Download emboj2009211-sup

Supplementary methods
Bioinformatics
The open reading frame, conserved domains, and chromosomal location of ZIP were
analyzed using the NCBI databases (www.ncbi.nlm.nih.gov). The theoretical molecular
weight and isoelectric point of ZIP were calculated using www.expacy.ch/tools. The
homologous alignment was analyzed by the ClustalW program (version 1.60) (Thompson
et al, 1994) and phylogenetic analysis was performed using the Jotun Hein method (Hein,
1990).
Antibodies and reagents
Antibodies used were:HDAC1, HDAC2, RbAp46/48, -actin from Sigma; MTA2
from Upstate Biotechnology; Mi-2 and mSin3a from Santa Cruz Biotechnology; and
EGFR from SAB (Signalway Antibody Co., Ltd., TX, USA). Trichostatin A (TSA) was
purchased from Sigma. Control siRNA and siRNAs for ZIP were synthesized by Shanghai
GeneChem
Inc
(Shanghai,
China).
The
sequence
for
control
siRNA
was:
5'-UUCUCCGAACGUGUCACGU-3'. The sequences for ZIP siRNA were: #1,
5'-GGACAACGGCUACUACACA-3'; #2, 5'-ACGGCUACUACACAGUCAA-3'; #3,
5'-CCAUCCAUGCUGUGGUGUU-3'.
Cyclic amplification and selection of target (CASTing) assay
A
library
of
single-stranded
oligonucleotides
containing
the
sequences
1
5’-GACTCGAGACTCCTAGGATGCGCA(N)20CGTCTATGTCAGTGAAGCTTCGAT-3
’ was generated. To produce double-stranded oligonucleotides, 400 picomoles of the library
was incubated in 100 l of polymerase reaction buffer containing 1,200 pmol of reverse
primer (5’-ATCGAAGCTTCACTGACATAGACG-3’), 200 M of each deoxynucleoside
triphosphate, and 10 units of Ex Taq polymerase (TaKaRa) and amplified with following
procedure: 5 min at 95oC, 20 min at 65oC, and 20 min at 72oC. The double-stranded
oligonucleotides were purified using the QIAquick Nucleotide Removal kit (Qiagen) and
incubated with GST-fused ZIP protein bound to glutathione beads in a binding buffer
containing 25 mM HEPES-KOH, pH 7.5, 100 mM KCl, 5 mM DTT, 5% glycerol, 1 mM
MgCl2, 100 M ZnCl2, 0.1% NP-40, 100 g/ml poly (dI-dC) and 1 mg/ml BSA. After a 30
min rotating incubation at room temperature, the beads were washed for eight times with
cold binding buffer without poly (dI-dC) and then boiled for 5 min in sterilized H2O. The
eluted oligonucleotides were used for PCR amplification. The amplified products were
subsequently used for a second round of selection. After nine rounds of amplification, PCR
products were cloned into pGEM-Teasy vector, transformed into DH5 competent host
cells, and sequenced.
Cell culture and reporter assay
The cell lines were obtained from the American Type Culture Collection (Manassas, VA).
MCF-7 and HeLa cells were maintained in DMEM supplemented with 10% FBS.
Transfections were carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA)
according to the manufacturer’s instructions. Luciferase activity was measured using a dual
2
luciferase kit (Promega, Madison, WI) according to the manufacturer’s protocol. Each
experiment was performed in triplicate and repeated at least three times.
Northern blotting
Human adult multiple tissue Northern blots (Clontech) were used and a 566-bp fragment
spanning bases 948 to 1514 of the human ZIP cDNA was labelled by random priming with
[-32P]dCTP (Amersham Biosciences) and used as a probe. Hybridization was performed
in ExpressHyb hybridization solution (Clontech) according to the manufacturer’s
instructions. The filter was exposed for autoradiography at -80°C after stringent washing.
