Supplemental methods

Isolation of total protein, soluble nuclear and chromatin fraction

After washing the cells twice with PBS, total protein extracts were isolated in RIPA buffer

(1× PBS, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxychelate, 0.1% (w/v) SDS) supplemented with 1 mM Pefabloc and 1 ng/µl Aprotinin/Leupeptin. Preparation of soluble and insoluble nuclear extract was performed as described (10). Briefly, cells were washed twice with PBS before resuspending in buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% (v/v) glycerol, 1 mM DTT, 0.1% (v/v) Triton X-100, supplemented with protease inhibitors: 1 mM Pefabloc, 1 ng/µl Aprotinin/Leupeptin and 10 mM β-glycerophosphate) and incubated on ice for 5 minutes. By centrifuging at 1,300×g for 5 minutes at 4 °C nuclei were isolated, washed once with buffer A (depleted of Triton X-

100) and subsequently lysed in buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT plus supplements as in buffer A). Soluble and insoluble (chromatin) fraction were separated via centrifugation at 1,700×g for 4 minutes at 4 °C. Chromatin samples were subsequently resuspended in buffer B.

Apoptosis assay

Apoptosis was analyzed using the Guava Nexin® assay (Guava Technologies, Millipore) according to the manufacturer’s instructions. Briefly, trypsinized cells were collected, centrifuged and the cell pellets were resuspended in 500 µl of medium. After diluting the cells to a concentration of 2× 105 - 1× 106 cells/ml, 100 µl of each diluted cell suspension and 100 µl of Guava Nexin solution were mixed. After incubation in the dark for

20 minutes, the samples were analyzed using the Guava FACScan.

1 Chromatin immunoprecipitation (ChIP)

Chromatin immunoprecipitation was performed as described by Nelson, et al. (11) with the modifications previously described (12, 13). After removing the medium, chromatin was crosslinked using 1.42% formaldehyde in PBS for 15 minutes at RT. Fixation was quenched by adding a final concentration of 156 mM glycine and incubating for 5 minutes.

Crosslinked cells were washed twice with cold PBS and scraped in IP buffer (150 mM

NaCl, 5 mM EDTA, 50 mM Tris (pH 8), 0.5% (v/v) NP-40, 1% (v/v) Triton X-100), supplemented with protease inhibitors: 1 mM Pefabloc, 1 ng/µl Aprotinin/Leupeptin, 10 mM

β-glycerophosphate and 1 mM N-ethylmaleimide. After washing once with IP buffer, the nuclear pellet was resuspended in 300 µl of the same buffer. Subsequently, samples were sonicated for 3× 10 minutes using a Bioruptor (Diagenode SA, Liège, Belgium) with high power setting with alternating pulses and pauses for 10 seconds each. Following centrifugation, the supernatant was pre-cleared with 100 µl Sepharose 4B (GE Healthcare,

Uppsala, Sweden) 50% slurry in IP buffer (plus supplements), rotating for 1 hour at 4 °C.

After a second centrifugation step, pre-cleared chromatin was diluted with IP buffer (plus supplements) and aliquoted in appropriate volumes. ChIP analysis was performed using

50 µl of chromatin diluted to a final volume of 500 µl in IP buffer (plus supplements) plus the indicated amounts of antibody (Supplemental Table 6) and incubated overnight at 4

°C. Chromatin complexes were captured by adding 30 µl of a 50% Protein-A or Protein-G

Sepharose slurry (GE Healthcare) and incubated for additional 2 hours rotating at 4 °C.

After centrifuging at 2,000×g for 2 minutes at 4 °C and washing six times with IP buffer, the immunoprecipitated complexes were reverse-crosslinked by adding 100 µl of 10% (w/v)

Chelex 100 slurry (Bio-Rad) and heating at 95 °C for 10 minutes. 2 µl Proteinase K (20

µg/µl, Invitrogen) were added to each sample, incubated at 55 °C with shaking at

1,000 rpm for 30 minutes and then inactivated by heating to 95 °C for 10 minutes. Finally,

DNA was recovered by centrifuging at 12,000×g for 1 minute at 4 °C.

2 Experimental background was determined by performing a ChIP assay with non-specific

IgG antibody. For normalization of ChIP samples, inputs were prepared as following: to 5

µl of chromatin, 0.5 µl of Pink Precipitant (5 mg/ml, Bioline, Luckenwalde, Germany) was added, precipitated by adding 50 µl of 100% EtOH and incubated overnight at -20 °C.

