The p150 subunit of CAF-1 Causes Association of SUMO2/3
with the DNA Replication Foci

Junsuke Uwada1, 2, Niina Tanaka1, Yutaro Yamaguchi1, Yasuhiro Uchimura1, 6,

Kei-ichi Shibahara 3, Mitsuyoshi Nakao4 and Hisato Saitoh1, 2, 5

1
2

Department of Biological Sciences, Graduate School of Science and Technology,

Global COE (Centers of Excellence) Program, Global Initiative Center for Pulsed Power Engineering,
Kumamoto University, Kumamoto, 2-39-1 Kurokami, Kumamoto 860-8555, Japan3

Department of Integrated Genetics, National Institute of Genetics, Research Organization ofInformation and Systems, Mishima, Japan

4

Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto
University, Kumamoto, Japan.
6

Present address: Medical Research Council Laboratory of Molecular Biology,
Cambridge, UK

5

To Whom correspondence should be addressed:
Email: hisa@kumamoto-u.ac.jp,

Full Postal Address: Department of Biological Sciences, Graduate School of Science and Technology,
Kumamoto University, Kumamoto, 2-39-1 Kurokami, Kumamoto 860-8555, Japan
Phone: +81-96-342-3450, Fax: +81-96-342-3450

1

Abstract

The small ubiquitin-related modifier 2/3 (SUMO2/3) can be post-translationally conjugated to awide variety of proteins constituting chromatin, the platform for genetic and epigenetic regulation.

Nevertheless, it is unclear how SUMO2/3 and SUMO2/3 modified proteins are delivered to thechromatin fibers. Here we report that the largest subunit of chromatin assembly factor 1 (CAF-1),

human p150, interacts directly and preferentially with SUMO2/3. Amino acid residue of 98-105 in p150

is essential and sufficient for SUMO2/3 interaction. p150-SUMO2/3 interaction coincided with regionsthat replicate chromatin fibers, because accumulation of the proliferating cell nuclear antigen (PCNA),

and incorporation of bromodeoxyuridine (BrdU) were detected at foci colocalized with both p150 and

SUMO2/3 during the S-phase in a cell line expressing epitope-tagged p150. Although inhibition of

SUMO2/3 expression had only a small effect on p150 deposition on the replication sites, depletion of

p150 led to delocalization of SUMO2/3 from the replication foci. Furthermore, p150 mutants deficientin SUMO2/3-interaction, caused a major reduction of SUMO2/3 at the replication foci. Thus, ourfindings suggest an expanded role of p150 as a SUMO2/3-interacting factor, and raise the intriguing

possibility that p150 plays a role in promoting delivery of SUMO2/3 or SUMO2/3 modified proteins (orboth) on chromatin fibers during replication.

Keywords: Posttranslational modification, SUMO, DNA replication, chromatin, CAF-1.

2

Introduction

Small ubiquitin-related modifiers (SUMOs) can be post-translationally conjugated to a wide

variety of cellular proteins; this is termed SUMOylation [1]. A large proportion of SUMOs, but not all,localize in the nucleus, implying that the SUMO modification pathway has an important role inregulating nuclear structures and functions [2]. So far, at least three vertebrate paralogs have beenreported: SUMO1, SUMO2 and SUMO3. SUMO2 and SUMO3 are more related to each other (95%amino acid identity) than they are to SUMO1 (~50% identity). Although SUMO1 and SUMO2/3 canbe equally conjugated to a subset of proteins, several lines of evidence indicate that SUMO1 andSUMO2/3 are conjugated to different proteins, and represent unique signals regulating different

cellular functions [3-6]. It is increasingly evident that SUMO2/3 modification plays a crucial role incontrolling many aspects of genetic and epigenetic events via regulating assembly and disassemblyof chromatin components in vertebrate cells. However, the mechanism by which different chromatinproteins are selectively modified or deposited (or both) on chromatin fibers by one paralog relative toanother is unknown.

The dynamics of eukaryotic chromatin fibers during the cell cycle have proven to be importantin regulating genetic and epigenetic stability of eukaryotic cells [7]. Spatiotemporally regulated3

delivery/deposition of histone and non-histone proteins on to DNA during replication is the criticalstep in establishment and maintenance of epigenetic marks on chromatin fibers. Among the crucial

factors for such regulation, it has been demonstrated that the chromatin assembly factor 1 (CAF-1),

composed of three subunits (p150, p60 and p48), assists in loading histone H3-H4 to the newly

replicated DNA strand [7]. Of particular interest, the CAF-1 chaperon complex is known to interact

with not only histones, but also with multiple non-histone proteins, including the homotrimericsliding clamp, proliferating cell nuclear antigen (PCNA) DNA polymerase accessory factor [8],methyl CpG DNA binding domain protein 1 (MBD1) [9, 10] and the specific heterochromatin protein

1 (HP1) [11, 12]. This implies that CAF-1 has a role in coordinating not only histone deposition, butalso non-histone protein association with chromatin fibers during DNA synthesis. However, in termsof chromatin assembly during DNA synthesis, the molecular basis of how this spectrum ofinteraction of CAF-1 can be regulated remains largely unknown.

