Insertion of human PPP1R26P1 into mouse Rb1

Genetic engineering by homologous recombination in mESCs was used to introduce the human PPP1R26P1 into intron 2 of the non-imprinted mouse Rb1 gene, positioned and oriented analogous to human RB1. To enable detection of allele specific expression, a single nucleotide variant (SNV) was introduced into one of the Rb1 alleles first. The stepwise generation of the modified ES cells is depicted in Figure 1B. The SNV was inserted into exon 3 using the targeting vector shown in Figure 1C, creating a new PstI restriction site but not altering the encoded amino acid. Successful homologous recombination in wildtype D3 mESCs was screended for by Southern blot. Three clones showed the expected restriction pattern consisting of a 6 kb and an 8 kb fragment for the wildtype and the targeted allele, respectively (Figure 1D). Of these three clones, only one had the SNV integrated in exon 3, as was determined by PCR followed by PstI digestion (Figure S1). A Southern blot hybridized with a probe internal to the targeting vector confirmed that this clone (designated Rb1_SNVneo) had a single integration at the Rb1 locus only (Figure S2A). In addition, a Southern blot on DNA digested with PstI and hybridized with a 3’ external probe, showed that the PstI site was correctly integrated in exon 3 at the Rb1 locus (Figure S2A). In this Rb1_SNVneo clone, removal of the selection cassette generated clones SNV / wt (Figure 1D). Note that the SNV allele is written to the left side throughout the text, this is not related to parental origin of the alleles, as this could not be determined.

One SNV / wt clone was used for the integration of PPP1R26P1 in intron 2 of Rb1 at the position corresponding to its location in human RB1. The sequences of intron 2 of RB1 and Rb1 show no high level of sequence similarity and also have different lengths of 35 kb and 23 kb, respectively. Two evolutionary conserved regions, termed ECR A and B, were identified using the ECR browser (http://ecrbrowser.dcode.org/) (Table S1, Figure 1A, C). In RB1, PPP1R26P1 integrated between these two ECRs, so they were used as anchor points to determine the insertion site in mouse Rb1, integrating PPP1R26P1 at the proportionally correct position between the two ECRs in the same orientation as in human RB1.

The targeting construct for PPP1R26P1, containing the 5.2 kb PPP1R26P1 is shown in Figure 1C. Correctly targeted independent clones (designated SNV_PPP1R26P1neo) were identified by Southern blot screening (Figure 1E). Phasing of the two recombination events, generation of the SNV in exon 3 and integration of PPP1R26P1, was determined in eight SNV_PPP1R26P1neo clones by pulsed-field gel electrophoresis followed by Southern blot (Figure 1F). For location of both modifications on the same allele (genotype SNV_PPP1R26P1/wt), a 23 kb fragment for the unmodified, wild type allele and a 27 kb fragment for the double-modified allele are expected. In case of the two modifications being located in trans (genotype SNV / PPP1R26P1), the predicted pattern is a 20 kb fragment for the allele carrying the SNV in exon 3 and a 30 kb fragment for the allele carrying PPP1R26P1 in intron 2 (Figure 1F). Six clones with genotype SNV / PPP1R26P1neo and two clones with genotype SNV_PPP1R26P1neo/wt (Figure 1F) were obtained. Three SNV / PPP1R26P1neo clones and one SNV_PPP1R26P1neo/wt clone were chosen for removal of the selection cassette. Two subclones each were used for further analysis, resulting in six independent clones with genotype SNV / PPP1R26P1 and two clones with genotype SNV_PPP1R26P1/wt in total. All clones were tested by Southern blot for correct, single integration events and successful removal of the selection cassette using 5’ and 3’ external probes, an internal probe and a probe hybridizing to the neomycin gene in the selection cassette (Figure S2B).

