Supplementary Materials
Plasmids, transfections and luciferase assays
Flag-tagged expression vectors for human HDAC1 to HDAC11, GFP-HDAC3 plasmid,
shRNA against human HDAC1-3 in pBS/U6 plasmid, the pBS/U6 Scrambled and the
reporter plasmid pGL3-waf1-LUC were described before (Friedlander et al., 1996;
Ramos et al., 2007; Zhang et al., 2004).The cloning of the ULBP promoters and ULBP1
deletion reporters and mutant constructs was performed by PCR amplification and
subsequent ligation of the obtained fragments into pGL2-Basic plasmid (Promega) as
previously described (López-Soto et al., 2006). The primers used are shown in
Supplementary Table 1. The identity of the constructs was verified by DNA sequencing
prior to use.
All transfections were done using Lipofectamine 2000 (Invitrogen). Exponential cells
were seeded into 24-well plates at a density of 2 x 105 cells/well the day before the
transfection. Cells were transfected with 200 ng of ULBP reporter constructs and 20 ng
of pRSV β-galactosidase as an internal control plasmid. Four hours after transfections,
cells were treated with TSA (Sigma) or DMSO and 18 hours later cell extracts were
collected and promoter activities were measured using a Dual Luciferase Reporter
Assay kit (Promega). The dose of TSA (100 nM) was chosen to reduce the toxicity due
to the transfection reagent. The luciferase activity was normalized against the βgalactosidase activity detected using a β-galactosidase Enzyme Assay kit (Promega). In
cotransfection experiments, cells were seeded in 12-well plates and 150 ng of ULBPs
reporter constructs were transfected in combination with 300 ng of pcDNA3.1 or
HDAC expression vectors plus 45 ng of pRSV β-galactosidase plasmid. Cells were
harvested 72 hours after transfections and luciferase and β-galactosidase activities were
measured. The same protocol was followed for siRNA-mediated knock-down of
HDAC1-3. For siRNA mediated silencing of Sp1 and Sp3, HeLa cells were
cotransfected with the ULBP1 (−137/+28) reporter construct plus Control siRNA (sc37007), Sp1 siRNA (sc-29487) or Sp3 siRNA (sc-29490) from Santa Cruz
Biotechnology following the manufacturer’s instructions. After 24 hours, 100 nM TSA
or DMSO were added. Cells were harvested 48 hours after transfection and luciferase
assays were performed. To investigate the role of HDAC3 on ULBPs protein
expression, HeLa cells were transfected in 6-well plates with 5 µg of shHDAC3 vector
or shScrambled in combination with 1 µg of pEGFP-C1 vector (Clontech). After 72
hours, ULBPs protein expression on the surface of GFP positive cells was evaluated by
flow cytometry analysis as described in Materials and methods. In some experiments,
HCT-116 cells were transfected with 4 µg of GFP-HDAC3 plasmid or pEGFP-C1
vector and ULBPs surface expression was measured 72 hours post-transfection as
described.
Semiquantitative and real-time PCR
Total RNA was extracted using the NucleoSpin kit (Macherey-Nagel) and 3 µg-sample
was reverse-transcribed using SuperScript III Reverse Transcriptase (Invitrogen) and
oligo(dT). cDNA samples were diluted 5-fold in water and 3 µl were used as template
in PCR reactions. Primers used are shown in Supplementary Table 1. For each gene, the
number of cycles and temperatures were optimized to detect differences in mRNA
amounts: ULBP1 and ULBP2; 95ºC for 2 min, 10 cycles at (94ºC for 20 sec, 70 ºC for 1
min), 20 cycles at (94ºC for 20 sec, 65ºC for 1 min, 72º for 1 min); the same program
was used for amplification of ULBP3 except that 35 cycles were included. PCR
products were resolved on 2% agarose gels. Real time-PCR was performed using SYBR
Green Master Mix (Applied Biosystems).
