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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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