2. Materials and Methods
2.2 Methods
2.2.1 Methods of Cell Biology
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bacterial culture dishes to avoid attachment of the EBs and the medium was replaced every four days.
2.2.1.5 Replating of EBs
For replating of cells from late EBs, EBs were washed three times with PBS and dissociated to single cells by repeated cycles of Accutase treatment at 37°C. The reaction was stopped by adding fresh EB medium (ES cell medium without LIF) to the dissociated cells. After centrifugation, dissociated cells were resuspended in fresh EB medium and equal numbers of cells were seeded in duplicate on gelatin- coated plates and LIF was added to one of the duplicate plates to a final concentration of 1000 U/ ml.
2.2.1.6 Transfection of plasmids
For transient reporter assays of oct4 dTALEs, HEK293T cells were transfected with polyethylenimine. Transfections of ogESCs and ogNSCs were carried out using Lipofectamine 2000 according to the manufacturer´s instructions.
2.2.1.7 Transient knock- down using small interfering RNAs (siRNAs)
For silencing of uhrf2 in ESCs, two different siRNAs targeting uhrf2 as well as a pool of non-targeting siRNA were used to control for off- target effects. Cells were plated at a density of 6-7*104 cells/ cm2 in normal ESC medium. After approximately one hour, siRNA diluted in OptiMEM with a final concentration of 50 nM was transfected using Lipofectamine 2000 according to the manufacturer´s conditions. Medium was changed after 24 hours and cells were split and transfected every second day for a total of 8-10 days. To monitor for knock-down efficiency, residual cells were harvested and lysed in RP1 buffer of the Triprep kit every second day. Lysates were stored at -80°C until the end of the experiments, so that all samples could be processed simultaneously.
2.2.1.8 Treatment of ogNSCs with epigenetic inhibitors
Valproic acid sodium salt (VPA) and 5-aza-2'-deoxycytidine (5-azadC) was dissolved in PBS at a concentration of 250 mM and 30 mM, respectively and sterile filtered. Trichostatin A (TSA) was dissolved in DMSO at a concentration of 5 mM. Prior to treatment with epigenetic inhibitors, ogNSCs were transfected with the T-83 construct (for details see 2.2.1.6) and after 12 hours, medium was exchanged with medium containing dilutions of the respective inhibitor or a combination thereof and cells were cultured for additional 36 hours. Final concentrations of the inhibitors were for TSA 30 nM, VPA 620 µM, 5-azadC 10 nM and for the combination of VPA 310 µM + 5-azadC 5 nM.
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To stain intracellular Oct4 protein levels during differentiation, EBs were washed twice in PBS and incubated in Cell Dissociation Buffer for 45 min at 37°C. An equal volume of EB medium was added and EBs were dissociated to single cells by vortexing. To distinguish dead from living cells, 7- amino- actinomycin D (7- AAD) was added at a concentration of 5 µg/ ml for 20 min at 4°C. After multiple washing steps in PBS, cells were fixed in Cytofix for 20 min at 4°C and washed twice in Perm/ Wash solution. Antibody staining was performed in Perm/ Wash buffer for 30 min at 4°C using Alexa Flour® 647 Mouse anti- Oct3/4 and Alexa Flour® 647 Mouse IgG1 K isotype control. After washing twice with Perm/ Wash buffer, cells were resuspended in Annexin buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) and analyzed with a FACS Aria II instrument. Data analysis was performed using FlowJo version 7.2.5.
2.2.1.10 Serum starvation of fibroblasts
To analyse whether uhrf2 shows an expression pattern that is dependent on the proliferative state of the cells, NIH3T3 cells were plated to about 60 % confluency one day before starvation. After washing twice with PBS, cells were cultured in starvation medium containing 0.5 % FBS and 1 % Gentamycin for 48 hours. Cells were released from starvation by adding fresh medium and samples were taken every 3, 6, 9, 12 and 24 hours after serum addition.
