Weber Suppl-revision-final E4894 - Genes &...
Transcript of Weber Suppl-revision-final E4894 - Genes &...
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Supplementary Information
PRMT1-mediated methylation of PIAS1 regulates inhibition of STAT1 signalling.
Susanne Weber, Florian Maaß, Michael Schuemann, Eberhard Krause, Guntram
Suske and Uta-Maria Bauer
Supplementary Information on Materials and Methods:
The sense strands of the in the manuscript used siRNA sequences are indicated below.
siPRMT1_1 5’-CGUGUAUGGCUUCGACAUG-3’,
siPRMT1_2 5’-UCAAAGAUGUGGCCAUUAA-3’,
siPRMT4 5’-GGACAUGUCUGCUUAUUGCUU-3’,
siPRMT6 5’-GCAAGACACGGACGUUUCA-3’,
siPIAS1 (against the PIAS1 ORF) 5’-GAACUAAA GCAAAUGGUUAUU-3’,
siPIAS1 UTR1 (against the PIAS1 3’UTR) 5’-AGAAAUGUACAGAGAACAA-3’,
siPIAS1 UTR2 (against the PIAS1 3’UTR) 5’-CGAAUGAACUUGGCAGAAA-3’,
siScr 5’-UAGCGACUAAACACAUCAA-3’.
For RT-QPCR, the following primers were used:
hCXCL9 forward 5’-TTGGGCATCATCTTGCTGGTTCT-3’
reverse 5’-TGGCTGACCTGTTTCTCCCACTT-3’,
hCXCL10 forward 5’-GAAGCAGTTAGCAAGGAAATGT-3’
reverse 5’-GACATATACTCCATGTAGGGAAGTGA-3’,
hGAPDH forward 5’-AGCCACATCGCTCAGACAC-3’
reverse 5’-GCCCAATACGACCAAATCC-3’,
hGBP1 forward 5’-TTCCAAAACTAAAACTCTTTCAGGA-3’
reverse 5’-GGTCAGCACCAGGCTCTCTA-3’,
hIRF1 forward 5’-GAGCTGGGCCATTCACAC-3’
reverse 5’-TTGGCCTTCCACGTCTTG-3’,
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hPIAS1 forward 5’-GGACCTGTCCTTCCCTATCTC-3’
reverse 5’-TGGAGATGCTTGATGTGGAA-3’,
hPRMT1 forward 5’-GAGAATTTTGTAGCCACCTTGG-3’
reverse 5’-CCTGGCCACAGGACACTT-3’,
hTAP1 forward 5’-CGGAAACCGTGTGTACTTATCC-3’
reverse 5’-TCAGGGCTTTCGTACAGGAG-3’.
Immunoprecipitated and eluted DNA from ChIP analysis was amplified by QPCR using the
following primers:
hCXCL9 promoter forward 5’-CTGGACTTTAATATTTCCCATCTGG-3’
reverse 5’-CTCTCCTAAACTCTGATTGGCTA-3’,
hCXCL10 promoter forward 5’-TACAATAACCCTAGGATAGCTATG-3
reverse 5’-CAGGGTCAAAGATCTGGAACTG-3’,
hGBP1 promoter forward 5’-TAGTTACAGTGTTATGATTTTAGACA-3’
reverse 5’-AGCTGCCTATTCTTTGAGAGG-3’,
hIRF1 promoter forward 5’-CCAGGGCTGGGGAATCC-3’
reverse 5’-TCGGGCGCACGTCTTGC-3’.
Suppl.Figure S1
Knockdown of PRMT1 or PIAS1 results in enhanced induction of PIAS1 target genes in
A375 melanoma cells.
A375 cells were transfected with control siRNA (siScr), siRNA against PRMT1
(siPRMT1_1) and PIAS1 (siPIAS1), respectively. 72 hours post transfection cells were
induced with 5 ng/ml IFNγ. Cells were harvested at the indicated time points and
subsequently total RNA was prepared. RT-QPCR was performed for detection of transcript
levels of STAT1 target genes, i.e. CXCL9, CXCL10, GBP1, IRF1 and TAP1. Results were
normalised using GAPDH mRNA level as reference. Transcript levels in uninduced control
cells were set to 1.