Input samples were centrifuged at 12,000×g at 4 °C, washed with 70% EtOH and then prepared by the Chelex method as described before.

One µl of each DNA (isolated ChIP-DNA or cDNA) sample was used for subsequent quantitative real-time PCR analysis in a final volume of 25 µl. A PCR reaction was setup as follows: 75 mM Tris-HCl (pH 8.8), 20 mM (NH4)2SO4, 0.01% (v/v) Tween-20, 3 mM

MgCl2, 200 µM dNTPs, 0.5 U/reaction Taq DNA Polymerase (Prime Tech, Minsk, Belarus),

0.25% (v/v) Triton X 100, 1:80,000 SYBR Green I (Roche, Mannheim, Germany), 300 mM

Trehalose and 300 nM primers (listed in supplemental table 2 and 3). Quantitative PCR was performed on a C1000TM Thermal Cycler and CFX96TM Optical Reaction Module

(Bio-Rad, München, Germany) with 40 cycles of a two-step amplification (95 °C for 15 s and 60 °C for 1 min) for each primer pair.

Chromosome conformation capture (3C)

Preparation of 3C template

Based on the protocol from Miele and Dekker (14) chromosome conformation capture analysis was adapted to our system as follows: Covalent crosslinking of interacting chromatin segments was achieved by adding 16 ml of 1.1% formaldehyde in PBS to

145×20 mm cell culture dishes and incubating on a shaker for 15 minutes at RT. In order to quench the crosslinking reaction, 863 µl of 2.5 M glycine were added to the formaldehyde-PBS solution and incubated on the shaker for additional 5 minutes. After washing twice with ice-cold PBS, cells were scraped in ice-cold lysis buffer (10 mM Tris-

HCl (pH 8), 10 mM NaCl, 0.2% NP-40), supplemented with 1 mM Pefabloc,

3 1 ng/µl Aprotinin/Leupeptin, 10 mM β-glycerophosphate and 1 mM N-ethylmaleimide and incubated on ice for 15 minutes. The fixed cells were then dounce homogenized with a pestle L (Sartorius, Göttingen, Germany) using 2× 15 strokes. Cells were pelleted at

2,500×g for 5 minutes, washed once with 1× NEBuffer 3 (New England Biolabs, Frankfurt,

Germany) and subsequently resuspended in 500 µl of 1× NEBuffer 3. Cells were centrifuged for 5 minutes at 2,500×g and after discarding the supernatant, pellets were shock frozen in liquid nitrogen and stored at -80 °C till further processing. Upon thawing, pellets were resuspended in 362 µl of 1× NEBuffer 3. For the digestion of the crosslinked chromatin, first 38 µl 1% SDS and 0.4 µl 10% Triton X-100 were added and incubated at

65 °C for 10 minutes. Afterwards, 44 µl 10% Triton X-100 and 400 U of BtgI restriction enzyme (New England Biolabs) as well as 100 µg/ml BSA were added, mixed and incubated at 37 °C overnight.

The next day, 86 µl 10% SDS were added for enzyme inactivation and incubated at 65 °C for 30 min. 745 µl of 10% Triton X-100, 745 µl of 10× ligation buffer (500 mM Tris-HCl (pH

7.5), 100 mM MgCl2, 100 mM DTT), 80 µl of 10 mg/ml BSA, 5,960 µl of ddH2O and 4,000 cohesive-end units of T4 DNA ligase (New England Biolabs) were added to each sample and incubated for 2 hours at 16 °C. In order to reverse crosslinking, 50 µl of 10 mg/ml

Proteinase K (Invitrogen) in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) were added and incubated at 65 °C overnight. After adding additional 50 µl of 10 mg/ml Proteinase K, samples were incubated at 42 °C for 2 hours and then transferred into 50 ml tubes. To recover DNA, a double phenol extraction followed by a PCIA (phenol : chloroform : isoamylacohol, 25 : 24 : 1) extraction was performed. 2 µl of GlycoBlue (Ambion, Austin,

USA), 1/10 volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of ice-cold 100%

EtOH were added before incubating at -20 °C overnight. After centrifuging at 12,000×g for

20 minutes at 4 °C, each pellet was redissolved in 1 ml TE buffer. A second DNA extraction was performed, by adding consecutively phenol, 2× PCIA and chloroform.

