HPV E6 oncoprotein targets histone methyltransferases for modulating specific. Chih-Hung Hsu, Kai-Lin Peng, Hua-Ci Jhang, Chia-Hui Lin, Shwu-Yuan Wu,

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1 1 HPV E oncoprotein targets histone methyltransferases for modulating specific gene transcription 3 5 Chih-Hung Hsu, Kai-Lin Peng, Hua-Ci Jhang, Chia-Hui Lin, Shwu-Yuan Wu, Cheng-Ming Chiang, Sheng-Chung Lee, Winston C.Y. Yu and Li-Jung Juan* Supplemental materials include Supplemental Materials and Methods, Supplemental References, Supplemental Figure Legends and Supplemental Figures Supplemental Materials and Methods In vitro chromatin assembly and transcription assays These procedures were performed as described (Thomas and Chiang 5). Briefly, HeLa core histones were mixed with the recombinant human histone chaperone NAP-1 on ice for 3 min, followed by the addition of DNA templates, Drosophila nucleosome assembly factor ACF and ATP. The mixtures were then incubated at 7 for hr. The formed chromatin was further incubated with and SET7, followed by addition of the HeLa nuclear extract, NTPs and the p MLT, the control plasmid which does not contain any binding site Antibodies Commercial Abs used for western are against (DO-1) (sc-1, Santa Cruz), CARM1 (A3-1A, Bethyl), PRMT1 (sc-1339, Santa Cruz), SET7 (-85, Millipore), actin (MAB151, Millipore), -tubulin (MAB137, Millipore), HA 1

2 1 3 5 (MMS-11R, Covance), Flag (M, Sigma), histone H3 (ab1791, Abcam), and p1 (C-19, Santa Cruz). Commercial Abs for IP and ChIP are against Asy-H3R17me (7-1, Millipore), Ace-H (-8, Millipore), Asy-HR3me (7-13, Millipore), (FL-393) (sc-3, Santa Cruz), Flag (M, Sigma), HA (MMS-11R, Covance), Normal Mouse IgG (1-371B, Millipore), and SET7 (-85, Millipore). 7 Supplemental References Thomas MC, Chiang CM (5). E oncoprotein represses -dependent gene activation via inhibition of protein acetylation independently of inducing degradation. Mol Cell 17: Supplemental Figure Legends Figure S1. si-18e efficiently knocks down 18E expression in HeLa. Cells were mock-transfected (lane 1), transfected with scramble RNA (lane ), or two independent si-18e (lanes 3 and ), followed by RT-PCR for measuring 18E mrna level and western using Abs against the indicated proteins Figure S. E represses histone methylation by CARM1 (A), PRMT1 (B), or SET7 (C) in vitro. Indicated proteins ( μg of each HMTs, 1,, or 5 μg of each E and 1 μg of core histones) were mixed with [ 3 H]-SAM (S-adenosyl-L-[methyl- 3 H] methionine) for the in vitro methyltransferase assays. The reaction products were separated by 15% SDS PAGE. The gels were stained with coomassie blue (upper panel) and analyzed by phosphoimaging (lower panel). The molar ratios of histone H3

3 1 3 5 to CARM1 to E are 1:.:.37 (lanes 3, and 9), 1:.:.7 (lanes, 7 and 1), and 1:.: 1.89 (lanes 5, 8 and 11) in (A), of histone H to PRMT1 to E are 1:.1:. (lanes 3, and 9), 1:.1:.8 (lanes, 7 and 1), and 1:.1: 1. (lanes 5, 8 and 11) in (B), of histone H3 to SET7 to E are 1:.8:.37 (lanes 3, ), 1:.8:.7 (lanes, 7), and 1:.8: 1.89 (lanes 5, 8) in (C) Figure S3. E represses CARM1- and PRMT1-stimulated transcriptional activity. The luciferase assays are identical to those in Figure 3A but the activities are displayed in different order. The western to indicate the protein levels of all factors involved is shown below the bar chart Figure S. E represses CARM1- and PRMT1-mediated induction of transcriptional activity independently of degradation. H199 cells were treated with MG13 and transfected with indicated expression plasmids in the presence of the p1 promoter-driven luciferase construct, followed by luciferase assays (upper bar chart) and western (lower panels) Figure S5. 11E, which does not interfere with protein level, inhibits p1 expression. UOS cells were transfected with indicated plasmids for 8 hr and then treated with Adr for another hr, followed by RT-PCR and western Figure S. E represses adriamycin-induced p1 mrna level depending on CARM1, PRMT1 and SET7. The experiments are identical to those in Figures 3D and 7A but the results are displayed differently. 3

4 1 3 Figure S7. Quantification of ChIP results of Figure A by real-time PCR using Roche Light Cycler 8 system with 38-well plates Figure S8. E inhibits Asy-HR3me and Asy-H3R17me around -responsive region of p1 and GADD5 promoters in the presence of Adr and MG13. UOS cells with or without 18E expression in the presence of MG13 and Adr were subjected to ChIP assays and western analysis Figure S9. Quantification of ChIP results of Figure A by real-time PCR using Roche Light Cycler 8 system with 38-well plates Figure S1. 18E does not affect binding to histone-free DNA. (A) E binds to p1 promoter depending on and E binding does not reduce association with histone-free DNA. The biotin-labeled DNA containing a 1 bp fragment of p1 promoter from -389 to -17 bp with a single binding site (illustrated on top) was linked to strepavidin beads and incubated with cell lysates of H199 with or without expressing or. As shown here, is pulled down by beads containing DNA, but not by beads alone, indicating that binds to the DNA fragment (lane 3). While E alone did not associate with the DNA (lane ), it appears in the pulled-down fraction in the presence of (lane ). -tubulin is not precipitated by this DNA fragment, suggesting that E recruitment is specific. Importantly, E binding does not interfere with the DNA binding ability of (lane ). (B) Cellular histone H3 does not associate with the DNA probe used in (A)., but not histone H3, is pulled down by DNA-conjugated beads (lane 3), indicating that

