Báo cáo khóa học: Interaction between the 2¢)5¢ oligoadenylate synthetase-like protein p59 OASL and the transcriptional repressor methyl CpG-binding protein 1 potx

Interaction between the 2¢)5¢ oligoadenylate synthetase-like protein
p59 OASL and the transcriptional repressor methyl CpG-binding
protein 1
Jesper B. Andersen*, Dorthe J. Strandbyga
˚
rd, Rune Hartmann† and Just Justesen
Department of Molecular Biology (MBI), University of Aarhus, Denmark
The human 2¢)5¢ oligoadenylate synthetases (OAS) form a
conserved family of interferon-induced proteins consisting
of four genes: OAS1, OAS2, OAS3 and the 2¢)5¢ oligo-
adenylate synthetase-like gene (OASL). When activated by
double-stranded RNA, OAS1–3 polymerize ATP into 2¢)5¢-
linked oligoadenylates; 2¢)5¢-linked oligoadenylates, in turn,
activate a latent endoribonuclease that degrades viral and
cellular RNAs. In contrast, while the p59 OASL protein is
highly homologous to the OAS family (45% identity), its 350
amino acid N-terminal domain lacks 2¢)5¢ oligoadenylate
synthetase activity. A C-terminal 164 amino acid domain,
which is 30% homologous to a tandem repeat of ubiquitin,
further distinguishes the p59 OASL protein and suggests
that it serves a biological role which is distinct from other
OAS family members. To dissect the function of p59 OASL,
we utilized the yeast two-hybrid system to identify interact-
ing proteins. Methyl CpG-binding protein 1 (MBD1), which
functions as a transcriptional repressor, was identified as a
strong p59 OASL interactor. Interestingly, like p59 OASL,
transcription of the MBD1 gene was induced by interferon,
indicating that these genes are co-ordinately regulated. The
interaction was confirmed in vitro and in vivo and was
mapped to the ubiquitin-like domain of p59 OASL. The p59
OASL–MBD1 interaction was specific, because p59 OASL

did not interact with any of the other MBD family members
and MBD1 did not interact with OAS1. These findings link
the p59 OASL with MBD1 transcriptional control in the
context of an interferon-stimulated cell, and provide the
basis for future studies to examine the functional role of this
interaction.
Keywords: interferon; MBD1; methylation; p59 OASL;
ubiquitin-like.
In 1957, Isaacs & Lindenmann identified interferon (IFN) as
the causative agent responsible for the phenomenon of viral
interference in animal viruses [1]. IFNs are potent cytokines
that play a key role in establishing resistance to viral
infections in vertebrates. In addition to the classical antiviral
response, IFNs also exhibit antitumor, antiproliferative,
antiparasitic, and immunomodulatory properties [2–4].
IFNs mediate their effects through activation of the JAK/
STAT signalling pathway, which results in the transcrip-
tional induction of a number of IFN-stimulated genes [4].
The 2¢)5¢ oligoadenylate synthetases (OAS) are part of a
regulated RNA decay pathway known as the 2–5A system.
The OAS proteins are produced as latent enzymes which
bind to double-stranded RNA (dsRNA) produced by
infecting viruses; the binding of dsRNA to OAS results in
enzyme activation [5]. Once activated, OAS polymerizes
ATP into 2¢)5¢-linked oligoadenylate, pppA(2¢p5¢A)
n
,
n ‡ 1, termed 2–5A [6–8]. The 2–5A oligomers bind to a
latent, monomeric endoribonuclease (RNase L), which
induces dimerization and activation [9]. Activated RNase L

mediates a general RNA degradation, leading to the
inhibition of viral protein synthesis [10].
In humans, the OAS gene family is composed of four
genes located on chromosome 12 [11]. The OAS1, OAS2
and OAS3 genes are encoded by a tightly coupled locus on
chromosome 12q24.1 [12]. The products of these three genes
are known, respectively, as the small (p42/p46), the medium
(p69/p71) and the large (p100) forms of OAS [13], all of
which are enzymatically active. The fourth member of the
OAS family is the OAS-like (OASL) gene that encodes
a 59 kDa protein (p59 OASL). In contrast to the other
members of the OAS family, p59 OASL is unable to
synthesize 2–5A [14,15]. However, it is still strongly induced
by IFN. The inability of p59 OASL to synthesize 2–5A is
ascribed to specific changes in three aspartic acid residues
Correspondence to J. Justesen, Department of Molecular Biology
(MBI), University of Aarhus, DK-8000 C, Aarhus, Denmark.
Fax: + 45 8942 2637, Tel.: + 45 8942 2682, E-mail:
Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase;
*GST, glutathione S-transferase; MBD1, methyl CpG-binding pro-
tein 1; MBD1v6, methyl CpG-binding protein 1 splice variant 6
(GenBank Accession Number AJ564845); NP-40, Nonidet P-40;
OAS, 2¢)5¢ oligoadenylate synthetase; p59 OASL, 2¢)5¢
oligoadenylate synthetase-like gene that encodes a 59 kDa protein;
Ub, ubiquitin; UbL, ubiquitin-like domain.
Present addresses: *Department of Microbiology & Immunology,
Greenebaum Cancer Center, University of Maryland at Baltimore,
MD 21201, USA; †Case Western Reserve University,
Department of Biochemistry, 10900 Euclid Avenue,
44106 Cleveland, OH 44195, USA.