4 Afterwards, DNA was precipitated by adding 2 µl of GlycoBlue, 1/10 volume of 3 M sodium acetate (pH 5.2) and 1.5 volumes of ice-cold 100% EtOH to the upper phase incubating overnight at -20 °C. The next day, samples were pelleted by centrifugation at 4 °C,

18,000×g for 20 minutes and washed five times with 70% EtOH. The air-dried DNA pellets were resuspended in 50 µl of TE buffer. After adding 1 µl of 10 mg/ml RNase A (Qiagen,

Hilden, Germany), 3C template samples were incubated at 37 °C for 15 minutes.

Preparation of control template

In order to compare PCR signal intensities of 3C samples in a quantitative manner, a control template containing all ligation products in equal amounts was used. DNA from bacterial artificial chromosome clones (BACs) covering the GREB1, CXCL12 or TFF1 loci were isolated and then digested, ligated and purified by phenol-chloroform extraction and ethanol precipitation as explained before. Subsequently, control DNA was serial diluted and applied as standard curve in the PCR analysis of 3C samples.

Quantitative PCR with TaqMan probes

One µl of each 3C sample was used for quantitative real-time PCR analysis. A 25 µl PCR reaction was setup as follows: 75 mM Tris-HCl (pH 8.8), 20 mM (NH4)2SO4, 0.01% Tween-

20, 3 mM MgCl2, 200 µM dNTPs, 1 U/reaction Taq DNA Polymerase, 0.25% Triton X-100,

30 nM primers, 300 mM Trehalose and 250 nM TaqMan probe (5’-FAM and

3’-BHQ1-labeled). PCR was performed with 45 cycles of a two-step amplification (95 °C for 15 s and 60 °C for 30 s).

3C values were normalized with values from an internal control site. The normalized levels were graphed relative to the non-treated control sample (set to one) and represented as

“normalized relative interaction”.

5 H2Bub1 antibody generation

Approximately 50 µg of ovalbumin-coupled peptide containing the C-terminal 13 amino acids of human histone H2B (N-CEGTKAVTKYTSSK-C) were conjugated through the ε- amino group of lysine 120 (underlined) to the C-terminal 5 amino acids of ubiquitin (N-

RLLGG-C) through the C-terminus of the final glycine residue (Peptide Specialty

Laboratories GmbH, Heidelberg, Germany) were injected both i.p. and s.c. into C57BL/6 mice using CPG 1668 (TIB MOLBIOL, Berlin, Germany) and incomplete Freund’s adjuvant. After a 6 week interval, a final boost was given i.p. and s.c. 3 days before fusion.

Fusion of the myeloma cell line P3X63Ag8.653 with the mouse immune spleen cells was performed according to standard procedures. Reactivity of hybridoma supernatants directed to BSA-coupled peptide was tested in a solid phase immunoassay. The hybridoma designated H2Bub1 (7B4) was stably subcloned and used for further analysis.

The immunoglobulin isotype was determined using mAbs against the mouse IgG heavy chains and light chains. The H2Bub1 (7B4) mAb has the IgG subclass IgG2a.

Immunohistochemical analysis

Paraffin-embedded sections were de-paraffinized and rehydrated according to standard procedures. Briefly, after incubating in Xylene for 20 min, the sections were rehydrated in a

100%, 90% and 70 % EtOH series before washing with PBS. Proteins were then unmasked by boiling slides in 10 mM citric acid/sodium-phosphate. After washing with

PBS, sections were quenched for endogenous peroxidase activity with 3% hydrogen peroxide in PBS for 45 minutes at room temperature and then blocked using 5% FCS diluted in PBS for 1 hour at room temperature. The anti-H2Bub1 antibody was diluted 1:10 in PBS containing 5% FCS was applied and incubated overnight at 4 °C in a humid chamber. Sections were washed using PBS before adding the biotinylated secondary antibody 1:200 diluted in PBS and incubated for 1 hour at RT. PBS washings were

6 followed by Avidin-Peroxidase (ExtrAvidin®-Peroxidase, Sigma-Aldrich, St. Louis, USA), incubation 1:1,000 diluted in PBS for 45 minutes. Staining signals were detected using

Diaminobenzidine substrate (ImmPACTTM DAB, SK-4105, Vector, Burlingame, USA).