In this paper, we show that CAF-1 p150 is a SUMO-interacting protein and propose its role inthe delivery of SUMO2/3 or SUMO2/3 modified proteins (or both) to chromatin fiber in the context ofDNA replication.

4

Materials and Methods

Plasmids

To generate p150 expression vectors, human CAF-1 p150 was amplified by PCR using theoligonucleotides listed in the Supplemental Materials.

Antibodies

Two rat monoclonal antibodies and two rabbit polyclonal antibodies, against SUMO1 andagainst SUMO2/3 were used as described previously [13]. The other antibodies used in this study

were anti-Flag M2 (Sigma), anti-His6 (Roche), anti-HA (Clontech), anti-PCNA (PC-10, Santa Cruz

Biotechnology), anti-p150 (ss1, abcam) and anti-BrdU (Ab-3, Thermo Scientific).

Recombinant protein expression and GST-pull-down assay

Expression and purification of recombinant proteins were carried out as previously described[13]. GST-pulldown assay was carried out according to Uchimura et al. [13].

Cell Cycle Synchronization, and Indirect Immunofluorescence Assay

Cells were synchronized with the double thymidine block protocol. In brief, cells were blocked5

by replacing the normal culture medium with fresh culture medium containing 2.5 mM thymidine

for 12 hr. Cells were released from the block by removing the thymidine-containing medium,

washing the monolayer twice, and incubating for 10 hr in fresh normal culture medium. The cells

were blocked again for 12 h, after that they were released to continue their cell cycle synchronously

from the G1/S border. Indirect immunofluorescence analysis was carried out according to Uchimura

et al. [13]. For BrdU staining, cells were treated with 10 μM BrdU for 15 min. Cells were fixed with 4%paraformaldehyde and permeabilized with 0.5% TritonX-100 followed by denaturing with 2N HCl

for 30 min. For PCNA staining, cells were fixed in methanol at -20 °C for 5 min.

Immunoprecipitation

For the immunoprecipitation described in Figure 2B, cultured cells were treated with dimethyl

3,3-dithiobispropionimidate-2HCl (5 mM) (Pierce) on ice for 30 min. Then cells were rinsed with an

ice-cold buffer (100 mM Tris-HCl (pH 8.0), 150 mM NaCl), and lysed in the buffer containing 50 mM

Tris-HCl (pH 7.5), 1% Triton X-100, 300 mM NaCl, 10 mM MgCl2, 2 mM NaF, 20mM N-ethyl

maleimide, 10% glycerol, the protease inhibitors, followed by sonication. The lysate was subjected toimmunoprecipitation as described previously [13]. The immunoprecipitated proteins were eluted

with SDS-sample buffer containing 300 mM DTT to cleave completely the cross-linked S-S-bonds.

6

Results and Discussion

Identification of CAF-1 p150 as a SUMO2/3-interacting protein

To identify a chromatin protein(s) that interacts with SUMO2/3, we performed a yeast

two-hybrid screening [13], and isolated the partial cDNA fragments of a mouse homologue of thelarge subunit of histone chaperone CAF-1 p150. As a step toward understanding the role of p150 inbinding SUMO2/3, we first obtained the full-length human p150 cDNA (Fig 1A) and generated therecombinant protein to perform a glutathione S-transferase (GST) pull-down assay (Fig. 1B).

His6-p150 appeared to associate more efficiently with GST-SUMO2- and GST-SUMO3-beads thanwith GST-SUMO1-beads. When a lysate prepared from the 293S cells expressing Flag- andhemagglutinin (HA)-epitope-tagged human p150, designated as FH-p150, was used in the GST

pull-down assay, the preferential interaction of FH-p150 with GST-SUMO2/3-beads was pronounced(Fig. 1C). Next, to determine the region responsible for binding to SUMO2/3, we generated a series ofdeletion mutants of recombinant p150. Eight amino acid residues of p150 (p15098-105) were found to be

essential and sufficient for binding to GST-SUMO2/3 (Fig. 1A and D). In addition, two kinds of p150

point mutants, designated His6-p150-I99A and His6-p150-D100A, eliminated the interaction with

GST-SUMO3, confirming the importance of p15098-105 for in vitro interaction between p150 and

SUMO2/3 (Fig. 1E).