CpG42 and CpG85 have different susceptibilities to DNA methylation

The fate of the two CpG islands introduced with PPP1R26P1 in the mESCs, CpG42 and CpG85, was analyzed by next-generation bisulfite sequencing (Figure 2, Figure S3A, Figure S4). As control for an unmethylated CpG island, CpG146 at the Rb1 promoter was included (UCSC genome browser, NCBI37/mm9). DNA methylation on the single read level is visualized in DNA methylation heat maps, showing one sequencing read per row and one CpG position in the amplicon per column. The sequencing reads are sorted from high to low methylation (Figure 2A). The DNA methylation heat maps indicate that CpG146 and CpG85 are sparsely methylated with a random pattern of methylated CpG positions. In contrast, CpG42 acquires DNA methylation to a higher degree and with an apparent preference for certain CpG positions. A summary of the amount of DNA methylation at all CpG positions in all analyzed reads of D3, SNV / wt and SNV / PPP1R26P1 clones in two biological replicates each shows that the Rb1 promoter (CpG146) is mostly free of methylation with a median methylation level of 1.85% (Figure 2A, B). The level of DNA methylation at CpG85 was low with a median level of 3.95% (Figure 2A, B). However, two clones showed up with elevated DNA methylation levels of 19% and 40%, respectively. Analysis at the single read level revealed that these two clones mainly contained either fully methylated or fully unmethylated reads (Figure S4A), indicating the presence of a mixed population of cells with some cells having a fully methylated CpG85 in their single PPP1R26P1 allele. At CpG42, the degree of total methylation is about five-fold higher than at CpG85 with a median level of 18.1% (Figure 2A, B).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 2. CpG42 and CpG85 have different susceptibility to DNA methylation.

A) Differences in DNA methylation levels of the Rb1 promoter (CpG146) and CpG42 and CpG85 in the integrated PPP1R26P1 pseudogene on the single read level. One representative DNA methylation heat map for each CpG island is shown. Single reads are in rows, CpG positions in the amplicon are in columns. Red: methylated, blue: unmethylated, white: not analyzed. CpG146 and CpG85 acquire a low level of random DNA methylation, CpG42 acquires DNA methylation at a considerable level, showing preference for certain CpG positions.

B) CpG42 has a higher median level of DNA methylation than CpG146 and CpG85. Beeswarm-boxplots show the median percentage of DNA methylation at CpG146, CpG42 and CpG85. The median is calculated over all CpG sites analyzed in all reads for a given CpG island. The boxplots show the median and the quartiles, single measurements are given as black dots. Percentage of median methylation is given below the labels.

C) CpG42 has a preference of sites for acquisition of DNA methylation. In pearl-necklace diagrams the degree of DNA methylation per CpG position in all analyzed reads is visualized in histograms. Coloring indicates the percentage of methylated (red), non-methylated (blue) and not-analyzed (white) CpGs. The position of CpG and % of methylation is indicated below the histogram. CpG146 and CpG85 have no preferential CpG position becoming methylated, CpG42 shows strong preference for DNA methylation at CpG positions 1, 7 and a medium preference for CpG positions 3, 8 and 18.

https://doi.org/10.1371/journal.pone.0074159.g002

The degree of methylation at a specific CpG position in the amplicon over all reads analyzed is visualized in the pearl-necklace diagram (Figure 2C). This shows that CpG146 and CpG85 have no preferential CpG position for acquisition of DNA methylation. For CpG42, the pearl-necklace diagram shows preferential DNA methylation at positions 1 and 7 of the CpG sites included in the amplicon, which are methylated in 40% of analyzed reads. The CpG sites at positions 3, 8 and 18 in the amplicon are methylated in more than 25% of reads. As methylation at these positions could serve as seed for acquisition of full DNA methylation at this CpG island, methylation levels were determined again in four clones at 15 passages later, representing about 30 to 40 cell divisions. However, the methylation level at CpG42, and at CpG146 and CpG85 as well, was stable over this time period and did not spread over the full CpG island (Figure S4B).

Analysis of two clones with the genomic arrangement SNV_PPP1R26P1/wt showed no difference in median methylation levels compared to the six clones with the genotype SNV / PPP1R26P1, indicating that the phase of the introduced modifications does not influence the DNA methylation levels at the three CpG islands analyzed (Figure S3A).

Transcription at CpG85 in mouse Rb1

In humans, expression of the alternative RB1 transcript 2B starts from an unmethylated CpG85 within PPP1R26P1. As we could show that CpG85 is largely unmethylated in the modified ESCs, expression of a transcript from CpG85 was measured. A transcript starting at the 3’-end of CpG85 could be amplified by standard RT-PCR. Using primers in CpG85, as depicted in Figure 3B, a transcript was detected with forward primers CpG85fwd_up3 and CpG85_fwd, but not with primers located further upstream (Figure S5). Expression of Rb1 and the house keeping gene Ppia was present in all samples (Figure S5). Quantitative RT-PCR revealed an elevated, but highly variable level of transcription at the position of probe CpG85_26 compared to probes CpG85_81, CpG85_67 and CpG85_2 (Figure 3A). The level of transcription measured with probe CpG85_26 was lower but comparable to the expression levels observed for the full-length Rb1 transcript, measured with probes spanning exons 4 to 6 and exons 18 to 19 (Figure 3A). The transcription start site of the detected transcript could not be determined, as 5’-RACE (rapid amplification of cDNA ends) experiments failed. In addition, no transcript connecting transcript 2B to downstream exons of Rb1 was detected by RT-PCR. Use of reverse primers in exons 3, 5 and 8 did not yield a product. Also, attempts to elongate the obtained PCR products by 3’-RACE failed.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 3. Transcription at CpG85.