Hematoxyline (Mayer’s hemalaun solution, Merck) was used for counterstaining.

7

Supplemental Reference List

(1) Kininis M, Isaacs GD, Core LJ, Hah N, Kraus WL. Post-Recruitment Regulation of RNA Polymerase II Directs Rapid Signaling Responses at the Promoters of Estrogen Target Genes. Mol Cell Biol 2008.

(2) Fan M, Nakshatri H, Nephew KP. Inhibiting proteasomal proteolysis sustains estrogen receptor-alpha activation. Mol Endocrinol 2004;18:2603-15.

(3) Fritah A, Saucier C, Mester J, Redeuilh G, Sabbah M. p21WAF1/CIP1 selectively controls the transcriptional activity of estrogen receptor alpha. Mol Cell Biol 2005;25:2419-30.

(4) So AY, Chaivorapol C, Bolton EC, Li H, Yamamoto KR. Determinants of cell- and gene-specific transcriptional regulation by the glucocorticoid receptor. PLoS Genet 2007;3:e94.

(5) Wang JC, Derynck MK, Nonaka DF, Khodabakhsh DB, Haqq C, Yamamoto KR. Chromatin immunoprecipitation (ChIP) scanning identifies primary glucocorticoid receptor target genes. Proc Natl Acad Sci U S A 2004;101:15603-8.

(6) Johnsen SA, Güngör C, Prenzel T, Riethdorf S, Riethdorf L, Taniguchi-Ishigaki N, et al. Regulation of estrogen-dependent transcription by the LIM cofactors CLIM and RLIM in breast cancer. Cancer Res 2009;69:128-36.

(7) Carroll JS, Meyer CA, Song J, Li W, Geistlinger TR, Eeckhoute J, et al. Genome- wide analysis of estrogen receptor binding sites. Nat Genet 2006;38:1289-97.

(8) Fullwood MJ, Liu MH, Pan YF, Liu J, Xu H, Mohamed YB, et al. An oestrogen- receptor-alpha-bound human chromatin interactome. Nature 2009;462:58-64.

(9) Chapman RD, Heidemann M, Albert TK, Mailhammer R, Flatley A, Meisterernst M, et al. Transcribing RNA polymerase II is phosphorylated at CTD residue serine-7. Science 2007;318:1780-2.

(10) Mendez J, Stillman B. Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis. Mol Cell Biol 2000;20:8602-12.

(11) Nelson JD, Denisenko O, Bomsztyk K. Protocol for the fast chromatin immunoprecipitation (ChIP) method. Nat Protoc 2006;1:179-85.

(12) Pirngruber J, Shchebet A, Schreiber L, Shema E, Minsky N, Chapman RD, et al. CDK9 directs H2B monoubiquitination and controls replication-dependent histone mRNA 3' end processing. EMBO Rep 2009;10:894-900.

(13) Pirngruber J, Johnsen SA. Induced G1 cell-cycle arrest controls replication- dependent histone mRNA 3' end processing through p21, NPAT and CDK9. Oncogene 2010;29:2853-63.

(14) Miele A, Dekker J. Mapping cis- and trans- chromatin interaction networks using chromosome conformation capture (3C). Methods Mol Biol 2009;464:105-21.

8

Supplemental Table 1

Target Gene siRNA Target Sequence Source Cat. No. Silencer® Select Negative Control #1 - Ambion 4390844 Silencer® Select Negative Control #2 - Ambion 4390847 PSMB3 Silencer® Select GGCUGAACCUGUAUGAGUUTT Ambion s11348 PSMB5 Silencer® Select UGAUAGAGAUCAACCCAUATT Ambion s11355 siGENOME Non-targeting siRNA pool #1 - Dharmacon D-001206-13 RNF40 AGAUGGAUGUGACAGGUCATT Ambion s18960 RNF40 siGENOME (#1) GAGATGCGCCACCTGATTATT Dharmacon D-006913-01 RNF40 siGENOME (#2) GATGCCAACTTTAAGCTAATT Dharmacon D-006913-02 RNF40 siGENOME (#3) GATCAAGGCCAACCAGATTTT Dharmacon D-006913-03 RNF40 siGENOME (#4) CAACGAGTCTCTGCAAGTGTT Dharmacon D-006913-04

For transfections the Dharmacon RNF40 siRNAs (#1 - #4) were pooled in a 1:1:1:1 ratio.