(Received 4 September 2003, revised 21 November 2003,
accepted 15 December 2003)
Eur. J. Biochem. 271, 628–636 (2004) Ó FEBS 2004 doi:10.1046/j.1432-1033.2003.03966.x
that are crucial for enzymatic activity to either glutamic acid
or threonine [16].
The N-terminus of the p59 OASL protein contains an
OAS core domain that is highly homologous to the rest of
the OAS family. In contrast, the C-terminus of the p59
OASL protein has sequence similarity to a tandem repeat
of ubiquitin (Ub), UbL1-UbL2 [14]. The Ub-like domain
(UbL) of p59 OASL lacks the C-terminal diglycine motif
that is critical for the covalent conjugation of Ub and UbL
to cellular proteins [17]. Accordingly, the role of the p59
OASL UbL is, as yet, unknown.
An orthologue of p59 OASL exists in mice that, like the
human p59 OASL, is devoid of 2–5A synthetase activity
[16]. As this class of proteins lack the enzymatic activity that
characterizes OAS family members and possesses a novel
UbL, it is probable that p59 OASL serves distinct biological
functions. To dissect the role of p59 OASL, we used the
yeast two-hybrid screening method to identify interaction
partners for the human p59 OASL protein. Our study
revealed that the methyl CpG-binding protein 1 (MBD1)
binds to the C-terminal UbL domain of p59 OASL, both
in vitro and in vivo. We also demonstrated that MBD1 is an
IFN-stimulated gene, thus the two genes are co-induced by
IFN. The implications of this interaction for the biological
functions of p59 OASL are discussed.
Methylation of DNA at CpG dinucleotides is pro-
grammed during embryogenesis and functions to silence

specific genes through development [18,19]. This can inhibit
an interaction between a sequence-specific DNA-binding
protein and its cognate promoter sequence, thus resulting in
an inactivation of the appropriate gene. Methylation of
mammalian DNA is specific for cytosine residues at the
5¢ position of CpG dinucleotide sequences. This epigenetic
modification is widespread in the eukaryotic genome, as
60–90% of all CpGs in vertebrates are methylated, leaving
the majority of nonmethylated CpGs to be found in CpG
islands of functionally active promoters [20]. The biological
consequences of DNA methylation have been implicated in
the regulation of cellular differentiation and embryogenesis.
DNA methylation has been observed to be involved in
tissue-specific gene transcription, X chromosome inactiva-
tion, genomic imprinting, cellular defense against viral
infection and tumorigenesis [21,22]. In addition, several
tumor-suppressor genes have been demonstrated to be
hypermethylated in cancer cells, resulting in transcriptional
repression [23,24].
Experimental procedures
Bait plasmid construction and yeast two-hybrid
screening
Full length p59 OASL and various deletions were amplified
by PCR and subcloned into the two-hybrid bait vector,
pBTM118, creating fusion proteins with the LexA DNA-
binding domain (Matchmaker; Clontech). The restriction
sites SmaI/SacII were used to subclone bait F and bait 1,
while SacII/XhoI were used to subclone baits 2, 3 and 4. A
human leukocyte cDNA library, constructed in the pACT2
GAL4 trans-activating vector, was used as prey (Match-

maker Two-Hybrid System; Clontech). To screen for p59
OASL interacting proteins, Saccharomyces cerevisiae L40
cells (MATa,trp1,leu2,ade2,GAL4,lexAops-HIS34,lexA-
ops-lacZ8) (Invitrogen) were transformed using the lithium
acetate/polyethylene glycol method, according to the sup-
plier’s manual (Matchmaker Two-Hybrid System; Clon-
tech). Selection in the L40 yeast strain is for the HIS
prototrophy and the reporter is an integrated LacZ gene.
Expression of each bait construct was verified by the
repression assay, and by Western blotting, using antibody
to LexA (Invitrogen). To suppress possible background
growth, triple selection plates (-Leu, -Trp, -His) were
supplemented with 20 m
M
3-amino-1,2,4-triazole (3-AT).
Positive clones were further tested for b-galactosidase
activity by growth on plates containing 5-bromo-4-chloro-
indol-3-yl b-
D
-galactoside. Positive interactions were further
assessed by using the b-galactosidase filter assay.
Plasmid identification of
p59 OASL
interacting partners
Plasmids from colonies 32 and 54 were transformed into the
Escherichia coli strain XL1-Blue for high yield plasmid
purification, using the plasmid Maxi kit (Qiagen) according
to the manufacturer’s instructions. Sequencing was under-
taken with the aid of a Thermo Sequenase II dye terminator
cycle sequencing kit (Applied Biosystems). Sequence ana-

