A glue for heterochromatin maintenance: stable SUV39H1 binding to heterochromatin is reinforced by the SET domain.

Abstract

Trimethylation of histone H3 lysine 9 and the subsequent binding of heterochromatin protein 1 (HP1) mediate the formation and maintenance of pericentromeric heterochromatin. Trimethylation of H3K9 is governed by the histone methyltransferase SUV39H1. Recent studies of HP1 dynamics revealed that HP1 is not a stable component of heterochromatin but is highly mobile (Cheutin, T., A.J. McNairn, T. Jenuwein, D.M. Gilbert, P.B. Singh, and T. Misteli. 2003. Science. 299:721-725; Festenstein, R., S.N. Pagakis, K. Hiragami, D. Lyon, A. Verreault, B. Sekkali, and D. Kioussis. 2003. Science. 299:719-721). Because the mechanism by which SUV39H1 is recruited to and interacts with heterochromatin is unknown, we studied the dynamic properties of SUV39H1 in living cells by using fluorescence recovery after photobleaching and fluorescence resonance energy transfer. Our results show that a substantial population of SUV39H1 is immobile at pericentromeric heterochromatin, suggesting that, in addition to its catalytic activity, SUV39H1 may also play a structural role at pericentromeric regions. Analysis of SUV39H1 deletion mutants indicated that the SET domain mediates this stable binding. Furthermore, our data suggest that the recruitment of SUV39H1 to heterochromatin is at least partly independent from that of HP1 and that HP1 transiently interacts with SUV39H1 at heterochromatin.

Full text

Page 1

T H E J O U R N A L O F C E L L B I O L O G Y
©
The Journal of Cell Biology, Vol. 170, No. 4, August 15, 2005 537–549
http://www.jcb.org/cgi/doi/10.1083/jcb.200502154
The Rockefeller University Press$8.00
JCB: ARTICLE
JCB537
A glue for heterochromatin maintenance: stable
SUV39H1 binding to heterochromatin is reinforced
by the SET domain
Ilke M. Krouwels, Karien Wiesmeijer, Tsion E. Abraham, Chris Molenaar, Nico P. Verwoerd, Hans J. Tanke,
and Roeland W. Dirks
Department of Molecular Cell Biology, Leiden University Medical Center, 2333 AL Leiden, Netherlands
T
rimethylation of histone H3 lysine 9 and the subse-
quent binding of heterochromatin protein 1 (HP1)
mediate the formation and maintenance of pericen-
tromeric heterochromatin. Trimethylation of H3K9 is gov-
erned by the histone methyltransferase SUV39H1. Recent
studies of HP1 dynamics revealed that HP1 is not a stable
component of heterochromatin but is highly mobile
(Cheutin, T., A.J. McNairn, T. Jenuwein, D.M. Gilbert,
P.B. Singh, and T. Misteli. 2003.
Festenstein, R., S.N. Pagakis, K. Hiragami, D. Lyon,
A. Verreault, B. Sekkali, and D. Kioussis. 2003.
ence.
299:719–721). Because the mechanism by which
SUV39H1 is recruited to and interacts with heterochro-
Science. 299:721–725;
Sci-
matin is unknown, we studied the dynamic properties of
SUV39H1 in living cells by using fluorescence recovery
after photobleaching and fluorescence resonance energy
transfer. Our results show that a substantial population of
SUV39H1 is immobile at pericentromeric heterochroma-
tin, suggesting that, in addition to its catalytic activity,
SUV39H1 may also play a structural role at pericentro-
meric regions. Analysis of SUV39H1 deletion mutants
indicated that the SET domain mediates this stable bind-
ing. Furthermore, our data suggest that the recruitment
of SUV39H1 to heterochromatin is at least partly inde-
pendent from that of HP1 and that HP1 transiently inter-
acts with SUV39H1 at heterochromatin.
Introduction
Heterochromatin is considered to be the part of the genome that
is gene poor, transcriptionally silent, and, furthermore, highly
condensed and static in interphase cells. In most eukaryotes, it
is characterized by DNA methylation at cytosine guanine dinu-
cleotides and by histone hypoacetylation and methylation.
These modifications play a pivotal role in the establishment
and function of heterochromatin by creating binding sites for
heterochromatin proteins (Jenuwein and Allis, 2001). HP1
(heterochromatin protein 1) is, with a few exceptions, typically
associated with heterochromatin and binds to histone H3,
which is methylated at lysine 9 (H3K9; Bannister et al., 2001;
Lachner et al., 2001). Because HP1 can form homodimers and
interacts with several other chromatin proteins, it is thought to
nucleate the formation of a higher order structure that is in-
compatible with transcription. An unexpected feature of HP1
is, however, that it is in continuous flux with chromatin. This
suggests that heterochromatin is not a static, inaccessible
higher order conformation but is a dynamic structure that has
the potential to rapidly adapt to various stimuli that influences
gene expression patterns or cell cycle progression (Cheutin et
al., 2003; Festenstein et al., 2003).
The histone methyltransferase SUV39H1 has been im-
plicated to play an essential role in the initial steps of hetero-
chromatin formation in mammals by selective methylation of
H3K9. In particular, trimethylation of H3K9 is mediated by
SUV39H1 and proved to be essential in the establishment of
constitutive heterochromatin at pericentromeric and telomeric
regions in the genome, both of which consist of tandem re-
peated DNA sequences (Peters et al., 2003; Garcia-Cao et al.,
2004). Mice that were deficient for SUV39H1 were shown to
display impaired pericentric H3K9 methylation and chromo-
somal instability (Peters et al., 2001). Furthermore, loss of
methylated H3 tails was shown to be accompanied by delo-
calization of HP1 from pericentric heterochromatin (Maison
et al., 2002). In
Drosophila melanogaster
the homologue of human SUV39H1 and of mouse Suv39h1
and Suv39h2. Similar to mouse mutants,
, SU(VAR)3-9 is
D. melanogaster
Correspondence to Roeland W. Dirks: r.w.dirks@lumc.nl
C. Molenaar’s
present address is Developmental Genetics Laboratory, Cancer
Research UK, London WC2A 3PX, England, UK.
Abbreviations used in this paper: 5-aza-C, 5-aza-2
pass; dn, double null; FLIM, fluorescence lifetime imaging microscopy;
fluorescence resonance energy transfer;
PMEF, primary mouse embryonic fibroblast; TSA, trichostatin A.

?
deoxycytidine;

BP, band-

FRET,

HP1, heterochromatin protein 1;

Page 2

JCB • VOLUME 170 • NUMBER 4 • 2005538
SU(VAR)3-9–null mutants revealed a strong reduction in
H3K9 methylation and an almost complete loss of HP1 from
chromocenter heterochromatin (Schotta et al., 2002). Together,
these findings underscore the essential role of SUV39H1 meth-
yltransferase activity in establishing heterochromatin.
The catalytic methyltransferase activity of SUV39H1 has
been mapped to the conserved COOH-terminal SET domain
and uses monomethylated H3K9 as a substrate (Peters et
al., 2003). The first 44 amino acids at the NH
SUV39H1 form an interaction domain for the chromoshadow
domain of HP1 and are, together with the adjacent chromo-
domain, required for binding to heterochromatin (Melcher et
al., 2000). Interestingly, in HP1-deficient
ivary gland nuclei SU(VAR)3-9 was shown to be lost from
chromocenter heterochromatin and to spread along euchromatic
regions, suggesting that an interaction between SU(VAR)3-9
and HP1 is essential for the association of SU(VAR)3-9 with
centromeric heterochromatin (Schotta et al., 2002). However,
the exact mechanism by which SUV39H1 interacts with het-
erochromatin is poorly understood.
Recently, the initial step in centromeric heterochromatin
formation was suggested to be mediated by direct or indirect
binding of SUV39H1 to components of an RNA interference
pathway (Maison et al., 2002), but the mechanism by which
SUV39H1 is initially recruited to centromeric repeat units is
still enigmatic. Also, it is unclear whether SUV39H1 interacts
only temporally with chromatin to methylate histone H3K9 or
participates in a more stable multiprotein complex together
with HP1 or other chromatin proteins to support a stable het-
erochromatic, pericentromeric chromatin structure. To gain
2
terminus of
D. melanogaster
,