9 Supplemental Table 2

Primers utilized in qPCR in 5’ to 3’ orientation. Primers not obtained from other previous studies were designed using a primer designing tool program (www.ncbi.nlm.nih.gov/tools/primer-blast/).

Gene Forward Primer (5’ to 3’) Reverse Primer (5’ to 3’) Source CXCL12 TGCCAGAGCCAACGTCAAGCATC CGGGTCAATGCACACTTGTCTGTTGT this study CXCL12 (hnRNA) GCCCCCTCCCCCACGTCTTT TCACAGAGCCCGGCTGAAGAACA this study FKBP5 TCCCTAAAATTCCCTCGAATG AAGGCAGCAAGGAGAAATGAT this study GILZ (hnRNA) TGAGGGAATGGGTGAAAAAG TGGAAATGCCCTAAAAGGTG this study GREB1 GTGGTAGCCGAGTGGACAAT ATTTGTTTCCAGCCCTCCTT (1) GREB1 (hnRNA) AACCTGGTGCCCAGGCCCAT ACACAGATAAAAGCAACGTGCGTCTCC this study h28SrRNA CTTTAAATGGGTAAGAAGCC ATCAACCAACACCTTTTCTG this study HNRNPK GACCGTTACGACGGCATGGTTGG ATCCGGAGCCACCCTGTGGTTC this study TFF1 (hnRNA) TTGGAGAAGGAAGCTGGATGG ACCACAATTCTGTCTTTCACGG (2) PGR TCCACCCCGGTCGCTGTAGG TAGAGCGGGCGGCTGGAAGT this study PKIB ACGTGGAGTCTGGGGTCGCC GAGAGCCTCCAGTTTGAGGGGCA this study RNF40 AGTACAAGGCGCGGTTGA GAAGCAGAAAACGTGGAAGC this study SGK1 AGGATGGGTCTGAACGACTTT CCAAGGTTGATTTGCTGAGAA this study SLC19A2 TGTTACTGACACCCCAGCTTC CACAGACCAGCAGAGAAGAGG this study WISP2 CATGCAGAACACCAATATTAAC TAGGCAGTGAGTTAGAGGAAAG (3) hnRNA = heterogeneous nuclear RNA

10 Supplemental Table 3

Primers utilized in ChIP studies.

Gene Genomic Position# Forward Primer (5’ to 3’) Reverse Primer (5’ to 3’) Source CXCL12 (11238) ER_hg18FDR20_11238 GGCCTCCAGCTGCCAGTCAGA CCTGGACCTACACCACGGGGG this study CXCL12 (11240) ER_hg18FDR20_11240 GAGTCACCCTGCCCCTCGACA AGGAGCCCTGTGCTCTCTGGC this study CXCL12 (11241) ER_hg18FDR20_11241 CAGCACCTGCTTCTCGCTTCCC GGAGTCGGCTCAGGGCCAACAA this study CXCL12-TSS - GCAGTGCGCTCCGGCCTTT CCTCACTGCAGACCGGGCCA this study CXCL12 3’ end - GTCCCCTGACACCACGCTGC TGTCCGGTCCTCCAAGCCCTC this study FKBP5 - TAACCACATCAAGCGAGCTG GCATGGTTTAGGGGTTCTTG (4) GILZ - TGGGTACTGGCCTTAACTTCA AATTTCCACCAGAAGGAGCA this study GREB1 (5367) ER_hg18FDR20_5367 GCTGACCTTGTGGTAGGCAC CAGGGGCTGACAACTGAAAT this study GREB1 (5368) ER_hg18FDR20_5368 CCTGGGAATGGAGATTTTGATA GAGCTGCGAGTCCCTAACAG this study GREB1-TSS - GCCAAATGGAAGAAGGACAG ACCACCTACCTCCAGTCACC (1) GREB1 3’end - GGGTGCCAAGTCGCTGCTGT CTGGATGGCAGAGGCGCCG this study PGR (12074) ER_hg18FDR20_12074 GGCCAGCAGTCCTGCAACAGTC CCCAAGCTTGTCCGCAGCCTT this study PKIB (8413) ER_hg18FDR20_8413 ACCTGACCATGTCGTTCCCTTGAGTTT TCCCGCAGTGATCTAATCCATCTGGTAGT this study PKIB (8414) ER_hg18FDR20_8414 GTGGGGGCTCACCCCTACCG GTGGGGATGTTTGGCACCCTGC this study SGK1 - CCACAGAGGAATCGAGGATG GTCCGTTCCGCATGTAATTT (4) SLC19A2 - GCATTCCCAACAGATGAGC GGAGGACATGTGGAACTCC (5) TFF1 3’ end - CCTCCTCCTCCCACCTGT CAGAGAGCAGGAGCTTTCCA this study TFF1 (4773) ER_hg18FDR20_4773 GACAGAGACGACATGTGGTGAGGTCA CACCCCGTGAGCCACTGTTGTC this study TFF1-TSS - CCTGGATTAAGGTCAGGTTGGA TCTTGGCTGAGGGATCTGAGA (1) WISP2 (4386) ER_hg18FDR20_4386 TGGCTTGACCCCATCATCTA GGTGTGACCCAGAGCAAAAC (6)