lysis was carried out using a 377 DNA sequencer (Perkin
Elmer). The DNA sequence for the methyl CpG-binding
protein 1 splice variant 6 (MBD1v6) has been submitted to
, having the EMBL/GenBank acces-
sion number AJ564845.
Cell culture and transfection
The human fibrosarcoma cell line, HT1080, was stably
transfected with either full length p59.F-V5 OASL or
p59DUbL-V5 OASL (a deletion mutant lacking the
C-terminal UbL domain) and an empty vector
(pcDNA3.1 V5/HisA; Invitrogen), as a control. Stable
transfectants were selected in 200 lgÆmL
)1
G418 (Geneticin
Sulphate; LifeTechnologies) and cultured in DMEM (Dul-
becco’s modified Eagle’s medium; GibcoBRL) supplemen-
ted with 10% fetal bovine serum (FBS) and 1% penicillin/
streptomycin. HeLa and T98G cell lines were grown
according to ATCC guidelines in DMEM supplemented
with 10% FBS and 1% penicillin/streptomycin.
RT-PCR analysis
Total RNA was purified from HeLa and T98G cells
using the Maxi RNEasy purification kit (Qiagen),
according to the manufacturer’s instructions. A 5 lg
aliquot of total RNA from each sample was reverse
transcribed using the First Strand cDNA synthesis kit
(Amersham Biosciences). For semiquantitative analysis of
the induction, by IFN, of MBD1 in HeLa cells, the PCR
was carried out for 20–35 cycles, comprising 2 min at
95 °C, 1 min at 95 °C, 1 min at 55 °C,and2minat

72 °C, and a final extension of 5 min at 72 °C, resulting
in a 550 bp PCR product for MBD1. The human
glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
gene was included as a control. The MBD1 and GAPDH
reactions were mixed in equal amounts before electro-
phoretic analysis on a 1% agarose gel.
Ó FEBS 2004 A novel interaction with p59 OASL (Eur. J. Biochem. 271) 629
Generation of a polyclonal antibody to p59 OASL
The p59 OASL was subcloned into a modified version of the
pET-9d vector (Novagen) having a 6 · His-tag. The protein
was expressed in E. coli BL21 (DE3) pRP4, pRI cells
and purified using Ni
2+
-nitrilotriacetic acid agarose beads
(Qiagen). The purified His-tagged protein was analysed on
10% SDS/PAGE and the band corresponding to p59
OASL was cut out. Rabbits were immunized twice and
antiserum was collected. To increase the specificity of the
antibody, precipitation was performed in saturated ammo-
nium sulphate, whereby buffer comprising saturated ammo-
nium sulphate (76 g ammonium sulphate in 100 mL of
ddH
2
O) was slowly added to the rabbit serum to a final
concentration of 47% (v/v). After stirring very slowly for
2hat4°C, the precipitate was collected by centrifugation
at 20 000 g and resuspended in NaCl/P
i
.Toremoveexcess
ammonium sulphate, the sample was dialyzed in NaCl/P

i
for 24 h. The protein concentration was measured using the
bicinchoninic acid protein assay (Pierce) and an ELISA
reader (lQuant; Bio-Tek Institute) at 562 nm. A 20 mg
sample of protein was further purified by gel filtration
chromatography (Highload 16/60 superdex 75; Pharmacia).
The column fractions (0.5 mL) were examined by 10%
PAGE and staining with Coomassie blue. Peak fractions
containing immunoglobulin antibodies were pooled and
stored at )80 °Cin200lL of NaCl/P
i
containing 0.1%
NaN
3
.
Glutathione
S
-transferase (GST) pull-down assay
Expression of the GST–MBD1 fusion protein. The
pGEH-GST-MBD1-HIS construct (A kind gift from
A. Bird, University of Edinburgh) was expressed in the
E. coli strain BL21(DE3) pRP4, pRI in 2 · YTG medium
containing 2% glucose (100 lgÆmL
)1
ampicillin, 20 lgÆmL
)1
kanamycin, 10 lgÆmL
)1
tetracycline). A 500 mL volume of
cells was cultured at 37 °C to reach an attenuance (D)of0.5

at 600 nm. To induce protein expression, 1.0 m
M
isopropyl
thio-b-
D
-galactoside (IPTG) (final concentration) was added
and culture continued for 2 h at 30 °C, then chilled for
15 min on ice. The cells were harvested by centrifugation
(8200 g,4°C, 15 min) and resuspended in 5 mL of NETN
buffer [20 m
M
Tris/HCl, pH 8.0; 100 m
M
NaCl; 1 m
M
EDTA; 0.5% Nonidet P-40 (NP-40); 1 m
M
dithiothreitol]
containing a protease inhibitor cocktail (Boehringer Mann-
heim GmbH). Sonication was performed on ice using a series
of 20 s bursts at amplitude 16, followed by a 30 s rest for
2 min. The cell debris was pelleted and the supernatant
stored at )80 °C in 20% (v/v) glycerol.
Purification of GST–MBD1. The fusion protein, GST–
MBD1, was purified on GST beads (Glutathione Seph-
arose
TM
4B fast flow; Amersham Pharmacia). For each
reaction, 150 lL of GST beads was washed three times in
an equal amount of NETN milk buffer (NETN buffer

