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 (email@example.com).
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
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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)
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