Volume 24 October 1, 2013
MBoC | ARTICLE
H4K20 methylation regulates quiescence
and chromatin compaction
Adam G. Everttsa, Amity L. Manningb, Xin Wanga, Nicholas J. Dysonb, Benjamin A. Garciac,
and Hilary A. Collerd
aDepartment of Molecular Biology, Princeton University, Princeton, NJ 08544; bMassachusetts General Hospital Cancer
Center, Harvard Medical School, Charlestown, MA 02129; cDepartment of Biochemistry and Biophysics, University of
Pennsylvania, Philadelphia, PA 19104; dDepartment of Molecular, Cell and Developmental Biology, University of
California, Los Angeles, and Department of Biological Chemistry, David Geffen School of Medicine, Los Angeles,
ABSTRACT The transition between proliferation and quiescence is frequently associated
with changes in gene expression, extent of chromatin compaction, and histone modifica-
tions, but whether changes in chromatin state actually regulate cell cycle exit with quies-
cence is unclear. We find that primary human fibroblasts induced into quiescence exhibit
tighter chromatin compaction. Mass spectrometry analysis of histone modifications reveals
that H4K20me2 and H4K20me3 increase in quiescence and other histone modifications are
present at similar levels in proliferating and quiescent cells. Analysis of cells in S, G2/M, and
G1 phases shows that H4K20me1 increases after S phase and is converted to H4K20me2 and
H4K20me3 in quiescence. Knockdown of the enzyme that creates H4K20me3 results in an
increased fraction of cells in S phase, a defect in exiting the cell cycle, and decreased chro-
matin compaction. Overexpression of Suv4-20h1, the enzyme that creates H4K20me2 from
H4K20me1, results in G2 arrest, consistent with a role for H4K20me1 in mitosis. The results
suggest that the same lysine on H4K20 may, in its different methylation states, facilitate mi-
totic functions in M phase and promote chromatin compaction and cell cycle exit in quiescent
Proper formation of tissues and organisms requires that cells have
the capacity to transition between a proliferative, cycling state and
a resting state outside the proliferative cell cycle termed quies-
cence. Cells integrate cues from growth factors, other cells, and
extracellular matrix proteins and interpret these signals as they
decide whether to commit to proliferation or quiescence. The abil-
ity of cells to properly exit the cell cycle, retain viability during
quiescence, and return to the cell cycle when needed is necessary
for complex multicellular processes such as growth and healing.
Cells that fail to quiesce properly can contribute to the formation
The transition between an out-of-cycle, quiescent state and a
proliferative state is associated with changes in gene expression
patterns (Schneider et al., 1988; Coppock et al., 1993; Venezia et al.,
2004; Coller et al., 2006) and, in some systems, changes in overall
transcription rates (Jaehning et al., 1975). These changes in gene
expression with quiescence may be accompanied and regulated by
alterations in the packing of DNA as chromatin (Tokuyasu et al.,
1968; Dardick et al., 1983; Setterfield et al., 1983). In particular,
modifications of lysines on the tails of histones H3 and H4 play an
important role in local control of transcriptional activation and si-
lencing, and the information encoded in these tails may constitute a
code interpreted by proteins that bind to specific modifications
(Jenuwein and Allis, 2001).
Ludwig Institute for Cancer
Received: Jul 18, 2012
Revised: Jul 8, 2013
Accepted: Jul 29, 2013
This article was published online ahead of print in MBoC in Press (http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E12-07-0529) on August 7, 2013.
The authors declare that they have no conflict of interest.
H.C., B.G., and A.E. designed experiments. A.M. performed the FISH experi-
ments reported in Figures 1 and 7. A.E. and X.W. performed the experiments
reported in Figure 5. A.E. performed the remaining experiments. H.C. and A.E.
wrote the manuscript, and all other authors contributed comments.
Address correspondence to: Hilary A. Coller (firstname.lastname@example.org).
Abbreviations used: 14dCI, 14 d of contact inhibition; P, proliferating.
© 2013 Evertts et al. This article is distributed by The American Society for Cell
Biology under license from the author(s). Two months after publication it is avail-
able to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
“ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of
the Cell®” are registered trademarks of The American Society of Cell Biology.
3026 | A. G. Evertts et al. Molecular Biology of the Cell
to measure the levels of ∼50 histone modifications. With these
approaches, we characterized changes in chromatin compaction
and histone modifications as fibroblasts transition from prolifera-
tion to contact inhibition–induced quiescence. We further defined
changes in histone modifications with time during quiescence,
over the course of the cell cycle, and in cells that are terminally
arrested and have entered senescence. Our findings highlight the
importance of methylation on H4K20. The singly methylated form
of H4K20 is enriched in mitotic cells, whereas in quiescent cells,
the levels of H4K20me2 and H4K20me3 increase. This specific
lysine, in its different forms, plays a role in mitosis and during
quiescence, facilitating compaction and the transition between
proliferative and quiescent cell cycle states.
Contact inhibition in fibroblasts is associated with chromatin
We established a model system of quiescence in which primary hu-
man dermal fibroblasts are sampled either proliferating or after be-
ing induced into quiescence by contact inhibition (Lemons et al.,
2010). We sought to determine whether the quiescent state
achieved when primary human fibroblasts are contact inhibited is
associated with a change in chromatin compaction. We collected
proliferating (P), 14 d contact–inhibited (14dCI), and restimulated
fibroblasts (Figure 1, A–C) and used dual-color FISH with probes for
two loci on chromosome 16 to monitor the extent of chromatin
compaction. This technique has been used as a global measure for
chromatin compaction (Bystricky et al., 2004; Chambeyron and Bick-
more, 2004; Centore et al., 2010). The distance between the two
probes was measured in ∼50 cells in each state, and the average
distance was calculated. In quiescent, contact-inhibited fibroblasts,
the average distance between these loci was shorter than in prolif-
erating fibroblasts (Figure 1, D and E). Fibroblasts that were restimu-
lated to enter the cell cycle contained the largest average interlocus
distance. Thus, entry into quiescence in response to contact inhibi-
tion results in more tightly packed chromatin that is reversibly un-
packed upon stimulation.
H4K20 is differentially methylated in quiescent fibroblasts
Given our observation that contact-inhibited fibroblasts pack chro-
matin more tightly than proliferating or restimulated fibroblasts, we
sought to uncover histone modification changes that are associated
with quiescence in this system. To quantitatively measure the global
changes in steady-state levels of histone modifications between P
and 14dCI fibroblasts, we analyzed histones by liquid chromatogra-
phy-mass spectrometry (LC-MS/MS). Mass spectrometry allows for a
highly quantitative analysis of ∼50 histone modifications in a single
experiment, eliminating the need to select specific modifications for
analysis before performing the experiment. Histones were extracted
from primary human fibroblasts using acid extraction (Shechter
et al., 2007) and modified with propionic anhydride to block un-
modified and monomethylated lysines. Histones were digested with
trypsin (cleaving only at arginine residues) and analyzed by LC-MS/
MS. The intensity values for all modification states of a given pep-
tide were used to calculate the fraction of the peptide in each indi-
vidual modification state. Differential labeling of peptide N-termini
with hydrogen-containing or deuterium-containing propionic anhy-
dride was used to analyze two samples simultaneously and calculate
fold changes between conditions, similar to microarray analysis.