sal-
further insight into the roles that SUV39H1 plays in hetero-
chromatin, we investigated the in vivo kinetics of SUV39H1 in
human osteosarcoma U2OS and in mouse NIH3T3 cells. Using
FRAP analysis, we showed that SUV39H1 has a significantly
slower exchange rate and a larger immobile fraction in hetero-
chromatic regions compared with HP1
that a substantial fraction of SUV39H1 is immobile at pericen-
tromeric heterochromatin, at least on the time scale of our ex-
periments, and may, therefore, play a structural role. Further-
more, our data indicate that at least a part of the dynamic
fraction of SUV39H1 is recruited to heterochromatin indepen-
dently from HP1 binding. In addition, interactions between
various SUV39H1 deletion mutants and HP1
in vivo by photobleaching and fluorescence resonance energy
transfer (FRET) techniques to characterize the binding of
HP1
?
to SUV39H1 at heterochromatin.
?
. Our results suggest
?
were analyzed
Results
EYFP-SUV39H1 localizes to
heterochromatin domains and
is catalytically active
To determine the in vivo kinetic properties of SUV39H1,
we made a construct coding for the fusion protein EYFP-
SUV39H1. This construct was transiently transfected into hu-
man U2OS and mouse NIH3T3 cells. Before analyzing the ki-
netic properties of the fusion protein, we first analyzed its spa-
tial distribution in moderately expressing cells after fixation. In
U2OS cells, EYFP-SUV39H1 localized to irregular shaped do-
mains, which were frequently observed to be present at the nu-
Figure 1.
mouse NIH3T3 as well as in human U2OS
cells. (A) After transfection, EYFP-SUV39H1 lo-
calizes in NIH3T3 cells to distinct nuclear re-
gions that are also visible by DAPI staining. In
U2OS cells, EYFP-SUV39H1 also localizes at
sites that are stained by DAPI, but these areas
are less well defined. (B) NIH3T3 cells trans-
fected with EYFP-SUV39H1 were labeled with
antibodies specific for trimethylated H3K9
and HP1?. (C) Human U2OS cells transfected
with EYFP-SUV39H1 were labeled with anti-
bodies against centromeres. Single optical
sections show the YFP-tagged protein, cen-
tromere labeling, and an overlay. Line scans
(diagonal lines through the images) show the
local intensity distributions of the EYFP fusion
protein in yellow and of the centromere label-
ing in red. Bars, 10 ?m.
Localization of EYFP-SUV39H1 in

Page 3

A GLUE FOR HETEROCHROMATIN MAINTENANCE • KROUWELS ET AL.539
clear periphery around nucleoli but were also dispersed
throughout the nucleoplasm (Fig. 1 A, right). In NIH3T3 cells,
a more defined distribution pattern of EYFP-SUV39H1 was
observed that corresponded to heterochromatic domains, as re-
vealed by DAPI staining as bright fluorescent regions (Fig. 1
A, left). In addition, a diffuse staining throughout euchromatic
regions was detected. These staining patterns are in agreement
with previous immunocytochemical studies on SUV39H1 lo-
calization that used a specific anti-SUV39H1 antibody (Aa-
gaard et al., 1999; Melcher et al., 2000). Notably, these studies
show that endogenous Suv39h1 is enriched at heterochromatic
foci in mouse interphase cell nuclei. Next, we compared the
staining patterns of EYFP-SUV39H1 with the immunocy-
tochemical staining patterns of trimethylated H3K9, HP1
HP1
?
. The latter two are heterochromatin proteins that are pre-
dominantly present in constitutive heterochromatin (Wreggett
et al., 1994). The results revealed very similar staining patterns
in U2OS as well as in NIH3T3 cells (Fig. 1 B), suggesting
that EYFP-SUV39H1 localizes mainly to heterochromatic do-
mains. Furthermore, immunocytochemical detection of cen-
tromeres in EYFP-SUV39H1–expressing U2OS cells revealed
that many, but not all, EYFP-SUV39H1 foci corresponded to
centromere localization (Fig. 1 C). Together, these experiments
confirm that low to moderate expression levels do not lead to
mislocalization of EYFP-SUV39H1. Previously, it was ob-
served that strong overexpression of SUV39H1 fusion proteins
led to aberrant localization patterns and to a redistribution of
HP1 (Melcher et al., 2000).
To confirm that EYFP-SUV39H1 is catalytically active
in vivo, Suv39h double-null (dn) primary mouse embryonic fi-
broblasts (PMEFs; Bannister et al., 2001) were transfected with
EYFP-SUV39H1 and were stained for trimethylated H3K9 and
HP1
?
by using specific antibodies. In nontransfected cells,
there appeared to be little trimethylated H3K9 present (Fig. 2
A, bottom; cells are indicated by arrows), and HP1
a diffuse nuclear staining (Fig. 2 B, bottom). In EYFP-
SUV39H1–expressing dn PMEFs, however, trimethylated
?
, and
?
displayed
H3K9 was found to be enriched in foci (Fig. 2 A, middle and
bottom), which are similar to the trimethylated H3K9–stained
foci that were observed in wild-type PMEFs (Fig. 2 A, top).
Also, HP1
?
relocated to heterochromatic foci in dn PMEFs ex-
pressing EYFP-SUV39H1 (Fig. 2 B, middle), which resembled
the HP1
?
-stained foci in wild-type PMEFs expressing EYFP-
SUV39H1 (Fig. 2 B, top). These results strongly indicate that
the Suv39h dn phenotype can be rescued by EYFP-SUV39H1
and that this fusion protein is functional.
SUV39H1 is a more stable component
of heterochromatin than HP1
To gain insight into the dynamic properties of SUV39H1 and
its recruitment to heterochromatin, FRAP experiments were
performed using NIH3T3 cells. Cells were transiently trans-
fected with EYFP-SUV39H1, and defined areas of
diameter were irreversibly photobleached for 2.5 s each in het-
erochromatic as well as in euchromatic regions. Fluorescence
recovery in the same areas was imaged at regular time points in
a time series (Fig. 3 A). The corresponding fluorescence inten-
?
?
1
?
m in
Figure 2.
type of Suv39h dn PMEFs. Suv39h dn and wild-type
PMEFs transfected with EYFP-SUV39H1 were labeled
with antibodies against trimethylated H3K9 (A) or HP1?
(B). In nontransfected Suv39h dn PMEFs, a little trimethy-
lated H3K9 is present throughout the nucleus (A, arrows).
Transfected cells show an increase in trimethylated H3K9
that is localized to heterochromatic areas (A, middle and
bottom). This localization is comparable to wild-type cells
(A, top). Bars, 5 ?m.
EYFP-SUV39H1 expression rescues the pheno-
Table I.
FRAP values measured in this study
Constructt
1/2
t
1/2
TSA t
1/2
5-aza-C
sss
EYFP-SUV39H1
(heterochromatin)
EYFP-SUV39H1
(euchromatin)
EYFP-SUV39H1
(heterochromatin U2OS)
EYFP-SUV39H1
(heterochromatin Suv39h
EYFP–SUV39H1-
EYFP–SUV39H1-Nchromo
EYFP–SUV39H1-chromo
EYFP–SUV39H1-
EYFP-HP1
?
19.07.7 14.6
11.8NDND
15.0 NDND
?
/
?
)
15.0ND ND
?
SET8.6
3.8
0.6
0.5
4.2
6.0
ND
ND
ND
1.8
3.0
ND
ND
ND
4.2
?
N89

Page 4

JCB • VOLUME 170 • NUMBER 4 • 2005 540
sity values that were collected from 37 cells were plotted in a
FRAP curve (after correction for photobleaching). The t
fluorescence recovery, which is a measure for the speed by
which molecules in the bleached area are replaced by mole-
cules from the environment, was
tin domains (Fig. 3 B and Table I). Remarkably, EYFP-
SUV39H1 recovery reached 70% as a maximum after 140 s,
suggesting that
?
30% of SUV39H1 is stably bound within het-
erochromatic domains on the time scale of our experiments.
To ensure the existence of an immobile or considerably
less mobile SUV39H1 fraction in heterochromatin domains,
we measured the dynamics of EYFP-SUV39H1 by FRAP us-
ing extended recovery periods in NIH3T3 and U2OS cells as
well as in Suv39h dn PMEFs. EYFP-SUV39H1 recovery
reached a maximum of 80% after 275 s in U2OS cells (t
?
15 s), a maximum of 70% after 140 s in NIH3T3 cells, and
only a maximum of 60% after 290 s in Suv39h dn PMEFs (t
of
?
15 s) in heterochromatic domains (Fig. 3 D and Table I).
1/2
of
?
19.0 s within heterochroma-
1/2
of
1/2
This suggests that 20–40% of SUV39H1 is stably bound within
heterochromatic domains depending on cell type. It should be
noted, however, that it proved difficult to obtain consistent
FRAP data from U2OS cells because its heterochromatin is too
dispersed to selectively photobleach with high accuracy. For
this reason, the mobile fraction of EYFP-SUV39H1 is probably
?
80%. Within euchromatic regions of NIH3T3 cells, we mea-
sured a t
1/2
of 11.8 s and no immobile fraction for EYFP-
SUV39H1 (Fig. 3 D and Table I).
The presence of an immobile or considerably less mobile
fraction of SUV39H1 in heterochromatin was further investi-
gated by performing two successive FRAP measurements on
the same heterochromatic domain in NIH3T3 cells. EYFP-
SUV39H1 was photobleached in a heterochromatic domain, and,
after a recovery period of 450 s, the same area was photobleached
for the second time, after which the fluorescence recovery was
measured again. During the second FRAP, an immobile fraction
would be invisible because it was bleached during the first
Figure 3.
and EYFP-HP1? shows that SUV39H1 is a less
dynamic component of heterochromatin.
NIH3T3 cells were transfected with EYFP-
SUV39H1 (A) or EYFP-HP1? (B). A heterochro-
matic area was selected and photobleached.
Images were recorded just before bleaching
and at different time intervals after bleaching.
Arrows indicate the photobleached region. To
illustrate the recovery of fluorescence more
clearly, pseudocolor images of the bleached
cells are shown. Fluorescent intensities range
from blue (low) to red (high). (C) Relative fluo-
rescence intensities are displayed in recovery
curves. Fluorescence recovery of EYFP-
SUV39H1 reached a plateau at ?70% after
140 s. Fluorescence recovery of EYFP-HP1?
reached a plateau at ?95% after 60 s. The
curves represent mean values from 37 and 24
cells, respectively. Error bars represent SD. (D)
FRAP curves calculated after extended recov-
ery periods of EYFP-SUV39H1 in heterochro-
matin of U2OS cells, of Suv39h dn PMEFs,
and of NIH3T3 cells and in euchromatin of
NIH3T3 cells. (E) FRAP curves obtained from
two successive FRAP measurements. After fluo-
rescence recovery after the first bleach, the
same region was bleached for the second
time, and the fluorescence recovery was mea-
sured. EYFP-SUV39H1 fully recovered after
the second bleach to 70% of the initial amount
of fluorescence measured before the first
bleach.
FRAP analysis of EYFP-SUV39H1