A) Expression of the transcript is elevated towards the 3’-end of CpG85. Quantitative RT-PCR with primer / probe assays (localization is shown in Figure 3B top) demonstrates significantly higher expression at the more 3’-end of CpG85. For comparison, expression of Rb1 is shown as measured with assays at exons 4/6 and exons 18/19. Analysis was conducted in six independent clones in two biological and two technical replicates, respectively. Expression levels are normalized to the level of β-actin. The p-value was calculated using the Welch two-sample test.

B) RNA polymerase II (RNAPII) is enriched at the 3’ end of CpG85. Presence of initiating and elongating RNAPII at the Rb1 and CpG85 promoters was tested for by ChIP. Top: schemes showing localization of primer / probe assays for qPCR over the Rb1 promoter (three assays) and CpG85 (four assays), as well as the mapped transcriptional start sites in CpG85. Results are depicted as % input and the non-related antibody (against PML protein) control is shown in black. Both types of RNAPII could be found at both promoters. Three independent experiments are shown, one in cells with genotype SNV_PPP1R26P1/wt and two in independent clones of genotype SNV / PPP1R26P1.

https://doi.org/10.1371/journal.pone.0074159.g003

Binding of initiating and elongating RNA polymerase II (RNAPII) at the 3’-end of CpG85 could be shown by chromatin immunoprecipitation (ChIP) in clones with the genotype SNV / PPP1R26P1 as well as the genotype SNV_PPP1R26P1/wt (Figure 3B). Enrichment of initiating RNAPII was more pronounced at the 3’- end of CpG85, corresponding to the data obtained by qPCR. Precipitation with an antibody against elongating RNAPII showed a similar pattern with enhanced enrichment at the 3’-end of CpG85. RNAPII occupancy of the Rb1 promoter in the same clones was determined as control. The obtained pattern was again similar for antibodies against the initiating and elongating forms of RNAPII. Specificity was confirmed with an antibody against promyelocytic leukemia protein (PML), showing no enrichment of both RNAPII types over the Rb1 promoter or CpG85 (Figure 3B). These results indicate active transcription at the 3’-end of CpG85. Cells with the genotype SNV_PPP1R26P1/wt showed the same result, with enhanced expression of transcript 2B and enrichment of initiating and elongating RNAPII at the 3’-end of CpG85 (Figure 3B, S3B).

Integration of PPP1R26P1 suppresses Rb1 in cis

As CpG85 remains mainly unmethylated and a transcript is initiated at the 3’-end of CpG85, just as in human RB1, skewing of allelic Rb1 expression was tested by single nucleotide primer extension (SNaPshot) analysis. The SNaPshot assay was validated using plasmids containing either an A or a G at the variant position (data not shown). Analysis of SNV / wt cells showed roughly balanced expression of the two alleles (Figure 4, left). In SNV / PPP1R26P1 cells carrying the PPP1R26P1 on the wildtype A allele, expression of this allele is suppressed, resulting in an about two-fold shift of expression in favor of the SNV G allele (Figure 4, middle). Suppression of expression of the allele carrying the PPP1R26P1 insertion was also observed in the SNV_PPP1R26P1/wt cells. In these cells, which carry the PPP1R26P1 insertion on the variant G allele, a two-fold stronger suppression of this allele was observed (Figure 4, right).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 4. Integration of PPP1R26P1 represses Rb1 in cis.