Surprisingly, after 14 d of contact inhibition, despite the fact that
the proliferating but not quiescent cells must replace their histone
modifications with each cell division, the majority of histone
When proliferating cells enter S phase, their histones are incor-
porated into chromatin unmodified. Most lysines are quickly modi-
fied, however, likely using the existing chromatin as a template to
transfer information. One lysine in particular does not become mod-
ified immediately after deposition—lysine 20 on H4 (Pesavento
et al., 2008; Zee et al., 2012). H4K20 becomes monomethylated
only during M phase as a result of mechanisms that limit the expres-
sion and activity of PR-Set7, the methyltransferase (Fang et al., 2002;
Nishioka et al., 2002). PR-Set7 is actively targeted for proteasome-
mediated degradation in S phase (Julien and Herr, 2004; Abbas
et al., 2010; Wu et al., 2010; Jorgensen et al., 2011). The M-phase
specific phosphorylation of PR-Set7 at serine 29 occurs at the onset
of mitosis and contributes to stabilization of the enzyme (Wu et al.,
2010). In addition, PHF8, an H4K20me1 demethylase, is removed
from chromatin in prophase, allowing for an accumulation of
H4K20me1 (Liu et al., 2010). Dimethylated and trimethylated H4K20
do not fluctuate as widely as the monomethyl form during the cell
cycle. H4K20me2 is the most abundant form in Drosophila (Yang
et al., 2008) and human cells (Young et al., 2009), existing on ∼80%
of histones. Trimethylated H4K20 is the least abundant of the three
forms and is associated with repeated sequences (Kourmouli et al.,
2004; Martens et al., 2005; Phalke et al., 2009). Knockouts of
the enzymes that modify H4K20 display a variety of phenotypes.
PR-Set7–knockout mice lose all H4K20 modifications and are em-
bryonic lethal. A conditional knockout in embryonic stem cells leads
to chromatin condensation defects (Oda et al., 2009). Knockout of
both Suv4-20h1 (the enzyme that converts the monomethylated
form to the dimethylated form) and Suv4-20h2 (the enzyme that
converts the dimethylated form to the trimethylated form) leads to
loss of the H4K20me2 and H4K20me3 histone forms and is perina-
tally lethal (Schotta et al., 2008). Loss of these modifications in
mouse cells leads to telomere elongation (Benetti et al., 2007;
Marion et al., 2011).
Studies examining the distribution of bulk levels of histone modi-
fications in quiescent and proliferating lymphocytes have revealed
differences in the levels of specific histone modifications between
the two states. In response to antigen, lymphocytes can be stimu-
lated from their quiescent state to divide. This reactivation is associ-
ated with an unpacking of the tightly wound heterochromatin and
physical relocalization of specific histone modifications within the
nucleus (Tokuyasu et al., 1968; Dardick et al., 1983; Setterfield et al.,
1983; Grigoryev et al., 2004). Activation of B cells is associated with
increased levels of multiple histone modifications (Baxter et al.,
2004). Lymphocyte activation is also associated with changes in the
intranuclear localization of specific histone modifications. H4K12Ac,
for instance, was excluded from centromeric heterochromatin in qui-
escent lymphoyctes and was redistributed more uniformly upon ac-
tivation (Grigoryev et al., 2004).
Primary dermal fibroblasts represent another cell type that can
transition between a quiescent and a proliferative state. Fibro-
blasts are often quiescent in vivo. Their physiological role is to se-
crete extracellular matrix proteins that give tissue its strength and
resilience. Fibroblasts can also proliferate, for instance, to replen-
ish dead cells, and upon activation in the context of a wound. Early
studies indicated a likely transition in chromatin structure upon
stimulation of quiescent fibroblasts based on circular dichroism
(Chiu and Baserga, 1975). We therefore set out to address whether
fibroblasts undergo changes in chromatin compaction and histone
modifications upon quiescence. We used fluorescence in situ hy-
bridization (FISH) for two loci on opposite arms of a single chromo-
some to quantitatively assess chromatin compaction. We also used
a mass spectrometry–based method (Plazas-Mayorca et al., 2009)
Volume 24 October 1, 2013 Chromatin regulation in quiescence | 3027
no methylation or one, two, or three methyl groups, each of which
has been reported to play a different cellular role. In quiescent fibro-
blasts, the fraction of H4K20 that is unmodified or contains a single
methyl group decreased >2-fold, whereas the fraction of H4K20
that is modified with two or three methyl groups increased 2- and
10-fold, respectively. We used an antibody specific for the trimethyl
form to confirm and validate the increase in H4K20me3 during qui-
escence and also found that the modification level is reversed after
48 h of restimulation (Figure 2B and Supplemental Figure S2). Im-
munofluorescence with the same antibody indicated that H4K20me3
was higher in abundance in the nucleus of quiescent versus prolifer-
ating fibroblasts, although a recognizable difference in the distribu-
tion pattern was not observed between the two states (Supplemen-
tal Figure S3). Thus, although most histone modifications are found
at similar steady-state levels in proliferating and contact-inhibited
fibroblasts, there were some histone lysines with reproducibly differ-
ent levels of methylation.
G1-enriched fibroblasts show differential H4K20 methylation
patterns compared with quiescent fibroblasts
The asynchronous proliferating cells that we monitored were a
mixture of cells in all phases of the cell cycle, whereas quiescent
cells were mostly in the G0/G1 cell cycle phase (Figure 1). To de-
termine whether there are histone modifications associated spe-
cifically with quiescence or G0, we compared histone modifica-
tions in quiescent cells with histone modifications in a purified
population of G1 cells, based on the expectation that quiescent
cells exit the cell cycle from G1 (Pardee, 1974). In this way, we
eliminated the effects of changes in histone modifications over
the cell cycle. Fibroblasts were synchronized with serum starva-
tion and hydroxyurea treatment. Cells were released from a G1/S
block via hydroxyurea and collected 12 h later in early G1. The
G1-enriched fibroblasts were compared with 14dCI fibroblasts us-
ing LC-MS/MS (Figure 2A). Most lysines had similar levels of mod-
ifications in G1-enriched fibroblasts compared with 14dCI fibro-
blasts, which is in accord with the finding that P and 14dCI cells
had similar levels of most histone modifications. The increase in
methylation on K27 observed in P versus 14dCI cells was reduced
with G1-enriched fibroblasts, suggesting that K27 methylation
fluctuates moderately over the cell cycle. We address this further
later. For H4K20, however, 14dCI fibroblasts contained higher lev-
els of dimethylated and trimethylated forms when compared not
only to proliferating cells but also to G1-enriched cells. Thus
these changes are associated with quiescence and do not reflect
a stalling in G1.