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Suppl.Figure S2
Double depletion of PRMT1 and PIAS1 reveals that both proteins cooperate in STAT1-
target gene regulation.
A, B: siRNA-mediated single or double knockdown of PRMT1 (siPRMT1_1 was used) and
PIAS1, respectively were carried out in HeLa cells. 48 hours post transfection cells were
induced with 5 ng/ml IFNγ for the indicated time points. Subsequently cells were harvested
for extraction of total RNA or protein. A, for detection of knockdown efficiency, protein
levels of PRMT1, PIAS1 and β tubulin were analysed by Western Blot in uninduced protein
extract. B, total RNA was analysed by RT-QPCR for transcript levels of various STAT1
target genes (CXCL9, CXCL10, GBP1, IRF1 and TAP1). Results were normalised to
GAPDH and transcript levels in uninduced control cells were set to 1.
Suppl.Figure S3
Kinetic of the transcriptional induction of STAT target genes in HeLa cells upon IFN
stimulation.
Hela cells were induced with IFNγ for 0, 1, 2, 4, 6 or 8 hours, respectively and total RNA was
prepared. RT-QPCR was performed for detection of transcript levels of the STAT1 target
genes CXCL9, CXCL10 and IRF1. Results were normalised using GAPDH mRNA level.
Transcript levels in uninduced control cells were set to 1.
Suppl.Figure S4
PRMT1 promoter recruitment requires IFNγ-induced activation and DNA-binding of
STAT1 to its target genes.
A: 2fTGH cells (parental cell line) and U3A cells (STAT1-deficient cell line) (McKendry et
al. 1991; Muller et al. 1993) were either left untreated or induced for 3, 6 and 9 hours with 5
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ng/ml IFNγ. Subsquently, cells were harvested and analysed by SDS-PAGE and Western
Blot with the antibodies against STAT1, P-STAT1, PRMT1 and PIAS1 for the expression of
the endogenous proteins, β tubulin served as loading control.
B: 2fTGH and U3A cells were treated as in A. Subsequently, RNA was prepared and
analysed by RT-QPCR for transcript levels of STAT1 target genes, as indicated. Results were
normalised to GAPDH and transcript levels in uninduced control cells were set to 1.
C: 2fTGH and U3A cells were treated as in A. Subsequently, cells were harvested and then
subjected to ChIP analysis. Recruitment of STAT1 and PRMT1 to CXCL9, CXCL10, GBP1
and IRF1 gene promoters was determined by QPCR. Results were displayed as percentage
input.
Suppl.Figure S5
Sumoylation status of STAT1 is not changed upon PRMT1 knockdown.
Hela cells were transfected with control siRNA (siScr) or siPRMT1 (siP1 = siPRMT1_1). 24
hours post transfection cells were transfected with Flag-EGFP-SUMO1 and HA-STAT1
(Ungureanu et al. 2005) as indicated. 24 hours post transfection of the plasmids, SDS-lysates
were prepared as follows: Cells were scraped in hot 1x SDS PAGE sample buffer (62.5 mM
Tris pH 6.8, 0.5% SDS, 10% Glycerol, 0.025% Bromphenolblue, 5% β-mercaptoethanol, 500
mM NEM), samples were sonified and centrifuged. Subsequently, samples were subjected to
SDS-PAGE and Western Blot analysis with antibodies recognizing HA-tag, PRMT1, Flag-
tag and β tubulin.
Suppl.Figure S6
The sumyolation activity of PIAS1 is not required for its repressive function of STAT1
target gene expression.
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Hela cells overexpressing either Flag-tagged EGFP (control), Flag-tagged PIAS1 wild type
(WT) or Flag-tagged PIAS1 W372A mutant (generated by mutagenesis PCR) were induced
with 5 ng/ml IFNγ for the indicated time points. Subsequently cells were harvested for the
preparation of total RNA or protein extracts. A, for detection of exogenous PIAS1
expression, protein levels were analysed by Western Blot analysis with anti-Flag and anti-β
tubulin antibody in uninduced protein extract. B, total RNA was analysed by RT-QPCR for
transcript levels of various STAT1 target genes (CXCL9, CXCL10, GBP1, IRF1 and TAP1).