Page 5

A GLUE FOR HETEROCHROMATIN MAINTENANCE • KROUWELS ET AL.541
FRAP. As shown in Fig. 3 E, after the second bleach, a full re-
covery of fluorescence was measured, which was 70% of the ini-
tial amount of fluorescence that was measured before the first
photobleaching. Thus, this experiment confirmed the existence of
an immobile fraction, at least on the time scale of the experiment.
Because various lines of evidence suggest that SUV39H1
interacts with HP1 proteins (Aagaard et al., 1999; Melcher et
al., 2000) and that HP1 proteins are very mobile in the nucleus
of living cells (Cheutin et al., 2003; Festenstein et al., 2003),
we sought to compare the dynamic properties of SUV39H1
with those of HP1 proteins in NIH3T3 cells. Fusion constructs
of HP1
?
and HP1
?
with EYFP were made and transiently
transfected into cells. Fusion proteins were shown to localize to
the same nuclear sites as their endogenous counterparts, as re-
vealed by immunocytochemistry with specific anti-HP1 anti-
bodies. Cells showing moderate expression levels were se-
lected, and, upon bleaching of small areas inside the nucleus
that corresponded to heterochromatin and euchromatin, re-
covery of fluorescence was recorded. Consistent with previ-
ous data, HP1 proteins revealed a very dynamic behavior in
NIH3T3 cells (Fig. 3 B). For EYFP-HP1
of
?
4.2 s and a maximum fluorescence recovery of
which was reached after 60 s from bleaching (Fig. 3 C and Ta-
?
, we measured a t
1/2
?
95%,
Figure 4.
tion mutants fused to EYFP. The chromodomain is shown as a gray shaded
box, and the SET domain is in black. The numbers refer to amino acid po-
sitions in the SUV39H1 protein.
Mutant SUV39H1 constructs. Overview of the SUV39H1 dele-
Figure 5.
Nchromo are more concentrated at cen-
tromeres than SUV39H1. (A) Localization of
the various SUV39H1 mutants in NIH3T3 cells.
Bar, 10 ?m. (B) Western blot showing expres-
sion of EYFP-SUV39H1 (lane 1), EYFP–
SUV39H1-?SET (lane
SUV39H1-Nchromo (lane 3). Ponceau stain-
ing shows equal loading of the gel. (C) The top
panel shows the distribution of EYFP–
SUV39H1-?SET, centromeres, and DNA in a
U2OS cell. The bottom panel shows a single
optical section of a U2OS cell expressing
EYFP–SUV39H1-?SET (left) and stained for
centromeres (middle). Colocalization of the
two is shown in the right image. The line scan
(diagonal lines through the images) shows the
local intensity distribution of EYFP–SUV39H1-
?SET in yellow and centromere labeling in
red. (D) Simultaneous detection of EYFP–
SUV39H1-Nchromo (left) and centromeres
(middle) in a U2OS cell. DNA is stained by
DAPI (top right). The bottom panel shows a sin-
gle optical section and a line scan (diagonal
lines through the images) giving the local inten-
sity distribution of EYFP–SUV39H1-Nchromo
(yellow) and centromere staining (red).
SUV39H1-?SET and SUV39H1-
2), and EYFP–

Page 6

JCB • VOLUME 170 • NUMBER 4 • 2005542
ble I). Similar kinetics of EYFP-HP1
CFP-SUV39H1 and EYFP-HP1
same cells (unpublished data). Together, these results suggest
that SUV39H1 is more stably bound to chromatin than HP1
proteins and that a significant fraction of SUV39H1 is recruited
to chromatin in an independent fashion or at least is not in a
stable complex together with HP1 proteins.
?
was measured when
were coexpressed in the
?
The SET domain with adjacent regions
mediates stable binding of SUV39H1
to heterochromatin
Three distinct protein domains have been identified in
SUV39H1, of which aa 3–44 at the NH
HP1
?
interaction surface that, together with the adjacent chro-
modomain (aa 44–88), direct accumulation at heterochromatic
regions (Melcher et al., 2000). The COOH-terminal SET do-
main (aa 249–412) has been shown to be responsible for H3K9
methylation but has also been suggested to modulate hetero-
chromatin association of SUV39H1 (Melcher et al., 2000;
Lachner et al., 2001). To investigate how the different domains
contribute to the kinetic behavior of SUV39H1 at chromatin in
vivo, we generated various deletion mutants of SUV39H1 ac-
cording to Melcher et al. (2000), fused them to EYFP, and tran-
siently expressed them in NIH3T3 cells. A cartoon depicting
2
terminus form the
the deletion mutants is shown in Fig. 4. First, we analyzed the
spatial distribution of fusion proteins in the cell nucleus. Cells
moderately expressing EYFP–SUV39H1-
Suv39H1 fused to EYFP) or EYFP–SUV39H1-Nchromo (aa
3–118 of Suv39H1 fused to EYFP) revealed the characteristic
heterochromatic localization pattern that has also been ob-
served for the full-length protein and after staining with DAPI
or HP1 antibodies (Fig. 5 A, top). However, when expressed in
U2OS cells, slightly different localization patterns were ob-
served. The patterns seemed more dotlike when compared with
localization of the full-length fusion protein (Fig. 5, C and D,
top). Nevertheless, the expressed EYFP–SUV39H1-
EYFP–SUV39H1-Nchromo fusion proteins were of the correct
size, as determined by Western blotting (Fig. 5 B). Confo-
cal analysis of U2OS cells expressing the fusion proteins that
were stained with an anticentromere antibody showed EYFP–
SUV39H1-
?
SET (Fig. 5 C, bottom) or EYFP–SUV39H1-
Nchromo (Fig. 5 D, bottom) localization only at the cen-
tromeres. This localization in interphase nuclei is consistent
with the finding that SUV39H1-
Nchromo localize more exclusively to the centromeres of
metaphase chromosomes (Melcher et al., 2000).
Next, we performed FRAP analysis to determine the ki-
netic properties of SUV39H1-
?
?
SET (aa 3–249 of
?
SET and
?
SET and SUV39H1-
SET and SUV39H1-Nchromo
Figure 6.
mobility rate than full-length SUV39H1. After transfection of NIH3T3
cells with EYFP–SUV39H1-?SET (A) or EYFP–SUV39H1-Nchromo
(B), a heterochromatic area was selected and photobleached. Im-
ages were recorded just before and at different time intervals after
bleaching. Arrows indicate the photobleached areas. (C) The corre-
sponding FRAP curves are plotted together with the FRAP curves for
EYFP-SUV39H1 and EYFP-HP1?. These curves indicate that EYFP–
SUV39H1-?SET and EYFP–SUV39H1-Nchromo are more dynamic
than the full-length protein. The FRAP curves for the two mutant pro-
teins represent means from 30 and 14 cells, respectively.
SUV39H1-?SET and SUV39H1-Nchromo have a higher