Single nucleotide primer extension analysis (SNaPshot) was used to determine the ratio of wildtype versus SNV allele expression in six clones with genotype SNV / PPP1R26P1 and two clones with genotype SNV_PPP1R26P1/wt in two biological and two technical replicates. Obtained values were normalized to values obtained for genomic DNA of the same clones. Beeswarm boxplots show the median and the quartiles, single measurements are given as black dots. The dotted line indicates the level expected for non-skewed expression. In SNV / wt cells, both alleles express about equal levels of Rb1. In cells with the genotype SNV / PPP1R26P1, this ratio shifts to preferential expression of the SNV allele (G) and suppression of the wildtype A allele carrying the PPP1R26P1 insertion. Cells with genotype SNV_PPP1R26P1/wt again show suppression of Rb1 on the allele carrying PPP1R26P1 (the G allele) versus the wildtype A allele. P-values were calculated using the Welch two-sample test.

https://doi.org/10.1371/journal.pone.0074159.g004

ES cell culture and gene targeting

Murine D3 ES cells (provided by Denise P. Barlow, CeMM, Vienna) were maintained on inactivated feeder cells under standard cell culture conditions, using KO-DMEM supplemented with 1/100 penicillin/streptomycin, 1/100 glutamine, 2 µM β-mercaptoethanol, 15% FCS, 1/100 non-essential amino acids, 1/1000 LIF (all ingredients from Life Technologies). Cells were kept in a humidified incubator at 37°C with 5% CO₂ and subcultured every second to third day.

For gene targeting, 5x10⁶ cells were electroporated with 50 µg linearized targeting vector using the BioRad electroporation device at 500 µF and 0.24 kV. After electroporation, cells were plated onto resistant feeder cells at a density of 1x10⁶ cells per 10 cm plate and selection with 400 µg/ml G418 (Roche Applied Science) started 24 h after electroporation, lasting for 7 days. Single resistant clones were picked, subcultured, frozen and screened by Southern blot. Positive clones were thawed and expanded, retested by Southern blot and the neomycin selection cassette was removed by Cre recombination using electroporation of 50 µg circular Cre expression plasmid. Cells were plated at low density; single clones were picked, expanded and tested by Southern blot for selection cassette removal. Introduction of the SNV was performed first (509 clones screened with probe SNP5’ext, targeting efficiency was 0.6%), followed by knock-in of PPP1R26P1 (452 clones screened with probe KIAA5’ext, targeting efficiency was 39%).

Construction of targeting vectors

The targeting vector for introduction of the SNV consists of a 6.5 kb homology region generated by SwaI digestion of BAC bMQ311j15 (obtained from Source BioScience, UK) (UCSC browser, build NCBI37/mm9, chr14:73,692,400-73,698,980), ligated into pBluescript opened with HincII. Before choosing exon 3 for integration of the SNV, absence of sequence motifs indicating exonic splice enhancers was confirmed using ESEfinder 3.0 (http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi [32]) to minimize the risk of aberrant splicing as a result of the modification. For introduction of the SNV, a fragment of 1.2 kb was generated by PCR using BAC bMQ311j15 as template and forward and reverse primers containing a PmeI and a BsiWI restriction site, respectively. The obtained PCR product was ligated into TOPO-vector pCR2.1 (Life Technologies). This vector was used as template in the mutagenesis reaction in combination with appropriately designed primers using the QuikChange II XL site-directed mutagenesis kit (Agilent). Nucleotide 96 in exon 3 of Rb1 (total size 116 nucleotides) was changed from A to G, creating a new PstI site. Successfully mutated plasmids were identified by PstI digestion and verified by sequencing. The 1.2 kb fragment was reintroduced into the homology vector using restriction enzymes PmeI / BsiWI, resulting in pBS_SNV. After pBS_SNV was linearized with HindII, the neomycin selection cassette, consisting of a loxP_PGK/neo_loxP array, was introduced into intron 3, resulting in the SNV targeting vector. This vector was linearized by NotI digestion before electroporation.

To determine the insertion site of PPP1R26P1 into mouse Rb1, the distances between ECR A and the 5’ end of PPP1R26P1 and between the 3’ end of PPP1R26P1 and ECR B were calculated as proportion of the total intronic sequence between the two ECRs. The nucleotides representing this percentage in the mouse intron between the two ECRs were calculated. The targeting vector for the introduction of PPP1R26P1 contains a 6.6 kb homology fragment (UCSC browser, build NCBI37/mm9, chr14:73,707,316-73,713,900) generated by long-range PCR using the Phusion Flash high-fidelity PCR master mix (Thermo Scientific) and BAC bMQ311j15 as template. XhoI and NotI restriction sites were introduced with the long-range PCR primers. The fragment was ligated into the vector pACYC_D4 opened by XhoI and NotI and sequence-verified by Sanger sequencing, generating pACYC_D4_ECR. PPP1R26P1 was generated by long-range PCR using the Phusion Flash high-fidelity PCR master mix (Thermo Scientific) using BAC RPCIB753O20259Q (obtained from imaGenes) as template and introducing SwaI restriction sites for cloning into pACYC_D4_ECR and the introduction of the neomycin selection cassette. Integrity of PPP1R26P1 was verified by Sanger sequencing. PPP1R26P1 was introduced into the unique XcmI site of pACYC_D4_ECR. The selection cassette, consisting of a loxP511_PGKneo_loxP511 array, was introduced directly upstream of PPP1R26P1 using the EcoRV site introduced by the PCR forward primer. The targeting vector was linearized by NotI before electroporation.

