate levels
of H3K79 di- and tri-methylation, indicating haploinsufficiency of
Dot1L (Figure 3A). Surprisingly, Western blot analysis using a
‘‘mono methyl H3K79’’ antibody (ab2886, Abcam) detected no
change in signal intensity in Dot1L mutant ES cell lines (data not
shown). To verify the results, we carried out mass spectrometry. In
wild-type ES cells, ,11% of histone H3 showed K79 methylation,
among which mono-, di-, and tri-methylation accounted for
,70%, ,30%, and less than 1%, respectively. In Dot1L1lox/1loxES
cells, H3K79 mono- and tri-methylation was absent although trace
amount of di-methylation was detected (Figure 3B and Figure S2).
We therefore concluded that the Western blot result showing no
alteration in H3K79 mono-methylation in the absence of Dot1L
was an artifact due to nonspecific recognition of histone H3 by the
‘‘mono methyl H3K79’’ antibody. The low level of H3K79 di-
Figure 3. Phenotypic analysis of Dot1L mutant ES cells. (A) Western blot analysis using extracts from ES cell lines of the indicated Dot1L
genotypes and antibodies specific for di-, and tri-methylated H3K79. Total histone H3 was used as a loading control. (B) Analysis of H3K79
methylation by mass spectrometry. Quantification of different forms of H3K79 methylation was obtained by comparing the extracted ion
chromatogram (EIC) intensity of the ion signals corresponding to the unmodified (Me0), mono-methylated (Me1), di-methylated (Me2), and tri-
methylated (Me3) K79-containing peptides. (C) The proliferation of Dot1L+/+, Dot1L1lox/+and Dot1L1lox/1loxES cells was determined by doing cell counts
every 24 hours for five days. Cells were grown in triplicate, and data shown is representative of three independent experiments. (D) The percentages
of apoptotic cells in Dot1L+/+, Dot1L1lox/+and Dot1L1lox/1loxES cell cultures. The asterisk indicates P,0.05 (Student t-test). ES cells were stained with
propidium iodide (PI) and PE conjugated anti-annexin V antibodies and analyzed by FACS. Apoptotic cells were annexin V positive and PI negative.
Cells were grown in triplicate, and data shown are representative of two independent experiments. (E) The percentages of each cell cycle stage in
Dot1L+/+, Dot1L1lox/+and Dot1L1lox/1loxES cell cultures as determined by PI staining and FACS.
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methylation detected in Dot1L1lox/1loxsamples could be from feeder
cells present in the culture or due to incomplete inactivation of
Dot1L. Taken together, these results indicated that Dot1L is most
likely the sole H3K79 methyltransferase in mice.
Dot1L mutant ES cells maintained an undifferentiated state, as
judged by morphology and ES cell markers such as Oct4 and
Nanog (data not shown). We investigated whether Dot1L
deficiency affects ES cell growth. We plated 36105Dot1L+/+,
Dot1L1lox/+, and Dot1L1lox/1loxES cells in standard ES cell medium
and monitored proliferation. By 24 hours, the number of Dot1L+/+
ES cells (7.36105) was significantly higher than the number of
Dot1L1lox/+or Dot1L1lox/1loxcells (4.26105and 3.66105respective-
ly, P,0.05, Figure 3C). Over the 5 days examined, Dot1L+/+,
Dot1L1lox/+, and Dot1L1lox/1loxES cells had average doubling
times of 16, 22, and 26 hours, respectively. The fact that both
Dot1L1lox/+and Dot1L1lox/1loxcells exhibited growth defects showed
the importance of Dot1L gene dosage in cellular function. The
reduction of H3K79 methylation in Dot1L1lox/+cells (haploinsuffi-
ciency) suggested that Dot1L level is relatively low. Interestingly,
Dot1L1lox/+mice were apparently normal despite the defects in
Dot1L1lox/+ES cells. It is possible that 50% of Dot1L can barely
maintain normal cellular function under ideal conditions (e.g. in
vivo) but is not sufficient to do so under suboptimal conditions (e.g.
in culture).