# Based on the data and nomenclature of Carroll, et al. (7).

11 Supplemental Table 4

Primers utilized in 3C. All primers were designed in this study using the interactions published by Fullwood, et al. (8). Hybridization Probe (5’ to 3’) Name Forward Primer (5’ to 3’) Reverse Primer (5’ to 3’) (5’-Fam / 3’-BHQ1) CXCL12 GAAGGAAGAAGAAACATGGACTCTGCTCCA ACAGAAGCTGGTTTACCGACTTGTCTGT GCCCCAGGGCACAACACACC Positive Interaction CXCL12 GAAGGAAGAAGAAACATGGACTCTGCTCCA CTCCCAGTGCAGAGGGAAGCATGT GCCCCAGGGCACAACACACC Negative Interaction GREB1 GGGCTGGGTGCCCGTTTTGT CCAGCAGCTGCACGCCACAT CCTGTGACATCTCTCCCAGCCCC Loading Control GREB1 GCCACTACATCCTTGGCTTTGTCCAC CCGCTGGTCAGCCGTTCAGG GTCAGGGCAAAGGACATGGCCAG Negative Interaction GREB1 CTGGGCCTCTCCAGGGGGTTTT CCGCTGGTCAGCCGTTCAGG GTCAGGGCAAAGGACATGGCCAG Positive Interaction TFF1 GCCAAGCACGCCCCCGAC AGGTCAGGTTGGAGGAGACTCCCA ACCGAGGGATGGACCCCTCCA Positive Interaction TFF1 GCCAAGCACGCCCCCGAC ACCAAGATGCCTGGGTCACAAACCAG ACCGAGGGATGGACCCCTCCA Loading Control

12 Supplemental Table 5

BAC clones CXCL12 RP13-309I17 (pBACe3.6), Bac Pac Resources, Oakland, USA GREB1 RPCIB753E0150Q (RPCI-11, pBACe3.6), imaGenes GmbH, Berlin TFF1 RPCIB753F01113Q (RPCI-11, pBACe3.6), imaGenes GmbH, Berlin

13 Supplemental Table 6

Antibodies used for ChIP and Western blot analyses and the respective dilutions. Target Protein Clone Cat. No° ChIP WB Source AKT 9272 - 1:1,000 Cell Signaling Beta-Actin ab6276 - 1:100,000 Abcam CDK9 C-20 sc-484 1 µg - Santa Cruz ERα HC-20 sc-543 1 µg 1:1,000 Santa Cruz ERK 9102 - 1:1,000 Cell Signaling GR E-20 sc-1003 1 µg - Santa Cruz GRIP1 M-343 sc-8996 1 µg - Santa Cruz Histone H2B 07-371 - 1:3,000 Millipore Histone H2Bub1 7B4 - - 1:10* - Histone H3 ab1791-100 - 1:5,000 Abcam Histone H3Ac 06-599 - 1:500 Upstate Histone H3K9/14Ac 06-599 1 µg - Upstate Histone H3K36me3 ab9050 - 1:1,000 Abcam HSC70 B-6 sc-7298 - 1:25,000 Santa Cruz IgG (non-specific) - ab46540 1 µg - Abcam MED12 ICH-00180 1 µg - Bethyl p-AKT (Ser 473) sc-7985-R - 1:500 Santa Cruz p-ERK 9101S - 1:1,000 Cell Signaling PSMB3 MCP102 PW8130-0100 - 1:1,000 Enzo/Biomol PSMB5 - ab3330 - 1:1,000 Abcam RNAPII N-20 sc-899 1 µg 1:1,000 Santa Cruz RNAPII P-Ser2 3E10 - 150 µl* 1:10* (9) RNAPII P-Ser5 3E8 - - 1:10* (9) RNAPII P-Ser7 4E12 - - 1:10* (9) RNF40 KA7-27 R9029 - 1:1,000 Sigma-Aldrich SSRP1 3E4 609802 - 1:500 BioLegend STAG1 ab4457 1 µg - Abcam