Page 7

A GLUE FOR HETEROCHROMATIN MAINTENANCE • KROUWELS ET AL.543
mutants in living cells at heterochromatic regions (Fig. 6, A
and B). In NIH3T3 cells, we measured a t
?
8.6 s for EYFP–SUV39H1-
?
SUV39H1-Nchromo. Notably, these proteins reached
recovery at 120 s and
?
95% at 40 s, respectively (Fig. 6 C and
Table I). These results indicate that both SUV39H1 mutants are
more dynamic than the full-length protein and that there is
no significant stably bound fraction. Furthermore, the t
fluorescence recovery and the mobile fraction of EYFP–
SUV39H1-Nchromo are comparable with that of HP1
pression experiments in NIH3T3 cells revealed that neither the
dynamics nor the localization of HP1 significantly changed as
a result of EYFP–SUV39H1-
Nchromo expression (unpublished data).
The mutants EYPF–SUV39H11-chromocore (aa 37–118
of Suv39H1 fused to EYFP) and EYPF–SUV39H1-
(aa 89–412 of Suv39H1 fused to EYFP) localized diffusely
throughout the nucleus in NIH3T3 cells (Fig. 5 A, bottom)
as well as in human U2OS cells (not depicted). FRAP analy-
sis revealed that EYPF–SUV39H1-chromocore and EYPF–
SUV39H1-
?
N89 gave very rapid recovery times (
?
0.5 s respectively; Table I). Expression of both fusion pro-
teins was checked by Western blotting and showed their ex-
pected sizes (not depicted). These data indicate that EYPF–
SUV39H1-chromocore and EYPF–SUV39H1-
diffuse freely throughout the nucleus, which is consistent with
immunocytochemical data (Melcher et al., 2000), and do not
(or to a minor extent) interact with chromatin.
1/2
?
of recovery of
3.8 s for EYFP–SET and of
?
90%
1/2
of
?
. Coex-
?
SET or EYFP–SUV39H1-
?
N89
?
0.6 and
?
N89 mutants
SUV39H1 dynamics at centromeric
heterochromatin is independent of its
methyltransferase activity
Our data and that of others suggest that targeting of SUV39H1
to centromeric heterochromatin is mediated by the HP1-bind-
ing domain together with the chromodomain and that this tar-
geting is even more profound in absence of the SET domain
(Melcher et al., 2000). Meanwhile, a SUV39H1 mutant lacking
the SET domain becomes more mobile at centromeric hetero-
chromatin. This suggests that the SET domain is not essential
for recruitment, but it still may play a role in stabilizing the in-
teraction of full-length SUV39H1 with centromeric heterochro-
matin. To investigate whether it is the methyltransferase enzy-
matic activity of the SET domain that mediates SUV39H1
binding to heterochromatin, we analyzed the mobility of two
point mutants; one with impaired methyltransferase activity
(SUV39H1-H324L) and one with enhanced methyltransferase
activity (SUV39H1-H320R). Both mutants were fused to
EYFP and revealed the same localization as EYFP-SUV39H1
when transiently transfected into NIH3T3 cells. FRAP analysis
of both point mutants revealed that the fluorescence recovery
times and the mobile fractions at heterochromatin did not sig-
nificantly deviate from that of wild-type SUV39H1 (Fig. 7), in-
dicating that the binding of SUV39H1 is not mediated by its
methyltransferase enzymatic activity.
SUV39H1 interacts with HP1 on
chromatin, as measured by FRET
FRAP analysis suggests that the binding of SUV39H1 to het-
erochromatin is strengthened by the SET domain (Fig. 6). Al-
though previous work suggested that the binding of SUV39H1
to chromatin was not governed by an interaction with HP1
(Melcher et al., 2000), this interaction may still play a role in
stabilizing the binding of SUV39H1 to pericentric heterochro-
matin in vivo. Thus, to examine the binding characteristics of
EYFP-SUV39H1 to chromatin in more detail, we analyzed the
interaction of full-length and mutant EYFP-SUV39H1 with
HP1 in vivo by using FRET. FRET only occurs when two fluo-
rescent proteins are at very close proximity (2–7 nm), meaning
that they have to be in direct interaction. At present, there are
various ways of measuring FRET in cells, each having their
specific requirements. We measured FRET by two independent
methods using the same experimental conditions and micro-
scope setup. The three-filter method measures the sensitized
emission of the acceptor molecule (Xia and Liu, 2001), and the
fluorescence lifetime imaging microscopy (FLIM) method
measures the decrease in donor fluorescence lifetime (Gadella,
1999), which occurs as a consequence of FRET.
U2OS cells were transiently cotransfected with EYFP-
HP1
?
and ECFP-SUV39H1, EYFP-HP1
SUV39H1-
?SET, or with EYFP-HP1? and ECFP–SUV39H1-
?N89. As a negative control, cells were transfected with
ECFP-SUV39H1 only. For each combination, 16–30 cells
were analyzed using the three-filter method as well as the
FLIM method. Cells expressing EYFP-HP1? together with
ECFP-SUV39H1 or ECFP–SUV39H1-?SET revealed sensi-
tized emission (Fig. 8, A and B), a significant decrease in mean
donor lifetime, respectively, and 2.2 and 2.1 against 2.5 ns
measured in control cells (Table II). Spatial analysis revealed
?
and ECFP–
Figure 7.
SUV39H1 dynamics. NIH3T3 cells were transfected with
EYFP–SUV39H1-H324L or with EYFP–SUV39H1-H320R.
Heterochromatic areas were selected and photo-
bleached. Images were recorded just before and at differ-
ent time intervals after photobleaching. The correspond-
ing FRAP curves are plotted together with the FRAP curve
for EYFP-SUV39H1. The curves represent mean values
from 28 and 25 cells, respectively.
Methyltranferase activity has no influence on

Page 8

JCB • VOLUME 170 • NUMBER 4 • 2005544
most prominent FRET at heterochromatic areas (Fig. 8, A and
B). Cells that were cotransfected with EYFP-HP1? and ECFP–
SUV39H1-?N89 did not show sensitized emission (Fig. 8 C)
or a decrease in donor lifetime (Table II). These results indicate
that SUV39H1 and SUV39H1-?SET, but not SUV39H1-
?N89, interact with HP1? in living cells. Moreover, the data
suggest that the potential role of the SET domain in binding of
SUV39H1 to heterochromatin is not mediated by an interaction
with HP1.
Previous in vitro studies have shown that the first 89
amino acids are indeed responsible for the interaction of
SUV39H1 with HP1? (Melcher et al., 2000). To confirm that
this interaction also occurs in living cells, we cotransfected
EYFP-HP1? together with ECFP–SUV39H1-Nchromo or
ECFP–SUV39H1-chromo. Again, cells were analyzed by the
three-filter as well as by the FLIM method. Cells cotransfected
with EYFP-HP1? and ECFP–SUV39H1-Nchromo showed a
clear, sensitized emission and a decrease in lifetime (2.2 ns, Ta-
ble II), whereas cells cotransfected with EYFP-HP1? and
ECFP–SUV39H1-chromocore did not show sensitized emis-
sion or a decrease in lifetime (2.6 ns, Table II). This confirms
the fact that it is also the most NH2-terminal part of SUV39H1
that interacts with HP1 in living cells.
DNA demethylation increases
SUV39H1 mobility
Evidence is accumulating that a tight interplay exists between
DNA methylation and histone modification. Interactions of
SUV39H1 with different DNA methyltransferases (Fuks et al.,
2003) as well as with methyl–cytosine guanine dinucleotide–
binding domain proteins (Fujita et al., 2003) have been de-
scribed previously. To explore the role of DNA methylation in
strengthening the interaction between SUV39H1 and hetero-
chromatin, we treated cells with the DNA demethylating agent
5-aza-2?deoxycytidine (5-aza-C). Cell sorting analysis revealed
that this treatment resulted in some decrease of cells that are in
S or G2 phase of the cell cycle (9 vs. 20% in untreated cells),
with a vast majority of cells in G1 (90 vs. 78%). Furthermore,
we showed by Western blotting that this treatment had no sig-
nificant influence on the trimethylation of H3K9 or acyetyla-
tion of histones (see Fig. 10 D). Next, we analyzed the mobility
of EYFP-SUV39H1 by FRAP, and the resulting curves show
that DNA demethylation increases the mobility of SUV39H1
(t1/2 of ?14.6 s compared with ?19.0 s in untreated cells; Fig.
9). This suggests that the interaction of SUV39H1 with compo-
nents of the DNA methylation machinery or with the unmeth-
ylated DNA itself has changed. However, the mobility of
SUV39H1 in 5-aza-C–treated cells was still less than deter-
mined for SUV39H1-?SET in untreated cells, indicating that
these interactions are not the sole mechanisms by which
SUV39H1 binds to pericentromeric heterochromatin. Further-
more, the observation that EYFP–SUV39H1-?SET becomes
more mobile in 5-aza-C–treated cells compared with non-
treated cells (Fig. 9) suggests that it is not the SET domain of
SUV39H1 that mediates the interaction with components of the
DNA methylation machinery or with DNA itself.
FRAP analysis of HP1? mobility in EYFP-HP1?–express-
ing cells treated with 5-aza-C showed that HP1? mobility is not
changed compared with nontreated cells (Fig. 9).
Trichostatin A (TSA) treatment affects
the binding of SUV39H1 but not
SUV39H1-?SET to chromatin
Heterochromatin is not only characterized by hypermethylation
but also by hypoacetylation of histones. The hyperacetylation
of histones is a hallmark of transcriptionally active genes and
has been suggested to prevent methylation of histone H3K9
and, thus, heterochromatin formation (Jenuwein and Allis,
2001). Therefore, histone deacetylases might be active at het-
Figure 8.
interaction between different autofluorescent
fusion proteins. U2OS cells were cotrans-
fected with ECFP-SUV39H1 and EYFP-HP1?
(A), with ECFP–SUV39H1-?SET and EYFP-
HP1? (B), or with ECFP–SUV39H1-?N89 and
EYFP-HP1? (C). A CFP, YFP, and FRET image
was acquired from all cells for spectral mea-
surements, and a FLIM stack was collected to
calculate fluorescent lifetimes. The CFP, YFP,
and FRET images were first corrected for pixel
shift and background. Then, the NFRET values
were calculated and displayed in pseudocol-
ors (Nfret). Absence of FRET is indicated by
blue as depicted in the scale. The fluorescent
lifetime values are also displayed in pseudo-
colors (FLIM). Blue (as depicted in the scale)
indicates a lifetime of 2.5 ns and an absence
of FRET.
FRET measurements confirm the
Table II. Mean ?? and ? mod
Construct
??
SD
? ModSD
ns ns
3.1
2.9
2.7
2.8
3.1
3.0
ECFP-SUV39H1
ECFP-SUV39H1/EYFP-HP1?
ECFP–SUV39-?SET/EYFP-HP1?
ECFP–SUV39H1-Nchromo/EYFP-HP1?
ECFP–SUV39H1-chromocore/EYFP-HP1?
ECFP–SUV39-?N89/EYFP-HP1?
2.5
2.2
2.1
2.2
2.6
2.5
0.1
0.1
0.1
0.2
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.2
Values are means from 16 to 30 cells.