We next examined apoptosis and cell cycle status of the Dot1L
mutant ES cells. Annexin V staining revealed that 4.0% of the
Dot11lox/1loxES cells and 3.1% of the Dot1L1lox/+ES cells were
annexin V positive, while only 1.2% of the Dot1L+/+ES cells were
annexin V positive (Figure 3D). This indicates that more than
twice as many of the Dot1L mutant ES cells were undergoing
apoptosis compared to wild-type ES cells. Furthermore, cell cycle
analysis by propidium iodide staining revealed an elevated
percentage of G2 cells and a reduced percentage of G1 cells
among the Dot1L mutant ES cells when compared to the wild-type
ES cells (Figure 3E). These results suggest that both elevated
apoptosis and G2 cell cycle arrest contribute to the reduced
growth rate of Dot1L mutant ES cells.
Dot1L-Deficient ES Cells Show Telomere Elongation and
Aneuploidy
Dot1-deficient S. cerevisiae show telomere elongation and defects
in telomere silencing [3]. We therefore evaluated the effect of
Dot1L inactivation on telomere length. First, we used Southern
blot terminal restriction fragment (TRF) analysis to estimate
telomere length in two ES cell lines of each Dot1L genotype. Both
Dot1L1lox/1loxlines and one of the Dot1L1lox/+lines showed telomere
elongation, as evidenced by the presence of high molecular weight
(MW) TRFs and the increase in the lengths of bulk TRFs
compared to wild-type controls (Figure 4A). Next, we carried out
quantitative fluorescence in situ hybridization (Q-FISH) using a
telomere-specific probe to determine the mean telomere length
(mtl) and the distribution of telomere lengths for each cell line
(Figure 4, B and C). Consistent with the TRF results, both
Dot1L1lox/1loxlines had higher mtl, greater percentages of elongated
(.100 kb) telomeres, and reduced percentages of short (,50 kb)
telomeres compared to Dot1L+/+lines (Figure 4C). The heterozy-
gous ES cells again showed an intermediate phenotype (Figure 4C).
Examination of the Q-FISH samples revealed frequent
aneuploidy in Dot1L-deficient cells (Figure 4B). To further
investigate this phenotype, we prepared metaphase chromosome
spreads from Dot1L1lox/1loxand Dot1L+/+ES cells and examined
them for chromosomal defects. Dot1L+/+cells were karyotypically
stable, as the vast majority had 40 chromosomes. In contrast, over
40% of metaphase Dot1L1lox/1loxcells were aneuploid. Most of the
aneuploid cells showed gain of chromosomes and some ended up
being tetraploid (Figure 4, B and D). Aside from aneuploidy, no
obvious chromosomal abnormalities were frequently observed in
Dot1L-deficient cells (Figure 4B). These results point to defects in
chromosome segregation in the absence of Dot1L.
Aberrant Telomere Elongation in Dot1L-Deficient Cells
Correlates with Activation of the ALT Pathway
Our data suggested a role for Dot1L in the homeostasis of
telomere length. Two main mechanisms have been described for
the maintenance of mammalian telomeres: the addition of
telomeric repeats by telomerase and the so-called alternative
lengthening of telomere (ALT) mechanism that relies on
homologousrecombination between
[15,16]. Dot1L mutant ES cells showed increased telomere
heterogeneity (Figure 4), which is a hallmark of ALT cells [17].
To determine whether the ALT pathway is activated in Dot1L-
deficient cells, we assessed the presence of ALT-associated PML
bodies (APBs, colocalization of PML and telomeres), another
hallmark of ALT [17]. Dot1L+/+, Dot1L1lox/1lox, as well as
Dnmt3a2/23b2/2 (positive control) ES cells were immuno-
stained with antibodies against TRF1 (a telomere-binding protein)
and PML. In the absence of Dot1L, both the frequency of cells
showing APBs and the number of APBs per cell were significantly
increased compared to wild-type cells (Figure 5, A–C, x2tests,
P,0.001), suggesting that aberrant elongation of telomeres in
Dot1L-deficient cells was due, at least in part, to activation of the
ALT pathway.
telomeric sequences
Dot1L Deficiency Results in Loss of Heterochromatin
Marks at Telomeres and Centromeres
Aneuploidy and telomere elongation can result from defects in
the chromatin structure at centromeres and telomeres, respectively
[18–23]. To evaluate changes in chromatin structure in Dot1L
mutant cells, we used chromatin immunoprecipitation (ChIP) to
examine histone modifications at major satellite repeats (present at
pericentric regions), minor satellite repeats (present at centromeric
regions), telomeric repeats, and subtelomeric regions (Figure 6).