Ubn FK1 PW8805-0500 - 1:1,000 Biomol Ubiquitin - Z0458 - 1:1,000 Dako

ChIP – Chromatin Immunoprecipitation WB – Western blot * From hybridoma supernatant

Secondary HRP-labeled antibodies were purchased from Santa Cruz Biotechnology, Heidelberg and Jackson ImmunoResearch Ltd., Suffolk, UK.

14 Supplemental Figure Legends

Supplemental Figure 1

Pharmacological proteasome inhibition and proteasome subunit depletion increase the amount of polyubiquitinated proteins in breast cancer cells and Bortezomib blocks estrogen-induced ERα downregulation. (A) MCF7 cells were treated with either vehicle

(ethanol, Cont) or increasing amounts of Bortezomib. Protein extracts were analyzed by

Western blot analysis using a specific antibody against polyubiquitinated proteins. (B)

Polyubiquitinated protein abundance was determined by immunoblotting upon treatment with 20 µM MG132, 50 nM Bortezomib or 1 µM epoxomicin for 4 hours. (C)

Polyubiquitinated and PSMB3/PSMB5 protein levels were detected by Western blot following PSMB3 and PSMB5 siRNA-mediated knockdown. (D - E) After 15 minutes pre- treatment with 50 nM Bortezomib (Bort), MCF7 cells were stimulated with 10 nM 17β-

Estradiol (E2) (D) or 1 µM Fulvestrant (Fulv) (E) for 6 hours. Protein extracts were analyzed by Western blot for polyubiquitinated proteins and ERα protein levels. Ubn indicates higher molecular weight ubiquitinated proteins. HSC70 serves as a loading control.

Supplemental Figure 2

Prolonged exposure to Bortezomib induces apoptosis. Cells undergoing apoptosis were defined via Annexin V-PE and 7-AAD staining and subsequent flow cytometry analysis.

Cells in the lower left gated quadrant (Annexin V-PE negative/7-AAD negative; red) reflect non-apoptotic cells, cells in the lower right quadrant (Annexin V-PE positive/7-AAD negative; blue) reflect early apoptotic cells, and cells in the upper right quadrant (Annexin

V-PE positive/7-AAD positive; green) reflect late apoptotic cells. (A) MCF7 cells were cultured in hormone-deprived growth medium and treated with either vehicle (ethanol,

15 Cont), 10 nM 17β-Estradiol (E2), 50 nM Bortezomib (Bort), 1 µM Hydroxytamoxifen (4-

OHT, Sigma) or 5 µM Doxorubicin (positive control, Doxo, Enzo Life Science, Lörrach,

Germany) for 24 hours or 48 hours. (B) Control, PSMB3 or PSMB5 siRNA-transfected

MCF7 cells were subjected to hormone-deprived medium for 24 hours. Cells were then treated with vehicle (ethanol, Cont), 10 nM 17β-Estradiol (E2) or 1 µM Tamoxifen (4-OHT) for another 24 hours. Representative samples from an experiment with n = 2 are shown.

The fractions of gated cells were quantified and are shown as “% cells” in bar graphs; mean values, n = 2.

Supplemental Figure 3

MCF7 cells were pre-treated for 15 minutes with 50 nM Bortezomib (Bort) or vehicle

(ethanol, Cont) and then incubated with 1 µM Fulvestrant (Fulv) for 2 hours. (A) Total mRNA was isolated and the expression levels of the two ERα target genes CXCL12 and

GREB1 (hnRNA) were normalized and graphically displayed as described in Figure 2D, mean values + s.d., n = 2. (B) ERα recruitment to the indicated sites was analyzed via

ChIP analysis as described in Figure 3B, mean values + s.d., n = 3.