We identified six modifications that changed significantly in
14dCI versus G1-enriched fibroblasts, including H4K20me2 and
H4K20me3 (Figure 2, C and D). All six modifications also change
significantly between 14dCI and P cells. Unmodified H3K9/K14 and
unmodified H3K27/K36 were statistically significant but very mod-
estly repressed in quiescent compared with G1 cells. H3K36me1
and H3K27me3K36me1 were statistically significantly but very mod-
estly increased in quiescent compared with G1 cells. In contrast,
large changes were observed for H4K20. 14dCI fibroblasts con-
tained a twofold increase in the fraction of lysines in the dimethy-
lated form. Because many histones in the cell are in the H4K20me2
form, this twofold change translates to ∼34% increase in the amount
of histones present in the dimethylated form in 14dCI cells
(Figure 2D). H4K20me3 is a rare modification representing only
0.2% of H4K20 in proliferating fibroblasts. Its levels were increased
approximately eightfold in 14dCI fibroblasts, in which it represented
∼1.4% of all H4K20.
modifications were present at similar levels in proliferating and qui-
escent fibroblasts (Figure 2A). Of the 48 modification states moni-
tored, there were 44 with less than a twofold change. This suggests
that when fibroblasts transition between proliferation and quies-
cence, they mostly maintain similar steady-state histone modifica-
tion levels. The results do not rule out a difference in the rates at
which these modifications are added and removed, and we explore
this question elsewhere (Evertts et al., 2013). It also does not rule
out changes in the locations of the modifications, which can be ad-
dressed using chromatin immunoprecipitation (ChIP) sequencing.
Some lysines did show differential levels of histone modifications
between P and 14dCI (Figure 2, C and D). On histone H3, K9 and
K27 were more likely to be methylated in quiescent fibroblasts than
proliferating fibroblasts. Lysine 9 methylation was increased in qui-
escent cells, as indicated by a loss of unmodified H3K9. For H3K27,
the most prominent change was that quiescent fibroblasts con-
tained higher levels of H3K27me2 and H3K27me3, especially in
combination with modified K36. The largest changes between P
and 14dCI histones occurred on H4K20 (Figure 2, C and D, and
Supplemental Figure S1). H4K20 can exist in four distinct forms with
FIGURE 1: Contact-inhibited fibroblasts exhibit increased chromatin
compaction. (A–C) Proliferating, contact-inhibited (14 d), and
restimulated fibroblasts were collected and analyzed by propidium
iodide staining and flow cytometry. FlowJo analysis was performed to
estimate the fraction of cells in G1, S, and G2/M phases. (D) Cells were
fixed, and dual-colored FISH probes were used to visualize 16q22 and
16p13. Approximately 30–100 cells were measured, and the average
distance between the foci was determined. Mean and SE are plotted.
Contact-inhibited fibroblasts exhibited a smaller interprobe distance
than proliferating (p = 7.5 × 10−4) and restimulated (p = 6.4 × 10−8) cells.
(E) Representative images of FISH on both copies of chromosome 16
for P, 14dCI, and restimulated fibroblasts. Each arm of chromosome 16
is marked with a different color to visualize the distance between the
arms. Scale bar, 2 μm.
3028 | A. G. Evertts et al. Molecular Biology of the Cell
Schulze et al., 2009). To address this question, we monitored histone
modifications over the cell cycle in primary fibroblasts using LC-MS/
MS (Figure 3A). Cells were synchronized with serum starvation and
hydroxyurea treatment and collected in S, G2/M, and G1 phases
(Figure 3, B–E). Histones were extracted and analyzed together with
14dCI histones to generate a fold-change difference between 14dCI
and each cell cycle phase. The fold change was normalized across
each modification so that decreases represent lower amounts of the
modification relative to the average of all phases, and increases rep-
resent higher amounts of the modification relative to the average of
all phases. The majority of modifications do not change as the cells
pass through S phase and into other phases (Figure 3A). This sug-
gests that most lysines are rapidly modified at the replication fork or
soon after and that the cell maintains comparable levels of most
modifications throughout the cell cycle. For instance, the different
modified forms of two lysines that have been studied extensively in
the literature for their roles in transcription, H3K9 and H3K27, are
present at relatively stable levels across cell cycle phases, with some
slight variations that are most pronounced in the dimethylated forms
(Figure 4, A and B).
Histone modification patterns are maintained during
We then tested whether the changes in histone modifications ob-
served at 14 d of contact inhibition would be maintained if the cells
remained contact inhibited for a longer period of time. We com-
pared the pattern of histone modifications in 14dCI fibroblasts with
the pattern in 21dCI fibroblasts (Figure 2A). An additional 7 d of
contact inhibition did not further increase or decrease histone modi-
fication levels, including on H4K20, suggesting that the changes in
histone modification levels achieved by 14 d of contact inhibition
were preserved when cells were maintained in a contact-inhibited
state for a longer period of time.
H4K20me1 levels increase over the course of the cell cycle
and are converted to H4K20me2 and H4K20me3 in
Our discovery that there were differences in histone modifications
between a G1-enriched cell population and an asynchronous prolif-
erating population suggested that there are changes in histone
modifications during cell cycle progression (Pesavento et al., 2008;
FIGURE 2: Modification status of H4K20 changes as fibroblasts become quiescent. The relative abundances of
detectable histone modifications on histones H3 and H4 were determined for P, G1-enriched, 14dCI, and 21dCI
fibroblasts using (LC-MS/MS). P, G1-enriched, and 21dCI histones were compared with 14dCI histones by labeling the
N-termini of histones with hydrogen- or deuterium-containing propionic anhydride. The values for all modification states
on each peptide were used to determine relative abundance for each individual modification state for both samples
analyzed together. (A) The log2 fold changes of 14dCI vs. proliferating, G1-enriched, and 21dCI are shown in heat map
format. Data represent averages from three independent experiments. (B) Western blotting shows a similar change in
abundance of H4K20me3 in noncycling states and cycling states. A pan-H4 antibody was used as a loading control. (C) Six
modifications exhibited statistically significant differences (t test, p < 0.05) between G1-enriched and 14dCI fibroblasts.
The data from A are plotted to show the fold change between 14dCI and proliferating, G1-enriched, and 21dCI
fibroblasts. Error bars indicate SE. (D) Percentage of total histones modified for each of the six significant modifications.
Volume 24 October 1, 2013 Chromatin regulation in quiescence | 3029
FIGURE 3: Cell cycle–dependent changes in histone modification levels. The relative abundances of histone
modifications were determined for S phase–enriched, G2/M-enriched, and G1-enriched fibroblasts using LC-MS/MS.
(A) The log2 fold change for each phase relative to the average of all phases for each modification in heat map format.
Data represent means from three independent experiments for S and G1 and two independent experiments for G2/M.
(B–E) Propidium iodide staining and flow cytometry were used to generate cell cycle profiles for (B) asynchronously
proliferating fibroblasts and cells enriched in (C) S phase, (D) G2/M phase, and (E) G1 phase. Data represent the mean of
three independent experiments. Error bars indicate SE.
