Protein - nucleic acid interaction analysis

A typical DNA or RNA binding reaction (25 μl) was performed in the presence of 0.2 g of dMi-2F and 80 ng of nucleic acid (DNA or ssRNA) in 40 mM KCl, 20 mM Tris pH 7.6, 1.5 mM MgCl2, 0.5 mM EGTA, 10 % glycerol, BSA (200 ng/ l), 1 mM DTT (supplemented with 0.4 units of RNAsin). For competition assays, samples were preincubated for 15 min at 26 C before the different amounts of competitor (PAR or DNA or RNA) were added. Reactions were further incubated at 26 C for 75 min. Products were analysed on 6 % native PAA gel and visualized with ethidium bromide (EtBr) staining.

ssRNA was synthesized by in vitro transcription using a fragment of hsp70 DNA as a template. This template (also used for the DNA EMSA assays) was produced by PCR amplification of cDNA derived from heat shocked Kc cells using the following primers:

T7-hsp70_f and hsp70_r (Table 3.12).

3.2.5.2 Native gel electrophoresis

For analysis of mononucleosome mobilization assay or EMSA products, native gel electrophoresis was conducted using the Novex system. Samples were separated on 6%

native polyacrylamide gel in 0,4 TBE buffer (chapter 3.1.2). The gel was pre-run at constant current of 80V for 45 min before samples were loaded and then the

94 electrophoresis was continued for 2 hrs at RT. Samples were run without a dye in 10%

glycerol. After electrophoresis, the gel was stained with ethidium bromide (0,5 μg/ml) in distilled water for 15 min on a horizontal rotating shaker at RT followed destaining by washing twice in water for 10 min. Gels were documented with the gel documentation system.

3.2.5.3 Chromatin immunoprecipitation (ChIP) Method principle

Chromatin immunoprecipitation assay (ChIP) was developed to determine whether a protein of interest binds to a specific DNA sequence in vivo (Solomon et al. 1988; Dedon et al. 1991; Orlando et al. 1997). Proteins are reversibly crosslinked to target DNA with formaldehyde. Chromatin is subsequently fragmented by sonication to short fragments, with an average of 500 bp. Subsequently protein-DNA complexes are precipitated using antibodies specific to the protein of interest. After extensive washing, crosslinking reversal and protein removal, DNA associated with the precipitated protein can be identified by conventional PCR, QPCR, by labelling and hybridization to DNA microarrays (ChIP-chip), or by direct high-throughput deep sequencing (ChIP-seq) (Collas 2010).

ChIP in cell culture

Kc cells were grown to 4-7x10⁶ cells/ml in Schneider‟s Drosophila medium, 1x10⁸ cells were usually processed for ChIP. Heat shock treatment was performed as described in chapter 3.2.8.4. In order to obtain temperature comparable to untreated cells for the crosslinking reaction, heat shock was stopped by adding 1/3 volume of 4°C medium. Cells were immediately crosslinked with 1% formaldehyde for 10 min at RT with slow agitation on a horizontal shaker. Crosslinking was quenched by adding 2 M Glycine to a final concentration of 240 mM. Cells were pelleted by centrifugation (1000 rpm, 10 min, 4 C, Heraeus Biofuge Pico) and washed once with 10 ml of cold PBS. Cell pellet was resuspended in 1 ml of SDS-Lysis Buffer and incubated on ice for 10 min. Cells were sonicated for 18 minutes (30 sec on/off) at the high intensity setting with a Bioruptor sonication device (Diagenode). To keep the temperature constant, ice was added to the ultrasound water bath after 9 minutes of sonication. Average length of sonified DNA was below 500 bp. Chromatin was spun down at 13000 rpm for 10 min at 4 C (Heraeus Biofuge Pico) and the lysate was diluted 1:10 in Chip Dilution Buffer. To remove DNA

95 and proteins which unspecifically bind to beads, chromatin was precleared with 80 μl of 50% protein G-sepharose beads (in 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 mg/ml BSA) and rotated on the wheel in the coldroom for 30 min.

1% of the diluted, precleared chromatin was taken as input sample. For each precipitation 1,3 ml of precleared chromatin was used and appropriate amounts of antibodies (Table 3.2) were added. As a negative control, -IgG were added to chromatin. Immunoprecipitation was carried out in siliconized tubes, overnight at 4°C on the rotating wheel. For each IP, 35 μl of 50% protein G-sepharose beads were added and incubated for 2 hours at 4°C. The immunoprecipitates were washed 3 times with Low Salt Wash buffer, 3 times with High Salt Wash Buffer, once with LiCl Wash Buffer and twice with TE buffer. Before the last wash with TE buffer, beads were transferred to fresh siliconized tubes. Each washing step was carried out on a rotating wheel for 10 min at 4 C and samples were spun down at 2000 rpm for 3 min at 4 C (Heraeus Biofuge Pico).

Protein-DNA complexes were eluted from the beads with 250 l of freshly prepared Elution Buffer for 30 minutes at RT on a rotating wheel. Beads were spun down and the supernatant was collected to a fresh tube. Elution was repeated and both supernatants were pooled. Appropriate amount of elution buffer was added to the input samples to adjust the volume to 500 μl. 20 μl of 5M NaCl were added to the eluates and input samples and decrosslinking was carried out at 65°C overnight in a thermomixer.