2.2.1.11 Analysis of cell cycle profile using FACS
After trypsinization and resuspension of the cell pellet in PBS, cells were fixed by slowly adding 100 % Ethanol while simultaneously vortexing to ensure homogenous fixation of the cells. Cells were incubated on ice for 15 min, washed twice in PBS and incubated in PBS containing 50 µg/ ml Propidium Iodide (PI), 0.1 mg/ ml RNAse A and 0.05 % Triton X-100 for 30 min at 37°C. Cells were then analyzed using a FACS Aria II instrument and data analysis was performed using FlowJo version 7.2.5.
2.2.1.12 Immunofluorescence staining
Immunostaining of Oct4 protein in ogNSCs was performed by Sebastian Bultmann. In brief, ogNSCs were grown on cover slips, transiently transfected with the oct4- dTALE T-83, fixed with 2 % paraformaldehyde in PBS and permeabilized in PBS containing 0.2 % Triton X-100.
Oct4 was stained using a primary goat- anti- Oct4 antibody (dilution 1:1000) and a secondary rabbit- anti- goat antibody coupled to Alexa Fluor 647 (dilution 1:2000). The antibodies were diluted in PBS containing 0.02 % Tween 20 and 2 % BSA. Cells were counterstained with DAPI and mounted in Vectashield (Vector Laboratories) Images were acquired with a Zeiss Axioplan 2 fluorescence microscope equipped with a Plan-NEOFLUAR 40x/1,3 oil objective.
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2.2.2.1 RNA extraction
Total RNA from cultured cells, EBs and tissue samples from 6 weeks old 129sv mice were isolated with TRIzol reagent or the NucleoSpin Triprep Kit according to the manufacturer´s instructions. To avoid genomic DNA contamination, isolated RNA was digested with recombinant RNase- free DNase I and further purified with the QIAGEN RNeasy kit. RNA concentration and purity was determined using a NanoVue spectrophotometer. Quality of RNA was assessed by agarose gel electrophoresis and RNA was stored at -20°C - -80C°.
2.2.2.2 cDNA synthesis
500 ng total RNA were used for cDNA synthesis with the High- Capacity cDNA Reverse Transcription Kit with RNase Inhibitor according to the manufacturer´s instructions and using random hexamer. Samples without reverse transcriptase were prepared in parallel to control for genomic DNA contamination. cDNA was stored at -20°C.
2.2.2.3 Quantification of mRNA levels using Real- time PCR
Transcript levels of genes of interest were quantified relative to transcript levels of stably expressed housekeeping genes with quantitative PCR using a 7500 Fast Real- Time PCR cycler with a 96 well block. For quantification, either TaqMan probes in combination with TaqMan Gene expression Master Mix or Primers designed with Primer Express were used together with the Power SYBR Green PCR Master Mix. TaqMan gene expression assay IDs and Primer sequences are listed in 2.1.5.1 and 2.1.5.2, respectively. Each sample was analyzed in technical triplicates for each primer set with a total volume of 10 µl. In every reaction, equal amounts of cDNA were diluted 1:25 – 1:50 and final concentrations for each SYBR green primer pair were 500 nM. TaqMan assays were used as recommended by the manufacturer. All primer pairs were tested for specificity and efficiency by using non template controls, melting curve analysis (in the case of SYBR green primers after each run), agarose gel electrophoresis and dilution series of cDNAs. The following program was used for amplification:
Pre-incubation 2min 50°C
Enzyme activation 10min 95°C
15sec 95°C
40x cycles of amplification 1min 60°C
5sec 95°C
Melting curve 1min 60°C
2.5°C/ min 95°C
Gene expression levels were normalized to gapdh and calculated with the sequence detection system software v1.3 using the comparative CT method (∆∆CT method).
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2.2.2.4 Genome- wide expression profiling using Microarrays
Microarray hybridization was carried out by our collaborators Dietmar Martin and Kerstin Maier at the Gene Center Affymetrix Microarray Platform (Laboratory of Patrick Cramer, LMU Munich). Sample preparation for microarray analysis was performed using the WT Expression Kit and WT Terminal Labeling and Controls Kit with 300 ng input RNA. Samples were hybridized to GeneChip Mouse Gene 1.0 ST microarrays according to the manufacturer’s instructions. Bioinformatical analysis including Principal Component Analysis (PCA) was performed by out collaborators Benedikt Zacher and Achim Tresch (Laboratory of Achim Tresch, LMU Munich). Quality control, normalization and further statistical analyses were carried out using the R/Bioconductor programming environment. Linear Models for Microarray Data (limma) was used to compute fold changes and p-values (Smyth, 2005).