7

We investigated the evolutionary conservation of the amino acid sequence of p15098-105. We

performed a database search and revealed that p15098-105 was highly conserved from yeast to humans(Supplemental Fig 1A). It should be noted that p15098-105 does not overlap with the previously

identified protein binding domains in p150, such as the binding domains for HP1 [11], PCNA [14],p60 [15, 16] and MBD1 [9] (Fig. 1A), and is not involved in the basic histone chaperone activity of

CAF-1 [11, 14], suggesting that p15098-105 has a (previously unknown) role among the CAF-1 p150

family. Furthermore, analysis of p15098-105 polypeptide sequence revealed a sequence motif that closely

matched the conserved SUMO-interacting/binding motif (SIM) found in other SUMO-binding

proteins [17], indicating that p15098-105 may represent a canonical SIM (Supplemental Fig 1B), and p150

is a SIM-containing SUMO2/3-interacting protein. To examine the affinity of p15098-105 (SIMp150) with

SUMO2/3, we performed a “Bead Halo” assay, which is a simple and rapid procedure that wasdeveloped to detect low-affinity interactions in real-time under equilibrium binding conditions(Supplemental Fig. 1C and D) [18], and revealed that that the affinity of p15098-105 with SUMO3 wasalmost similar to, although less pronounced, than that of SIM derived from MCAF1 (GST-SIMMCAF1), a

previously well-characterized SIM (Kd = ~1.3 µM) [19].

p150-SUMO2/3 interaction occurs in vivo8

To assess whether p150 interacts preferentially with SUMO2/3 in vivo, we observed subcellular

localization of transiently expressed Flag-tagged p150 and endogenous SUMO2/3 in HeLa cells. As

shown in Fig 2A, a substantial number, but not all, of the cells showed multiple foci in which both

Flag-p150 and endogenous SUMO2/3 were enriched. We detected few cells containing foci that wereco-stained with both anti-SUMO1 and anti-Flag antibodies. In addition, an immunoprecipitation

analysis revealed that multiple SUMO-modified proteins were co-precipitated with both anti-SUMO1

and -SUMO2/3 antibodies. However, the signals detected by anti-SUMO2/3 antibody appeared

significantly greater than those by anti-SUMO1 antibody (Fig. 2B).

Collectively, these results

indicate the preferential binding of p150 to SUMO2/3 over SUMO1 in vivo.

It should be noted that, when proteins immunoprecipitated with anti-Flag antibodies wereprobed with anti-Flag antibody, we failed to detect the SUMOylated FH-p150 bands that were

expected to migrate above the 150 kDa band of the non-modified full-length FH-p150 (Figure 2B and

Supplemenrtal Fig. 2A). In addition, we tested SUMOylation of p150 using the E. coli SUMOylation

system [20]; however, the results were negative (Supplemental Fig. 2B). Thus, these results indicatethat p150 was not an efficient SUMOylation substrate. Rather, it is feasible that p150 has an importantrole in regulating the interaction with other chromatin proteins containing SUMO2/3 moiety.

9p150-SUMO2/3 interaction appears in DNA replication fociConsidering that p150 is a component of histone delivery/deposition factor and is concentrated

at the sites of DNA replication during the S-phase [15, 21], we expected that the p150-SUMO2/3 focirepresented the regulated accumulation of SUMO2/3 at the sites of DNA replication via the interaction

with p150 in cells undergoing S-phase progression. To investigate whether p150-SUMO2/3 interaction

occurred at the replication sites, we established a cell line in which Flag-HA-tagged p150 (FH-p150)

was stably expressed. As shown in Supplemental Fig. 3, the cell line, designated as NFH-p150 293S,

expressed FH-p150 approximately 6-times more than did endogenous p150, and grew normally incomparison with the control 293S cell line. Because this cell line allowed us to detect the precise

distribution of p150 as well as p150-SUMO2/3 interaction in vivo, we examined whether p150-SUMO2/3

interaction appeared during the S-phase in this cell line using a conventional method: pulse labelingwith bromodeoxyuridine (BrdU) and immune-staining with the antibody specific for PCNA, a protein

constituting the replication machinery at sites of ongoing replication, were combined [21, 22].

To identify cells undergoing S-phase, we labeled cells with BrdU for 15 min followed byimmunofluorescence analysis using antibody against BrdU. Among the BrdU-positive cells(approximately 40% of the total cell population), we found multiple foci that were triple-stained with

antibodies for BrdU, FH-p150 and SUMO2/3 antibodies, suggesting that p150-SUMO2/3 interaction

10

appeared in the cells undergoing S-phase (Fig. 3A). To determine whether FH-p150 and SUMO2/3 werelocated at the replication machinery, we used an antibody against PCNA and found that many of the

PCNA-positive cells in which p150 and SUMO2/3 were co-localized with punctuate nuclear foci (Fig.

3A). These data indicate that both FH-p150 and SUMO2/3 accumulated at active sites for DNA

synthesis, and suggest the existence of p150-SUMO interaction during the S-phase in NFH-p150 293S

cells.

