E.2 Methods
E.2.9 Preparation of nuclear extracts
E.2.9.1 Nuclear extracts from human placenta
The following protocol for the preparation of a nuclear extract from human placenta was established on a protocol provided by Dr. J. Griesenbeck and on published data for Dnmt1 preparations from human placenta (Wang et al. 1984; Pfeifer et al. 1985; Gorski et al. 1986;
Yoo et al. 1987).
Purification was performed in the cold room and samples were always kept on ice. Protease inhibitors PMSF (1mM), Leupeptin (10µg/ml), Aprotinin, Pepstatin (1µg/ml) were added to buffers prior to use. (Since Leupeptin, Aprotinin and Pepstatin are very expensive, they were used only for buffer volumes > 100ml)
Fresh human placenta was received directly after birth and placed into ice-cold wash buffer 1x PBS supplemented with 1mM DTT and 1mM PMSF.
Human placenta is extensively rinsed three times with wash buffer to wash away blood.
Fibrous membrane, fat and connective tissue are removed and remaining soft tissue is cut into small pieces (approximately 1-2cm3, referred to as nuggets) that are directly shock frozen in liquid nitrogen and stored at -80°C until use.
Nuclei preparation
Approximately 1l of buffer A (20mM Hepes pH 7.6, 2mM EDTA, 1mM EGTA, 15mM KCl, 10% glycerol, 0.01% NP-40, 0.2mM spermidine, 0.5mM spermine, 1mM DTT) is added to 300g of placenta nuggets in a plastic bag. Nuggets are pre-thawed under running water to
facilitate homogenization and subsequently homogenized three times in a Waring Blender for 20sec each at full speed.
The homogenate is successively filtered through metal gauze of two different pore sizes (1500µm and 500µm respectively; (Uchida et al. 1993)) and centrifuged (@10min, 1300g, 4°C)
The pellet is resuspended in 100ml buffer A, filtered through metal gauze of two different pore sizes (200µm and 75µm respectively) and centrifuged. Again the pellet is resuspended in 100ml buffer A (supplemented with leupeptin, aprotinin, peptstatin) using a donce homogenizer (loose pestle) and filtered through three layers of Miracloth to remove fat).
The filtrate is centrifuged (@ 5min, 4500rpm, 4°C, 50ml falcon tube) and roughly 25ml NPV (nuclear packed volume) of crude nuclei are recovered (white color, fat containing).
The quality of prepared nuclei can be checked visually performing a DAPI stain.
A tip of a small spatula of nuclei are resuspended in 1x PBS, DAPI (stock 40µg/µl, stored in the dark) is added to a 1:100 dilution and incubated on ice for 3min. Nuclei are washed 3x times with 1x PBS (@2min, 2000rpm, 4°C) and resuspended in 200µl 1x PBS. A small drop is put on a microscope slide and covered with a glass slip. Visualization is performed with confocal microscope (Zeiss, Axiovert200).
Figure 58 Nuclei from human placenta
Nuclei prepared from human placenta are DAPI stained. Size bar is indicated in red.
Nuclear extraction
0,9 volumes of buffer C (20mM Hepes pH 7.9, 20% glycerol, 420mM NaCl, 1.5mM MgCl2, 0.2mM EDTA, 1mM DTT) are added to the nuclear pellet followed by douncing 3-5 times with a loose pestle (L-type) and 3 times with a tight pestle (S-type). The solution is transferred into Beckman Ti45 centrifugation tubes and while stirring, 5M NaCl is slowly added drop wise to raise the salt concentration by 100mM to approximately 300mM. The nuclear extract is incubated on a rotating device with 3-5x vortexing for 30-45 minutes and subsequently centrifuged (Beckman ultracentrifuge, 4°C, 120,000g, 90min)
Fat on top of the supernatant (referred to as nuclear extract) is removed with kimwipe and chromatin on the bottom of the tube is avoided as much as possible.
The nuclear extract is saturated with ammonium sulfate to 60-65% (0.33g AS/ml extract) over a period of 15min followed by a 30min incubation time.
After centrifugation (@30000g, 4°C, Kontron A8.24, 30min) the pellet (rather slimy) is properly resuspended with Dounce homogenizer in 3.5ml buffer D50 (20mM Hepes pH 7.9, 20% glycerol, 50mM NaCl, 2mM MgCl2, 1mM EDTA, 1mM DTT with all protease inhibitors).
Dialysis is performed twice for 2h against 2l of buffer D50 (do not dialyze overnight) and centrifuged (30min, 13000g, 4°C). The resulting nuclear extract (referred to as NucD60) with approximately 4ml and an average yield of 20mg/ml was stored in aliquots at -80°C.
E.2.9.2 Nuclear extracts from HelaS3 cells
The protocol for the preparation of nuclear extracts from Hela cells was done as described by F. Pugh (Pugh 1995).
HelaS3 cells (E.1.11) were grown in spinner flasks in RPMI 1640 supplemented with glutamax, 10% FBS and penicillin/streptomycin at 37°C, 5% CO2. Cells of a 10 liter culture were harvested with a cell density of approximately 1x106 cells/ml (@ 15min, 4°C 4650g).
The quality of the prepared nuclei was checked visually with DAPI stain (see nuclei preparation from placenta, E.2.9.1).
Figure 59 Nuclei prepared from HelaS3 cells
Nuclei prepared from human placenta are DAPI stained. Size bar is indicated in red.
E.2.9.3 MNase prepared nuclear extracts from HelaS3 cells
In order to prepare a nuclear extract that is also enriched in protein associated with chromatin, HelaS3 nuclei (prepared as described above) were enzymatically treated with MNase (Micrococcal nuclease) and mechanically treated with a Dounce homogenizer to solubilise nuclear proteins normally tightly bound to nucleosomes.
The following protocol was modified according to G. Längst (Langst et al. 1997).
The nuclear pellet (10ml) is washed twice with buffer A (15mM Hepes pH 7.6, 15mM NaCl, 60mM KCl, 2mM MgCl2 suplemented with 1mM PMSF, 10µg/ml leupeptin, 1µg/ml aprotinin and pepstatin.). After centrifugation (5min, 1000g, 4°C) the nuclear pellet is resuspended in 2 nuclear packed volumes final (add 1 NPV) in buffer A, CaCl2 is added to a final concentration of 1mM, followed by incubation in a water bath until the temperature of the nuclei solution reaches 25°C. A 100µl aliquot for later DNA analysis is removed.
After addition of 3000U MNase/ml NPV (Sigma, N5386, 3U/µl of NPV), the nuclei solution is incubated for 20min at 25°C on a rotating device. The MNase reaction is stopped with the addition of ¼ volume of 4x Stop solution (20mM EDTA, 20mM Hepes pH 7.6). A 100µl aliquot is withdrawn for DNA analysis.
The nuclei are dounced with 20-40 strokes in a Dounce homogenizer (S=B-type pestle) and subsequently centrifuged (@ 30min, 13000g, 4°C). Fat and fluffy stuff floating on top are avoided.
DNA analysis
The 100µl aliquots are dounced 30x and centrifuged (@13000g, 30min). 300µl of 4x stop buffer and 100µl of 4% SDS are added. RNAse digest (50µg) is performed for 1 hour at 37°C and proteinaseK digest (50µg) for 1h at 55°C. DNA is precipitated and analyzed on 1.5%
agarose gel.