Results were normalised to GAPDH and transcript levels in uninduced control cells were set
to 1.
Suppl.Figure S7
Knockdown of PRMT4 or PRMT6 does not influence PRMT1/PIAS1-dependent
STAT1 target gene expression.
A-D: siRNA-mediated knockdown of PRMT4 (A, B) or PRMT6 (C, D) was performed in
HeLa cells. Subsequently cells were induced with 5 ng/ml IFNγ for the indicated time points
and harvested for the preparation of total RNA or protein extracts. A, C: Knockdown of
PRMT4 (A) and PRMT6 (C) was monitored by Western blot analysis of uninduced cell
extracts. B, D: RT-QPCR of total RNA from PRMT4 (B) or PRMT6 (D) knockdown was
performed for transcript levels of various STAT1 target genes. Results were normalised to
GAPDH and transcript levels in uninduced control cells were set to 1.
Suppl.Figure S8
Validation of the procedure for metabolical labelling.
Translational block with cycloheximide and chloramphenicol (CHX/CAM) was investigated
by incubation of HeLa cells with 35S-methionine. Subsequent to incubation with CHX/CAM
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for 30 minutes, 35S-methionine was added to the medium. After an incubation of 3 hours cells
were harvested, protein extracts resolved on SDS-PAGE, blotted and visualised by
fluorography.
Suppl.Figure S9
In vitro sumoylation assay with PIAS1 WT and PIAS1 R303 mutants.
In vitro SUMO modification was performed in the presence of recombinant E1 enzyme
(Aos1/Uba2), E2 enzyme (Ubc9) and 100 ng of purified HA/Flag-tagged Sp3 protein, which
is a well-established target of sumoylation by PIAS1 (Sapetschnig et al. 2002). The SUMO
E3 ligase PIAS1 proteins were expressed by IVT (Promega) according to the supplier’s
instructions from a pBluescript plasmid, which contained the full-length cDNAs under the
control of a T3 promoter. PIAS1 - either WT, R303K or R303F mutant - or as control
(pBluescript empty vector programmed IVT) was included into the reactions. Furthermore,
recombinant SUMO1 protein was either added (+SUMO1) or not (-SUMO1). The assay
conditions are described by Sapetschnig et al. (2002). Reactions were incubated at 30°C for 3
hours and subsequently analysed by SDS-PAGE and anti-Flag and anti-PIAS1 Western Blot.
The sumoylated Sp3 species are indicated and reveal equal sumoylation capacity of PIAS1
WT and the R303 mutants.
Suppl.Figure S10
Intracellular localisation/distribution of STAT1, PIAS1 WT and R303 mutants in HeLa
cells using immunofluoresence staining.
HEK293 cells were plated on cover slips and transiently transfected with EGFP, EGFP-
PIAS1 WT, EGFP-PIAS1 R3030K and EGFP-PIAS1 R303F. 48 hours after transfection cells
were either left untreated or treated with 5 ng/ml IFNγ for 1 and 8 hours. Subsequently, cells
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were rinsed in PBS and fixed for 10 min. in PBS/2% formaldehyde. Afterwards cells were
permeabilised in PBS/0.2 % TritonX100 and then stained with the anti-STAT1 antibody (sc-
346, Santa Cruz) in the presence of PBS/4 % BSA for 2 hours at room temperature.
Afterwards cells were rinsed three times in PBS and stained with the secondary antibody
anti-rabbit Cy3 (Jackson Immuno Research). For nuclear/DNA staining cells were incubated
with 1 µg/ml DAPI (4’,6-Diamidin-2’-phenylindol-dihydrochlorid) in PBS for 1 min. at room
temperature. After the final washes in PBS cells were mounted (Mowiol containing 25 mg/ml
DAPCO) and analysed by fluorescence microscopy (Axioskop 2, Zeiss) for EGFP,
STAT1/Cy3 and DAPI staining. The EGFP-PIAS1 mutant proteins revealed a similar
exclusive nuclear localisation and similar intranuclear distribution as PIAS1 WT. Further, the
presence of the PIAS1 WT or mutants did not change the IFNγ-induced translocation of
STAT1 into the nucleus (1 hours IFNγ treatment) or its shuttling out of the nucleus in the late
phase of the IFN response (8 hours IFNγ treatment).
