FLAG and/or HA tags. 3x ERE-TATA luc vector and TATA-luc vector were made with pgl

Transcription

1 Supplementary information Methods Plasmid construction Full-length her and hma were inserted into the pcdna vector (Invitrogen) with FLAG and/or HA tags. x ERE-AA luc vector and AA-luc vector were made with pgl.7 (Promega) and AA pgl.80 vector (Promega), respectively, for luciferase assay (Hill et al., 00). GS-ER (LBD) was prepared as previously described (Ohtake et al., 00; Yanagisawa et al., 00). Human MA, cdnas for in vitro translation/gs-pull down assay were purchased from Open Biosystems. Antibodies he antibodies used in this study are: For western blotting; ER (sc-5), BP (sc-0), -actin (sc-66), BAF70 (sc-0757), BAF7 (sc-055), Mi (sc-78), and SinB (sc-768); (Santa Cruz Biotechnology), MA (BL80), MA (BL808), MA (BL88); (Bethyl laboratory), Brg- (07-78); (Upstate), HDAC (PA-860) and HDAC (PA-68); (ABR). For Immunoprecipitation and ChIP assay; Msx Hu Estrogen receptor (MAB6) (CHEMICON), Mi (sc-78) (Santa Cruz Biotechnology), Brg- (07-78) (Upstate), MA (BL80) (Bethyl laboratory), HDAC (PA-860) (ABR), anti-acetyl-histone H (06-599) (Upstate) and Mouse IgGa (ab8-500) (abcam).

2 For Immunofluorescence; Msx Hu Estrogen receptor (MAB6) (CHEMICON), Mi (sc-78) (Santa Cruz Biotechnology), and Brg- (07-78) (Upstate) Cell culture Approximately x0 6 cells of HeLa S (both wild type and HeLa-ER ), MCF7 or MDA-MB cells were spread on a 0 cm-dish and cultured as proliferating mono-layers in DMEM or RPMI supplemented with 0% FBS, %Antibiotic-antimycotic (Invitrogen). HeLa-ER stable cells were cultured in the medium containing 00 g/ml G8 (SIGMA). For large-scale culture, HeLa -ER cells were transferred to a suspension culture as previously described (Yanagisawa et al., 00). Luciferase assay he luciferase assay was performed using the Dual-luciferase reporter assay system (Promega). 00 ng of x ERE-AA luc vector and AA pgl.80 vector (Promega), used as an internal control for transfection efficiency, were included in all transfections. Immunoprecipitation (IP) Nuclear extracts were prepared with the CelLytic NuCLEAR Extraction Kit (Sigma) from asynchronous or synchronized cells according to the manufacturer s instructions. E was treated for.5 h. he nuclear extracts of MCF7 cells were used for IP using anti-er antibody (CHEMICON) with protein A Sepharose (Ohtake et al, 007).

3 ChIP analysis ChIP analysis was performed using the ChIP assay kit (Upstate) (akezawa et al, 007; Ohtake et al, 007), according to the manufacturer s instructions. Asynchronous or synchronized HeLa-ER cells were treated with E from 0 to 0 min. Soluble chromatins, prepared from 0 6 cells, were immunoprecipitated with antibodies against the indicated proteins. For PCR, we used primer pairs against ER -target gene promoter regions as follows: ebag9: (5 -AGCGCCCCGCCG- and 5 -GGAGGCGCGGC- ) for ERE region, (5 -AGACCGGCCCAGCAGA- and 5 -GAGGCCGGCCG- ) for distal region c-fos: (5 -GAAGAGGGAGAAGGG- and 5 -GAAGCGGCACGG- ) for ERE region PCR products were visualized on % agarose/ris-acetate-eda (AE) gels. sirna Knockdown was induced using sirna duplexes targeting MA//. All transfections were performed using lipofectamine 000 (Invitrogen) 6 h before synchronization with a sirna concentration of 50 nm. sirna pools for sima (M-007-0), sima (M-0088), sima (M ) and nonspecific control (D ) were purchased from Dharmacon Inc. qr-pcr For the quantitative real-time reverse transcription-pcr (qr-pcr), MCF7 cells were

4 transfected with sirna 6 h before synchronization. E and/ or SA were treated for h. otal RNA was extracted from the MCF7 cells using an ISOGEN Kit (Nippon Gene). One microgram of total RNA from each sample was reverse transcribed into first-strand cdna with random hexamers through the use of Superscript III reverse transcriptase (Invitrogen). Primer sets for all genes were purchased from akara Bio Inc. Real-time PCR was performed as previously described (Nakamura et al., 007). For PCR, the following primers were used: ebag9: 5'-CCGGACCAGCCCAGCAA -' and 5'-AGGCCAGGGGGAACA -', ps: 5'-GGCAAAAAGGGCGCGC -' and 5'-CGGGACAACACCGGCG-', gapdh: 5'-GCACCGCAAGGCGAGAAC -' and 5'-GGGAAGACGCCAGGGA -' Cell-proliferation assays Cell-proliferation assays were performed by using Cell-Counting Kit (CCK)-8 (Dojindo, Kumamoto, Japan). he cells were harvested and plated in 96-well plates at 0 cells/well and maintained at 7 C in a humidified incubator. At the indicated time points, 0 μl of the CCK-8 solution was added into the triplicate wells and incubated for hours and the absorbance at 50 nm was measured to calculate the numbers of vital cells in each well. GS-pull down assay GS-fusion proteins were prepared as previously described (Yanagisawa et al., 00). he in vitro-translated proteins were then incubated with beads in NE-N buffer (0 mm ris-hcl, ph 7.5, 00 mm NaCl, mm EDA, and 0.5% NP0) with mm PMSF. Bound proteins were separated by 8% SDS-PAGE, lightly stained with Coomassie brilliant blue to verify