FIGURE 4: Modification states of H4K20 exhibit the greatest fluctuation across the cell cycle among histone lysines.
The relative percentage of modified histones was calculated using mass spectrometry data for histone modifications in
S, G2/M, G1, and 14dCI. The relative distribution of methylated forms is plotted for (A) H3K9, (B) H3K27, (C) H4K20, and
(D) acetylated H4 on lysines 5–16. Error bars indicate SE.
3030 | A. G. Evertts et al. Molecular Biology of the Cell
Suv4-20h2 did not alter the cell cycle profile significantly (Figure 6F).
Suv4-20h1–overexpressing cells were also stained with a histone 3
phospho-S10 antibody, an M-phase marker. Fewer phosho-S10–
positive cells were observed in Suv4-20h1–overexpressing cells, in-
dicating G2 arrest (Supplemental Figure S5). Finally, the same cells
were stained with X-gal to measure the level of senescence-associ-
ated β-galactosidase expression and were determined to have
higher levels of senescence than control cells (Supplemental
Figure S6). Thus a shift in the fraction of histone H4K20 containing
the dimethylated rather than monomethylated form had a signifi-
cant effect on cell cycle progression.
Knockdown of Suv4-20h1 and Suv4-20h2 results in loss
To further explore the possible importance of the trimethylated form
of H4K20, the form that displays the largest increase in quiescent
One lysine, H4K20—the same lysine that exhibited the largest
changes between proliferating and quiescent fibroblasts—did dis-
play large changes in modification levels across the cell cycle
(Figure 4C). This is consistent with reports that H4K20 methylation
status is cell cycle dependent (Jorgensen et al., 2007; Pesavento
et al., 2008; Oda et al., 2009; Abbas et al., 2010; Centore et al.,
2010; Wu et al., 2010). The unmodified form of histone H4K20 was
present at its highest level among the cell cycle states during
S phase. It decreased as the cells moved into G2/M and G1 and
remained low in quiescent cells. Levels of H4K20me1 were low in
S phase and progressively increased in G2/M. H4K20me1 levels
were lower in 14dCI than in G1 cells, although the result is not
statistically significant. Levels of H4K20me2 were relatively stable
throughout the cell cycle but increased in quiescence. Levels of
H4K20me3 were also relatively constant and very low over the
course of the cell cycle but rose in quiescent fibroblasts. Other
lysines on H4, such as H4K5-K16, did not exhibit the same pattern
of increased modification over the course of the cell cycle as
H4K20 (Figure 4D). The results indicate that H4K20, in its unmodi-
fied and monomethylated states, is the lysine that exhibits the
greatest changes over the course of the cell cycle and exhibits the
largest changes with quiescence.
Modification levels in senescence are similar to those
Quiescent fibroblasts have the ability to reenter the cell cycle and
proliferate after a period of cell cycle arrest. Other cells, such as
senescent or terminally differentiated cells, remain in a state of cell
cycle arrest and do not routinely reexpress proliferation-associated
genes. We sought to compare histone modifications in reversibly
arrested cells with those of permanently arrested cells by extend-
ing our analysis to senescent fibroblasts. A retroviral vector ex-
pressing an oncogenic form of the Ras protein (G12V) was intro-
duced into fibroblasts. RasG12V-overexpressing fibroblasts grew
rapidly and then ceased division and became senescent. Immuno-
blotting confirmed higher Ras levels in engineered fibroblasts
compared with cells transduced with a control vector (Figure 5A).
β-Galactosidase staining confirmed that the RasG12V-overexpress-
ing cells had entered senescence (Figure 5B). LC-MS/MS analysis
of histones in senescent fibroblasts indicated that the levels of
H4K20 modifications are similar between quiescence and senes-
cence (Figure 5C). This suggests that changes in the pattern of
H4K20 methylation occur during other types of cell cycle arrest
and not just during quiescence.
Overexpression of Suv4-20h1 causes an increase in the
fraction of cells in G2
Most H4K20 lysines are in a dimethylated form in fibroblasts that
were contact inhibited for 14 d. To assess whether a shift from the
monomethylated to dimethylated form of H4K20 contributes to cell
cycle exit, we overexpressed the enzyme that catalyzes the transi-
tion from H4K20me1 to H4K20me2 (Suv4-20h1) in fibroblasts
(Figure 6). Fibroblasts were transduced with a retrovirus containing
Suv4-20h1 driven by a cytomegalovirus promoter and allowed to
recover for 24 h after selection with puromycin. Fibroblasts overex-
pressing Suv4-20h1 were larger and more flattened than control
cells (Figure 6, B and D). The cell cycle profile of control and Suv4-
20h1–overexpressing fibroblasts was determined by staining cells
with propidium iodide, followed by flow cytometry analysis. Overex-
pression of Suv4-20h1 caused a decrease in the fraction of cells in S
phase and an increase in the fraction of cells in G2/M (p = 0.02,
paired t test; Figure 6, A, C, and E), whereas overexpression of
FIGURE 5: Histone analysis of senescent fibroblasts. Fibroblasts were
transduced with a retrovirus containing an empty pBABE vector or
pBABE-RasG12V. (A) Immunoblotting was performed to detect the
expression of Ras. (B) β-Galactosidase expression was detected in
cells transduced with a control vector and cells expressing RasG12V by
fixing cells and incubating with X-gal. (C) Mass spectrometry was
performed on control cells that were 14dCI and RasG12V-expressing
cells that were senescent. Relative percentage of modifications on
H4K20 are plotted. Error bars indicate SE.
Volume 24 October 1, 2013 Chromatin regulation in quiescence | 3031
Suv4-20h1 siRNA increased the distance between FISH probes but
did not achieve statistical significance, whereas treatment with the
Suv4-20h2 siRNA did produce a statistically significant change in
compaction (p = 0.01; paired t test). These results are consistent with
previous reports that the trimethyl form of H4K20 promotes com-
paction (Lu et al., 2008), as intrachromosomal locus distances in-
creased in cells with lower levels of this modification.