H3K79 di-methylation was detected in all these heterochromatin
regions in Dot1L+/+cells (Figure 6, A and B). As expected, this
modification was reduced in Dot1L1lox/+cells and almost absent in
Dot1L1lox/1loxcells (Figure 6, A and B), validating our experimental
procedures. As further controls, the levels of centromere- and
telomere-bound histone H3 were similar in wild-type and Dot1L
mutant cells (Figure 6, A and B), and the telomere-binding protein
TRF1 associated with telomeric repeats, but not with major
satellite repeats (Figure 6B).
In Dot1L1lox/1loxcells, H4K20 tri-methylation, a hallmark of
constitutive heterochromatin, was greatly reduced at minor
satellite repeats and sub-telomere regions, and moderately reduced
at major satellite repeats (Figure 6A). Consistent with this
observation, immunofluorescence analysis revealed the loss of
enrichment of H4K20 tri-methylation at pericentric heterochro-
matin in the absence of Dot1L (Figure S3). H3K9 di-methylation,
but not H3K9 tri-methylation, was reduced in all regions
examined in Dot1L1lox/1loxcells (Figure 6, A and B). Concomitantly,
H3K9 mono-methylation showed marked increases at major
satellite repeats and minor satellite repeats (Figure 6A), and H3K9
acetylation, a mark of euchromatin, was elevated in all regions
examined (Figure 6, A and B). These changes appeared to be
heterochromatin-specific, as all histone modifications examined,
except H3K79 methylation, showed no global changes in Dot1L
mutant cells (Figure S4). Dot1L deficiency did not cause
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alterations in DNA methylation at major satellite repeats and
minor satellite repeats as well as other genomic regions such as the
intracisternal A-particle (IAP) retroviral elements (Figure S5).
Altogether, these results suggest that loss of H3K79 methylation
results in a less compacted (or more open) chromatin state at
centromeres and telomeres.
Discussion
In this report, we provide genetic evidence that Dot1L and, by
implication, H3K79 methylation are essential for mammalian
development and normal cellular function. We show that loss of
Dot1L results in yolk sac angiogenesis defects and embryonic
Figure 4. Telomere elongation and aneuploidy in Dot1L-deficient ES cells. (A) Telomere restriction fragment (TRF) analysis upon MboI
digestion of genomic DNA from two independent ES cell clones of each of the genotypes: Dot1L+/+, Dot1L1lox/+and Dot1L1lox/1lox. Note the presence of
high molecular weight TRFs in Dot1L1lox/1loxcells, which correspond to longer telomeres. (B) Representative images generated during the Q-FISH
assay showing metaphase spreads from Dot1L+/+and Dot1L1lox/1loxES cells labeled with a telomere-specific fluorescent probe. (C) Telomere length
distribution of two independent ES cell clones of each of the Dot1L genotypes as determined by Q-FISH. Twenty metaphases of each ES cell clone
were analyzed. Note the increase in mean telomere length (Mtl) in both clones of Dot11lox/1loxcells and the intermediate phenotype of Dot11lox/+lines.
The percentages of telomeres below 50 kb and above 100 kb in length are indicated. (D) Scatter plot of the chromosome number of a Dot1L+/+ES
cell line and two Dot1L1lox/1loxcell lines. Chromosome number was determined by manually counting chromosomes in chromosome spreads. Each
point represents the chromosome number of a single cell (n represents the number of metaphase cells counted).
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lethality. Furthermore, our characterization of Dot1L-deficient ES
cells reveals that, like in yeast, H3K79 methylation plays a critical
role in heterochromatin structure in mammalian cells. Consider-
ing the differences in chromatin structure between yeast and
mammals, the phenotypic similarities in mutants of these
organisms are both striking and surprising. For example, both
mutant organisms exhibit telomere elongation, but mammalian
telomeres contain H3K79 methylated histones, while S. cerevisiae
telomeres contain no histones at all. Furthermore, while ,7% of
the budding yeast genome is packaged as heterochromatin
(including rDNA), ,55% of the mammalian genome is composed
of heterochromatin [24].