5 equal amounts of fusion protein, and then visualized by autoradiography (Endoh et al., 999). References Endoh, H., Maruyama, K., Masuhiro, Y., Kobayashi, Y., Goto, M., ai, H., Yanagisawa, J., Metzger, D., Hashimoto, S. and Kato, S. (999) Purification and identification of p68 RNA helicase acting as a transcriptional coactivator specific for the activation function of human estrogen receptor alpha. Mol Cell Biol, 9, Nakamura, Imai Y, Matsumoto, Sato S, akeuchi K, Igarashi K, Harada Y, Azuma Y, Krust A, Yamamoto Y, Nishina H, akeda S, akayanagi H, Metzger D, Kanno J, akaoka K, Martin J, Chambon P, and Kato S. (007) Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts. Cell 0, 8-. Ohtake, F., akeyama, K., Matsumoto,., Kitagawa, H., Yamamoto, Y., Nohara, K., ohyama, C., Krust, A., Mimura, J., Chambon, P., Yanagisawa, J., Fujii-Kuriyama, Y. and Kato, S. (00) Modulation of oestrogen receptor signalling by association with the activated dioxin receptor. Nature,, Yanagisawa, J., Kitagawa, H., Yanagida, M., Wada, O., Ogawa, S., Nakagomi, M., Oishi, H., Yamamoto, Y., Nagasawa, H., McMahon, S.B., Cole, M.D., ora, L., akahashi, N. and Kato, S. (00) Nuclear receptor function requires a FC-type histone acetyl transferase complex. Mol Cell, 9,

6 Legend to supplementary figures Figure S Confirmation of cell cycle synchronization in HeLa-ER, MCF7 and MDA-MB cells. DNA content analysis by Flow Cytometry in each cell cycle stage is shown. Figure S (A) he E-induced transactivation functions of ER in asynchronous cells or synchronized cells at G/S or G/M phases. Luciferase assays were performed in HeLa-ER cells. (B) he protein abundance and nuclear localization of ER across the cell cycle in HeLa-ER cells. Nuclear extracts were prepared from asynchronous or synchronized HeLa-ER cells treated with or without E, and subjected to SDS-PAGE for Western blotting (left panel). Localization of ER in HeLa-ER treated with or without E (for 0 minutes) was assessed by immunocytochemistry (right panel). he cells were stained with either PRO (blue) for DNA or red for anti-er. (C) Cell cycle-dependent recruitment of chromatin-remodeling complex components on the ebag9 promoter in HeLa-ER. ChIP assays were performed in the same way as described in Figure B. (D) he association of Mi with ER partially mediated by MA//, respectively, at G/M. sirna for MA// was transfected into MCF7 cells 6h before synchronization at G/M. ER interactants were co-immunoprecipitated (IP) with anti-er antibody from nuclear extracts of the MCF cells treated with E for.5 h, and subjected to Western Blotting. (E) Direct interaction between liganded ER and MA//. GS-fused proteins were 6

7 prepared as previously described (Yanagisawa et al., 00) (left panel). In vitro-translated MA// or SRC- protein was incubated with GS ER (LBD) in the presence of E (0 6 M). Bound proteins were subjected to SDS-PAGE followed by autoradiography (right panel) 7

8 Figure S Asy G/S G/M E - E + E - E + E - E + HeLa- G 69.% S.8% G/M 7.% G 69.% S.% G/M 7.% G 7.7% S 55.% G/M 7.% G 7.8% S 5.6% G/M 8.6% G.% S 8.% G/M 88.6% G.% S 7.5% G/M 89.% MCF7 G 88.7% S.% G/M 8.% G 88.0% S.% G/M 8.6% G 9.6% S 9.% G/M.% G 0.% S 8.% G/M.6% G 5.5% S 7.% G/M 87.% G 5.6% S 7.% G/M 87.0% MDA-MB G 5.7% S.% G/M 6.% G 58.5% S 7.% G/M.% G 8.9 % S 8.8 % G/M. % G 5.% S.% G/M.% G 5.5% S 5.% G/M 69.% G 6.5% S 5.% G/M 68.%

9 Figure S A ERE-AA LucP B HeLa- G/S G/M DNA Merged E- BP G/S E- G/M E+ G/S BP E+ 0 + Asy 5 + G/S 6 HeLa- + G/M HeLa- C D E si C o si nt M si A M si A M si A M A / / E+ E- G/M Asy G/S G/M Asy G/S G/M (KDa) 0 MA 0 Input MA 00 MA 80 MA ebag9 IP: AcH 5 6 HeLa- SRC- 0 Mi 5 MCF7 E- E+ E- E+ MA 50 In pu 60 Mi HDAC MA {ERE} Mi t Input Brg- GS (L -E BD R ) α GS E GS (L -E BD R ) α IP Asy WB : GS Fold activity CBB staining 5 GS pull-down