Knockdown of Suv4-20h1 and Suv4-20h2 results in
increased S-phase cells and defects in quiescence entry
To determine the effect of the loss of H4K20me2 and H4K20me3
on proliferation and quiescence, we transfected cells with a pool of
four siRNAs targeting Suv4-20h1 and Suv4-20h2 (four sequences for
each transcript). Cells were transfected twice, which resulted in a
reduction in the levels of the targeted transcripts (Supplemental
Figure S7). At 24 h after the second transfection, cells were treated
with the modified nucleotide 5-ethynyl-2’-deoxyuridine (EdU) for
2 h. The incorporated EdU was detected using a chemically bound
fluorophore and analyzed by flow cytometry (Click-iT EdU). The per-
centage of cells that were in S phase or entered S phase in the 2-h
labeling was significantly higher for fibroblasts transfected with
siSuv4-20h1/h2 than for a control set of siRNAs (Figure 8A). We next
assessed whether knockdown of Suv4-20h1/h2 affects the ability of
fibroblasts to exit the proliferative cell cycle in response to quies-
cence cues. We serum starved fibroblasts and monitored the frac-
tion in S phase 24 h later with Click-iT EdU. We discovered that cell
populations in which Suv4-20h1/h2 was depleted contained a
greater fraction of cells in S phase than controls (Figure 8A). By per-
forming the experiment with pools of siRNAs specific for either
Suv4-20h1 or Suv4-20h2, we discovered that Suv4-20h2 was the en-
zyme responsible for the S-phase phenotype (Figure 8A), as a reduc-
tion in Suv4-20h1 showed similar levels of S-phase cells, and knock-
down of Suv4-20h2 resulted in more S-phase cells. We then tested
whether Suv4-20h2 knockdown alone could lead to resistance to
contact inhibition and found that after 48 h of contact inhibition, the
population of cells transfected with the siSuv4-20h2 contained more
cells in S phase (Figure 8A). To eliminate the possibility of off-target
effects from the siRNA pools, we performed knockdown experi-
ments with individual siRNAs for both the control and Suv4-20h2
(Figure 8B). Transfection with three of the four Suv4-20h2 sequences
resulted in more S-phase cells compared with the control sequences,
suggesting that the results are not a consequence of off-target
effects of the siRNAs. These findings indicate that not only do the
levels of the higher methylated forms of H4K20 increase with quies-
cence, but the methylation state of H4K20 plays a functional role in
the control of cell cycle progression, with the trimethyl H4K20 main-
taining cells in a noncycling state (Figure 9).
Previous studies in lymphocytes (Baxter et al., 2004; Grigoryev et al.,
2004) and mesodermal precursors (Schubeler et al., 2000; Zhang
et al., 2002; Caretti et al., 2004; Mal, 2006) revealed that quiescence
in these cell types is associated with large changes in the levels of
many histone modifications. Our data, in contrast, demonstrate that
proliferating and contact-inhibited fibroblasts contain similar steady-
state, global levels of most histone modifications. Our data are in
accord with a recent study in which quiescence in T-lymphocytes
was not associated with changes in the global levels of histone mod-
ifications but instead with chromatin condensation by the condensin
II complex (Rawlings et al., 2011). Our results indicating similar levels
of histone modifications in proliferating and quiescent fibroblasts
for most modifications are also consistent with our previous data
cells, we also generated fibroblasts with retroviral vectors containing
knockdown of both Suv4-20h1 and Suv4-20h2. Mass spectrometry
was used to monitor the reduction of both modifications. H4K20me2
was reduced by ∼30%, and H4K20me3 was reduced by ∼70%
(Figure 7A). We analyzed intrachromosome distances using dual-
color FISH, as described earlier, in shControl and shSuv4-20h1/h2
cells and discovered that this distance increased 1.2-fold in the
knockdown cells, p = 1.2 × 10−6 (analysis of variance [ANOVA]; Figure
7B). Representative images show the decreased compaction ob-
served in shSuv4-20h1/h2 cells (Figure 7C). A more extreme exam-
ple of the decreased compaction is shown in Figure 7C, right, and
was observed only in shSuv4-20h1/h2 cells. We also monitored the
amount of compaction in cells with individual knockdowns of Suv4-
20h1 and Suv4-20h2 using small interfering RNAs (siRNAs). We
found that a reduction of either methyltransferase caused an ∼15%
increase in the distance between FISH probes, which is similar to the
effect observed with the shSuv4-20h1/h2 double knockdown. The
FIGURE 6: Overexpression of Suv4-20h1 induces G2/M arrest.
(A–D) Fibroblasts were transduced with a retrovirus containing an
empty expression vector or vectors expressing Suv4-20h1 or Suv4-
20h2. (A, C) Control and Suv4-20h1–expressing cells were stained with
propidium iodide and analyzed by flow cytometry. (B, D) Light
microscopy images (4×) were taken to show overall cell morphology.
The experiment was performed three times; representative data are
shown. (E, F) Percentages of cells in G1, S, and G2/M were determined
using FlowJo software and plotted for control and Suv4-20h1– and
Suv4-20h2–expressing cells. Error bars indicate SE.
3032 | A. G. Evertts et al. Molecular Biology of the Cell
in either H4K79me2 or H4K79me3 with our methods in human
In contrast, the methylation status of H4K20, a lysine that is not
conserved in S. cerevisiae, varied dramatically over the course of the
cell cycle in the human fibroblasts we studied. Previous reports
based on mass spectrometry analysis in HeLa cells showed that new
H4K20 is unmodified in S phase and becomes monomethylated
only in M phase (Pesavento et al., 2008; Zee et al., 2012) via specific
regulation of PR-Set7. We also show an increase of H4K20me1 dur-
ing G2/M and early G1 relative to S phase. H4K20 then shifts from
being highly enriched with monomethyl to predominantly dimethyl
and trimethyl when cells become quiescent. Although H4K20me1
has a critical function in M phase, quiescent cells contain lower lev-
els of the monomethylated form and higher levels of dimethylated
and trimethylated forms.
The H4K20 methylation profile is distinct in quiescent fibroblasts
from its profile at any point in the cell cycle. In quiescent fibroblasts,
the dimethylated and especially trimethylated forms accumulate
(Sarg et al., 2002; Kourmouli et al., 2004). This methylation pattern
indicating that contact-inhibited fibroblasts both repress and acti-
vate genes upon entering quiescence (Coller et al., 2006; Pollina
et al., 2008; Lemons et al., 2010) and maintain high metabolic rates
(Lemons et al., 2010). Although global levels do not change for
most modifications, however, the positions of the modifications
within chromatin could shift dramatically between proliferating and
quiescent fibroblasts. ChIP-sequencing experiments using antibod-
ies that bind these histone modifications would address whether
quiescence is associated with a redistribution of activating or repres-
sive marks among promoters to accommodate a new transcriptional
Histone modifications have been shown to vary across the cell
cycle. One report shows that H3K79me2 levels peak in Saccharo-
myces cerevisiae at G2/M and the enzyme responsible for creating
the mark (Dot1) is dependent on SBF, a cell cycle–regulated pro-
tein complex (Schulze et al., 2009). Knockdown of Dot1L in small-
cell lung cancer cells resulted in a proliferation block and display of
senescence characteristics, further linking this modification to the
cell cycle (Kim et al., 2012). We did not measure a large change
FIGURE 7: Suv4-20h1 and Suv4-20h2 knockdown results in decreased compaction. shRNAs were stably integrated and
expressed in fibroblasts. Nonspecific sequences were expressed in the control cells, and sequences targeting Suv4-20h1
and Suv4-20h2 were used to reduce H4K20me2 and H4K20me3. (A) Histones were analyzed by mass spectrometry, and
the fold change was plotted for shSuv4-20h1/h2 cells vs. shControl. (B) Dual-colored FISH was used to measure the
distance between both arms of chromosome 16 in shControl and shSuv4-20h1/h2 cells (p = 1.2 × 10−6; ANOVA).