Furthermore, we demonstrated that the cells expressing either HA-p150-I99A (Fig. 3B) or

HA-p150101-938 (data not shown), two of the SUMO-interaction-deficient mutants, significantly impaired

the accumulation of SUMO2/3 at the PCNA-foci; this provides further evidence that p150 is a necessarycomponent for deposition of SUMO2/3 to sites of DNA replication, and confirms the importance ofSIMp150 for interaction with SUMO2/3 at the replication foci.

Dynamics of p150-SUMO2/3 interaction during S-phase

The number, distribution and size of p150-SUMO2/3 foci varied from cell to cell (Fig. 3A anddata not shown), implicating the spatiotemporal regulation of p150-SUMO2/3 interaction withreplication machinery during the S-phase. Therefore, we aimed to locate sites of DNA replication more

precisely by PCNA staining, and investigate the interaction dynamics of p150 and SUMO2/3 with11

replication machinery. We first synchronized NFH-p150 293S cells at the beginning of the S-phase bythymidine block and then released them for various times to prepare cell populations at different stagesof the S-phase. Based on the well defined spatiotemporal organization of sites of DNA replication,

which were identified by either the time period after release from the thymidine block, PCNA stainingor BrdU incorporation (or both) [21-25], we grouped the immunostaining patterns of the synchronizedcell population during the S-phase progression into three categories (Fig. 3C): early S-phase patterns

with a high density of small foci spread throughout the nucleus detected after 1~4 hr release; mid

S-phase patterns with a scattered medium foci throughout the nucleus, and a faint concentration on the

nuclear rim observed after 4~6 hr release; and late S-phase patterns with a few large dots locatedmainly at the nuclear and nucleolar periphery, detected after 8~10 hr release.

In early S-phase, the number and fluorescence intensity of PCNA dots rapidly increased as thecells were allowed to progress into S-phase and begin replication around the euchromatic regions.

During this time, anti-HA and –SUMO2/3 antibodies detected many small dots spread throughout thenucleus and co-localized with the PCNA dots, suggesting that p150-SUMO2/3 interaction occurred at

the PCNA-containing replication machinery (upper panel). The pattern of p150-SUMO2/3 interaction

detected by anti-HA and –SUMO2/3 antibodies changed during the mid (middle panel) and late S-phase(bottom panel); that is, the nuclear interior at these stages appeared to be filled with p150-SUMO2/3 foci12coincident with PCNA foci. The small number of large, homogeneously stained PCNA foci detectable

at the nuclear and nucleolar periphery during mid or late S-phase (or both), may correspond toconstitutive heterochromatin [22-25]; the p150-SUMO2/3 foci co-localized with PCNA foci during mid

and late S-phase in this cell line, would represent the constitutive heterochromatin regions. These

results indicate dynamic associations of p150 and SUMO2/3 with PCNA-containing replication

machinery, and imply that p150-SUMO interaction may be regulated spatiotemporally during theS-phase.

p150 is required for delivery/deposition of SUMO2/3 at the replication fociTo assess the role of p150 in SUMO2/3 delivery/deposition on chromatin fibers undergoing

DNA replication, we transfected NFH-p150 293S cells with two independent pSilencer plasmids

containing siRNAs against p150 (#1 and #2) to down regulate its expression. As shown in Fig. 4A,

immunoblot analysis indicated efficient reduction of FH-p150 by both pSilencer plasmids. After 48

hr treatment with p150 pSilencer plasmids, we performed immunostaining with anti-SUMO2/3

antibody; there were many cells with a remarkable reduction in SUMO2/3 accumulation at the focidetected by both anti-FH-p150 and -PCNA antibodies (Fig. 4B and C). In contrast, when we

transfected NFH-p150 293S cells with two independent siRNAs against SUMO2/3 to down regulate13their expression, many of the SUMO2/3-knockdown cells showed accumulation of the p150 focico-stained with anti-PCNA antibody (Fig 4D). Taken together, these results indicate that SUMO2/3

was not a necessary component for p150 localization, but the level of p150 in the cells has significant

effects on the deposition of SUMO2/3 to the sites of DNA replication, suggesting a p150-dependent

mechanism to deliver preferentially SUMO2/3 or SUMO2/3 modified proteins (or both) to thechromatin fibers during the S-phase.

The exact target chromatin protein(s) of p150 (i.e. SUMOylation-dependent andreplication-coupled) remain unexplained. However, several proteins are feasible. For instance,

many studies have demonstrated that p150 may act to initiate heterochromatin assembly byinteracting with heterochromatin associated proteins such as HP1, MBD1 and SETDB1 [10, 12, 22, 26,27]; these appear to be efficient SUMOylation substrates or interacting partners for SUMO [13, 26, 28,29]; thus, we assume that one or some of the components involved in the assembly of

HP1-MBD1-SETDB1-containing heterochromatin are candidates for the SUMOylation-dependentdelivery to replicating heterochromatin by p150. More work is needed to identify SUMO2/3

modified chromatin protein(s) which associates with p150 and explore the precise functional role of

p150-SUMO2/3 interaction at the replication sites.