Dot1L recruitment is coupled with gene transcription [25] and
H3K79 methylation is enriched in euchromatin, which seem to be
counterintuitive to the heterochromatin phenotype of Dot1L-
deficient cells. One possible explanation is that Dot1L inactivation
alters the expression of specific factors involved in heterochromatin
assembly.Alternatively,globallossofH3K79methylationmayresult
in redistribution of heterochromatin factors, thereby reducing their
relative abundance at constitutive heterochromatin. Indeed, loss of
Dot1 in yeast leads to mislocalization of the Sir proteins, which
promote heterochromatin formation and telomere silencing [7,8].
H3K9 tri-methylation and H4K20 tri-methylation are hallmarks
of constitutive heterochromatin, such as that at centromeres and
Figure 5. Increased APBs in Dot1L-deficient cells. (A) Confocal microscopy images showing either TRF1 (telomere marker, green), PML (marker for
PML bodies, red), or combined fluorescence (yellow if colocalize, indicated by arrows) in wild-type (+/+) and Dot1L-deficient (1lox/1lox) ES cells. Late-
passage (p120)Dnmt3a/3b-deficient (3a2/23b2/2) ES cells were usedas a positive control. Circled are nuclei of cells. (B) Quantificationof percentage of
cells showing colocalization of telomeres with PML bodies. A cell was considered positive when it showed 2 or more colocalization events. An increased
frequency of cells showing APBs was observed in Dot11lox/1loxcultures compared towild-type controls (x2test, P,0.001). (C) Quantification ofthe number
of APBs per cell. Dot11lox/1loxcells showed a significant increase in the number of APBs compared to wild-type cells (x2test, P,0.001).
doi:10.1371/journal.pgen.1000190.g005
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telomeres [20,21,23,26,27]. Based on the observation that H3K9tri-
methylation by the Suv39h methyltransferases is required for the
induction of H4K20 tri-methylation by the Suv4-20h methyltrans-
ferases at pericentric heterochromatin, a sequential model of
chromatin assembly at constitutive heterochromatin has been
proposed in which Suv4-20h enzymes act downstream of the
Suv39h enzymes [26]. Dot1L-deficient cells show loss of H4K20 tri-
methylation at telomeres and centromeres, suggesting that Dot1L
functions upstream of the Suv20h enzymes. Given that H3K9 tri-
methylation showsno obviousalterations inDot1L-deficientcells,itis
possible that Dot1L acts in parallel or downstream of the Suv39h
enzymes. Interestingly, despite the relatively normal levels of H3K9
tri-methylation, H3K9 di-methylation is severely reduced at
constitutive heterochromatin in the absence of Dot1L. Because the
Figure 6. Changes of heterochromatin structure in Dot1L-deficient ES cells. (A) Quantitative real-time PCR results using DNA from Dot1L
mutant and wild-type ES cells immunoprecipitated with antibodies specific for the indicated histone modifications or without an antibody (No Ab)
and normalized using input DNA values. PCR primers specific for major satellite repeats, minor satellite repeats, the subtelomere region of
chromosome 1 or the subtelomere region of chromosome 2 were used. (B) Dot blot analysis of ChIP DNA using either a telomere-specific probe or a
major satellite repeat-specific probe. Input DNA at 1:10, 1:100 and 1:1000 dilutions was used as a positive control. DNA precipitated from
2.56106cells were used for each assay.
doi:10.1371/journal.pgen.1000190.g006
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total levels of H3K9 di-methylation and of several H3K9
methyltransferases (Suv39h1, ESET, and G9a) are not altered in
Dot1L-deficient cells (Figure S4), we speculate that Dot1L deficiency
may affect the targeting of one or more H3K9 methyltransferases or
demethylases to constitutive heterochromatin. Furtherstudies willbe
required to elucidate the mechanisms by which Dot1L and H3K79
methylation regulate heterochromatin.