(C) Representative images from FISH experiments depicting both copies of chromosome 16 with each arm identified
with a different color. Right, shSuv4-20h1/h2, an example of more extreme decompaction. (D) siRNA sequences were
used to reduce the expression of Suv4-20h1 or Suv4-20h2 individually. FISH was used to measure chromosome 16
compaction in siControl, siSuv4-20h1 (p = 0.055; paired t test), and siSuv4-20h2 cells (p = 0.01; paired t test).
Volume 24 October 1, 2013 Chromatin regulation in quiescence | 3033
involves cellular senescence within an organism, and H4K20me3
was found to increase in 450-d-old rat livers (Sarg et al., 2002).
H4K20me1 was shown to decrease in 12-mo-old mouse brains
(Wang et al., 2009), which could be indicative of a shift to H4K20me2
and H4K20me3. These studies further highlight the link between
nondivision and increases in H4K20me2 and H4K20me3.
We discovered that overexpression of Suv4-20h1, the methyl-
transferase that creates the dimethylated form of H4K20 at the ex-
pense of the monomethylated form, caused cell cycle arrest in G2.
This phenotype could reflect an overabundance of dimethylated
H4K20 or a relative depletion of monomethylated H4K20 (Supple-
mental Figure S4). Previous reports indicated that a lack of
H4K20me1 caused by inactivation of PR-Set7 results in activation of
a G2/M checkpoint (Abbas et al., 2010) and abnormal chromosomes
during mitosis (Rice et al., 2002; Oda et al., 2009). The phenotypes
associated with a lack of H4K20me1 could reflect the importance of
H4K20me1 in chromatin condensation, as subunits of the condensin
II complex and other proteins have been found to bind H4K20me1
and can induce chromatin condensation in vitro (Trojer et al., 2007;
Liu et al., 2010). Further, H4K20me1 can recruit L3MBTL1, which
preferentially binds monomethylated and dimethylated lysines and
induces chromatin compaction to negatively regulate gene expres-
sion (Trojer et al., 2007; Kalakonda et al., 2008).
A reduction in Suv4-20h2 resulted in defects in both S-phase cell
number and cell cycle exit. Knockdown cells cultured in the pres-
ence of full serum contained an increased fraction of cells in S phase
compared with controls. Further, in response to serum starvation or
contact inhibition, there were more S-phase cells in knockdown than
control cell populations. Increased numbers of cells in S phase could
result from cells progressing through S phase more slowly or cells
being blocked in S phase. Recent reports suggest a role for H4K20
in origin licensing and specifically demonstrate binding of H4K20me3
to ORC components (Vermeulen et al., 2011; Beck et al., 2012).
Therefore it is possible that a loss of H4K20me3 may interfere with
normal origin firing and lead to defects in S-phase progression.
Alternatively, the propensity to proliferate in cells in which Suv4-
20h2 is knocked down may be due to a loss of H4K20me3 at critical
regions of the genome, such as cell cycle regulatory genes.
H4K20me3 deposition has been shown to be dependent on Rb
(Gonzalo et al., 2005), and loss of the modification may affect ex-
pression of E2F genes. We observed additional S-phase cells at the
expense of G1/G0 cells, suggesting that cells are likely to be exiting
G1/G0 and progressing into S phase faster.
is likely associated with both reversible and irreversible cell cycle
exit. Other nondividing states, such as differentiation of murine
myogenic and neural lineages, also show increased levels of
H4K20me3 (Biron et al., 2004; Tsang et al., 2010), and mass spec-
trometry analysis of differentiating embryonic stem cells showed a
correlation between H4K20 methylation and loss of pluripotency
(Phanstiel et al., 2008). In our experiments, contact-inhibited and
senescent fibroblasts had a similar H4K20 profile despite the fact
that senescent and quiescent cells have very different chromatin
structures (Rai and Adams, 2012). Recent analysis of senescence-
associated heterochromatin foci, however, demonstrate that these
structures are not dependent on changes in the levels of repressive
chromatin marks such as H3K9me3 (Chandra et al., 2012; Chandra
and Narita, 2013), making increases in H4K20 unlikely to drive chro-
matin changes associated with senescence. The aging process often
FIGURE 8: Knockdown of Suv4-20h2 results in a higher fraction of
cells in S phase in full serum and after induction into quiescence.
Fibroblasts were treated with the nucleotide analogue EdU for 2 h,
stained with DAPI, and analyzed by flow cytometry to determine the
fraction of cells in S phase. (A) A pool of 4 siRNAs was used to reduce
the expression of Suv4-20h1 and Suv4-20h2. A nontargeting pool was
used as a control. Cells were analyzed in full serum and after 24 h of
serum starvation. Cells were also analyzed after transfection with
either a pool of siRNAs targeted against Suv4-20h1 or a pool of
siRNAs targeted against Suv4-20h2. Contact-inhibited cells were
analyzed with Suv4-20h2 siRNAs only. (B) Cells with full serum were
treated with one of four individual control siRNAs or one of four
siRNAs targeting Suv4-20h2, and the fraction of EdU-positive cells
FIGURE 9: Model for H4K20 methylation with cell cycle exit. The
relative levels of H4K20 methylation are shown for the four possible
modification states: unmodified (0), monomethyl (1), dimethyl (2), and
trimethyl (3). Modification levels are shown for the four phases of the
cell cycle, as well as for quiescence. Functions are assigned to some
modifications in particular phases with information from the literature
(black) and data from this study (red).
3034 | A. G. Evertts et al. Molecular Biology of the Cell
HFFs were plated at 30–40% confluence and maintained in a 37°C
incubator for 16 h. Cells were initially washed 2X with PBS and se-
rum-starved for 24 h in DMEM with 0.1% fetal bovine serum (FBS) to
synchronize them in G1. Cells were then washed 2× with PBS and
incubated with DMEM + 10% FBS + 2 mM hydroxyurea to release
them from serum starvation–induced arrest and block them at the
G1/S transition. After incubation for 18 h, cells were washed 2× with
PBS, and fresh DMEM with 10% FBS was added to allow a synchro-
nized exit into other cell cycle phases. Cells were harvested at 3 h
(S phase), 6.5 h (G2/M phases), and 12 h (G1 phase).
A total of 5 × 106 Phoenix cells was transfected with 5 μg of Ampho
helper plasmid (Imgenex, San Diego, CA) and 5 μg of either pBABE
or pBABE-RasG12V using Arrest-In (Open Biosystems, Huntsville, AL).
Viral supernatant was collected 48 h posttransfection and filtered
with a 0.45-μm filter. Cycling human fibroblasts were infected for
24 h with viral supernatant plus Polybrene at 2.6 μg/ml. Cells were
selected for 48 h in DMEM + 10% FBS + 2 μg/ml puromycin. After
selection, cells were passaged approximately three times until cell
division ceased. Cells were fixed between 1 and 14 d after cell cycle
arrest and stained with X-gal for 8 h to visualize β-galactosidase–
HFFs were removed from plates with PBS + 0.05% trypsin-EDTA.