14

Conclusion

We demonstrated that p150 interacts directly with SUMO2/3, and found that the binding of

p150 with SUMO2/3 is dependent on SIM located near its N-terminus. When p150 was

over-expressed in cultured human cells, we found that SUMO2/3 foci dramatically changed their

number, distribution and size as the S-phase progressed, and most of these foci, but not all,appeared at the regions where the DNA and chromatin fibers actively replicate. In addition, we

revealed that reduced expression of p150 failed in the delivery of SUMO2/3 to the DNA replicationfoci. Thus, in the context of DNA /chromatin synthesis, our identification of CAF-1 p150 as a

SIM-containing SUMO2/3-interacting protein shows that it is a novel regulatory component in the

SUMO modification pathway, and should warrant continued studies on the functional significanceof p150-SUMO2/3 interaction in the deposition of chromatin proteins.

Acknowledgements

We appreciate the helpful discussions with the members of the Saitoh Laboratory . This work

was supported by grants to Hisato Saitoh from the Naito Foundation, the Novartis Foundation, theMinistry of Education, Culture, Sport, Science and Technology (MEXT), and the Japan Society for

the Promotion of Science (JSPS). Junsuke Uwada is a Junior Research Associate of the global COE

15

Program at the Global Initiative Center for Pulsed Power Engineering.

16

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Figure Legends
Figure 1. CAF-1 p150 is a SUMO-interacting protein(A) Schematic representation of human CAF-1 p150, and a summary of the GST pull-down assay.

p150 deletion constructs are represented as thin black lines. The Table on the right summarizes thepull-down assay. + indicates sufficient binding, - indicates negligible binding.

(B) 500 µl of the lysate from E. coli expressing recombinant full-length His6-p150 was incubated withbeads bound to 5 µg of GST-only, GST-SUMO1, GST-SUMO2 and GST-SUMO3. Proteins associatedwith the beads were analyzed by immunoblot analysis using anti-His6 antibody.

(C) 500 µl of the lysate prepared from the 293S cells expressing Flag-HA-tagged p150 (FH-p150),

was incubated with beads bound to 5 µg of GST-only, GST-SUMO1, GST-SUMO2 and GST-SUMO3.

Proteins associated with the beads were analyzed by immunoblot assay, using the anti-Flag

antibody.

(D) 500 µl of the lysate from E. coli expressing recombinant full-length His6-SUMO3 was incubatedwith beads bound to 5 µg of GST-only, GST-15098-105 and GST-15098-102. Proteins associated with thebeads were analyzed by immunoblot analysis using anti-His6 antibody.

(E) 500 µl of the lysate from E. coli expressing recombinant full-length His6-p150-wild type, - I99A

and -D100A mutants were incubated with beads containing GST-only or GST-SUMO3. Proteins23

associated with the beads were analyzed by immunoblot analysis using anti-His6 antibody.

Figure 2. SUMO2/3-p150 interaction is detectable in vivo.

(A) HeLa cells expressing Flag-p150 were fixed, and subjected to indirect immunofluorescenceanalysis using anti-Flag antibody either with anti-SUMO2/3 (upper panel) or anti-SUMO1 (bottom

panel) antibody. Insets on the right side of each picture show 2.5-fold magnification of selected regions(white boxes). Merged images are shown on the right side. Bar = 10 µm.

(B)Exponentiallygrowing

NFH-p150

293S

cells

and

293S

cells

were

subjected

toimmunoprecipitation analysis with anti-Flag antibody as described in Materials and Methods.

Western-blot analyses using anti-Flag (left panel), anti-SUMO1 (middle panel) or anti-SUMO2/3 (rightpanel)

antibody,

areshown.

Arrowheads

indicate

SUMO

modified

proteins

that

co-immunoprecipitated with FH-p150.

Figure 3. p150-SUMO2/3 interaction occurs at/around the sites of DNA synthesis during S-phase

in NFH-p150 293S cells.

(A) Exponentially growing NFH-p150 293S cells in which FH-p150 was stably expressed wereincubated in the presence of BrdU for 15 min followed by indirect immunofluorescence analysis

24

using antibodies as indicated. Bar = 10 µm.

(B) HeLa cells expressing HA-p150wt and HA-p150-I99A were expressed were subjected to indirect

immunofluorescence analysis using antibodies as indicated. Bar = 10 µm.

(C) Exponentially growing NFH-p150 293S cells were arrested at the G1/S boundary by the double

thymidine-bock procedure. After culturing in standard medium for 1 hr (upper panel), 6 hr (middle

panel) and 8 hr (bottom panel), the cells were fixed and subjected to indirect immunofluorescenceanalysis using antibodies as indicated. Bar = 10 µm.

Figure 4. SUMO2/3 localization at replication foci is reduced in p150-depleted cells.