Perturbation of epigenetic marks at constitutive heterochroma-
tin has been shown to cause chromosome instability and telomere
elongation [20–23]. Therefore, aberrant changes in chromatin at
centromeres and telomeres most likely underlie the aneuploidy
and telomere elongation observed in Dot1L-deficient ES cells. How
the observed alterations in chromatin structure and cellular
function contribute to the developmental abnormalities in Dot1L-
deficient embryos is less clear. The requirement of Dot1L for
normal cellular function does not appear to be ES cell-specific, as
RNAi-mediated Dot1L knock-down in somatic cell lines also leads
to growth arrest and cell death [13]. It is thus probable that
intrinsic defects in cellular proliferation and viability, which
themselves are likely the result of heterochromatin alterations,
contribute to the growth defects and apoptosis observed in Dot1L
mutant embryos. However, we believe that yolk sac defects are a
major cause of embryonic lethality. In the absence of Dot1L, yolk
sac angiogenesis is severely impaired. As both the endoderm and
mesoderm cell layers of the visceral yolk sac are critical for blood
vessel development [28,29] and both express Dot1L, we speculate
that, in the absence of Dot1L, aberrant changes in gene expression
and chromatin structure in one or both cell layers may underlie
the yolk sac vascular defects. Some embryonic abnormalities, such
as cardiac dilation, could be secondary to yolk sac vascular defects.
Although Dot1L is also expressed in primitive erythrocytes, loss of
Dot1L does not appear to have an obvious impact on
erythropoiesis. It remains to be determined, however, whether
the erythrocyte function is impaired.
Materials and Methods
Construction of the Gene Targeting Vector
The Dot1L conditional targeting vector, in which a 2.3-kb
genomic region containing exons 5 and 6 was flanked by loxP sites,
was constructed by sequentially subcloning Dot1L genomic
fragments and a floxed bGeo cassette into pBluescript SK
(Stratagene). The Dot1L genomic fragments were generated by
PCR using mouse genomic DNA as the template. The primer
pairs used were: 59-TTC ACT AGT CCC CAC CTT TGG ATT
G-39 and 59-GGC ACT AGT GTC ACA CAC CTT TA-39 for
the 59 arm, 59-CAT GTC GAC ACC GTG TAG TCC TGG
TGG GA-39 and 59-CTC GGC CGG CCT TGC CTG TGG
CTG ACG-39 for the 39 arm, and 59-GAC ACC GGT GCC
TGG CAA CCT TTT GG-39 and 59-CTG GGC GCG CCA
CCA GGA ACA CAC AGG TAC-39 for the floxed region
(underlined are the restriction sites used for cloning). The identity
of the vector was verified by DNA sequencing.
Generation of Dot1L Mutant Mice
The Dot1L conditional targeting vector was transfected into ES
cells via electroporation, and transfected cells were selected with
G418. Clones with homologous recombination (Dot1L3lox/+) were
identified using Southern blot. Genomic DNA was digested with
EcoRI and hybridized with a 59 external probe (The probe was
generated by PCR using the following primers: 59-CTC TGG
TAC CTT TGT TGT TAT ACA G-39 and 59-CTC TCA AGT
CGA CTG TAA GAT GAA G-39). Multiple Dot1L3lox/+clones
were used to generate chimeric mice and F1 heterozygotes.
Deletion of exons 5 and 6 as well as the bGeo cassette was
achieved by crossing Dot1L3lox/+mice with Zp3-Cre transgenic
mice, which express the Cre recombinase in the germline. Mutant
mice were maintained on a C57BL/6 inbred or a C57BL/6-
129Sv hybrid background. Primers used for PCR genotyping
were: DF1: 59-GGA ACT CAA GCT ATA GAC AG-39, DR1:
59-CAC TGC CCA GGT CGA CAA ACA G-39, and DR2: 59-
ATC CTC TCT CCT GAG GAG GCA GC-39 (Figure 1).
Embryo Collection, X-Gal Staining, and Histology
Female mice in Dot1L1lox/+intercrosses were examined for plug
formation to establish the timing of copulation. Deciduas were
isolated from euthanized females at various time points following
copulation, and embryos were examined under a dissecting
microscope. DNA from the yolk sac was used for genotyping by
PCR using the primers described above. X-gal staining was
performed on 7.5- to 12.5-dpc Dot1L3lox/+embryos and littermates
as previously described [30]. Embryo, yolk sac, and placental tissue
specimens, which were harvested at 9.5-dpc, were fixed in Bouin’s
solution, washed extensively in 70% ethanol, processed routinely for
paraffin embedding, sectioned at 5 mm, stained with hematoxylin
and eosin, and then evaluated by bright field microscopy.