Genes with a fold change of 2 and a false discovery rate below 0.05 were considered differentially expressed. These genes are listed in the appendix (chapter 6). A one-sided Kolmogorov-Smirnov test was used to compare gene expression changes in wt and knockout EBs globally as well as for bivalent and non-bivalent genes between day 0 and 4 of differentiation. I performed Gene ontology (GO), tissue expression and chromosomal location analysis using DAVID (Huang et al., 2008, 2009). For GO enrichment analysis the lowest level of GO categories for biological process (GO_BP_ALL) was used. To identify significantly enriched GO categories, the corrected p-value calculated according to the Benjamini- Hochberg method was used for multiple testing corrections. Microarray data have been deposited into the GEO database under the accession number GSE36679.
2.2.2.5 DNA extraction
Genomic DNA from ESCs, EBs and tissue samples from 6 weeks old 129sv mice were extracted using either the NucleoSpin Triprep Kit or the QIAmp DNA Mini Kit according to the manufacturer´s instructions. Shearing of fragments and concentration measurements with Hoechst was done by Sebastian Bultmann. Isolated genomic DNA samples were sheared to 500 - 1500 bp fragments by sonication to reduce the viscosity and improve homogeneity and the concentration was measured by fluorometry. 50 µl of diluted sample were mixed with 50 µl of 2x TNE (Tris 20 mM, pH 7.4; NaCl 400 mM and EDTA 2mM) containing 200 ng/ µl of Hoechst 33258 and fluorescence was measured in a TECAN infinite M1000 plate reader (Ex:
350/10; Em: 455/10). Serial dilutions ranging from 20 to 2000 ng/ ml of the 5hmC containing reference DNA fragment (see below) were used as standard for quantification.
2.2.2.6 Preparation of reference DNA fragments for 5hmC glucosylation assay
Reference DNA fragments (1139 bp) containing 0 % and 100 % 5hmC were prepared by PCR, using dCTP and 5-hydroxymethyl- dCTP, respectively. As a template, T4 phage DNA
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was used with the following primers: 5’-TGG AGA AGG AGA ATG AAG AAT AAT-3’ and 5’-GTG AAG TAA GTA ATA AAT GGA TTG-3’, Phusion HF DNA Polymerase and the following cycling profile: one cycle of 98°C, 2 min and 35 cycles of 98°C, 10s; 58°C, 10s;
72°C, 30s. Note that the Primer sequences do not contain any cytosine residues. PCR products were purified by gel electrophoresis and silica column purification.
2.2.2.7 Quantitative 5hmC glucosylation assay
Assays for quantification of 5hmC glucosylation were performed by Alexandra Szwagierczak and Sebastian Bultmann. Reactions contained 150 mM NaCl, 20 mM Tris, pH 8.0, 25 mM CaCl2, 1 mM DTT, 2.8 μM “cold” UDP-glucose, 0.86 nM UDP- [3H]glucose (glucose-6-3H; 60 Ci / mmol;), 1 µg of DNA substrate and 36 nM recombinant β- gt in a total volume of 50 µl.
After incubation for 20 min at RT, reactions were terminated by heating at 65°C for 10 min.
From each reaction, 20 µl were spotted in duplicate on paper filters and DNA was precipitated by incubation in 5 % TCA for 15 min at RT. Afterwards, filters were washed twice with 5 % TCA and once with 70 % ethanol. Remaining radioactivity was measured using a Liquid Scintillation Analyzer Tri- Carb 2100TR with quench indicating parameter set on tSIE/
AEC (transformed spectral index of the external standard / automatic efficiency control) in 4 ml of Rotiszint Eco Plus scintillation liquid in snaptwist vials. Samples were measured for 30 min or until the 2 σ value reached 2 %. To calculate the percentage of 5hmC per total cytosine from the incorporation of [3H]glucose, a calibration curve was used which was measured with the reference fragments for every experiment. The percentage of 5hmC was then corrected for the difference in C abundance between reference fragment (35 %) and mouse genome (42 %).
