Direct Interaction between SET8
and Proliferating Cell Nuclear
Antigen Couples H4-K20
Methylation with DNA
Michael S. Y. Huen‡1,2, Shirley M.-H. Sy‡1,3, Jan M. van Deursen§,
and Junjie Chen‡4
Pediatrics,MayoClinicCollegeofMedicine,Rochester, Minnesota 55905
Chromatin endowed by histone modifications governs chro-
matin structure, which in turn represents a means to regulate
formation. Recent evidence revealed a plethora of enzymes that
catalyze specific histone modifications for epigenetic mainte-
and developmental defects. The histone methyltransferase
SET8 (also known as Pr-Set7) was previously reported to
spatial control of SET8 activity remains elusive. Here, we pro-
vide evidence to support that SET8 monomethylates Lys20of
histone H4 during S phase by tethering to proliferating cell
interacting protein box. In addition, we show that SET8 func-
tion is required for S phase progression. Finally, deletion of
SET8 in mice causes embryonic lethality, suggesting that SET8
plays an important role in mammalian embryogenesis.
Covalent modifications of histone tails play important roles
in regulating chromatin dynamics. Recent genetic and immu-
nochemical analyses suggest that these histone “marks” are of
fundamental importance in controlling the recruitment of reg-
ulatory factors that ensure cell proliferation and survival (1–3).
In particular, histone methylation, which occurs predomi-
nantly on histones H3 and H4 at arginine and lysine residues,
has been implicated in DNA repair, maintenance of telomere
length homeostasis, heterochromatin formation, and mitotic
regulation (4–9). Accordingly, a number of modules have been
such that effector complexes are brought to close proximity of
Histone methyltransferases, including SET8, Suv4-20h1/2,
NSD1, and Ash1, have been shown previously to methylate
histone H4 at Lys20(H4-K20) (8, 13–19). Specifically, H4-K20
trimethylation have been associated with constitutive pericen-
tromeric heterochromatin formation, whereas mono- and di-
malignant brain tumor protein L3MBTL1, which is important
for chromatin condensation (20–22). In addition, we proposed
previously that dimethylated H4-K20 might play a role in the
recruitment of the DNA damage-response protein 53BP1 to
chromatin (4). Despite mounting evidence for the biological
Given that SET8 expression is cell cycle-regulated, we
decided to further probe how SET8 might be required for cell
cycle progression. Using a TAP5scheme, we identified PCNA
by its physical tethering to PCNA, failure of which results in
improper S phase progression, S/G2checkpoint arrest, and
embryonic lethality in mice.
MATERIALS AND METHODS
Antibodies—Anti-histone H4 and anti-monomethylated
H4-K20 antibodies were purchased from Upstate Cell Signal-
ing. Anti-SET8 antibodies were from Abcam and Upstate Cell
Signaling. Anti-actin and anti-FLAG (M2) antibodies were
was from Santa Cruz Biotechnology Inc. Anti-PCNA (PC10)
and anti-glyceraldehyde-3-phosphate dehydrogenase antibod-
ies were from Chemicon.
Cell Culture and Transfection—HeLa and 293T cells were
cultured in RPMI 1640 medium supplemented with 5% fetal
calf serum, 5% bovine serum, 100 units/ml penicillin, and 100
?g/ml streptomycin and maintained in 5% CO2at 37 °C. Cell
transfection was performed using FuGENE 6 (Roche Applied
Science) and Lipofectamine 2000 (Invitrogen) following the
Construction of set8 and Its Mutants—The set8 cDNA was
obtained from American Type Culture Collection and sub-
cloned into the entry vector pDONR201 (Gateway Technol-
ogy). Site-directed mutagenesis was performed according to
standard procedures to obtain the YMAA mutant using prim-
ers 5?-ACC GGG CAG TCA AAG ATC TAT TCC GCC GCG
AGC CCG AAC AAA TGC TCT GGA ATG-3? and 5?-CAT
TCC AGA GCA TTT GTT CGG GCT CGC GGC GGA ATA
* This work was in part supported by National Institutes of Health Grant
indicate this fact.
1Both authors contributed equally to this work.
2Supported by an Anna Fuller fellowship from the Yale Cancer Center.
3Supported by the Croucher Foundation, Hong Kong.
ment of Defense and a member of the Mayo Clinic Breast Specialized
Program of Research Excellence (supported by NCI Grant P50
CA116201 from the National Institutes of Health). To whom corre-
spondence should be addressed. Tel.: 203-785-3758; Fax: 203-785-
7482; E-mail: firstname.lastname@example.org.
5The abbreviations used are: TAP, tandem affinity purification; PCNA, prolif-
erating cell nuclear antigen; GST, glutathione S-transferase; SFB, S tag/
FLAG tag/streptavidin-binding peptide; siRNA, small interfering RNA;
FACS, fluorescence-activated cell sorter; PIP, PCNA-interacting protein.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 17, pp. 11073–11077, April 25, 2008
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
APRIL 25, 2008•VOLUME 283•NUMBER 17 JOURNAL OF BIOLOGICAL CHEMISTRY 11073
This paper is available online at www.jbc.org
by guest on November 3, 2015
van Deursen and Junjie Chen
2008, 283:11073-11077. J. Biol. Chem.
Michael S. Y. Huen, Shirley M.-H. Sy, Jan M.
H4-K20 Methylation with DNA Replication
Proliferating Cell Nuclear Antigen Couples
Direct Interaction between SET8 and
doi: 10.1074/jbc.C700242200 originally published online March 3, 2008
Find articles, minireviews, Reflections and Classics on similar topics on the
10.1074/jbc.C700242200Access the most updated version of this article at doi:
. JBC Affinity Sites
When a correction for this article is posted •
When this article is cited •
to choose from all of JBC's e-mail alerts Click here
This article cites 33 references, 11 of which can be accessed free at
by guest on November 3, 2015