5 1 the DNA probe, which can be recognized and bound by, maintains a nucleosome-free structure in DAPA reaction Figure S11. The enzymatic activity of SET7 is critical for stress-induced stability and its downstream gene expression. UOS cells were transfected with control scramble RNA (sc-rna), sirna against SET7 (si-set7), or plasmid encoding methylase-dead SET7 (mt SET7), in the absence (lanes 1 to ) or presence (lanes 5 to 8) of Adr for 3 hr, followed by western. The protein levels of and p1 were quantified and shown in the bar chart below the western blots Figure S1. 18E associates with CARM1, PRMT1 and SET7 independently of. -null H199 cells transiently expressing HA-tagged HMT, Flag-tagged E of 18E or both were subjected to IP and western. As shown here, HA Ab, but not IgG, pulls down HA-CARM1 and (A), as well as HA-PRMT1 and (B). The interaction of and the endogenous SET7 is also demonstrated using Flag Ab (C, lanes 1 to ) or SET7 Ab (lanes 5 to 8) for IP and Abs against indicated proteins for western. These results demonstrate that in the absence of, HPV E still associates with CARM1, PRMT1, and SET7 in cells Figure S13. SET7 does not stimulate activity in the absence of stress. (A) The -responsive transcription can not be induced by SET7 in in vitro transcription experiment (compare lane 3 to ). (B) The luciferase reporter assays indicate that the ectopically expressed SET7 does not enhance -dependent p1 promoter activity in H199 cells. (C) Over-expressed SET7 does not increase the level of -induced p1 mrna in H199 (compare lanes and 5 to lanes and 3). (D) ChIP assays show that 5

6 1 exogenous SET7 does not increase the level of chromatin-bound nor local H3 acetylation (compare lane to 1) in UOS cells.

7 Relative 18E mrna level Hsu et al. _Supplementary Figure S mock sc si-18e KD1. si-18e KD 1 3 CARM1 PRMT1 SET7 actin

8 Hsu et al. _Supplementary Figure S (A) E CARM1 Core Histones Coomassie 11E 1E 18E GST - Core histones Autoradiography methylated Histone H3 (B) (C) E PRMT1 Core Histones Coomassie Autoradiography E SET7 Core Histones Coomassie Autoradiography 11E 1E 18E GST E 1E BSA Core histones Core histones -methylated Histone H -methylated Histone H3

9 Relative luciferase activity Hsu et al. _Supplementary Figure S HA-CARM1 HA-PRMT1 HA-CARM1 HA-PRMT1 18E actin

10 Relative luciferase activity Hsu et al. _Supplementary Figure S MG HA-CARM1 HA-PRMT1 HA-CARM1 HA-PRMT1 18E actin

11 Relative p1 mrna level Hsu et al. _Supplementary Figure S5 Adr Flag-E actin 1 3

12 Relative p1 mrna level Hsu et al. _Supplementary Figure S 1. Adr si-carm1 si-prmt1 si-set7

13 Hsu et al. _Supplementary Figure S7 15 MG13 18E HA-CARM HA-PRMT HA-CARM1 E/Q HA-PRMT1 E/Q Asy-H3R17me Asy-HR3me Ace-H

14 Hsu et al. _Supplementary Figure S8 p Adr/MG13 18E GADD5 (-binding region) 1..5 Asy-HR3me GADD5 (-unrelated region) 1..5 p1 GADD5 (-binding region) GADD5 (-unrelated region) Asy-H3R17me 1

15 Hsu et al. _Supplementary Figure S9 MG13 18E (mg) (mg) p1 promoter.5 1. GADD5 promoter.5 1. p1 promoter.5 1. GADD5 promoter.5 1 3

16 Hsu et al. _Supplementary Figure S1 (A) (B) bead S bead S B -389 Distal binding site of p1 promoter GAACATGTCCCAACATGTTG -17 DNA *S: Streptavidin B: Biotin MG13 18E (mg) (mg) BSDNA 1 3 Histone H3 b-tubulin BS b-tubulin Input b-tubulin 1 3

17 Signal normalized to β-actin Hsu et al. _Supplementary Figure S11 Adr - - SET7 p1 actin p

18 Hsu et al. _Supplementary Figure S1 (A) (B) (C) Input IP: anti-flag Flag -18E - - IP: HA IP: IgG HA-CARM1 HA-CARM1 IP: HA IP: IgG HA-PRMT1 HA-PRMT1 SET7 Light chain 1 3 Input IP: anti-set7 - - SET7 Input 1 3 HA-CARM1 actin Input 1 3 HA-PRMT1 actin actin

19 Relative p1 mrna level Hsu et al. _Supplementary Figure S13 Relative luciferase activity (A) (B) 3 p5x REMLP (chromatin) p MLP (DNA) SET7 actin (C) (D) 1 3 SET ChIP p1 promoter Ace-H