Proteins were removed from the samples by proteolytic digestion with Proteinase K. For this, 20 μl of 1M Tris-HCl pH 6.5, 10 μl of 0,5M EDTA and 2 μl of Proteinase K (10 mg/ml) were added and samples were incubated at 45 C for 1 hour. DNA was purified using PeqGold Cycle Pure DNA isolation kit according to the manufacturer's instructions.

DNA was eluted from columns with 30 μl PeqLab elution buffer. The relative amount of DNA compared to the IP input was determined via QPCR, using 1 μl DNA per reaction and specific primer pairs (Table 3.10 and chapter 3.2.2.3).

SDS-Lysis Buffer 50 mM Tris-HCl, pH 8,0 1% (w/v) SDS

10 mM EDTA Proteinase inhibitors Chip Dilution Buffer 16,7 mM Tris-HCl, pH 8,0

16,7 M NaCl

96 QPCR calculations for ChIP

For ChIPs, all samples were referenced to the respective IP-input:

ΔCt sample = Ct input - Ct sample and displayed as a percentage of input: %input = 2 ΔCt sample

Standard deviations s from triplicate technical measurements were used to determine the error for input percentages:

1,2 mM EDTA

1,1% (v/v)Triton X-100 0,01% (v/w) SDS Proteinase inhibitors Low Salt Wash Buffer 20 mM Tris-HCl, pH 8,0

150 mM NaCl 2 mM EDTA

1% (v/v) Trion X-100 0,1% (w/v) SDS

High Salt Wash Buffer 20 mM Tris-HCl, pH 8,0 500 mM NaCl

2 mM EDTA

1% (v/v) Trion X-100 0,1% (w/v) SDS

LiCl Wash Buffer 10 mM Tris-HCl, pH 8,0 0,25M LiCl

1 mM EDTA 1% (v/v) NP-40 1% (w/v) Sodium Deoxycholate

TE Buffer 10 mM Tris-HCl, pH 8,0

1 mM EDTA Elution Buffer 0,1 M NaHCO3

1% (w/v) SDS

97 3.2.5.4 RNA immunoprecipitation (RIP)

RNA immunoprecipitation method (RIP) is used to identify RNA-protein interactions which occur in vivo. In the immunoprecipitation methodology, an RNA binding protein is immunoprecipitated from a cell lysate, followed by reverse transcription of the immunoprecipitated RNA and PCR with primers targeting the interacting RNA (Peritz et al. 2006).

RNA immunoprecipitation was performed as described in (Gilbert and Svejstrup 2006). Kc cells were heat shocked and crosslinked as for ChIP with 1% formaldehyde for 10 min.

Crosslinking was quenched by adding 2 M Glycine to a final concentration of 240 mM.

Cells were pelleted by centrifugation (1000 rpm, 10 min, 4 C, Heraeus Megafuge 1.0) and washed once with 10 ml of cold PBS. 75x10⁶ cells were resuspended in 800 μl of FA Buffer and lysed on ice for 15 min. Cells were sonicated as for ChIP, spun down and chromatin was digested with DNAse I. For this, chromatin solution was adjusted to 25 mM MgCl2 and 5 mM CaCl2, 1 μl of DNAse I (Fermentas) was added and reaction was incubated for 10 min at RT and then stopped with 20 mM EDTA. Chromatin was spun down at 13000 rpm for 10 min at 4 C (Heraeus Biofuge Pico). 100 μl of chromatin were taken as input and froze down at -20 C. 300 μl of chromatin were used for each precipitation, filled up to 500 μl with FA buffer. Appropriate amounts of antibodies were added, in addition 2 μl of rabbit pre-immunoserum from unrelated antibody were used as a negative control. Samples were incubated in siliconized tubes overnight at 4°C on the rotating wheel.

RNA-protein complexes were precipitated with 30 μl of 50% protein G-sepharose beads (equilibrated in FA buffer and 1 mg/ml BSA) for 2 hrs at 4°C. IPs were washed 5 times in FA buffer and twice with TE buffer. Before the last wash with TE buffer, beads were transferred to fresh siliconized tubes. Each washing step was carried out on a rotating wheel for 10 min at 4 C and samples were spun down at 2000 rpm for 3 min at 4 C (Heraeus Biofuge Pico).

RNA-protein complexes were eluted twice with 100 μl of RIP Elution Buffer - first by incubation on the rotating wheel for 10 min at RT and second by incubation in a thermomixer at 1000 rpm at 65°C for 10 min.

100 μl of elution buffer were added to the input samples to adjust the volume to 200 μl.

Eluates and input samples were digested with proteinase K (10 mg/ml) for 1 hr at 42°C and decrosslinking was performed at 65°C overnight. Immunoprecipitated RNA was purified

98 using PeqGold Total RNA Kit (PeqLab) according to the manufacturer's instructions. All samples were additionally digested with DNAse I on the columns and eluted with 30 μl of RNAse free dH20. cDNA was synthesized with 10 μl of eluted RNA and 2 μl of input RNA with random hexamers and analysed by QPCR with appropriate primer pairs.

Amount of precipitated RNA was calculated as for ChIP. For IP specificity, abundant transcripts of rp49 and actin5c were detected.

FA Buffer 50 mM Hepes-KOH, pH 7,6

140 mM NaCl

1% (v/v) Triton X-100

0,1% (w/v) Sodium Deoxycholate RNAsin (100 U/ml)

Proteinase inhibitors (added freshly) RIP Elution Buffer 100 mM Tris-HCl, pH 8,0

10 mM EDTA 1% (w/v) SDS RNAsin (40 U/ml)

3.2.6 Poly(ADP-ribose) binding analysis