Suppl.Figure S11
Characterisation of HeLa cells depleted for endogenous PIAS1, but overexpressing
PIAS1 wild type and R303 mutant.
A: HeLa cells were transfected with control siRNA (siScr) or siRNA against the ORF
(siPIAS1 ORF) and the 3’UTR (siPIAS1 UTR1, siPIAS1 UTR2 and a mixture of both,
siPIAS1 UTR1+2). 24 hours later the siRNA treated cells were transfected with empty vector
(control), Flag-tagged PIAS1 wildtype (WT), R303K mutant or R303F mutant. 48 hours after
this second transfection cells were harvested and analysed by SDS-PAGE and Western Blot
with the anti-PIAS1 antibody for the expression of endogenous and exogenous PIAS1
protein, β tubulin served as loading control.
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B: HeLa cells were treated as in A. After the second transfection the cells were either left
untreated or induced for 4 hours with 5 ng/ml IFNγ. Subsequently, RNA was prepared and
analysed by RT-QPCR for transcript levels of STAT1 target genes, as indicated. Results were
normalised to GAPDH and transcript levels in uninduced control cells were set to 1.
Suppl.Figure S12
ChIP analysis of the recruitment of STAT1 and PIAS1 to the GBP1 or IRF1 gene
promoter in cells depleted for endogenous PIAS1, but overexpressing wild type or
mutant PIAS1.
A, B: HeLa cells were treated as described in Suppl.Fig. S10. Subsequent to the second
transfection cells were stimulated with 5 ng/ml IFNγ for 3 hours or left unstimulated and then
subjected to ChIP analysis. Recruitment of STAT1 (A) to the GBP1 gene promoters and of
Flag-PIAS1 (B) to the GBP1 and IRF1 gene promoters was determined by QPCR. Results
were displayed as percentage input.
Suppl.Figure S13
Analysis of the concentration-dependent influence of IFNγ on the proliferation capacity
of HeLa cells.
For growth curve analysis, HeLa cells were seeded (10.000 cells per well) and after 4 hours
either were not induced or induced with 7.5 ng/ml and 15 ng/ml IFNγ, respectively. Cell
numbers were determined after 2, 3, 4, 5 or 6 days. For each condition and time point
triplicates were counted. Error bars are depicted accordingly.
Suppl.Figure S14
Knockdown of PRMT1 and PIAS1 sustains in HeLa cells for up to 6 days.
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A, B: HeLa cells were transfected with control siRNA (siScr), siRNA against PRMT1
(siPRMT1_1) or against PIAS1 (siPIAS1). 48 hours post transfection cells were trypsinised
and again seeded. After 2, 4, and 6 days cells were harvested and protein extracts were
prepared. Western Blot analysis for endogenous PRMT1 (A) and PIAS1 (B) confirmed the
efficient and robust knockdown.
References of the Supplementary Information:
McKendry, R., John, J., Flavell, D., Muller, M., Kerr, I.M., and Stark, G.R. 1991. High-frequency mutagenesis of human cells and characterization of a mutant unresponsive to both alpha and gamma interferons. Proc Natl Acad Sci U S A 88(24): 11455-11459.
Muller, M., Laxton, C., Briscoe, J., Schindler, C., Improta, T., Darnell, J.E., Jr., Stark, G.R., and Kerr, I.M. 1993. Complementation of a mutant cell line: central role of the 91 kDa polypeptide of ISGF3 in the interferon-alpha and -gamma signal transduction pathways. Embo J 12(11): 4221-4228.
Sapetschnig, A., Rischitor, G., Braun, H., Doll, A., Schergaut, M., Melchior, F., and Suske, G. 2002. Transcription factor Sp3 is silenced through SUMO modification by PIAS1. Embo J 21(19): 5206-5215.
Ungureanu, D., Vanhatupa, S., Gronholm, J., Palvimo, J.J., and Silvennoinen, O. 2005. SUMO-1 conjugation selectively modulates STAT1-mediated gene responses. Blood 106(1): 224-226.
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