Page 9

A GLUE FOR HETEROCHROMATIN MAINTENANCE • KROUWELS ET AL.545
erochromatin to maintain a hypoacetylated state. Treatment of
cells with the histone deacetylase inhibitor TSA has been
shown to result in the dissociation of HP1 from pericentro-
meric heterochromatin domains, probably by preventing meth-
ylation of H3 by SUV39H1 (Taddei et al., 2001). To test
whether the in vivo kinetics of SUV39H1 binding to chromatin
alters as a consequence of hyperacetylation, NIH3T3 cells ex-
pressing EYFP-SUV39H1 were treated with TSA. As shown
by Western blotting, this treatment indeed resulted in an in-
creased amount of acetylated histones and in a decrease in tri-
methylated H3K9 (Fig. 10 D). Cell sorting analysis revealed
that this treatment resulted in some decrease of cells that were
in S or G2 phase of the cell cycle (12 vs. 20% in untreated
cells), with a vast majority of cells in G1 (87 vs. 78%). TSA-
treated cells revealed a less pronounced localization of EYFP-
SUV39H1 at heterochromatic regions, which is consistent with
previous data (Maison et al., 2002; Cheutin et al., 2003). Fur-
thermore, FRAP analysis revealed a faster fluorescence recov-
ery of EYFP-SUV39H1 in TSA-treated cells (t1/2 of ?7.7 s)
compared with untreated cells (t1/2 of ?19.0 s). Also, the total
mobile fraction of EYFP-SUV39H1 at chromatin increased
from ?70 to ?90% (Fig. 10, A and C, and Table I). TSA treat-
ment had no significant effect on the kinetics of SUV39H1-
?SET (t1/2 of ?8.6 s). However, consistent with previous data
(Cheutin et al., 2003), EYFP-HP1? mobility increased after
TSA treatment (Fig. 10 B) to a t1/2 of ?1.8 s compared with 4.2 s
in untreated cells (Fig. 10 C). These data show that hyperacety-
lation of histones reduces the binding of EYFP-SUV39H1 and
EYFP-HP1? to heterochromatin but does not prevent a tran-
sient SET-independent interaction of EYFP-SUV39H1 with
chromatin.
Discussion
SUV39H1, the human homologue of the D. melanogaster posi-
tion effect variegation modifier Su(var)3-9, catalyzes the meth-
ylation of histone H3K9, thereby creating binding sites for HP1.
In particular, SUV39H1 catalyzes trimethylation events, which
are characteristic of constitutive pericentromeric heterochroma-
tin formation. Despite the fact that much is known about the cat-
alytic activity of SUV39H1, little is known about how this pro-
tein is recruited to and interacts with chromatin. In this study, we
report that SUV39H1 that is localized at pericentromeric hetero-
chromatin consists of two populations: a mobile and an immo-
bile one. Furthermore, the mobile population of SUV39H1 ap-
pears to be significantly less mobile than HP1. These findings
suggest that SUV39H1 may, in addition to its catalytic activity,
also serve a structural role in chromatin. In addition, our data
support the idea that HP1 is not necessarily recruited in a com-
plex with SUV39H1 to pericentromeric chromatin.
Initially, HP1 had been considered a stable component of
heterochromatin in order to maintain a compact, inaccessible
chromatin conformation that would exclude transcriptional ac-
tivity. This concept has changed because HP1 proteins were
shown by FRAP analysis to be highly mobile in living cells
(Cheutin et al., 2003; Festenstein et al., 2003). Recently, vari-
ous other structural chromatin-binding proteins were shown to
interact transiently, indicating that transient interactions are a
common feature of chromatin proteins (Christensen et al.,
2002; Harrer et al., 2004; Phair et al., 2004). These findings led
to the suggestion that the dynamic nature of architectural chro-
matin-associated proteins would be essential to regulate gene-
related processes (Harrer et al., 2004; Phair et al., 2004). This
idea is consistent with recent findings showing that condensed
chromatin domains are accessible for high molecular weight
dextrans (Verschure et al., 2003) as well as for gene regulatory
and chromatin proteins (Chen et al., 2005). Our observation
that a substantial fraction of SUV39H1 is stably associated
with chromatin during an extended period of time suggests,
however, that not all chromatin-associated proteins are neces-
sarily in continuous flux with the nucleoplasm. The significant
immobile fraction of SUV39H1 might be indicative of chroma-
tin conformations that are not prone to processes regulating
transcriptional activity but that play a role in the structural or-
ganization of the cell nucleus. This might be particularly true
for centromeric and telomeric regions, which have been impli-
cated to play roles in spatial nuclear organization and generally
show constrained movements in live cells (Molenaar et al.,
2003). Consistent with this idea is the observation that changes
in DNA methylation and histone acetylation have significant
effects on the architecture of interphase chromosome arms but
not on the positioning of centromeres and telomeres in wheat
cell nuclei (Santos et al., 2002).
SUV39H1, however, is not only found at constitutive si-
lent regions but also at genomic regions displaying transcrip-
tional activity (Greil et al., 2003). Furthermore, transcription-
Figure 9.
SUV39H1 and SUV39H1-?SET but not that of HP1?. NIH3T3
cells were transfected with EYFP-SUV39H1, EYFP–SUV39H1-
?SET, or with EYFP-HP1? and were treated with 5 ?M 5-aza-C.
After 48–52 h of treatment, a heterochromatic area was selected
and photobleached. Images were recorded just before and at
different time intervals after photobleaching. The corresponding
FRAP curves are plotted together with the FRAP curves for EYFP-
SUV39H1, EYFP–SUV39H1-?SET, and EYFP-HP1? obtained
from nontreated cells. Curves represent the means from 18, 12,
and 16 cells, respectively, and show that both EYFP-SUV39H1
and EYFP–SUV39H1-?SET become more dynamic after 5-aza-C
treatment.
DNA demethylation increases the dynamics of both

Page 10

JCB • VOLUME 170 • NUMBER 4 • 2005546
ally inactive genes are found in open chromatin structures,
whereas transcriptionally active genes are found in domains of
compact chromatin (Gilbert et al., 2004). Hence, the classical
strict distinction between heterochromatin (that it is supposed
to be transcriptionally inactive) and euchromatin (that it is sup-
posed to be transcriptionally active) does not exist. Because it
is not possible to spatially define and distinguish small tran-
scriptionally active and inactive regions in living cells express-
ing EYFP-SUV39H1, it was not possible to reproducibly
analyze and compare the dynamic properties of SUV39H1 as-
sociation at sites that were distinct from the relatively large
heterochromatin domains. Therefore, the FRAP data obtained
from nuclear regions that were adjacent to the pericentromeric
heterochromatin in NIH3T3 cells is likely a reflection of
SUV39H1 mobility rates at various levels of chromatin com-
paction. Nevertheless, the fact that we, on average, measured
higher mobility rates in regions that we defined as euchromatic
suggests that SUV39H1 is more dynamic at less condensed
chromatin than at pericentromeric heterochromatin.
Recently, a slow moving fraction of HP1 has been iden-
tified at pericentromeric heterochromatin by using fluores-
cence correlation microscopy and FRAP (Schmiedeberg et
al., 2004). However, this fraction appears to be relatively
small (7% in NIH3T3 cells) when compared with the rela-
tively large immobile fraction of SUV39H1 (?30% in
NIH3T3 cells) that we measured. Furthermore, the fast fluo-
rescence recovery rate of HP1 (t1/2 of ?4 s) compared with
the slow recovery rate of SUV39H1 (t1/2 of ?19 s) support the
idea that both proteins are not necessarily recruited as a com-
plex to chromatin, which is in contradiction with current
models (Lachner et al., 2001; Maison and Almouzni, 2004).
Our findings and the data of others are consistent with a
model that SUV39H1 is a structural component of hetero-
chromatin and is able to recruit HP1 by direct protein–protein
interaction (Stewart et al., 2005). Interestingly, another SET
domain–containing protein, multiple myeloma SET II, has re-
cently been shown to bind to a nucleoplasmic structure (pos-
sibly chromatin) with even higher affinity than SUV39H1
(Keats et al., 2005). This finding suggests that the tight asso-
ciation of SUV30H1 with chromatin is a more common fea-
ture that is shared by other SET proteins. However, multiple
myeloma SET II belongs to another subfamily than does
SUV39, whose function is unknown and may not even pos-
sess histone methyltransferase activity.
Figure 10.
the dynamics of both SUV39H1 and HP1?. NIH3T3 cells were
transfected with EYFP-SUV39H1 (A) or EYFP-HP1? (B) and
treated with 50 ng/ml TSA. After 18–22 h of TSA treatment, a
heterochromatic area was selected and photobleached. Images
were recorded just before and at different time intervals after
photobleaching. Arrows indicate the photobleached areas. (C)
The corresponding FRAP curves are plotted together with the
FRAP curves for EYFP-SUV39H1 and EYFP-HP1? obtained from
nontreated cells. Curves represent means from 22 and 8 cells, re-
spectively. (D) Western blots showing the levels of acetylation
and trimethylation in nontreated (lane 1), TSA-treated (lane 2),
and 5-aza-C–treated (lane 3) NIH3T3 cells. Ponceau staining
shows equal protein loading.
Inhibition of histone deacetylase activity increases