(A) NFH-p150 293S cells were transfected with pSilencer-p150 (#1 and #2) or control vector. 48 hours

post-transfection, total cell lysates were subjected immunoblot analysis, indicating efficient reductionof p150 by both pSilencer plasmids.

(B) NFH-p150 293S cells were transfected with control (upper panel) or pSilencer-p150 #1 vector(bottom panel). 48 hours post-transfection, cells were triple-stained with antibodies as indicated. The

merged image is indicated on the right. Bar = 10 µm.

(C) The result in (B) was quantified by scoring 50 cells from two independent experiments. Bar chart

depicts the ratio of cells in which SUMO2/3 accumulated at the PCNA-containing replication foci.

25

(D) NFH-p150 293S cells were transfected with pSilencer-SUMO2 and pSilencer-SUMO3 vector. 48

hours post-transfection, cells were triple-stained with antibodies as indicated (bottom panel). The

merged images are indicated on the right. A representative of the non-transfected cells is also shown(upper panel). Bar = 10 µm.

26SUMO2/3
p60

B

D
88
101

1
rich

HP1
PCNA

240

His6-SUMO3C

His6-p150

E

αHis6

27

ut
(
G S 10%)T
GS
T
GS – SUM
T
GS – SU O 1
T – MO
SU 2
MO3

CAF-1 p150
motif

In p

SIM 105
IIDLTEDS PxVxL Arg/Glu/Lysinput
G S (5%)TGS
T -S
inp UM
O3
ut
G S (5%)TGS
Tinp SUM
utO
G S (5% 3
)
T
GS
T -S
UM
O3

1

ut
G S (10%T)GS
T–
GS SUM
T–O
SU 1
GS
T – MO
SU 2
MO
3

A

ut
G S (20%
)
T
I9 8
-S
I98 105
-T
10
2

In p

In p

Glu/Asprich

Wild Type

His6-p150
αHis6
I99A

MBD1
938

S
Inte UMO
ra ction

Figure 198467

FH-p150

αHis6
αFlag
D100A

+
+
−
−
+

Figure 2

A
Flag-p150

SUMO2/3

Flag-p150

SUMO1

Flag-IP
input

Flag-IP

input

Flag-IP
input

input
Flag-IP

Flag-IP
input

NFH-p150
293S
293S

Flag-IP

input

NFH-p150
NFH-p150
293S 293S 293S 293S

B

160
100

FH-p150
αFlag

50

20
(kDa)
αSUMO1αSUMO2/3

28

Figure 3

A

B

BrdU

SUMO2/3

FH-p150

PCNA

SUMO2/3

FH-p150

PCNA

SUMO2/3

Wild Type

Merge

PCNA

SUMO2/3

I99A

Merge

PCNA

SUMO2/3

FH-p150

Merge

C
G1/S release
1hr

6hr

8hr

29

s iR

FH-p150γ-tubulin

BC

The cells containg PCNA-SUMO2/3 foci/ the cells containing PCNA foci.

50
(p1

NA

50
s iR

s iR

NA

NA

(p1

)#

1

A

)#
2
(c o
n tr
ol )

Figure 4
100
80
60
40
20
0

PCNA

SUMO2/3

FH-p150

PCNA

SUMO2/3

FH-p150control siRNA(p150)

Merge

controlsi p150D

Mergecontrolsi SUMO2/3

30

Supplemental Methods

Plasmids

To generate p150 expression vectors, human CAF-1 p150 was amplified by PCR using thefollowing oligonucleotides: forward primer,

5’-ATCTCTAGAGAATTCATGGATTGCAAAGATAGAC-3’ (an EcoRI site is underlined); reverse

primer, 5’-ATCCTCGAGTCTAGATCAGGATGCACCCAGTGG-3’(an XhoI site is underlined). The

generated PCR fragments were digested by EcoRI and XhoI and cloned with pET28 (Novagen),

pcDNA3-Flag and pcDNA-HA predigested with EcoRI and XhoI. The deletion mutants of p150 weregenerated with the following oligonucleotides: forward primer for 88aa-,

5’-CGGAATTCATCGAAACCAGTATTGGCCAG-3’; forward primer for 101aa-,

5’-CGGAATTCTTGACAGAGGACTCGAATGAG-3’; forward primer for 240aa-,

5’-CGGAATTCGCCACACCCCAAGGCAAGAAC-3’; reverse primer for -467aa,

5’-ATCCTCGAGTCTAGATCAGGTCCGACGCCGAGGGGC-3’. To generate pGEX 4T-1

p150-(I98-S105) and p150-(I98-T102), the following origonucleotides were used and subcloned into

pGEX 4T-1(GE Healthcare): forward primer, 5’-CGGAATTCATCATTGATTTGACA-3’: reverse primer(I98-S105), 5’-CTCTCGAGCTAGTCCTCTGTCAAATCAAT-3’: reverse primer (I98-T102),5’-CTCTCGAGCTATGTCAAATCAATGAT-3’. To construct plasmids expressing p150-I99A and