Immunoprecipitation and Western blotting
MCF-7 or HeLa cells were transfected with ZIP expression plasmid. Forty-eight hours after
the transfection, cellular lysates were prepared by incubating the cells in lysis buffer (50
mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% NP40) for 20 min at 4ºC. This was followed by
centrifugation at 14,000 g for 15 min at 4ºC. For immunoprecipitation, 500 g of protein
was incubated with specific antibodies (1-2 g) for 12 h at 4ºC with constant rotation; 50 l
of 50% protein A or G agarose beads was then added and the incubation was continued for
an additional 2 h. Beads were then washed five times using the lysis buffer. Between
washes, the beads were collected by centrifugation at 3,000 g for 30 s at 4ºC. The
precipitated proteins were eluted from the beads by resuspending the beads in 2 x
SDS-PAGE loading buffer and boiling for 5 min. The resultant materials from
immunoprecipitation or cell lysates were resolved using 10% SDS-PAGE gels and
3
transferred onto nitrocellulose membranes. For Western blot analysis, membranes were
incubated with appropriate antibodies for 1 h at room temperature or overnight at 4ºC
followed by incubation with a secondary antibody. Immunoreactive bands were visualized
using Western blotting Luminol reagent (Santa Cruz Biotechnology) according to the
manufacturer’s recommendation.
GST pull-down assays
GST fusion constructs were expressed in BL21 Escherichia coli cells, and crude bacterial
lysates were prepared by sonication in TEDGN (50 mM Tris-HCl, pH 7.4, 1.5 mM EDTA,
1 mM dithiothreitol, 10% (v/v) glycerol, 0.4 M NaCl) in the presence of the protease
inhibitor mixture. The in vitro transcription and translation experiments were done with
rabbit reticulocyte lysate (TNT systems, Promega) and L-[35S]methionine (Amersham
Biosciences) according to the manufacturer’s recommendation. In GST pull-down assays,
about 10 g of the appropriate GST fusion proteins was mixed with 5-8 l of the in vitro
transcribed/translated products and incubated in binding buffer (75 mM NaCl, 50 mM
HEPES, pH 7.9) at room temperature for 30 min in the presence of the protease inhibitor
mixture. The binding reaction was then added to 30 l of glutathione-Sepharose beads and
mixed at 4°C for 2 h. The beads were washed three times with binding buffer, resuspended
in 30 l of 2×SDS-PAGE loading buffer, and resolved on 10% gels. The gels were then
fixed in 50% methanol, 10% acetic acid for 30 min and dried. Protein bands were detected
by autoradiography at -80°C for 4-24 h.
4
RT-PCR and real-time RT-PCR (qPCR)
Total cellular RNAs were isolated with the TRIzol reagent (Invitrogen) and used for first
strand cDNA synthesis with the Reverse Transcription System (Promega, A3500).
Quantitation of all gene transcripts was done by qPCR using Power SYBR Green PCR
Master Mix and an ABI PRISM 7300 sequence detection system (Applied Biosystems,
Foster City, CA) with the expression of -actin as the internal control. The primer pairs
used were as follows: EGFR forward primer: 5'-CAGGAGGTGGCTGGTTATG-3', EGFR
reverse
primer:
5'-GCACAGGGCAGGGTTGTT-3';
-actin
forward
primer:
5'-CGTGGACATCCGCAAAGAC-3',
-actin
5'-CTCGTCATACTCCTGCTTGC-3';
ZIP
forward
primer:
5'-TGAAGCCGTGCCCGTTCTT-3',
ZIP
reverse
primer:
reverse
primer:
5'-CCGTTGTCCACATCGGTGAT-3'.
HDAC assays
Immunoprecipitates from HeLa were incubated with 1 ml of [3H]acetate-labelled HeLa
histones that had been isolated from butyrate-treated HeLa cells by acid extraction for 2 h
at 37°C and deacetylase activity was determined as described (Xue et al, 1998). The
released [3H]acetate was extracted with ethyl acetate and quantified by liquid scintillation
counting.
ChIP and Re-ChIP
ChIP experiments were performed according to the procedure described previously (Shang
5
et al, 2000; Sun et al, 2004; Wu et al, 2005; Wu et al, 2006). The following primer pairs
were used for EGFR gene sequence: 5'-AGACCCAGGCACAAACAT-3' (forward) and
5'-GTCCATGTTACAGCCAGAC-3' (reverse). Re-ChIPs were done essentially the same
as primary IPs. Bead eluates from the first immunoprecipitation were incubated with 10
mM DTT at 370C for 30 min and diluted 1:50 in dilution buffer (1% Triton X-100, 2 mM
EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 8.1) followed by re-immunoprecipitation with
the second antibodies.