Page 11

A GLUE FOR HETEROCHROMATIN MAINTENANCE • KROUWELS ET AL.547
To identify the protein domain that is responsible for sta-
ble binding of the immobile population of SUV39H1 to peri-
centromeric heterochromatin, we characterized the exchange
rate of various deletion mutants of SUV39H1 at pericentro-
meric heterochromatin in living cells by FRAP analysis. This
analysis revealed that in addition to the NH2 terminus and the
adjacent chromodomain of SUV39H1, the SET domain also
plays a role in chromatin binding. Consistent with previous
data (Melcher et al., 2000), we observed that SUV39H1-?SET
localization was mainly restricted to pericentromeric hetero-
chromatin regions compared with wide-spread chromatin asso-
ciations of full-length SUV39H1. This suggests that the SET
domain plays a role in chromatin association. Our finding that
SUV39H1-?SET is highly mobile at the pericentromeric het-
erochromatin regions in living cells reinforces this suggestion
and indicates that the SET domain mediates the stable interac-
tion of SUV39H1 with pericentromeric chromatin, whereas the
other domains are responsible for the recruitment of SUV39H1
to chromatin. Furthermore, FRAP analysis of the point mu-
tants SUV39H1-H324L and SUV39H1-H320R revealed that it
is not the histone methyltransferase activity of the SET domain
that stabilizes binding of SUV39H1. This is consistent with
previous localization data showing that heterochromatin asso-
ciation of SUV39H1 can be uncoupled from its intrinsic his-
tone methyltransferase activity (Lachner et al., 2001).
The mechanism that mediates the stable interaction of
SUV39H1 via the SET domain with pericentromeric chroma-
tin remains elusive. It has been described previously that
SUV39H1 not only interacts with HP1 (Aagaard et al., 1999)
but also interacts with components of the DNA methylation
pathway (Fujita et al., 2003; Fuks et al., 2003) and with his-
tone deacetylases (Vaute et al., 2002). Our FRET analysis re-
veals that SUV39H1 and HP1 indeed interact at pericentro-
meric chromatin in live cells but that this interaction does not
mediate the stable binding of SUV39H1 with heterochroma-
tin. Furthermore, we could find no indication that binding of
the immobile SUV39H1 population to pericentromeric chro-
matin is mediated by DNA methylation. Interestingly, treat-
ment of cells with the deacetylation inhibitor TSA resulted in
a higher mobility of SUV39H1 and in a significant reduction
of the immobile SUV39H1 fraction at pericentromeric hetero-
chromatin, whereas the kinetic behavior of SUV39H1-?SET
did not change. This suggests that histone deacetylase activity
is required for the stable interaction of SUV39H1 to pericen-
tromeric chromatin via the SET domain. Remarkably, expo-
sure of HeLa cells to TSA was previously reported to relocate
centromeres from a more internal position to the nuclear pe-
riphery (Taddei et al., 2001; Maison et al., 2002), supporting
the idea that the immobile population of SUV39H1 may con-
tribute to maintaining nuclear architecture. By immunoprecip-
itation and activity assays, it has been demonstrated that the
NH2-terminal part of SUV39H1 can interact with histone
deacetylase 1 and 2 (Vaute et al., 2002). This interaction,
which might be direct, may help to recruit SUV39H1 to
heterochromatin but does not, however, explain the role of
the SET domain in maintaining an immobile population of
SUV39H1. Recent studies indicate that a yet unidentified
RNA may contribute to HP1 and may also contribute to
SUV39H1 binding to pericentromeric heterochromatin (Mai-
son et al., 2002; Muchardt et al., 2002). We are currently in-
vestigating the interesting possibility that an RNA component
contributes to the establishment of an immobile population of
SUV39H1 at pericentromeric heterochromatin.
In summary, our data have important implications for
understanding the mechanisms through which SUV39H1 ex-
erts its biological functions. It is intriguing that the SET do-
main not only displays histone methyltransferase activity but
also plays a role in the binding of SUV39H1 to heterochroma-
tin. In particular, the SET domain is likely to be involved in
stabilizing the association of a population of SUV39H1 pro-
teins to heterochromatin. We speculate that this stable, immo-
bile population of SUV39H1 plays an essential role in the
maintenance of a functional nuclear architecture and may also
act as an interaction platform for other heterochromatin pro-
teins. Future studies will address how the stable association of
SUV39H1 at heterochromatin is maintained. This is particu-
larly relevant because the SET domain of SUV39H1 has also
been identified as being a protein–protein interaction domain
with possible implications in cancer development (Schneider
et al., 2002). For example, the SET domain was shown to in-
teract with Sbf-1, a protein with the ability to transform fibro-
blasts (Cui et al., 1998). It can be speculated that this interac-
tion may interfere with the functioning of SUV39H1 either by
deregulating methyltransferase activity or possibly by direct
destabilization of the chromatin conformation by weakening
the binding of SUV39H1 to chromatin.
Materials and methods
Construction of autofluorescent fusion proteins
SUV39H1 (nt 46–1284 of NM_003173) was amplified from cDNA that
was generated from RNA isolated from human osteosarcoma cells
(U2OS). This was done by using the forward primer 5?-GCGCGCGAAT-
TCTATGGCGGAAAATTTAAAAGG-3? containing the EcoRI site and the
reverse primer 5?-GCGCGCGGTACCCTAGAAGAGGTATTTGCGGC-3?
containing the KpnI site. SUV39H1 deletion mutants were generated ac-
cording to Melcher et al. (2000). SUV39H11-?SET was amplified from
pEYFP-SUV39H1 with the full-length forward primer and the reverse
primer 5?-GCGCGCGGTACCCCGGAAGATGCAGAGGTCAT-3? con-
taining the KpnI site. SUV39H1-Nchromo was made by using the full-
length forward primer and the reverse primer 5?-GCGCGCGGTAC-
CCAGGTAGTTGGCCAAGCTTG-3? containing the KpnI site. SUV39H1-
chromocore was made by using the forward primer 5?-GCGCGCGAAT-
TCTAGGAACCTCTATGACTTTGA-3? containing the EcoRI site and the
Nchromo reverse primer. SUV39H11-?N89 was made by using forward
primer 5?-GCGCGCGAATTCTCACAAGGACTTAGAAAGGGA-3? con-
taining the EcoRI site and the full-length reverse primer for. Purified PCR
fragments were inserted in frame into the EcoRI-KpnI fragment of pECFP-
C1 and pEYFP-C1 (CLONTECH Laboratories, Inc.).
HP1? was amplified with the forward primer 5?-GCGCGGTAC-
CCATGGGGAAAAAACAAAACAAG-3? containing the KpnI site and the
reverse primer 5?-GCGCCCCGGGCGTTCTTGTCATCTTTTT-3? containing
the XmaI site and was cloned in the KpnI-XmaI fragment of pECFP-N1 and
pEYFP-N1 (CLONTECH Laboratories, Inc.). Two SUV39H1 point mutants
were made using the QuikChange II Site-Directed Mutagenesis Kit (Strat-
agene). For generating point mutant SUV39H11-H324L, we used the
sense primer 5?-CTATGGCAACATCTCCCGCTTTGTCAACCACAGTTG-3?
and the antisense primer 5?-CAACTGTGGTTGACAAAGCGGGAGATGT-
TGCCATAG-3?. For generating SUV39H1-H320R, the sense primer was
5?-CATCTCCCACTTTGTCAACCTCAGTTGTGACCCCAAC-3? and the an-
tisense primer was 5?-GTTGGGGTCACAACTGAGGTTGACAAAGTGG-
GAGATG-3?. All constructs were verified by sequencing.

