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.. Immunoprecipitation p59.F-V5 O
Trang 1Interaction 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¢-2¢)5¢-linked oligoadenylates, in turn,
activate a latent endoribonuclease that degrades viral and
cellular RNAs In contrast, while the p59 OASLprotein 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 OASLprotein 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-bindinteract-ing protein 1 (MBD1), which
functions as a transcriptional repressor, was identified as a strong p59 OASLinteractor 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 OASLwith 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 OASLis unable to synthesize 2–5A [14,15] However, it is still strongly induced
by IFN The inability of p59 OASLto 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: JJ@mb.au.dk
Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase;
*GST, glutathione S-transferase; MBD1, methyl CpG-binding
pro-tein 1; MBD1v6, methyl CpG-binding propro-tein 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)
Trang 2that are crucial for enzymatic activity to either glutamic acid
or threonine [16]
The N-terminus of the p59 OASLprotein contains an
OAS core domain that is highly homologous to the rest of
the OAS family In contrast, the C-terminus of the p59
OASLprotein 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
OASLUbLis, as yet, unknown
An orthologue of p59 OASLexists 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 OASLprotein Our study
revealed that the methyl CpG-binding protein 1 (MBD1)
binds to the C-terminal UbLdomain of p59 OASL, both
in vitroand 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 OASLare 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 mM 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 ofp59 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 http://www.ebi.ac.uk, 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 OASLor p59DUbL-V5 OASL (a deletion mutant lacking the
(pcDNA3.1 V5/HisA; Invitrogen), as a control Stable transfectants were selected in 200 lgÆmL)1G418 (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
95C, 1 min at 95 C, 1 min at 55 C, and 2 min at
72C, 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
Trang 3Generation 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 Ni2+-nitrilotriacetic acid agarose beads
(Qiagen) The purified His-tagged protein was analysed on
10% SDS/PAGE and the band corresponding to p59
OASLwas cut out Rabbits were immunized twice and
antiserum was collected To increase the specificity of the
antibody, precipitation was performed in saturated
nium sulphate, whereby buffer comprising saturated
ammo-nium sulphate (76 g ammoammo-nium sulphate in 100 mLof
ddH2O) was slowly added to the rabbit serum to a final
concentration of 47% (v/v) After stirring very slowly for
2 h at 4C, the precipitate was collected by centrifugation
at 20 000 g and resuspended in NaCl/Pi To remove excess
ammonium sulphate, the sample was dialyzed in NaCl/Pi
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 C in 200 lLof NaCl/Pi containing 0.1%
NaN3
GlutathioneS-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 colistrain BL21(DE3) pRP4, pRI in 2· YTG medium
containing 2% glucose (100 lgÆmL)1ampicillin, 20 lgÆmL)1
kanamycin, 10 lgÆmL)1tetracycline) A 500 mLvolume of
cells was cultured at 37C to reach an attenuance (D) of 0.5
at 600 nm To induce protein expression, 1.0 mMisopropyl
thio-b-D-galactoside (IPTG) (final concentration) was added
and culture continued for 2 h at 30C, then chilled for
15 min on ice The cells were harvested by centrifugation
(8200 g, 4C, 15 min) and resuspended in 5 mLof NETN
buffer [20 mM Tris/HCl, pH 8.0; 100 mM NaCl; 1 mM
EDTA; 0.5% Nonidet P-40 (NP-40); 1 mMdithiothreitol]
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-aroseTM 4B fast flow; Amersham Pharmacia) For each
reaction, 150 lLof 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 lLof NETN milk buffer and 2.6 mLof
supernatant, and rotated for 1 h at 4C After incubation,
the beads were pelleted (1200 g, 4C, 10 min), and washed
five times in 1 mLof 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 OASLin a total volume of
250 lLNETN buffer and 10% v/v glycerol for 18 h at 4C The reaction mixture was washed four times in 500 lL
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 LipofectAMINETM
Plus reagents, according to the manufacturer’s instructions (LifeTechnologies, Inc.) The cells were grown in a T150 culture tank and transfected with 45 lg of pCS-MT-MBD1 5xMyc tagged plasmid At 24 h post-transfection, the cells were lysed in 0.5 mLof RIPA lysis buffer containing a protease inhibitor cocktail [50 mM Tris/HCl (pH 7.4), 150 mMNaCl, 1 mMEDTA, 0.5% NP-40, 15% glycerol, 1 mM NaF) The cells were mechanically lysed, using 20 strokes, with a Dounce-Homogenizer The lysate was then cleared with protein G–beads (protein G–SepharoseTM 4 fast flow; Amersham Pharmacia), for
3 h at 4C, to minimize nonspecific binding The precleared lysate was incubated with 100 lLof washed sepharose–protein G anti-V5 immunoglobulin (1 : 500; Invitrogen) complex, in a total volume of 0.5 mLof NaCl/Pi, for 1 h at room temperature After incubation, the beads were washed five times in 0.5 mLof RIPA wash buffer [50 mM Tris/HCl (pH 7.4), 100 mM NaCl, 0.1% NP-40, 1 mM 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 OASLand MBD1 The reaction mixture was prepared according to the supplier’s manual and incubated
at 30C for 1.5 h
Immunoprecipitation p59.F-V5 OASLand p59DUbL-V5 OASL containing a V5 epitope tag were expressed using unlabeled methionine in the in vitro translation reactions, and 15 lLof each reaction was incubated with 50 lLof precoupled V5 protein G beads (Protein G–SepharoseTM 4 fast flow; Amersham Pharmacia; anti-V5 immunoglobulin, 1 : 500 dilution, Invitrogen) in a total volume of 0.5 mLof ice-cold IP buffer [20 mMTris/HCl (pH 7.9), 10% glycerol, 0.1MKCl,
5 mMdithiothreitol, 0.1% NP-40]
To minimize nonspecific binding to the beads, 10 lLof 10% BSA was added to each reaction After 1 h, each reaction was supplemented with [35S]methionine in vitro translated full-length MBD1 (15 lL), and the incubation was continued for 3 h at 4C The beads were washed five times in 0.5 mLof IP buffer containing 100 mMNaCl, and the immunoprecipitated proteins were analyzed by SDS/ PAGE (10% gel) and autoradiography
Trang 4Identification 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 OASLis 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· 107
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
LacZreporter) 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 NCBIBLASTserver 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 OASLwas 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 OASLto interact with other mem-bers of the methyl CpG-binding protein family (MBD2, MBD3 and MBD4) by introducing the prey constructs
MBD4–GAL4 into an L40 yeast strain expressing the bait LexA–p59.4 OASL Only the LexA–p59.4 OASLstrain
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 LexA–p59.4 OASLpro-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).