Colony formation assay
MCF-7 cells were stably transfected with ZIP expression plasmid. The cells were
maintained in culture media for 14 days supplemented with 1 mg/ml G418 and were then
stained with crystal violet.
Cell flow cytometry
MCF-7 cells stably transfected with ZIP were synchronized by serum starvation for 24 h,
and released with 10% FBS or addition of EGF for an appropriate period of time; cells were
then trypsinized, washed with PBS, and fixed in 70% ethanol at 4°C overnight. After being
washed with PBS, cells were incubated with RNAase A (Sigma) in PBS for 30 min at 37°C
and then stained with 50 mg/ml propidium iodide (PI). Cell cycle data were collected with
FACSCalibur (Becton Dickinson) and analyzed with ModFit LT 3.0 (Verity Software
House Inc., Topsham, ME).
Lentiviral production and infection
6
Recombinant lentiviruses were constructed by subcloning human ZIP into the iDuet101
shuttle vector (Ye et al, 2008). The recombinant construct as well as two assistant vectors,
pUOG and pCMV, were then transiently transfected into HEK 293T cells. Viral
supernatants were collected 48 h later, clarified by filtration, and concentrated by
ultracentrifugation. The concentrated virus was used to infect 5 x 105 cells in a 60-mm dish
with 8 g/ml polybrene. Infected MCF-7 cells were selected with 2 g/ml hydromycin
(Merck). The construction of RNAi lentivirus system using pLL3.7 and other LentiLox
vectors
were
carried
out
according
to
a
protocol
described
online
(http://web.mit.edu/jacks-lab/protocols/pll37.htm). In brief, siRNA sequences targeting ZIP
were designed and cloned into the pLL3.7 shuttle vector. The recombinant construct as well
as three assistant vectors, pREE, VSUG, and RSU/REU, were then transiently transfected
into HEK 293T cells. Viral supernatants were collected 48 h later, clarified by filtration, and
concentrated by ultracentrifugation. The concentrated virus was used to infect 5 x 105 cells
in a 60-mm dish with 8 g/ml polybrene. Infected MCF-7 cells were then subjected to
sorting by EGFP expression.
Supplementary references:
Hein J (1990) Unified approach to alignment and phylogenies. Methods Enzymol 183: 626-645
Shang Y, Hu X, DiRenzo J, Lazar MA, Brown M (2000) Cofactor dynamics and sufficiency in estrogen
receptor-regulated transcription. Cell 103: 843-852.
Sun X, Zhang H, Wang D, Ma D, Shen Y, Shang Y (2004) DLP, a Novel Dim1 Family Protein Implicated in
Pre-mRNA Splicing and Cell Cycle Progression. J Biol Chem 279: 32839-32847
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive
multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix
choice. Nucleic Acids Res 22: 4673-4680
7
Wu H, Chen Y, Liang J, Shi B, Wu G, Zhang Y, Wang D, Li R, Yi X, Zhang H, Sun L, Shang Y (2005)
Hypomethylation-linked activation of PAX2 mediates tamoxifen-stimulated endometrial carcinogenesis.
Nature 438: 981-987
Wu H, Sun L, Zhang Y, Chen Y, Shi B, Li R, Wang Y, Liang J, Fan D, Wu G, Wang D, Li S, Shang Y (2006)
Coordinated regulation of AIB1 transcriptional activity by sumoylation and phosphorylation. J Biol Chem 281:
21848-21856
Xue Y, Wong J, Moreno GT, Young MK, Cote J, Wang W (1998) NURD, a novel complex with both
ATP-dependent chromatin-remodeling and histone deacetylase activities. Mol Cell 2: 851-861
Ye Z, Yu X, Cheng L (2008) Lentiviral gene transduction of mouse and human stem cells. Methods Mol Biol
430: 243-253
8