Bisulfite genomic sequencing
The procedure was performed as described with minor modifications (Clark et al.,
1994). Genomic DNA isolated from HeLa and U937 cell lines were digested using 20 U
of DdeI restriction enzyme (New England Biolabs) and denatured by treatment with
NaOH to a final concentration of 0.3 M. Then, 1.04 ml of 3.6 M sodium bisulfite (pH 5)
and 60 µl of 10 mM hydroquinone (Sigma) were added to the denatured DNA. The
solution was incubated in the dark at 55º for 16 hours with a jolt to 95º for 5 minutes
every three hours. Converted DNA was desalted and purified using the QIAEX II kit
(Qiagen), treated with 0.3 M NaOH, ethanol precipitated and eluted in 100 µl of TE
buffer (10 mM Tris-HCl [pH 7.5], 0.1 mM EDTA). The EMBOSS program CpGplot
was used with default settings to search for CpG islands within the promoter and first
exon of ULBP1 gene (http://www.ebi.ac.uk/emboss/cpgplot/). A fragment of 412-bp
encompassing the ULBP1 CpG island was amplified by PCR using the following
oligonucleotides: 5’-GTATTAGTGTTTATATTGGGGTAATGATAAAT-3’ (forward)
and 5’-AACCAAACAACAAATACAAAAAC-3’ (reverse). The product of PCR was
electrophoresed, gel-purified, cloned in pBluescript vector and four clones for each cell
line were sequenced.
ChIP Assays
Chromatin immunoprecipitation (ChIP) experiments were performed using the ChIP
assay kit from Upstate Biotechnology. In experiments with HDACi, exponential cells
were first treated or untreated with 250 nM TSA for 4 or 18 hours. In HDAC3 and Sp3
knock-down ChIPs, HeLa cells were transfected with shHDAC3 plasmid for 72 hours
or with an siRNA against Sp3 for 48 hours. Afterwards, cells were cross-linked by
treatment with 1% formaldehyde for 20 minutes (in ChIPs for HDACs and transcription
factors) or 7 minutes (in ChIPs for histones) at room temperature. Then, cells were
lysed by incubation with SDS Lysis Buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl
[pH 8.1]) with protease inhibitors at 4ºC and, after centrifugation, the supernatant was
diluted 10-fold in ChIP Dilution Buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM
EDTA, 16.7 mM Tris-HCl [pH 8.1], 167 mM NaCl). The soluble chromatin was
immunoprecipitated 4 hours at 4ºC using 4 µg of the following antibodies: anti-HDAC1
(sc-7872X), -HDAC2 (sc-7899X), -Sp1 (sc-420X), -Sp3 (sc-644X), -AP-2α (sc-184X)
or normal rabbit IgG (sc-2027) all from Santa Cruz Biotechnology; -HDAC3 (ab7030)
provided by abcam; anti-acetyl-histone H3 (06-599) and anti-acetyl-histone H4 (06598) were from Upstate. Antibody-protein complexes were recovered by incubating
with Protein A-Agarose slurry at 4ºC with rotation and histone complex was eluted by
adding 500 µl of elution buffer (1% SDS, 0.1 M NaHCO3). The histone-DNA crosslinks were reversed by adding 5 M NaCl and incubating at 65ºC overnight. DNA was
extracted, washed and diluted in 50 µl of TE buffer and 3µl were used as template in
semiquantitative RT-PCR. Primers for amplification of ULBP promoters are provided
in Supplementary Table 1. The products obtained were electrophoresed on 2% agarose
gels and visualized by ethidium bromide staining. In some experiments, signal
intensities were analyzed by densitometry using the ImageJ software (National
Institutes of Health).
Coimmunoprecipitation assay
For coimmunoprecipitations, HeLa cells were transfected with FLAG-tagged full-length
HDAC3 (1-428) and C-terminus deleted HDAC3 (1-373) expression vectors (Yang et
al., 2002) or empty plasmid in 60 mm plates. After 48 hours, 250 nM TSA or DMSO
were added for 24 hours. Then, cells were washed and harvested with cold PBS,
centrifuged at 1,000 rpm for 10 min at 4ºC and resuspended in cold hypotonic lysis
buffer [10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 2 mM EDTA, 0.25% Triton X-100,
1mM PMSF, complete inhibitor mixture (Roche Applied Science) and phosphatase
inhibitor cocktails I and II (Sigma)]. After 10 min of incubation on ice, 100 mM NaCl
was added to the samples which were maintained on ice for 5 more min. Then, samples
were centrifuged at 13,000 rpm for 15 min at 4ºC. An aliquot of the supernatant was
kept as total extract control and the remaining supernatant was incubated overnight at
4ºC with anti-FLAG-M2 agarose-conjugated (Sigma-Aldrich). Beads were washed 8
times with 1 ml of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl and 0.05% Triton X-100,
and were centrifuged at 1,000 rpm for 1 min in each wash. Immunoprecipitated proteins
were eluted by incubation at 4ºC for 2 h with an excess of FLAG peptide (1 mg/ml;
Sigma-Aldrich). After centrifugation, supernatants were load on 10% SDS-PAGE gels
and transferred to nitrocellulose membranes. Blots were probed with the Sp3 antibody
as described previously.
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