For propidium iodide staining, cells were fixed and permeabilized
by adding one volume of PBS to two volumes of 100% ethanol
and stored at 4°C for >24 h. Ethanol was removed, and cells were
incubated with propidium iodide (PI; 40 μg/ml; EMD Chemicals,
Gibbstown, NJ) and RNase A (200 μg/ml; Roche, Basel, Switzerland)
in PBS for 1 h in the dark. For Click-iT analysis, cells were incubated
for 2 h in 10 mM EdU. Cells were pelleted, fixed with 4% paraform-
aldehyde, and treated with Alexa 488 azide (Invitrogen). DNA was
stained with 4′,6-diamidino-2-phenylindole (DAPI). All cells were
analyzed with a FACScaliber flow cytometer (BD Biosciences, San
Jose, CA). At least 20,000 cells were analyzed per sample. The soft-
ware FlowJo (version 8.8.2, Watson algorithm) was used to estimate
the fraction of cells in G1, S, and G2/M for PI-stained cells. Paired t
tests were used to determine whether the fraction of cells in differ-
ent phases of the cell cycle were significantly different in knockdown
cells and controls.
Histone isolation and preparation for MS
Histones were purified using acid extraction as previously described
(Shechter et al., 2007). Briefly, cell pellets were thawed and resus-
pended in 10 volumes (for every volume of cell pellet) of nuclear
isolation buffer (15 mM Tris-HCl at pH 7.5; 60 mM KCl; 15 mM NaCl;
5 mM MgCl2; 1 mM CaCl2; 250 mM sucrose; 1 mM DTT; 5 μM micro-
cystin; 500 μM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochlo-
ride; 10 mM sodium butyrate) with 10% NP-40 and incubated for
5 min. Nuclei were washed 2× with nuclear isolation buffer lacking
NP-40 and then mixed with five volumes (for every volume of nuclei)
of 0.4 N H2SO4 and incubated for 3–4 h at 4°C. Histones were pre-
cipitated with trichloroacetic acid and washed once with acetone
containing 0.1% HCl, followed by two washes with 100% acetone.
Histones were air dried overnight and resuspended in H2O. Histones
were processed using methods previously described (Plazas-
Mayorca et al., 2009). A solution containing 100 μg of purified his-
tones was reduced to 40 μl using a vacuum concentrator and mixed
with 20 μl of 3:1 anhydrous isopropanol to propionic anhydride
Modifications of H4K20 may be functionally important for qui-
escence. Dimethylated H4K20 serves as a binding site for the
DNA-damage recognition protein 53BP1 and thereby helps to me-
diate repair of double-strand breaks (Sanders et al., 2004; Botuyan
et al., 2006). Mouse embryonic fibroblasts containing null alleles
for the two Suv4-20h histone methyltransferases exhibit increased
sensitivity to damaging stress as a result of inadequate double-
strand break repair (Schotta et al., 2008). Quiescent cells cannot
engage in the high-fidelity DNA repair mechanism of homologous
recombination because there are no sister chromosomes from
which to perform homologous recombination (Blanpain et al.,
2011). Instead, they must rely on the error-prone pathway of non-
homologous end joining. Higher H4K20me2 levels in quiescent
cells could help them to initiate DNA repair events, which might
help to protect them from DNA damage.
H4K20me3, the mark most significantly enriched in quiescent
chromatin, is colocalized with H3K9me3 at centromeres
(Martens et al., 2005), telomeres (Benetti et al., 2007), and peri-
centric heterochromatin (Schotta et al., 2004). The methyltrans-
ferase for H4K20me3 may be recruited to sites of H3K9me3 via
interaction with the H3K9me3-binding protein Hp1 (Schotta
et al., 2004). Depletion of H4K20me3 by knockdown of Suv4-
20h1 and Suv4-20h2 results in depletion of dimethyl and trim-
ethylated H4K20, and chromosomes are less compactly orga-
nized, consistent with such a role for this modification. We did
not detect significant changes in the levels of H3K9me3 between
proliferating and quiescent cells, but with H4K20me3 constitut-
ing <2% of H4, we might not have detected such a small change
Loss of trimethylation at H4K20 is a common hallmark of human
cancer (Fraga et al., 2005; Pogribny et al., 2006; Van Den Broeck
et al., 2008; Schneider et al., 2011). In a large panel of cancer cells
and matched tumors and normal tissue, histone H4 consistently
exhibited decreased trimethylation in cells derived from tumors
(Fraga et al., 2005). In one study of bladder cancer, H4K20me3
levels decreased with increasing tumor grade (Schneider et al.,
2011). In lung tumors, an association was observed between low
levels of H4K20me3 and decreased levels of Suv4-20h2 (Van Den
Broeck et al., 2008). Decreased expression of H4K20me3 could
reflect the proliferative state of the tumor cells. Alternatively, lower
levels of H4K20me3 could promote tumorigenesis by preventing
the repression of genes that control cell cycle progression and
thereby inhibiting formation of a proper out-of-cycle state. A bet-
ter understanding of the changes in chromatin dynamics as cells
enter, maintain, and exit a quiescent state is likely to provide im-
portant insights into the control of cellular proliferation and how
this process is altered in developmental abnormalities, aging, and
MATERIALS AND METHODS
Primary human foreskin fibroblasts (HFFs) were isolated from donor
foreskins as previously described (Legesse-Miller et al., 2009). All
experiments were performed in cells with a passage number of <13.
Cells were cultured in DMEM (Invitrogen, Carlsbad, CA) with 10%
fetal bovine serum unless otherwise indicated. Proliferating cells
were plated at 50% confluence and harvested after 24 h to avoid
contact inhibition. Contact-inhibited cells were plated at 50% con-
fluence and incubated for the indicated number of days with me-
dium changes every 3 d. Cells used for histone analysis were scraped
in phosphate-buffered saline (PBS) from tissue culture plates and
flash frozen in liquid nitrogen.
Volume 24 October 1, 2013 Chromatin regulation in quiescence | 3035
Littleton, CO) was incubated for 16 h at 4°C. A secondary antibody
was incubated for 1 h at room temperature at 1:10,000 dilution, and
the membrane was exposed using an enhanced chemiluminescence
detection kit. For antibody testing, modified peptides (AnaSpec,
Fremont, CA) were spotted onto a polyvinylidene fluoride membrane.
Primary and secondary conditions were identical to those described
earlier except that streptavidin–horseradish peroxidase (PerkinElmer,
Waltham, MA) was used instead of a secondary antibody. For immu-
nofluorescence, cells were grown on glass slides (EZ slide; EMD
Millipore, Billerica, MA) and fixed using 4% formaldehyde for 15 min.