Page 12

JCB • VOLUME 170 • NUMBER 4 • 2005548
Cell culture and transfection
U2OS cells and NIH3T3 mouse fibroblast cells were cultured on 3.5-cm
glass bottom petri dishes (MatTek) in DME without phenol red and contain-
ing 1.0 mg/ml glucose, 4% FBS, 2 mM glutamine, 100 U/ml penicillin,
and 100 ?g/ml streptomycin buffered with 25 mM Hepes, pH 7.2 (all from
Invitrogen). Wild-type and Suv39h dn PMEFs (provided by T. Jenuwein, Re-
search Institute of Molecular Pathology, Vienna, Austria) were cultured as
described previously (Lehnertz et al., 2003). Transient transfections were
performed at ?70–80% confluence using 1 ?l LipofectAMINE 2000 (Invi-
trogen) and 0.75–1.5 ?g DNA. For TSA treatment, cells were incubated
with 50 ng/ml TSA (Sigma-Aldrich) for 18–22 h. For 5-aza-C treatment,
cells were incubated with 5 ?M 5-aza-C (Sigma-Aldrich) for 48–54 h.
Immunocytochemistry
Antibodies that were used in this study are listed as follows: human anti-
centromere (Antibodies, Inc.), mouse antiheterochromatin protein 1? and
? (both from Euromedex), rabbit anti–2x-trimethylated H3K9 (provided by
T. Jenuwein), and the appropriate secondary antibodies. These antibodies
were diluted 1:100, 1:500, 1:500, and 1:250, respectively, in TBS con-
taining 0.5% (wt/vol) blocking reagent (Roche) and 0.1% Tween 20.
Cells that were grown on microscopic glass slides were washed in
PBS and fixed in 2% formaldehyde in PBS for 5 min for incubation with
the anticentromere antibody or with 4% formaldehyde and 0.5% Triton
X-100 (Sigma-Aldrich) in 0.1? PBS for 5 min. Subsequently, cells were per-
meabilized in PBS containing 1% Triton X-100 for 15 min, washed three
times in PBS, and washed once in TBS containing 0.1% Tween 20. Then,
cells were incubated with the first antibody for 45 min for the anticen-
tromere or for 3 h at 37?C followed by three washes in TBS/0.1% Tween
20. Finally, cells were incubated with the secondary antibody for 45 min
at 37?C, washed in TBS/0.1% Tween 20, and mounted in Citifluor (Agar
Scientific, Ltd.) containing 400 ?g/ml DAPI (Sigma-Aldrich).
Cell sorting
Cells were stained with propidium iodide according to the DNAcon3 kit
(DakoCytomation). Cells were sorted in FACS (Becton Dickinson) and ana-
lyzed with Cell Quest software (BD Biosciences).
Protein blot analysis
Cells were lysed in NuPAGE LDS Sample Preparation Buffer (Invitrogen).
Protein samples were then size fractionated on Novex 4–12% BisTris gra-
dient gels using MOPS buffer (Invitrogen) and were subsequently trans-
ferred onto Hybond-C extra membranes (GE Healthcare) using a subma-
rine system (Invitrogen). Blots were stained for total protein using Ponceau
S (Sigma-Aldrich). After blocking with PBS containing 0.1% Tween 20
and 5% milk powder, the membranes were incubated with anti-GFP anti-
body (1:500; Roche), anti-H3Ac antibody (1:1,000; Upstate Biotechnol-
ogy), and anti–2x-trimethylated H3K9 (1:500). The secondary antibodies
that were used were anti–rabbit (1:2,000) and anti–mouse (1:5,000)
HRP-conjugated antibodies (Pierce Chemical Co.). Bound antibodies were
detected by chemiluminescence using ECL Plus (GE Healthcare).
Microscopy
Wide-field microscopy was performed on a microscope (model DMRXA;
Leica) equipped with a 100-W mercury arc lamp and a 100? NA 1.3
plan Apo objective. Colocalization studies were performed on a confocal
microscope system (model TCS/SP2; Leica). Image stacks were acquired
with a 100? NA 1.4 plan Apo objective and were analyzed with Leica
confocal software.
FRAP
Photobleaching experiments were performed on a confocal microscope.
The temperature of the cells was maintained at 37?C by a heating ring sur-
rounding the culture chamber (Harvard App., Inc.) and a microscope ob-
jective heater (Bioptechs). A 100? NA 1.4 plan Apo lens and a 514-nm
laser line was used for photobleaching. Selected areas were subjected to
five excitation pulses of 500 ms each at high laser power. Images were
collected at time intervals of 450 ms from 5 s for 1.5–10 min after bleach-
ing. The first postbleach image was acquired 2 s after photobleaching. To
better quantify the recovery of fast moving SUV39H1 deletion mutants, se-
lected areas were bleached once for 500 ms, and the first postbleach im-
age was acquired after 500 ms. Quantitation of the fluorescent recovery
was performed using Leica confocal software and Microsoft Excel. The re-
covery curves were corrected for background, fluorescence fading, and
decrease in fluorescence during photobleaching. The t1/2 value was de-
fined as the time required for reaching half-maximum recovery and was
calculated from the corrected recovery curves.
FRET imaging
FRET was measured using a microscope (Axiovert 135 TV; Carl Zeiss Mi-
croImaging, Inc.) equipped with a 100-W mercury arc lamp, a 100?
plan Neofluar NA 1.3 phase objective, and a CCD camera (Micromax;
Princeton Instruments). The spectral FRET measurements using three-filter
sets were performed essentially as described by Xia and Liu (2001). CFP
images were obtained with a filter set containing a 436/20-nm bandpass
(BP) excitation filter, a 510-nm dichroic mirror, and a 480/200-nm BP
emission filter. YFP images were obtained with a filter set containing a
500/20-nm BP filter, a 520-nm dichroic mirror, and a 535/30-nm BP
emission filter (all from Chroma Technology Corp.). FRET images were ac-
quired with a filter set containing a 436/20-nm BP excitation filter, a 510-
nm dichroic mirror, and a 535/30-nm BP emission filter. The set of three
images was corrected for background and pixel shift. Images were further
processed, and the relative FRET efficiencies were calculated on a pixel-
by-pixel basis. For the calculation of relative FRET values, the formula as
described previously by Xia and Liu (2001) was used:
(1)
where NFRET is the normalized FRET value and FRET, Dfd, and Afa are in-
tensities measured through FRET, CFP, and YFP filter sets. a–d are bleed-
through percentages measured in cells expressing CFP or YFP only. a and
b are the percentages of YFP and CFP, respectively, that bleed through the
FRET filter set. c is the percentage of CFP that bleeds through the YFP filter
set, and d is the percentage of YFP that bleeds through the CFP filter set.
Values for a–d were 22.6, 55.4, 0.4, and 0.6%, respectively. Calculated
FRET values were displayed in pseudocolors.
FLIM was performed essentially as described previously (Gadella,
1999) using a microscope (Axiovert 135 TV; Carl Zeiss MicroImaging,
Inc.) equipped with a 100-W mercury arc lamp, a 100? plan Neofluar
NA 1.3 phase objective, and an image intensifier (model C5825;
Hamamatsu) modulated at 40,000 MHz. ECFP was excited with a 458-nm
argon/krypton laser line (Innova 70C; Coherent) modulated at 80,000
MHz using an acousto-optic modulator and a filter set containing a
458/10-mn BP excitation filter, a 470-nm dichroic mirror, and a 480/
20-nm BP emission filter. 20 phase images (one every 36?C; 10 forward
and 10 back to correct for bleaching) were acquired using a CCD cam-
era (CoolSNAP fx; Photometrics). Reference phase settings and modula-
tion were set every 15 min using a freshly prepared solution of 100 nM
rhodamine 6G in 100% ethanol with a single component lifetime of 4.0
ns. Fluorescence lifetime values ?? and ?mod were calculated with the
equations ?? ? (1/?) tan?? and ?M ? (1/?)?(1/M2 ? 1). Lifetime values
were calculated for each pixel and were displayed in pseudocolors.
We are particularly grateful to Thomas Jenuwein for supplying reagents and
advice and to Prim Singh for critical reading of the manuscript. We thank
Dorus Gadella for his help building the FLIM system and Shosh Knaan for help
with the cell sorting experiments.
This work was partially supported by Cyttron (grant BSIK03036).
Submitted: 25 February 2005
Accepted: 1 July 2005
References
Aagaard, L., G. Laible, P. Selenko, M. Schmid, R. Dorn, G. Schotta, S. Kuhfit-
tig, A. Wolf, A. Lebersorger, and P.B. Singh, et al. 1999. Functional
mammalian homologues of the Drosophila PEV-modifier Su(var)3-9 en-
code centromere-associated proteins which complex with the heterochro-
matin component M31. EMBO J. 18:1923–1938.
Bannister, A.J., P. Zegerman, J.F. Partridge, E.A. Miska, J.O. Thomas, R.C.
Allshire, and T. Kouzarides. 2001. Selective recognition of methylated
lysine 9 on histone H3 by the HP1 chromo domain. Nature. 410:120–124.
Chen, D., M. Dundr, C. Wang, A. Leung, A. Lamond, T. Misteli, and S. Huang.
2005. Condensed mitotic chromatin is accessible to transcription factors
and chromatin structural proteins. J. Cell Biol. 168:41–54.
Cheutin, T., A.J. McNairn, T. Jenuwein, D.M. Gilbert, P.B. Singh, and T. Mis-
teli. 2003. Maintenance of stable heterochromatin domains by dynamic
HP1 binding. Science. 299:721–725.
Christensen, M.O., M.K. Larsen, H.U. Barthelmes, R. Hock, C.L. Andersen, E.
Kjeldsen, B.R. Knudsen, O. Westergaard, F. Boege, and C. Mielke.
2002. Dynamics of human DNA topoisomerases II? and II? in living
cells. J. Cell Biol. 157:31–44.
NFRET
FRETaDfd cAfa
×
–
()×
–b AfadDfd
×
–
()×
–AfaDfd
×⁄
=