ES Cell Derivation and Culture
Dot1L mutant ES cells were derived from blastocysts produced
from intercrosses of Dot1L1lox/+mice, as previously described [31].
Established ES lines were maintained in ES cell medium [32].
Apoptosis was analyzed using an Annexin V-PE apoptosis
detection kit (BD Pharmingen). Cell cycle analysis was done using
a PI/RNase Staining Buffer (BD Pharmingen).
Immunofluorescence and Immunoblot Analyses
Immunoblot and indirect immunofluorescence analyses were
carried out using standard procedures. The following antibodies
were used: anti-H3K79Me1 (Abcam), anti-H3K79Me2 (Abcam),
anti-H3K79Me3 (Abcam), anti-H3 (Millipore), anti-H3K4Me2
(Millipore), anti-H3K4Me3 (Millipore), anti-H3K9Me1 (Milli-
pore), anti-H3K9Me2 (Millipore), anti-H3K9Me3 (Millipore),
anti-H3K27Me1 (Millipore), anti-H3K27Me3 (Millipore), anti-
H3K9Ac (Millipore), anti-H4K20Me3 (Millipore), anti-H4Ac
(Millipore), anti-Suv39h1 (Upstate), anti-ESET (Upstate), anti-
G9a (Cell Signaling), anti-TRF1 (Abcam), anti-PML (Chemicon),
Alexa 488-conjugated goat anti-rabbit IgG (Molecular probes),
Alexa 555-conjugated goat anti-mouse IgG (Molecular Probes),
and peroxidase-conjugated goat anti-rabbit and goat anti-mouse
IgG (Jackson ImmunoResearch Laboratories).
Mass Spectrometry Analysis
Histone H3 purified from ES cells was digested with trypsin, and
the resulting peptides were analyzed using a LTQ-FT mass
spectrometer (Thermo Fisher Scientific Inc.) hyphenated with an
Agilent 1200 HPLC system (Agilent). Identification of the peptides
was performed bysearching the MS/MS fragmentation data against
the histone H3 sequence using MASCOT search software (Matrix
Science, version 2.1). All identifications were manually inspected for
correctness. The abundance of each identified and validated peptide
was calculated from its peak intensity using extracted ion
chromatogram (XIC) of LC/MS spectra. Relative quantification
of different forms of H3K79 methylation was performed by
comparingthe signalintensities
EIAQDFKTDLR at m/z 668.35 ([MH2]2+), EIAQDFKmeTDLR
at m/z 675.36 ([MH2]2+), EIAQDFK2meTDLR at m/z 682.35
([MH2]2+), and EIAQDFK3meTDLR at m/z 689.35 ([MH2]2+).
ofthe trypticpeptide
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Telomere Length Analysis
To analyze telomere length, we performed Q-FISH and TRF
analyses according to procedures described previously [22].
Metaphase Spread Analysis
To prepare metaphase spreads, cells were incubated with
0.1 mg/ml of colcemid for 4 hours and then harvested and
resuspended in 200 ml PBS. 10 ml of 75 mM KCl solution was
added dropwise with constant gentle agitation. Cells were fixed by
slow addition of 3:1 methanol/acetic acid solution, and then
dropped onto a microscope slide. Slides were washed in 70%
acetic acid, stained with DAPI and mounted. Chromosome
spreads were observed using a Zeiss fluorescence microscope.
Chromatin Immunoprecipitation Assays
ChIP was performed using 206106ES cells as described in the
online protocol provided byUpstate. Antibodysources are described
above. Purified DNA was either analyzed with quantitative real-time
PCR (qPCR) using Applied Biosystems SYBR PCR mastermix or
used in a dot blot assay as described [22]. qPCR primers used were
specific for major satellite repeats, minor satellite repeats [33], and
subtelomeric regions of chromosome 1 (forward: 59-TTA GGA
CTT CTG GCT TCG GTA G-39, reverse: 59-AGC TGT GGC
AGG CAT CGT GGC-39) and chromosome 2 (forward: 59-GAA
TCC TCC CTG TAG CAG GG-39, reverse: 59-GTA CAT AAC
CGATCCAGGTGTG-39).Relativeenrichmentwascalculatedas
2ˆ(CT(control CHIP) - CT(experimental CHIP)), where CTis equal
to the CT(immunoprecipitated sample) - CT(input) and normalized
sothat the wild-type value was1,with the exception of H3K9Me1 at
major satellite repeats where the Dot1L1lox/1loxvalue was 1. Each
sample used in the dot blot contained DNA precipitated from
2.56106cells. Probes used were
specific for telomeric repeats ((TTAGGG)x11) and major satellite
repeats (59-TAT GGC GAG GAA AAC TGA AAA AGG TGG
AAA ATT TAG AAA TGT CCA CTG TAG GAC GTG GAA
TAT GGC AAG-39), respectively.