After permeabilization for 20 min with methanol, cells were treated
with blocking buffer and antibodies as described for Western blot-
ting. Anti–rabbit-488 was used for detection of antibodies, and cells
were stained with DAPI at 1 μg/ml to visualize the nucleus.
The CDS of Suv4-20h1 (variant 1) was PCR amplified from human
cDNA (forward, 5′-CCCGGGttaattaaATGAAGTGGTTGGGAGAA-
TCCAAGA-3′; reverse, 5′-CCCGGGggatccTTAGGCATTAAGCCT-
TAAAGACTGA-3′) and cloned into retroviral vector pQCXIP using
PacI and BamHI restriction sites. Vectors that express short hairpin
RNAs (shRNAs) against Suv4-20h1 (GTTTGTGTCAACTGGTCGA-
GATACAGCAT) and Suv4-20h2 (CGACCTGGATGTCGGCGGT-
GAAGAGCTGT) were purchased from OriGene (Rockville, MD).
Virus was generated and collected as described (senescence model).
For overexpression, fibroblasts were transduced with pQCXIP– or
pQCXIP-Suv4-20h1–derived virus and selected for 48 h with 2 μg/ml
puromycin. For the Suv4-20h1/h2 knockdowns, an shRNA against
green fluorescent protein (puromycin resistance gene) and a scram-
bled shRNA (blasticidin resistance gene) were used as controls. Both
control viruses or viruses derived from shRNAs to Suv4-20h1/h2
were used to coinfect fibroblasts. Infected cells were selected with
2 μg/ml puromycin and 30 μg/ml blasticidin for 5 d. Cells expressing
shRNAs were passaged three or more times to sufficiently reduce
histone modification levels before analysis. Cells expressing Suv4-
20h1 were maintained for 24 h without selection media and
analyzed. siRNAs were purchased from Thermo Scientific (Lafayette,
CO) as siGENOME SMARTpools targeting Suv4-20h1 and Suv4-
20h2. We transfected 100 nM of each siRNA pool or 200 nM of
control siRNAs into HFFs using Oligofectamine (Invitrogen, Carlsbad,
CA). Cells were expanded for 48 h, transfected again, and either
analyzed 24 h later as proliferating cells or serum starved for 24 h
and then analyzed.
(Sigma-Aldrich, Basel, Switzerland). The mixture was incubated at
37°C for 15 min and reduced back to 40 μl, constituting one round
of “propionylation.” The process was repeated one additional time,
and the solution was then reduced to near dryness. Histones were
resuspended in 100 μl of 100 mM ammonium bicarbonate (pH 8.0)
and digested with trypsin (Promega, Madison, WI) at a ratio of 1:50
trypsin to histones for 6–8 h at 37°C, followed by quenching of the
reaction by acidifying with glacial acetic acid (to pH <5) and freezing
at −80°C. Two additional rounds of propionylation were performed
to modify the newly exposed N-termini of histone peptides. To
quantify two samples in the mass spectrometer, one sample was
modified by propionic anhydride containing deuterium instead of
hydrogen (Cambridge Isotope Laboratories, Andover, MA), causing
a mass shift of +5 Da. Histone peptides from two samples were then
mixed and purified of salts with C18 STAGE-Tips constructed as de-
scribed previously (Rappsilber et al., 2003).
Histones were separated by reversed-phase high-performance liquid
chromatography (HPLC) on an Agilent 1200 series HPLC system
(Agilent, Santa Clara, CA) using a 75-μm–inner diameter fused silica
column packed with 10–15 cm of 5-μm C18 (Michrom, Auburn, CA).
A gradient of 0.7–30% buffer B in buffer A (buffer A, 0.1 M acetic
acid; buffer B, 95% acetonitrile in 0.1 M acetic acid) for 35 min fol-
lowed by 30–98% buffer B for 30 min was used to elute peptides,
which were ionized via electrospray ionization. Peptides were ana-
lyzed in a LTQ-Orbitrap mass spectrometer (ThermoFisher Scientific,
San Jose, CA). Full scans of m/z = 290–1000 with a resolution of
30,000 were measured in the Orbitrap. Collision-induced dissocia-
tion was used to fragment ions corresponding to isobaric acetylated
peptides (H3K9ac or K14ac [528.296, z = +2] and H3K18ac or K23ac
[570.841, z = +2]) in segments where those peptides elute; in other
segments data-dependent fragmentation was performed on the
seven most intense ions. Extracted ion chromatograms from Orbit-
rap data were integrated to yield intensity values for all histone pep-
tides of interest. The values for all modified forms of a particular pep-
tide were used to find the relative abundance of individual modified
forms for that peptide.
Dual-color fluorescence in situ hybridization
Cells were harvested and incubated at 37°C for 30 min in 0.59%
KCl. Cells were fixed in ice-cold methanol:acetic acid at a 3:1 ratio
and spread on glass slides. Slides were prepared for FISH using fluo-
rescently labeled probes specific for the arms of chromosome 16
(16q22, red; 16p13, green) according to the manufacturer’s instruc-
tions (LPH 022; Cytocell, Cambridge, United Kingdom). Coverslips
were mounted, and DNA was detected with 0.2 μg/ml DAPI/anti-
fade solution (Cytocell). Fluorescence images were captured with an
Orca AG cooled charge-coupled device camera (Hamamatsu,
Hamamatsu, Japan) mounted on a Nikon TI (Melville, NY)/Yokagawa
(Tokyo, Japan) CSU-10 spinning disk confocal microscope with a
100×, 1.4 numerical aperture objective. A series of 0.25-μm optical
sections was collected in the z-axis for each channel (DAPI, fluores-
cein, and Texas red). Intrachromosome distances under each condi-
tion were measured with SlideBook analysis software (SlideBook,
Denver, CO). Approximately 30–100 cells were used to measure in-
trachromosome distances for each condition, and three biological
replicates were scored.
Immunoblotting and immunofluorescence
We separated 10 μg of acid-extracted histones on a 15% polyacryl-
amide gel. Primary rabbit anti-H4K20me3 (Novus Biologicals,
We thank Eric Neeley (Novus Biologicals) for providing reagents and
all of the members of the Coller and Garcia laboratories for helpful
discussions. A.L.M. is supported in part by an American Cancer
Society Postdoctoral Fellowship. A.L.M. and N.J.D. are supported
by National Institutes of Health Grant R01 CA155202. B.A.G. is sup-
ported by a National Science Foundation Early Faculty CAREER
award, National Science Foundation Grant CBET-0941143, and a
grant supported by Award DP2OD007447 from the Office of the
Director, National Institutes of Health. H.A.C. is supported by
National Institute of General Medical Sciences Center of Excellence
Grant P50 GM071508, the Cancer Institute of New Jersey, the New
Jersey Commission on Cancer Research, National Cancer Institute
1RC1 CA147961-01, a Focused Funding Grant from the Johnson &
Johnson Foundation, and National Institutes of Health/National In-
stitute of General Medical Sciences Grants 1R01 GM081686 and
1R01 GM086465. H.A.C. was a Milton E. Cassel Scholar of the Rita
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