Supplemental Figure 4

Proteasome blockage using Bortezomib decreases the expression of most estrogen- induced genes already after 6 hours. (A) Heatmap showing log2-fold-changes in experiments 17β-Estradiol vs. control (E2) and Bortezomib + 17β Estradiol vs. 17β-

Estradiol (Bort + E2) (columns) for genes which are significantly (q < 0.05; q = p values adjusted to FDR) regulated by 17β-Estradiol (FC < -log2 (1.3) or FC > log2 (1.3)) (rows).

Upon growing MCF7 cells in hormone-depleted medium for 24 hours and pre-treating with

50 nM Bortezomib or vehicle (ethanol) for 15 minutes, cells were exposed to 10 nM 17β-

Estradiol for 6 hours. Gene expression was analyzed (red = downregulated and blue =

16 upregulated genes); mean values, n = 3. (B) Significantly estrogen-affected genes which are influenced by Bortezomib. E2 genes: ↓ = q < 0.05, FC < -log2 (1.3); ↑ = q < 0.05, FC > log2 (1.3); Bort + E2 genes: ↓ = q < 0.1, negative FC; ↑ = q < 0.1, positive FC. (C)

Estrogen-induced genes which are super-induced by either Bortezomib alone (Bort) or the combined treatment of Bortezomib and 17β-Estradiol (Bort + E2). Heatmap shows log2- fold-changes in experiments 17β-Estradiol vs. control (E2), Bortezomib vs. control (Bort) and Bortezomib + 17β-Estradiol vs. control (Bort + E2) (columns) for genes which are significantly (q < 0.05; q = p values adjusted to FDR) upregulated by 17β-Estradiol (FC > log2 (1.3)) (rows). Gene expression was analyzed (intensity of blue indicates the degree of upregulation); mean values, n = 3.

Supplemental Figure 5

Inhibition of proteasomal activity with Bortezomib shows no effect on ERα recruitment to various estrogen-activated target genes. ChIP analysis using a specific ERα antibody was performed as described in Figure 3B, mean values + s.d., n = 3.

Supplemental Figure 6

Proteasome inhibition using Bortezomib has no effect on ERα, GRIP1, MED12 and

STAG1 recruitment to two other ERα target genes and does not affect estrogen-induced long-range interaction. MCF7 cells were treated as in Figure 4. ERα (A), GRIP1 (B),

MED12 (C) and STAG1 (D) binding to the indicated EREs on GREB1 and TFF1 genes was analyzed via ChIP analysis as described in Figure 3B; mean values, n = 3. (E)

Graphical schemes of the tested 3C sites on the GREB1 and TFF1 loci which were chosen as described in Figure 4E. (F) 3C assay was performed and analyzed as described in

Figure 4E; mean values + s.d., n = 3.

17 Supplemental Figure 7

Chromosomal long-range interactions were analyzed as described in Figure 4E. Shown are interactions of the respective anchor site with a negative control site (labeled C in

Figure 4E; Supplemental Figure 6E), mean values + s.d., n = 3.

Supplemental Figure 8

Bortezomib treatment does not affect transcriptional initiation complex assembly but decreases RNAPII P-Ser2 elongation on estrogen target genes. H3Ac (A), RNAPII (B),

CDK9 (C) and RNAPII P-Ser2 (D) binding to the indicated gene sites was analyzed via

ChIP analysis. MCF7 cells were treated as in Figure 4 and ChIP analysis was performed as in Figure 3B, mean values, n = 3.

Supplemental Figure 9

(A, C) MCF7 (A) and A549 (C) cells were transfected with control or RNF40 siRNAs for 24 hours, incubated in hormone-free medium for 48 hours and then stimulated with 10 nM

17β-Estradiol (E2) (A) or 100 nM Dexamethasone (Dex) (C) for 2 hours. Hormone-induced gene expression was analyzed as described in Figure 5C, D, mean values + s.d., n = 3.

(B) MCF7 cells were treated as in Figure 5E, F. MED12, GRIP1 and CDK9 protein levels were analyzed by Western blot analysis. HSC70 serves as loading control.

Supplemental Figure 10

H2B and H2Bub1 expression in human normal breast tissue, malignant tumor and metastasis. Representative pictures of a TMA showing immunohistochemical H2B and

H2Bub1 staining in malignant, metastatic breast tumor and normal adjacent tissue samples. Samples were categorized into negative or positive for H2B or H2Bub1 (shown in left upper corners of the images); bar = 100 µm.

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