Isolation of genomic DNA and RNA

Prior to isolation of genomic DNA and RNA of successfully targeted ES cell clones, ES cells were cultivated feeder-free on gelatinized dishes for three passages.

Genomic DNA was isolated by alkaline lysis using standard methods [33]. RNA was isolated using QIAzol (Qiagen) and the standard protocol provided by the manufacturer.

Southern blot and PFGE

Restriction enzyme digests for Southern blot were performed with HincII, HindIII or BamHI enzymes for detection by SNP and KIAA 5’ external probes. PstI was used for SNP and KIAA 3’ external and internal probes, and BamHI for the neo probe (all restriction enzymes from New England Biolabs). Southern blotting was performed according to standard procedures by capillary transfer [33]. Probes were radioactively labeled using the Megaprime kit (GE Healthcare) and blots were hybridized overnight and washed at 65°C with Church buffer. PCR primers for probe generation are listed in Table S2.

To analyze phasing of the two targeting events, pulsed-field gel electrophoresis (PFGE) using the CHEF Mapper XA system (BioRad) was applied. Genomic DNA was digested with EcoRV and resolved on a 0.8% TBE gel in 0.5x TBE. Settings were lower limit 15 kb, upper limit 35 kb and run time 27 h 18 min. After electrophoresis, Southern blotting and hybridization was done as described using the KIAA 5’ external probe.

Next generation bisulfite sequencing

Next generation bisulfite sequencing on the Roche 454 Junior system was carried out as described [34]. Briefly, bisulfite conversion of genomic DNA was performed using the EZ DNA Methylation-Gold Kit (Zymo Research) according to the manufacturer’s instructions. Converted DNA was amplified in a first round of PCR using tagged primers and the Qiagen HotStarTaq Master Mix (Qiagen). In a second round of PCR, sample-specific bar code sequences (MIDs – multiplex identifiers) and universal linker adapters for the 454 system were added using primers complementary to the first-round PCR tags. Amplicon libraries were purified, diluted, clonally amplified by emulsion PCR and sequenced on the Roche 454 GS junior system according to the manufacturer’s protocol.

Data analysis included stringent quality score filtering, sorting according to MIDs and separation of amplicons with same MIDs using the Geneious 6 software (Biomatters) and methylation analysis using the BiQAnalyzer HT (http://biq-analyzer-ht.bioinf.mpi-inf.mpg.de [35]). Raw data are available on request. The mean methylation over all CpGs analyzed in all amplicons in all clones studied, the number of analyzed reads and the bisulfite conversion rate are listed in Table S3. The bisulfite conversion rate was on average 96.3%, 96.6% and 88% for CpG146, CpG42 and CpG85, respectively.

RT-PCR and qPCR

RNA was digested with RNase-free DNase I RQ1 (Promega) and 5 µg were reverse transcribed into cDNA using the GeneAmp RNA PCR kit (Applied Biosystems) with random hexamers. PCR was performed using GoTaq polymerase (Promega) under following conditions: 95°C 2 min, 35 cycles: (95°C 30 sec, annealing temperature 58-62°C for 30 sec, 72°C 30 sec), 72°C 5 min, hold at 4°C. Amplification products were visualized by gel electrophoresis. Primer sequences are listed in Table S2.

Quantitative PCR was conducted on the LightCycler 480 II (Roche Applied Science) using the LightCycler480 probes master mix with corresponding primers and universal probe library (UPL) probes (Roche Applied Science). Primer / probe combinations are listed in Table S2. Cycling conditions were 96°C 1 min, 45 cycles: (96°C 10 sec, 60°C 15 sec, 72°C 1 sec), hold at 4°C.