31

-D100A, site-directed mutagenesis was performed. SUMO-1/2/3 expression plasmids were used as

described previously (Uchimura et al., 2006). A shRNA expression vector targeting SUMO2, SUMO3

and CAF-1 p150 were constructed by inserting duplex oligonucleotides into a pSilencer-3.1- H1-purovector (Ambion). The oligonucleotides used were as follows:

pSilencer SUMO2,

5’-GATCCGGCCTACTGCGAGAGGCAGGGCTTGTTCAAGAGACAAGCCCTGCCTCTCGCAGTA

GGCCTTTTTTGGAAA-3’ and5’-AGCTTTCCAAAAAAGGCCTACTGCGAGAGGCAGGGCTTGTCTCTTGAACAAGCCCTGCCTCTCGCAGTAGGCCG-3’; pSlencer SUMO-3,5’-GATCCAGCCTATTGTGAACGACAGGGATTGTTCAAGAGACAATCCCTGTCGTTCACAATAG

GCTTTTTTTGGAAA-3’ and5’-AGCTTTCCAAAAAAAGCCTATTGTGAACGACAGGGATTGTCTCTTGAACAATCCCTGTCGTTCACAATAGGCTG-3’; pSilencer p150 #1,

5’-GATCCGCAACTGTCATGTGGGTTCTTTCAAGAGAAGAACCCACATGACAGTTGTTTTTTGGA

AA-3’ and5’-AGCTTTTCCAAAAAACAACTGTCATGTGGGTTCTTCTCTTGAAAGAACCCACATGACAGTT

GCG-3’; pSilencer p150 #2,

5’-GATCCGAACAAGCTCAGACTGCAATTCAAGAGATTGCAGTCTGAGCTTGTTCTTTTTTGGA32

AA-3’ and5’-AGCTTTTCCAAAAAAGAACAAGCTCAGACTGCAATCTCTTGAATTGCAGTCTGAGCTTGTT

CG -3’.

Bead halo assay

This method is based on a modification of a low-affinity protein interaction procedure “BeadHallo assay”, described by Rexach and colleagues (Patel and Rexach, 2008). The detailed procedure is

as follows. The SIMp150(I98-S105), SIMMCAF1(G965-E975) and SIMSETDB1(T120-D131) were expressed in E.

coli as a recombinant GST-fusion protein and were then incubated with glutathione-Sepharose 4B

beads (GE Healthcare). We used 10 µl of the packed beads for bacterial lysate prepared from 50 ml ofbacterial culture. This procedure results in GST-fusion proteins immobilized at high concentrations on

the surface of Sepharose beads. (Approximately 80-90 µg of GST-fusion protein were bound to 10 µl of

packed beads). The beads were resuspended as a 50% slurry in PBS and a 2.0 µl portion of the slurry, inwhich approximately 20 particles of beads were included, was mixed, on a 76 x 26 mm Micro Slide

Glass, (Thikness 1.2 mm, Matsunami), with 1.0 µl of bacterial lysate containing recombinant

GFP-SUMO1/3. As soon as the incubation started, the beads can be viewed in real-time underequilibrium conditions using a fluorescence microscope with 10x air objective (Eclipse E600, Nikon).

33

When a GFP-SUMO1/3 binds to an immobilized GST-fusion protein of interest, the interaction isvisible through GFP filters as a halo of fluorescence around the dark beads.

Supplemental Figure 1. Comparison of SIMp150 with other SIMs.

(A) The amino acid sequence of human CAF-1 p15098-105 is highly conserved among vertebrate andinvertebrate species. The sequences within the gray region indicate amino acid residues similar tohuman p15098-105. The corresponding Accession Nos. are human p150, AAA76736; mouse p150,

NP_038761; chicken p150, NP_001073220; zebrafish p150, NP_001038478; fruit fly (D. melanogaster),

NP_572495; and yeast (S. cerevisiae), NP_015343. The numbers on the left side indicate the position of

the amino acid residue in each protein.

(B) Sequence comparison with previously identified SUMO-interacting polypeptides. The sequences

the gray region indicate amino acid residues similar to the canonical SIM’s hydrophobic core

(V/I-X-V/I-V/I or V/I-V/I-X-V/I). The black underline represents the region similar to a negatively

charged cluster of amino acids that often follows the hydrophobic core sequences of SIM. The

corresponding Accession Nos. are p150, AAA76736; MCAF1, AAO91864; SETDB1, NP036564; PIAS1,

NP057250; DAXX, NP001135441; PML, NP150241; SP100, NP001073860; TOPORS, NP005793. The

numbers on the left side indicate the position of the amino acid residue in each protein.