containing 0.5% milk powder). The beads were incubated
with 200 lL of NETN milk buffer and 2.6 mL of
supernatant, and rotated for 1 h at 4 °C. After incubation,
the beads were pelleted (1200 g,4°C, 10 min), and washed
five times in 1 mL of NETN buffer containing a protease
inhibitor cocktail.
GST-MBD1 pull-down assay. Ten micrograms of GST-
MBD1 fusion protein, immobilized on GST beads, was
incubated with 4 lg of p59 OASL in a total volume of
250 lL NETN buffer and 10% v/v glycerol for 18 h at 4 °C.
Thereactionmixturewaswashedfourtimesin500lL
of NETN buffer and the immobilized proteins were assayed
by SDS/PAGE (10% gel) and Western blotting using
antibody to p59 (diluted 1: 15 000).
Co-immunoprecipitation of p59 OASL and MBD1
Transfections were performed using LipofectAMINE
TM
Plus reagents, according to the manufacturer’s instructions
(LifeTechnologies, Inc.). The cells were grown in a T150
culture tank and transfected with 45 lgofpCS-MT-
MBD1 5xMyc tagged plasmid. At 24 h post-transfection,
the cells were lysed in 0.5 mL of RIPA lysis buffer
containing a protease inhibitor cocktail [50 m
M
Tris/HCl
(pH 7.4), 150 m
M
NaCl, 1 m
M
EDTA, 0.5% NP-40, 15%

glycerol, 1 m
M
NaF). The cells were mechanically lysed,
using 20 strokes, with a Dounce-Homogenizer. The lysate
was then cleared with protein G–beads (protein
G–Sepharose
TM
4 fast flow; Amersham Pharmacia), for
3h at 4°C, to minimize nonspecific binding. The
precleared lysate was incubated with 100 lL of washed
sepharose–protein G anti-V5 immunoglobulin (1 : 500;
Invitrogen) complex, in a total volume of 0.5 mL of
NaCl/P
i
, for 1 h at room temperature. After incubation,
the beads were washed five times in 0.5 mL of RIPA
wash buffer [50 m
M
Tris/HCl (pH 7.4), 100 m
M
NaCl,
0.1% NP-40, 1 m
M
EDTA, 15% glycerol] and the
complex-bound proteins were isolated by centrifugation.
The immunoprecipitated proteins were analysed by SDS/
PAGE (10% gel) and Western blotting using a polyclonal
MBD1 antibody from sheep (1 : 2000 dilution).
In vitro
translation

The TNT Quick in vitro Translation kit (Promega) was used
to express p59 OASL and MBD1. The reaction mixture was
prepared according to the supplier’s manual and incubated
at 30 °Cfor1.5h.
Immunoprecipitation
p59.F-V5 OASL and p59DUbL-V5 OASL containing a V5
epitope tag were expressed using unlabeled methionine in
the in vitro translation reactions, and 15 lL of each reaction
was incubated with 50 lL of precoupled V5 protein G
beads (Protein G–Sepharose
TM
4 fast flow; Amersham
Pharmacia; anti-V5 immunoglobulin, 1 : 500 dilution,
Invitrogen) in a total volume of 0.5 mL of ice-cold IP
buffer [20 m
M
Tris/HCl (pH 7.9), 10% glycerol, 0.1
M
KCl,
5m
M
dithiothreitol, 0.1% NP-40].
To minimize nonspecific binding to the beads, 10 lL of
10% BSA was added to each reaction. After 1 h, each
reaction was supplemented with [
35
S]methionine in vitro
translated full-length MBD1 (15 lL), and the incubation
was continued for 3 h at 4 °C. The beads were washed five
times in 0.5 mL of IP buffer containing 100 m

M
NaCl, and
the immunoprecipitated proteins were analyzed by SDS/
PAGE (10% gel) and autoradiography.
630 J. B. Andersen et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Results
Identification of a novel p59 OASL interaction partner
using the yeast two-hybrid system
To study the function of p59 OASL, we sought to identify
partners using the yeast two-hybrid system, a powerful
genetic technique for identifying protein–protein inter-
actions [25]. The bait applied in this study was a fusion
between the DNA-binding domain of the bacterial LexA
gene and the human p59 OASL. To identify proteins
that interact with specific domains of p59 OASL, deletion
mutants containing the P-loop, ATP-binding, and UbL
domains, individually or in combination, were also used as
bait (Fig. 1). p59 OASL is highly expressed in leukocytes;
therefore, to maximize the possibility of identifying phys-
iologically relevant interactions partners, we chose a prey
library from human leukocyte cDNA fused to the trans-
activating domain of GAL4. The yeast strain L40 was used
for screening the library, enabling selection of bait and prey
plasmids by the TRP1 and LEU2 selection marker genes,
respectively. The different bait constructs were transformed
into the yeast strain L40 and the expression of the fusion
protein was confirmed by Western blotting (data not
shown).
Of the five bait constructs screened, baits F, 1 and 2 failed
to produce any positive clones. Bait 3 produced numerous