32P-labelled oligonucleotides
DNA Methylation Assay
Genomic DNA isolated from ES cells was digested with
methylation-sensitive restriction enzymes and analyzed by South-
ern hybridization using probes specific for the major satellite
repeats, the minor satellite repeats, and the intracisternal A
particle retrovirus [32,34].
Supporting Information
Figure S1
sentative hematoxylin and eosin-stained sections from 9.5-dpc
Dot1L+/+(left) and Dot1L1lox/1lox(right) embryos illustrating focal
Elevated apoptosis in Dot1L1lox/1loxembryos. Repre-
areas of extensive apoptosis in the Dot1L1lox/1loxembryo (asterisk).
Scale bars=100 mm.
Found at: doi:10.1371/journal.pgen.1000190.s001 (0.5 MB PDF)
Figure S2
MS/MS. Tryptic digest mixtures were analyzed by ESI MS using
an LTQ-FT instrument. The precursor ion (m/z=668.35),
corresponding to the doubly charged (z=2) version of peptide
ion was selected for collision-induced dissociation (CID)-based
MS/MS analysis. The fragment ion spectrum was inspected for y
ions and the deduced sequence is indicated. The double
methylation on K79 was identified from the spectrum. Unmod-
ified, mono-methylated, and tri-methylated peptides were identi-
fied in the same way (data not shown).
Found at: doi:10.1371/journal.pgen.1000190.s002 (0.5 MB PDF)
Identification of H3K79 methylation by Nano-ESI
Figure S3
heterochromatin in Dot1L1lox/1loxcells. Dot1L+/+, Dot1L1lox/+and
Dot1L1lox/1loxES cells were immunostained with antibodies specific
for the indicated histone modifications and examined using a
fluorescent microscope. Dot1L1lox/1loxcells showed no obvious
alterations in the level and localization pattern of all modifications
tested, with the exception of H4K20 tri-methylation, which
displayed a more diffused nuclear pattern compared to Dot1L+/+
and Dot1L1lox/+cells.
Found at: doi:10.1371/journal.pgen.1000190.s003 (0.2 MB PDF)
Loss of H4K20Me3 enrichment at pericentric
Figure S4
H3K79 methylation in Dot1L1lox/1loxcells. Lysates from Dot1L+/+,
Dot1L1lox/+
and Dot1L1lox/1lox
ES cells were analyzed with
immunoblotting using antibodies specific for the indicated histone
modifications (A) or H3K9 methyltransferases (B).
Found at: doi:10.1371/journal.pgen.1000190.s004 (0.1 MB PDF)
No global changes in histone modifications besides
Figure S5
Dot1L. Genomic DNA from Dot1L+/+, Dot1L1lox/+, Dot1L1lox/1lox,
and Dnmt12/2 (c/c) ES cells were digested with MaeII (for major
satellite repeats) or HpaII (for minor satellite repeats and IAP) and
analyzed by Southern blot using the indicated probes.
Found at: doi:10.1371/journal.pgen.1000190.s005 (0.02 MB PDF)
No alteration in DNA methylation in the absence of
Acknowledgments
We thank F. Black and M. Constant for technical assistance, Y. Liu for
statistical analyses, T. de Lange for providing the telomere probe for TRF
analysis, R. Bronson for consultation on histological analysis, E. George
and colleagues in the Epigenetics Program at Novartis Institutes for
Biomedical Research for helpful discussions.
Author Contributions
Conceived and designed the experiments: BJ YZ EL TC. Performed the
experiments: BJ HS AB HL JB SH GAB SK HZ RV TC. Analyzed the
data: BJ SK SG TC. Wrote the paper: BJ TC.
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