34

(C) Bead Halo experiments for analyses of the SUMO3 interaction with SIMp150, SIMMCAF1 or SIMSETDB1.
Bacterial lysate containing 0.1 µg/ml (1st column; 100x), 0.01 µg/ml (2nd column; 10x) and 0.001 µg/ml (3rdcolumn; 1x) of EGFP-SUMO3 were mixed with the bead-immobilized GST-SIMp150 (1st lane),
GST-SIMMCAF1 (2nd lane), GST-SIMSETDB1 (3rd lane) and GST-alone (4th lane). An aliquot of theimmobilized protein was resolved by SDS-PAGE, stained with Coomassie blue, and is shown on theleft. The mixtures were treated as described in the Materials and Methods, and then imaged under afluorescent microscope using an appropriate GFP-filter. The GFP signal localized to the beads

indicates a positive SUMO-SIM interaction. The affinity of SIMMCAF1 was measured and calculated aspreviously described [16].

(D) The GFP signals on the beads-immobilized GST-SIMp150 (dark gray), GST-SIMMCAF1 (light gray),

GST-SIMSETDB1 (gray) and GST-alone (black), were quantified using an image-analyzing system. a.u.,

arbitrary relative signal intensity units.

Supplemental Figure 2. p150 may not be efficiently modified with SUMO2/3.

(A) Failure of detection of endogenously SUMOylated p150. Using NFH-p150 293S cell lysate, an

immunoprecipitation assay was performed using control IgG (lanes 1) and anti-Flag antibody (lanes 2)

under high salt (stringent) conditions. Immunoprecipitated fractions were subjected immunoblot assay

35

using anti-Flag antibody. The arrowhead indicates the position at FH-p150.

(B) A small amount of SUMO modification of p150 in the bacterial SUMOylation system. GST, fused

either with MBD1 (left panel) or p150 (right panel) protein, was subjected to bacterial SUMOylation[17] and purified with glutathione Sepharose beads. Purified products were subjected to SDS-PAGE

and stained with Coomassie blue (CB). The asterisks represent the positions of SUMO modified

GST-MBD1. Molecular size markers are indicated on the right side.

Supplemental Figure 3. Characterization of the NFH-p150 293S cell line(A) FH-p150 was stably over-expressed in the NFH-p150 293S cell line. Immunoblot analysis of 293S

cells and NFH-p150 293S cells using antibodies against p150 (upper panel) and Flag (middle panel) are

shown. For the loading control, immunoblot analysis using anti-gamma tubulin antibody is shown(bottom panel). The numbers at the bottom of each lane represent the relative amounts of the band

intensities at around 150 kDa detected by anti-p150 antibody.

(B) The cell cycle progression is not perturbed in NFH-p150 293S cells. Growth curves of 293S cells

and NFH-p150 293S cells are compared. The cell numbers were counted at 24 hr intervals for 5 days

and the relative ratio at each point was plotted.

36

Supplemental Figure 1

A
Human CAF-1 p150
Mouse CAF-1 p150
Zebrafish CAF-1 p150
Fruit Fly CAF-1 p180
Yeast CAC1

88- IETSIGQSTV IIDLTEDS NEQPDSLVDH -115
99- ATIKPVPSVV IIDLTENC SDIPDSPEGH -126
101- RSPLRSAPEA TIDLTEDS NDSAKQQPAP -128
123- VKTTDSVVED VIELDEDE ADKEIEDQDQ -150
31- SNKFLTKEKD VITLDDPK EDVSGPMIET -58

B Human
CAF-1 p150
MCAF1
SETDB1
PIAS1
DAXX
PML
SP100
TOPORS

93- GQSTV IIDL TEDSNEQPDSLVD -114
961- SDSSG VIDL TMDDEESGASQDP -982
117- SRPTE IIEI PDEDDDVLSIDSG -138454- NKKVE VIDL TIDSSSDEEEEEP -475
728- CDPEE IIVL SDSD
-740
551- EAEER VVVI SSSEDSDAENSSS -572
318- NQASD IIVI SSEDSEGSTDVDE -339
902- SRSPV VITI DSDSDKDSEVKED -923
E/D richC
100X

EGFP-SUMO3
10X

1X

GST-SIM

p150

MCAF1
( Kd = 1.3± 0.11 μM )SETDB1

GST

D

asd
EGFP-SUMO3
37

+

-1
AF

−

+

E1E2SUMO3

p1
50

-1
AF

T -C
GS

−

GS

BD
1
BD
1
GS

(kDa)

GS

(kDa)

T-M

AG
FL

ti-

T -M

Ig G
ol

B

an

n tr
Co

inp

ut

A

T -C

p1
50

Supplemental Figure 2

175
*

160

FH- p150

83

100

220
GSTMBD1

GSTCAF-1 p150

160

coomassie stained

αFlag

α CAF1 p150
1.0
αFlag
α γTubulin

6.1

B
35
30
Relative cell number

293S

A

NFH-p150 293

Supplemental Figure 3

25
20

293S
NFH-p150 293

15
10
5
0
1

2

3

4

Culture time (days)

38

5