false positive results and further analyses were therefore
abandoned. However, a screen with bait 4, of 2.4 · 10
7
transformants that covered the library more than six times,
detected 54 colonies capable of growing on triple selection
plates. Out of the 54 possible positive interactions, only two
colonies showed positive staining on plates containing
5-bromo-4-chloroindol-3-yl b-
D
-galactoside (positive for the
LacZ reporter). Plasmids from these colonies were isolated
and their inserts sequenced. The two independently isolated
colonies contained an identical insert. A search of GenBank
using the NCBI
BLAST
server identified the 3 kb insert to be
homologous to MBD1.
p59 OASL interacts specifically with MBD1
The specificity of the interaction was tested by retransfor-
mation of the prey constructs into the L40 strain expressing
different control bait plasmids; positive interactions were
detected by the ability of these transformants to grow on
triple selection plates and to activate the LacZ reporter
gene (Fig. 2). To examine the specificity of the interaction
in the yeast two hybrid system, we utilized two sets of
controls (a) an empty bait vector and a bait vector
containing the unrelated Fhit cDNA as general negative
controls and (b) a bait vector containing the p42 OAS
cDNA that addressed the interaction with another OAS
family member. The original bait LexA–p59.4 OASL was

used as a positive control. This set of controls showed that
the MBD1 reacted specifically with the p59.4 construct, but
not with the empty bait vector, Fhit or a different member
of the OAS family, p42 OAS (Fig. 2A). We also tested the
ability of LexA–p59.4 OASL to interact with other mem-
bers of the methyl CpG-binding protein family (MBD2,
MBD3 and MBD4) by introducing the prey constructs
MBD2a–GAL4, MBD2b–GAL4, MBD3–GAL4 and
MBD4–GAL4 into an L40 yeast strain expressing the bait
LexA–p59.4 OASL. Only the LexA–p59.4 OASL strain
Fig. 1. The bait constructs used in the p59 2¢)5¢-oligoadenylate syn-
thetase-like (OASL) yeast two hybrid screenings. (Numbers refer to
exons; Mw, molecular mass.) Bait designations F and 1–4 refer to the
following constructs, respectively: LexA-p59.F OASL, LexA-p59.1
OASL, LexA-p59.2 OASL, LexA-p59.3 OASL (grey) and LexA-p59.4
OASL (black).
Fig. 2. Specificity of the interaction between Le xA–p59.4 OASL pro-
tein and prey MBD1–GAL4AD. (A) The L40 yeast strain was trans-
formed with the indicated baits and preys and assayed on double and
triple selection plates. Prey32 and Prey54 denote the preys identified in
the yeast two-hybrid screen. LexA–p42 OAS and LexA–Fhit were used
as controls. (B) The MBD family prey constructs MBD2a–GAL4,
MBD2b–GAL4, MBD3–GAL4 and MBD4–GAL4, were a kind gift
from F. Ishikawa (Tokyo Institute of Technology, Japan).
Ó FEBS 2004 A novel interaction with p59 OASL (Eur. J. Biochem. 271) 631
transformed together with MBD1v6 (MBD1–GAL4AD)
was able to grow on triple selection, showing that p59
OASL specifically interacts with MBD1 of the MBD
family (Fig. 2B).
To further verify the p59 OASL–MBD1 interaction, we

employed an in vitro GST pull-down assay. MBD1 fusion
protein was expressed in E. coli and purified using gluta-
thione sepharose beads. The purified MBD1 fusion protein,
or GST alone, were incubated together with purified
recombinant p59 OASL (Fig. 3). The beads were prepared
for SDS/PAGE and analysed, by Western blotting, for the
presence of the p59 OASL using a p59 OASL specific
antibody (Fig. 3). Only the MBD1 fusion protein was able
to pull down p59 OASL, while the GST control was
negative.
The p59 OASL interacts with MBD1 via the UbL
To map the domain of p59 OASL that interacts with
MBD1, the prey construct was introduced into L40 yeast
strains expressing the different bait constructs shown in
Fig. 1. Only baits 3 and 4 grew on triple selection plates and
stained positive for b-galactosidase (Fig. 4). The two baits
that showed an interaction with MBD1 both contain the
C-terminal part of p59 OASL where the UbL is located,
suggesting that the UbL of p59 OASL is required for the
interaction with MBD1. However, MBD1 did not interact
with the full-length p59.F OASL (bait F). MBD1 interacts
with bait 4, but pull-down assays clearly show that it can
interact with full length p59 OASL. The lack of an
interaction with full length p59 OASL in yeast can be
explained by difficulties in introducing large, full size
mammalian proteins into the nuclei of yeast. In fact, the
repression assay indicated that the full length bait construct
did not express as well as the other constructs tested (data
not shown); in contrast, bait 4 was expressed at the highest
level of all the bait constructs.