Trang 5transformed together with MBD1v6 (MBD1–GAL4AD)
was able to grow on triple selection, showing that p59
OASLspecifically 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 OASLusing a p59 OASLspecific
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 OASLthat 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 OASLwhere the UbLis located, suggesting that the UbLof p59 OASLis 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 OASLin 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 UbLof p59 OASL, we expressed a full length p59.F-V5 OASLand 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 OASLand p59DUbL -V5 OASL via their C-terminal V5 epitope tag These beads were then used
in pull-down assays, together with [35S]methionine-labeled MBD1 (Fig 5A) As seen in Fig 5, full length MBD1 did not interact with the beads alone or with the p59 OASLdeletion 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 [35S]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
OASLand the bound proteins were analysed by SDS/PAGE (10%
gel) and Western blotting using anti-p59 OASLimmunoglobulin
(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
of p59 OASL using GST–MBD1 beads; lanes 5 and 6 (WASH), GST
beads; lane 7 (control), pull-down of p59 OASLusing 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.
Trang 6Verifying the interactionin vivo in HT1080
fibrosarcoma cells
The p59 OASL–MBD1 interaction was further verified
in vivoby 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 UbLdeletion 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 UbLis 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 OASLwere
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 UbLof p59 OASLand not Ub in general, we
performed a pull-down assay between monomeric Ub and
MBD1 (Fig 7).35S-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 OASLis 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, 500 UÆmL)1; IFN-c, 100 UÆmL)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
coded protein G beads; lane 2, MBD1 to p59DUbL-V5 OASL protein G
beads; lane 3, MBD1 to p59-V5 OASLprotein 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 lLof crude MBD1.F; lane 2, 2 lLof crude p59DUbL-V5 OASL;
lane 3, 2 lLof crude p59.F-V5 OASL.
Fig 6 In vivo interaction between p59 OASLprotein and MBD1.
In vivo pull-down of MBD1 transfected into p59.F-V5 OASLand p59DUbL-V5 OASL stably transfected human fibrosarcoma HT1080 cells The full length p59 OASLand 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 OASLbeads (total precipitate loaded); lane 2,
5 lLof p59.F-V5 OASLHT1080 crude lysate; lane 3, empty; lane 4, MBD1 pull-down using p59DUbL-V5 OASL beads (total precipitate loaded); lane 5, 5 lLof 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).
Trang 7Poly(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 (http://www.transfac.gbf.de/cgi-bin/mat
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 OASLwas 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 lLof crude 35 S-methionine labeled MBD1; lane 2, 10 lg of monomeric Ub; lane 3, positive control, 10 lLof35S-methionine labeled MBD1 bound to 50 lLof anti-(MBD1) immunoglobulin coated protein G beads incubated overnight at
4 C; lane 4, 10 lLof 35 S-methionine labeled MBD1 incubated overnight at 4 C with 10 lg of monomeric Ub using 50 lLof anti-MBD1 immunoglobulin coated protein G beads; lane 5, empty; lane 6, negative control, 10 lg of monomeric Ub incubated ovenight at 4 C with 50 lLof 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 lLof cDNA was used [RT-PCR from 5 lg of total RNA purified from HeLa cells: uninduced samples (NT); IFN-a, 500 UÆmL)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.
Trang 8(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 (http://www.expasy.org/
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 OASLprotein, 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 OASLand 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 vivoby 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 OASLis 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
and MBD1, which is mediated through the UbL domain of
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 OASLas an antiviral protein,
despite the missing OAS activity, was suggested and
supported by preliminary data, where cells transfected
with p59 OASLexhibit 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
and Signe Eskildsen Nielsen for the Fhit and p42 OAS bait
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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