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1 Supplemental Methods Isolation of Total Protein, Soluble Nuclear And 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.
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