To verify that the interaction with MBD1 requires the
UbL of p59 OASL, we expressed a full length p59.F-V5
OASL and the deletion mutant lacking the entire UbL,
p59DUbL-V5 OASL, using a nonradioactive in vitro
translation system. Precoupled anti-V5 antibody protein
G–sepharose beads were used to immunoprecipitate
p59.F-V5 OASL and p59DUbL-V5OASLviatheir
C-terminal V5 epitope tag. These beads were then used
in pull-down assays, together with [
35
S]methionine-labeled
MBD1 (Fig. 5A). As seen in Fig. 5, full length MBD1
did not interact with the beads alone or with the p59
OASL deletion mutant (Fig. 5A, lanes 2 and 3), whereas
a strong interaction was observed with the full length p59
OASL (Fig. 5A, lane 3). As a control, the expression of
all three constructs was translated using [
35
S]methionine
(Fig. 5B).
Fig. 3. Verification of the p59 OASL–MBD1 interaction by glutathione
S-transferase (GST) pull-down. In vitro GST pull-down assay. GST–
MBD1 bound to GST beads was incubated with recombinant p59
OASL and the bound proteins were analysed by SDS/PAGE (10%
gel) and Western blotting using anti-p59 OASL immunoglobulin
(1 : 15 000 dilution). Lane 1 (control), 4 lg of recombinant p59
OASL; lanes 2 and 3 (WASH), GST–MBD1 beads; lane 4, pull-down
ofp59OASLusingGST–MBD1beads;lanes5and6(WASH),GST
beads; lane 7 (control), pull-down of p59 OASL using GST beads. The
pGEH–GST–MBD1 construct was a kind gift from A. Bird (Institute

of Cell and Molecular Biology, University of Edinburgh, UK).
(B) Purification of the GST–MBD1 fusion protein. A total of 0.2 lg
of protein was applied to SDS/PAGE (10% gel) then stained with
Coomassie blue.
Fig. 4. Retransformation. The prey, methyl
CpG-binding protein 1 (MBD1)–GAL4AD
was transformed into each of the five, LexA–
p59 OASL, bait expressing L40 strains. These
cells were plated on double selection plates for
3 days and replated for 3–5 days on triple
selection plates supplemented with 20 m
M
3-AT. Activation of the second reporter gene,
LacZ, was analyzed using the b-galactosidase
filter assay for blue coloring.
632 J. B. Andersen et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Verifying the interaction
in vivo
in HT1080
fibrosarcoma cells
The p59 OASL–MBD1 interaction was further verified
in vivo by co-immunoprecipitation. MBD1 was transiently
transfected into HT1080 human fibrosarcoma cells that
were stably transfected with either full length p59 OASL
(p59.F-V5) or with a C-terminal UbL deletion mutant of
p59 OASL (p59DUbL-V5). After transfection, the cells were
cultured for 24 h to permit expression of MBD1. p59.F-V5
and p59DUbL-V5 were precipitated using precoupled anti-
V5 protein G–sepharose beads. To identify the interaction,
we analysed the precipitates by Western blotting using a

polyclonal antibody raised against full length MBD1
(Fig. 6). MBD1 only interacted with the full length p59
OASL, confirming the findings, of previous assays, indica-
ting that UbL is required for the interaction between the two
proteins (Fig. 6, lane 1). Expression of MBD1 in HT1080
cells was verified in lane 2 and lane 5. To confirm that both
the full length and the deletion mutant of p59 OASL were
stably expressed in the HT1080 cells used in the immuno-
precipitation assay, we performed Western blot analysis
using anti-V5 immunoglobulin (data not shown). Empty
pcDNA3.1-V5 stably transfected HT1080 cells were used as
a negative control in this assay.
MBD1 does not interact with human Ub
To investigate whether the p59 OASL–MBD1 interaction is
specific for the UbL of p59 OASL and not Ub in general, we
performed a pull-down assay between monomeric Ub and
MBD1 (Fig. 7).
35
S-labeled MBD1 was expressed by in vitro
translation (Fig. 7, lane 1). Immunoprecipitation of MBD1
was performed using anti-MBD1 immunoglobulin coupled
to protein G–beads (lanes 3 and 4). The precipitates were
then visualized by autoradiography. Co-immunoprecipita-
tion of monomeric Ub, together with labeled MBD1
precipitate, was assayed by Western blotting using a Ub-
specific antibody (Fig. 7B, lanes 2, 4, and 6). MBD1 did not
interact with monomeric Ub, demonstrating the specificity
of the interaction with the Ub-like domain of p59 OASL
(lane 4).
MBD1