Page 13

A GLUE FOR HETEROCHROMATIN MAINTENANCE • KROUWELS ET AL. 549
Cui, X., I. De Vivo, R. Slany, A. Miyamoto, R. Firestein, and M.L. Cleary.
1998. Association of SET domain and myotubularin-related proteins
modulates growth control. Nat. Genet. 18:331–337.
Festenstein, R., S.N. Pagakis, K. Hiragami, D. Lyon, A. Verreault, B. Sekkali,
and D. Kioussis. 2003. Modulation of heterochromatin protein 1 dynam-
ics in primary mammalian cells. Science. 299:719–721.
Fujita, N., S. Watanabe, T. Ichimura, S. Tsuruzoe, Y. Shinkai, M. Tachibana, T.
Chiba, and M. Nakao. 2003. Methyl-CpG binding domain 1 (MBD1) in-
teracts with the Suv39h1-HP1 heterochromatic complex for DNA methyl-
ation-based transcriptional repression. J. Biol. Chem. 278:24132–24138.
Fuks, F., P.J. Hurd, R. Deplus, and T. Kouzarides. 2003. The DNA methyltrans-
ferases associate with HP1 and the SUV39H1 histone methyltransferase.
Nucleic Acids Res. 31:2305–2312.
Gadella, T.W.J. 1999. Fluorescence lifetime imaging microscopy (FLIM):instru-
mentation and applications. In Fluorescent and Luminescent Probes for
Biological Activity. A Practical Guide for Technology for Quantitative
Real-Time Analysis. W.T. Mason, editor. Academic Press, New York.
467–489.
Garcia-Cao, M., R. O’Sullivan, A.H. Peters, T. Jenuwein, and M.A. Blasco.
2004. Epigenetic regulation of telomere length in mammalian cells by the
Suv39h1 and Suv39h2 histone methyltransferases. Nat. Genet. 36:94–99.
Gilbert, N., S. Boyle, H. Fiegler, K. Woodfine, N.P. Carter, and W.A. Bick-
more. 2004. Chromatin architecture of the human genome: gene-rich do-
mains are enriched in open chromatin fibers. Cell. 118:555–566.
Greil, F., I. van der Kraan, J. Delrow, J.F. Smothers, E. de Wit, H.J. Bussemaker,
R. van Driel, S. Henikoff, and B. van Steensel. 2003. Distinct HP1 and
Su(var)3-9 complexes bind to sets of developmentally coexpressed genes
depending on chromosomal location. Genes Dev. 17:2825–2838.
Harrer, M., H. Luhrs, M. Bustin, U. Scheer, and R. Hock. 2004. Dynamic inter-
action of HMGA1a proteins with chromatin. J. Cell Sci. 117:3459–3471.
Jenuwein, T., and C.D. Allis. 2001. Translating the histone code. Science.
293:1074–1080.
Keats, J.J., C.A. Maxwell, B.J. Taylor, M.J. Hendzel, M. Chesi, P.L. Bergsagel,
L.M. Larratt, M.J. Mant, T. Reiman, A.R. Belch, and L.M. Pilarski.
2005. Overexpression of transcripts originating from the MMSET locus
characterizes all t(4;14)(p16;q32)-positive multiple myeloma patients.
Blood. 105:4060–4069.
Lachner, M., N. O’Carroll, S. Rea, K. Mechtler, and T. Jenuwein. 2001. Methyla-
tion of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature.
410:116–120.
Lehnertz, B., Y. Ueda, A.A. Derijck, U. Braunschweig, L. Perez-Burgos, S. Ku-
bicek, T. Chen, E. Li, T. Jenuwein, and A.H. Peters. 2003. Suv39h-medi-
ated histone H3 lysine 9 methylation directs DNA methylation to major
satellite repeats at pericentric heterochromatin. Curr. Biol. 13:1192–1200.
Maison, C., and G. Almouzni. 2004. HP1 and the dynamics of heterochromatin
maintenance. Nat. Rev. Mol. Cell Biol. 5:296–304.
Maison, C., D. Bailly, A.H. Peters, J.P. Quivy, D. Roche, A. Taddei, M. Lach-
ner, T. Jenuwein, and G. Almouzni. 2002. Higher-order structure in peri-
centric heterochromatin involves a distinct pattern of histone modifica-
tion and an RNA component. Nat. Genet. 30:329–334.
Melcher, M., M. Schmid, L. Aagaard, P. Selenko, G. Laible, and T. Jenuwein.
2000. Structure-function analysis of SUV39H1 reveals a dominant role
in heterochromatin organization, chromosome segregation, and mitotic
progression. Mol. Cell. Biol. 20:3728–3741.
Molenaar, C., K. Wiesmeijer, N.P. Verwoerd, S. Khazen, R. Eils, H.J. Tanke,
and R.W. Dirks. 2003. Visualizing telomere dynamics in living mamma-
lian cells using PNA probes. EMBO J. 22:6631–6641.
Muchardt, C., M. Guilleme, J.S. Seeler, D. Trouche, A. Dejean, and M. Yaniv.
2002. Coordinated methyl and RNA binding is required for heterochro-
matin localization of mammalian HP1alpha. EMBO Rep. 3:975–981.
Peters, A.H., D. O’Carroll, H. Scherthan, K. Mechtler, S. Sauer, C. Schofer, K.
Weipoltshammer, M. Pagani, M. Lachner, and A. Kohlmaier, et al. 2001.
Loss of the Suv39h histone methyltransferases impairs mammalian het-
erochromatin and genome stability. Cell. 107:323–337.
Peters, A.H., S. Kubicek, K. Mechtler, R.J. O’Sullivan, A.A. Derijck, L. Perez-
Burgos, A. Kohlmaier, S. Opravil, M. Tachibana, and Y. Shinkai, et al.
2003. Partitioning and plasticity of repressive histone methylation states
in mammalian chromatin. Mol. Cell. 12:1577–1589.
Phair, R.D., P. Scaffidi, C. Elbi, J. Vecerova, A. Dey, K. Ozato, D.T. Brown, G.
Hager, M. Bustin, and T. Misteli. 2004. Global nature of dynamic pro-
tein-chromatin interactions in vivo: three-dimensional genome scanning
and dynamic interaction networks of chromatin proteins. Mol. Cell. Biol.
24:6393–6402.
Santos, A.P., R. Abranches, E. Stoger, A. Beven, W. Viegas, and P.J. Shaw.
2002. The architecture of interphase chromosomes and gene positioning
are altered by changes in DNA methylation and histone acetylation. J.
Cell Sci. 115:4597–4605.
Schmiedeberg, L., K. Weisshart, S. Diekmann, G. Meyer Zu Hoerste, and P.
Hemmerich. 2004. High- and low-mobility populations of HP1 in het-
erochromatin of mammalian cells. Mol. Biol. Cell. 15:2819–2833.
Schneider, R., A.J. Bannister, and T. Kouzarides. 2002. Unsafe SETs: histone
lysine methyltransferases and cancer. Trends Biochem. Sci. 27:396–402.
Schotta, G., A. Ebert, V. Krauss, A. Fischer, J. Hoffmann, S. Rea, T. Jenuwein,
R. Dorn, and G. Reuter. 2002. Central role of Drosophila SU(VAR)3-9
in histone H3-K9 methylation and heterochromatic gene silencing.
EMBO J. 21:1121–1131.
Stewart, M.D., J. Li, and J. Wong. 2005. Relationship between histone H3
lysine 9 methylation, transcription repression, and heterochromatin pro-
tein 1 recruitment. Mol. Cell. Biol. 25:2525–2538.
Taddei, A., C. Maison, D. Roche, and G. Almouzni. 2001. Reversible disruption
of pericentric heterochromatin and centromere function by inhibiting
deacetylases. Nat. Cell Biol. 3:114–120.
Vaute, O., E. Nicolas, L. Vandel, and D. Trouche. 2002. Functional and physi-
cal interaction between the histone methyl transferase Suv39H1 and his-
tone deacetylases. Nucleic Acids Res. 30:475–481.
Verschure, P.J., I. van der Kraan, E.M. Manders, D. Hoogstraten, A.B. Houts-
muller, and R. van Driel. 2003. Condensed chromatin domains in the
mammalian nucleus are accessible to large macromolecules. EMBO Rep.
4:861–866.
Wreggett, K.A., F. Hill, P.S. James, A. Hutchings, G.W. Butcher, and P.B. Singh.
1994. A mammalian homologue of Drosophila heterochromatin protein 1
(HP1) is a component of constitutive heterochromatin. Cytogenet. Cell
Genet. 66:99–103.
Xia, Z., and Y. Liu. 2001. Reliable and global measurement of fluorescence
resonance energy transfer using fluorescence microscopes. Biophys. J.
81:2395–2402.