FASE 2 Validación de la propuesta

Crosslinking

U937 cells were induced with 4–hydroxytamoxifen (OHT) at a final concentration of 1 µM. 1 x 108 – 1 x 109 cells were used while cell density was around 5 x 105 / ml. After 24 hours cells were harvested by centrifugation in 250 ml buckets (Corning, 300 g, 5 min, RT). Pellets were resuspended in 40 ml of pre–warmed medium containing fetal calf serum (10%) and pooled in a 50 ml Falcon tube. After another centrifugation step the pellet was resuspended in 43.78 ml of serum–free medium (RT). 1.22 ml of formaldehyde (37%, Roth, cat. no. 4979.1) were added and Falcon tubes were incubated on a roller table for 9 min at RT. Glycine was added to a final concentration of 125 mM (3 ml of a 2 M solution) and the samples were mixed quickly and put on ice for 10 min. From that point on all steps of the protocol were carried out on ice. Cells were centrifuged (300 g, 5 min, 4°C) and the pellets were washed thrice in cold PBS and transferred to .15 ml Falcon tubes. At this point the

procedure can be interrupted by shock–freezing the pellet and storage at –80°C. Alternatively one can procede directly.

Cell lysis and sonication

To isolate nuclei pellets were resuspended in 10 ml of Lysis Buffer 1 (LB1) and rocked gently at 4°C. Nuclei were centrifuged (4.000 g, 10 min, 4°C) and resuspended in 10 ml of Lysis Buffer 2 (LB2). Again the samples were rocked gently at 4°C for 10 min before spinning them down again (4.000 g, 10 min, 4°C). Pellets were resuspended in 5 ml of Lysis Buffer 3 (LB3) without Triton X–100 and glycerol and sonicated in an ice/ethanol bath using a Branson 250 sonifier with the microtip (40% output, 6 min total: 15 sec on / 45 sec off). Resulting fragment lengths were ranging from 300 to 500 bp.

After sonication 1/20th of the total volume of 10% Triton X–100 (0.5% final concentration) was added to the samples, which were then transferred to 1.5 ml tubes. Samples were centrifuged (15.000 g, 15 min, 4°C) and supernatants were transferred to new 15 ml Falcon tubes. Sample volumes were adjusted with LB3 in order to reach a concentration of double-stranded DNA (as obtained through optical measurement) of 1–2 mg/ml. Glycerol was added to a final concentration of 10%. 1 ml aliquots were snap–frozen and stored at –80°C. 10 µl of the sample were

digested with 2 µl of Proteinase K (PCR grade, Roche) for 2 h at 65°C. After DNA

purification (Qiaquick, Qiagen) fragment lengths were checked on an agarose gel. If necessary, further sonication was carried out.

Lysis Buffers: Complete (Roche, cat. no. 11697498001) protease inhibitor mix was added to all lysis buffers before use.

25x Complete

• Grind 1 tablet in between 2 sheets of balance paper • Dissolve fine powder in 2 ml of H2O

Lysis Buffer 1 (LB1) final conc. 5,0 ml 1M Hepes-KOH, pH 7.5 50mM 2,8 ml 5M NaCl 140mM 0,2 ml 0,5M EDTA, pH 8.0 1mM 10,0 ml 100% glycerol 10% 5,0 ml 10% NP-40 0,5% 2,5 ml 10% Triton X-100 0,25% 74,5 ml H2O --- 100,0 ml • store at 4oC

Lysis Buffer 2 (LB2) final conc.

4,0 ml 5M NaCl 200mM 0,2 ml 0,5M EDTA 1mM 0,1 ml 0,5M EGTA 0,5mM 1,0 ml 1M Tris-HCl pH 8 10mM 94,7 ml H2O --- 100 ml store at 4°C

2x Lysis Buffer 3 (LB3) final conc. (1x)

2,8 ml 5M NaCl 140mM 0,4 ml 0,5M EDTA 1mM 0,2 ml 0,5M EGTA 0,5mM 2,0 ml 1M Tris-HCl pH 8 10mM 10,0 ml 10% N-lauroyl sarcosine 0,5% 84,6 ml H2O --- 100,0 ml • store at 4°C

• before use, dilute to 1x LB3 and supplement with: before sonication:

10% Na-deoxycholate 0,1% (freshly prepared) 1x Complete after sonication:

10% Triton X-100 (at step 2.5) 0,5% 100% glycerol (at step 2.6) 10%

Immunoprecipitation and isolation of DNA fragments

For one IP 100 µl of chromatin extract (2 mg/ml) were used. For preclearing 10 µl of blocked beads were used per 100 µl of extract. Samples were incubated under constant agitation at 4°C for 30–60 min. In parallel blocked beads were pre–incubated with the appropriate antibody. Typically 2–10 µg of antibody were

used per IP. In the case of hybridoma supernatants 100–200 µl were used. For each

IP 15–20 µl of beads were used. The pre–incubation was done in a total volume of

400 µl and volumes were adjusted with cold PBS. After incubation at 4°C for 30–60 min tubes were centrifuged (200 g, 2 min, 4°C) and washed once with cold PBS.

100 µl of pre–cleared chromatin extract was added to 20 µl of antibody–coated beads. The total volume was adjusted to 400 µl using LB3 without glycerol. IP reactions were incubated under constant agitation at 4°C overnight.

Beads were washed six times with 1 ml of RIPA buffer and once with 1 ml of

TE buffer containing 50 mM NaCl. For elution 100 µl of pre–warmed (65°C)

elution buffer has been added to the beads and samples were rocked for 10 min at 1400 rpm in a thermomixer (65°C). After centrifugation supernatants were transferred to new tubes (RT). Crosslink reversal took place overnight at 65°C.

1 volume of TE buffer containing RNase (0.2 µg/µl final concentration) was added to the samples followed by incubation at 37°C for 1–2 hours. Proteinase K was added to a final concentration of 0.2 µg/µl and samples were incubated at 56°C for 2 hours. DNA was isolated by PCI extraction and ethanol precipitation in the presence of

10 µg of glycogen. Pellets were resuspended in 50 µl Tris/HCl. DNA concentrations

were determined and 0.5–1 µg were loaded onto an agarose gel to check for fragment length distribution. 20 ng of this DNA were used as a sample for PCR reactions. For detection of MLL–VP16–ER–HA bound to the chromosomal Hoxa9 promoter primers "hoxa9end2" and "hoxaend_r" were used for PCR, for the episomal Hoxa9 promoter the primers "hoxa9F–65" and "lucR_short".

Wash buffer (RIPA buffer): final concentration 5ml 1M Hepes (pH 7.6) 50 mM

200µl 1M EDTA 1 mM 10 ml 10% NP-40 (IPGEL) 1%

10 ml 5M LiC 0.5 M

7 ml 10% DOC (Na deoxycholate) 0.7% (freshly prepared) ---

100 ml

Elution buffer: 50mM Tris pH8 10mM EDTA 1% SDS