is induced by IFN
The p59 OASL is expressed at low basal levels and is
dramatically induced by type I and type II IFNs; therefore
we sought to determine whether MBD1 was also regulated
by IFN. A database of IFN-stimulated genes (http://
www.lerner.ccf.org), which is based upon gene expression
profiling using oligonucleotide DNA arrays, currently lists
1351 IFN-regulated genes. In this database, MBD2 was
reported to be induced by type I IFN, but the regulation of
other family members, including MBD1, had not been
investigated.
To investigate whether MBD1 is induced by IFN, we
used cDNA from HeLa cells treated with IFN-a,-c or
dsRNA (IFN-a,500UÆmL
)1
;IFN-c,100UÆmL
)1
;or
Fig. 5. The ubiquitin-like (UbL) domain of the p59 OASL protein is
required for MBD1 interaction. (A) Pull-down assay. P59.F-V5 OASL
and p59DUbL-V5 OASL were expressed using rabbit reticulocyte
lysate (RRL). The expressed p59 variants were precipitated using anti-
V5 Ig coded protein G–beads. Each reaction was supplemented with
BSA. Lane 1, negative control: MBD1 incubated with anti-V5 Ig
codedproteinGbeads;lane2,MBD1top59DUbL-V5 OASL protein G
beads; lane 3, MBD1 to p59-V5 OASL protein G beads. The samples
were separated by SDS/PAGE (10% gel) and visualized by autoradi-
ography. (B) Control, in vitro translation using [
35
S]methionine. Lane

1, 2 lL of crude MBD1.F; lane 2, 2 lL of crude p59DUbL-V5 OASL;
lane 3, 2 lL of crude p59.F-V5 OASL.
Fig. 6. In vivo interaction between p59 OASL protein and MBD1.
In vivo pull-down of MBD1 transfected into p59.F-V5 OASL and
p59DUbL-V5 OASL stably transfected human fibrosarcoma HT1080
cells. The full length p59 OASL and the deletion mutant were preci-
pitated using anti-V5 tagged protein G beads. Precipitates were sepa-
rated by SDS/PAGE (10% gel) and analysed by Western blotting
using antibody to MBD1 (1 : 1000 dilution). Lane 1, MBD1 pull-
down using p59.F-V5 OASL beads (total precipitate loaded); lane 2,
5 lL of p59.F-V5 OASL HT1080 crude lysate; lane 3, empty; lane 4,
MBD1 pull-down using p59DUbL-V5 OASL beads (total precipitate
loaded); lane 5, 5 lL of p59DUbL-V5 OASL HT1080 crude lysate.
The plasmid pCS–MT–MBD1–5x Myc and the antibody to MBD1
were kind gifts from A. Bird (Institute of Cell and Molecular Biology,
University of Edinburgh, UK).
Ó FEBS 2004 A novel interaction with p59 OASL (Eur. J. Biochem. 271) 633
Poly(I)•Poly(C), 10 lgÆmL
)1
) for 24 h (Fig. 8). Expression
of MBD1 mRNA was monitored in a semiquantitative
PCR assay using a primer set spanning a 500 bp region
in the N-terminus, which is identical in all MBD1 splice
variants. As a control, we used a specific primer set
identifying GADPH. MBD1 is clearly induced by IFN-a,
IFN-c and the synthetic dsRNA [Poly(I)
•Poly(C)]; how-
ever, IFN-a is the strongest inducer. This gene regulation
profile is identical to that observed for p59 OASL (data
not shown). Consistent with this regulation, we identified

a gamma activated sequence (GAS), TTCCctgaa, in the
MBD1 promoter ( />Search/), located 1628 bp upstream of the start codon,
but did not find any IFN-stimulated response elements
(ISRE) in the 2 kb region upstream of the transcriptional
start site.
MBD1v6
: a novel splice variant
The prey cDNA sequences isolated from colonies 32 and 54
were identical and both represented a novel splice variant of
the MBD1 gene, named MBD1v6 (GenBank accession no.:
AJ564845). This alternative splice variant lacks exon 9
(HPRALAPSPPAEFIYYCVDEDEL) and exon 13 (ITE
IFSLGGTRFRDTAVWLP) compared with MBD1v1.
Translation of the MBD1v6 cDNA sequence predicts a
protein of 550 amino acids with a novel C-terminus of 24
amino acids, resulting in a novel stop codon prior to exon 14
Fig. 7. MBD1 does not interact with human ubiquitin (Ub). The specificity of the interaction between MBD1 and the ubiquitin-like domain (UbL) of
p59 OASL was analyzed by co-immunoprecipitation of monomeric Ub with MBD1. (A) Autoradiography of a 15% SDS/PAGE gel. MBD1 was
labeled using [
35
S]methionine in RRL. Lane 1, 2 lL of crude
35
S-methionine labeled MBD1; lane 2, 10 lg of monomeric Ub; lane 3, positive
control, 10 lL of
35
S-methionine labeled MBD1 bound to 50 lL of anti-(MBD1) immunoglobulin coated protein G beads incubated overnight at
4 °C; lane 4, 10 lL of
35
S-methionine labeled MBD1 incubated overnight at 4 °Cwith10lg of monomeric Ub using 50 lL of anti-MBD1
immunoglobulin coated protein G beads; lane 5, empty; lane 6, negative control, 10 lg of monomeric Ub incubated ovenight at 4 °Cwith50lL of