SNaPshot analysis

SNaPshot analysis for determination of allelic ratios of mRNA (after reverse transcription into cDNA) and genomic DNA as reference was conducted using the ABI Prism SNaPshot ddNTP Primer extension kit (Applied Biosystems) according to the manufacturer’s instructions. The reaction products were analyzed on an ABI 3700 sequencer (Applied Biosystems) and electropherograms were analyzed using Gene Mapper 4.0 software (Applied Biosystems). Allelic ratios of cDNA were normalized to ratios obtained with genomic DNA from the same sample. Primer sequences are listed in Table S2. Two, two and six clones in two biological and two technical replicates each were included in the analysis for SNV / wt, SNV_PPP1R26P1/wt and SNV / PPP1R26P1, respectively.

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) was carried out on formaldehyde-fixed chromatin of modified and wildtype ES cells. Cells were harvested by trypsinization and adjusted to 1x10⁷ cells in 500 µl PBS, and crosslinked using formaldehyde at 1% final concentration for 10 min at room temperature with agitation. Glycine was added up to 0.125 M final concentration to stop crosslinking and cells were incubated at room temperature for 5 minutes with agitation. Cells were washed once with cold PBS and once with ChIP lysis buffer (20 mM Tris-Cl pH8.0, 85 mM KCl, 0.5% NP-40). Cell pellets were snap-frozen in liquid nitrogen and stored at -80°C until further use. Cell pellets were thawed on ice and resuspended in 1 ml ChIP lysis buffer, incubated for 10 min at 4°C with agitation. Cells were pelleted by centrifugation (250g, 5 min, 4°C) and resuspended in nuclei lysis buffer (50 mM Tris-Cl pH8.0, 10 mM EDTA, 1% SDS). Chromatin was sheared using the Covaris S220 instrument with 6x16mm microtubes (Covaris) at following conditions: duty cycle 5%, intensity 4, peak incident power 140 Watts, 200 cycles per burst, time 3 min. After shearing, samples were centrifuged (18,000g, 4°C, 10 min) to remove unsoluble debris. 20 µl of sheared chromatin were used for reverse crosslink and analysis of fragment size by gel electrophoresis. Fragment size ranged from 200 to 600 base pairs. For immunoprecipitation 50 µl of sheared chromatin was set aside as input and 20-40 µg of sheared chromatin in a volume of 100 µl was used for antibody incubation. The chromatin was diluted 1:10 with dilution buffer (20 mM Tris-Cl pH8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) and precleared with 50 µl of diluted Dynabeads Protein A (Life Technologies) for 1 h at 4°C with agitation. Before use, Dynabeads were washed three times with dilution buffer and blocked with 1 mg/ml BSA overnight. Per sample, 4 µg of antibody were added and incubated overnight at 4°C with agitation. Antibodies used were: rabbit polyclonal to PML (abcam #ab53773) as non-related control, rabbit polyclonal to RNAPII CTD repeat YSPTSPS (phospho S5) (abcam #5131 or Bethyl laboratories # A300-655A), rabbit polyclonal to RNAPII CTD repeat YSPTSPS (phospho S2) (abcam #5095). Antibody-bound chromatin was coupled to blocked Dynabeads for 4 h at 4°C with agitation. Afterwards, beads were washed once with 1 ml of each of the following buffers: low salt buffer (20 mM Tris-Cl pH8.0, 2 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100), high salt buffer (20 mM Tris-Cl pH8.0, 2 mM EDTA, 500 mM NaCl, 0.1% SDS, 1% Triton X-100), LiCl buffer (20 mM Tris-Cl pH8.0, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% Sodiumdeoxycholate), and twice with TE buffer (10 mM Tris pH8.0, 1 mM EDTA). Each cycle of washing was conducted for 10 min at 4°C with agitation. After washing, beads were resuspended in 200 µl elution buffer (50 mM Tris-Cl pH8.0, 10 mM EDTA, 1% SDS) and incubated for 45 min at 65°C at 700 rpm in a thermomixer (Eppendorf). The input sample was diluted with elution buffer up to 200 µl final volume. Afterwards, supernatant was transferred to a new tube and 10 µl of proteinase K (10 mg/ml) was added to precipitated samples and input for incubation overnight at 55°C and 300 rpm in a thermomixer. Then 1 µl of RNase A (10 mg/ml) was added and samples were incubated at 37°C for 1 h. Samples were extracted with the MinElute reaction cleanup kit (Qiagen) and eluted in 20 µl buffer. qPCR analysis was performed using 1 µl of eluted chromatin and input (diluted 1/20 before) as template. Primer/probe combinations are listed in Table S2. Results are calculated and represented as % input.