anti-MBD1 immunoglobulin coated protein G beads. (B) 15% SDS/PAGE gel. Western blot using anti-Ub specific Ig (Dako).
Fig. 8. Interferon (IFN) induction of MBD1. A semiquantitative PCR assay performed using 20–35 cycles of PCR comprising 2 min at 95 °C, 1 min
at 95 °C, 1 min at 55 °C and 2 min at 72 °C, followed by 5 min at 72 °C. In each reaction, 0.5 lL of cDNA was used [RT-PCR from 5 lgoftotal
RNA purified from HeLa cells: uninduced samples (ÔNTÕ); IFN-a,500UÆmL
)1
;IFN-c, 100 UÆmL
)1
;Poly(I)•Poly(C), 10 lgÆmL
)1
(pIC)]. PCR
reactions of MBD1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were performed separately and mixed prior to application and 1%
agarose gel electrophoresis.
Fig. 9. Translation of the methyl CpG-binding
protein 1 splice variant 6 (MBD1v6). The methyl
CpG-binding domain (MBD or TAM),
Zn-finger domains (CxxC1-3), nuclear local-
ization signal (NLS) and the transcriptional
repression domain (TRD) are indicated by
black boxes. The novel 24 amino acid C-ter-
minus is indicated by grey letters. The possible
myristyl N-myristylation site in the novel
C-terminus is indicated with bold grey letters.
634 J. B. Andersen et al. (Eur. J. Biochem. 271) Ó FEBS 2004
(Fig. 9). To confirm the existence of MBD1v6 in vivo,we
performed RT-PCR analysis, with primers specific for
MBD1v6, on total RNA from either HeLa or T98G cells
using a primer set spanning exon 13. The PCR product was
analysed by gel electrophoresis, purified and sequenced. The
sequence confirms the novel splice variant (data not shown).
The novel C-terminal sequence was investigated using the

ExPASy protein motif database ( />cgi-bin/scanprosite). A sequence – GNfdND – was identi-
fied as a possible myristyl N-myristylation site, having
the consensus sequence, G-{EDRKHPFYW}-x(2)-[STAG
CN]-{P}.
Discussion
Using the yeast two-hybrid system, we identified a novel
interaction partner for the human OASL protein, namely
MBD1. MBD1 is a transcriptional repressor that selectively
binds methylated 5¢ ends of CpG dinucleotides and silences
gene expression [26–28]. Furthermore, the interaction was
specific for MBD1, as we failed to detect any interaction
between p59 OASL and other members of the MBD family
using the yeast two-hybrid system. The interaction was
demonstrated both in an in vitro GST pull-down assay and
in vivo by co-immunoprecipitation. By testing a number of
deletion mutants of p59 OASL, as well as by using the yeast
two-hybrid system and in vitro and in vivo co-immunopreci-
pitations, we have shown that the C-terminal Ub-like
domain of p59 OASL is required for interaction with
MBD1. However, we did not detect any interaction between
MBD1 and monomeric Ub in vitro. Taken together, our
data demonstrate a specific interaction between p59 OASL
andMBD1,whichismediatedthroughtheUbLdomainof
p59 OASL. Our RT-PCR study demonstrated that MBD1
was induced by IFN-a and -c, and synthetic dsRNA
poly(I)
•poly(C), thus both proteins are present at high levels
during IFN stimulation of cells.
A putative role for p59 OASL as an antiviral protein,
despite the missing OAS activity, was suggested and

supported by preliminary data, where cells transfected
with p59 OASL exhibit an increased resistance to encepha-
lomyocarditis virus (EMCV) infection [29] (R. Hartmann,
unpublished). Recent work, by Zhao et al., has shown
that genetically modified mice, which lack a functional
MBD1 gene, exhibit increased transcription of endog-
enous provirus, an effect that was not seen in MBD2
knockout mice [30]. It is thus possible that MBD1 can act
as an inhibitor of viral transcription via its interaction
with p59 OASL. We are currently conducting experiments
to clarify the role played by both MBD1 and p59 OASL
in the antiviral state induced by IFN.
Acknowledgements
We thank Dr Adrian Bird (University of Edinburgh, Edinburgh,
UK) and Dr Fuyuki Ishikawa (Tokyo Institute of Technology,
Tokyo, Japan) for clones and antibody of the methyl CpG-binding
protein 1; Dr Dominique Rebouillat and Dr Bryan Williams
(Department of Cancer Biology, Cleveland Clinic Foundation,
Cleveland, OH, USA) for providing an OAS panel of stably
transfected HT1080 fibrosarcoma cells; and Morten Mulig Nielsen
andSigneEskildsenNielsenfortheFhitandp42OASbait
constructs, respectively. We thank Dr Bret A. Hassel for critical
reading of this manuscript. This work was supported by the
Danish Natural Science Research Council and the Danish Cancer
Society.
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