2.2.2.8 Preparation of reference DNA fragments for testing PvuRts1I specificity
Reference DNA fragments containing exclusively 5hmC, 5mC or unmodified cytosine residues were prepared by PCR using 5-hydroxymethyl- dCTP, 5-methyl- and dCTP, respectively. As a template for all reference fragments, T4 phage DNA was used for PCR amplification by Phusion HF DNA Polymerase and primer 5’-GTG AAG TAA GTA ATA AAT GGA TTG-3’, which does not contain cytosine residues. To generate the reference 1139 bp fragment with 100 % 5hmC for restriction with PvuRts1I the second primer was 5’-TGG AGA AGG AGA ATG AAG AAT AAT- 3’, which also does not contain cytosine residues. To generate the 800 and 500 bp control substrates containing only 5mC and only unmodified cytosine the second primer was 5’-GCC ATA TTG ATA ATG AAA TTA AAT GTA-3’ and 5’-TCA GCA ATT TTA ATA TTT CCA TCT TC-3’, respectively. After PCR amplification, products were purified by gel electrophoresis followed by silica column purification.
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2.2.2.9 Preparation of DNA substrates for Linker – PvuRTS1I – analysis
To prepare substrates containing different 5hmC levels (0 %, 1 %, 2,5 %, 5 %, 10 %), genomic DNA from wt JM8A3.N1 was used as a template to amplify the Region III of the nanog promoter (Hattori et al., 2007) by PCR using Phusion HF DNA Polymerase (Finnzymes) and the following primers: For 5´ TCA GGA GTT TGG GAC CAG CTA 3´ and Rev 5´CCC CCC TCA AGC CTC CTA 3´, resulting in a 867 bp fragment. To generate nanog promoter substrates containing increasing 5hmC levels, 5-hydroxymethyl- dCTP and dCTP were added to the PCR reactions at appropriate ratios and PCR products were purified by silica column purification. Successful incorporation of 5hmC levels was confirmed by the quantificative 5hmC glucosylation assay. The radioactive assay was conducted by Sebastian Bultmann.
2.2.2.10 Preparation of linker
A linker containing a random 3´overhang was generated by annealing the following primer:
For 5´CTC GTA GAC TGC GTA CCA TG NN 3´ and Rev 5´CA TGG TAC GCA GTC TAC CAG 3´. Successful annealing of oligos was checked by loading 100 ng of each oligo (annealed and unannealed) together with the loading dye xylenol mixed with bromphenol blue (ratio 1:1) on a 15 % non- denaturating PAGE. Ultra low range marker was used to control for size, gel was stained with SYBR green (1:1000) for 20 mins and scanned on a Typhoon imaging system using filter sets for excitation at 488 nm and emission of 520 nm.
2.2.2.11 Digestion with PvuRts1I
Sheared genomic DNA of wt and TKO ESCs or 250 ng of each fragment with increasing 5hmC content were digested with 2 U of PvuRts1I in reaction buffer containing 150 mM NaCl, 20 mM Tris, pH 8.0, 5 mM MgCl2, 1 mM DTT for 15 min at 22°C followed by a heat inactivation of 20 min at 60°C. To control for successful digestion, reference substrates (see 2.2.2.8) were digested using the same conditions and analyzed on a 1 % agarose gel electrophoresis.
2.2.2.12 Detection of PvuRts1I digested fragments using qPCR
10 ng of digested or mock treated genomic DNA was amplified by qPCR using two different primer pairs specific for the nanog upstream regulatory region at standard cycling conditions (see 2.1.5.3). To ensure that equal amounts of gDNA were added to each well, a primer pair specifically amplifying the oct4 locus was used to correct for pipetting mistakes. Each sample was analyzed in technical triplates and amplification of single products was tested by melting curve analysis after each run.
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2.2.2.12 Detection of PvuRts1I digested fragments using linker - PCR strategy
25 ng of each digested fragment was ligated to a linker containing the random 3´ overhang.
The ligation reaction was carried out using T4 DNA Ligase overnight at 16°C. As a control for the specificity of the ligation, each fragment was Mock- ligated, where no linker was added to the reaction. To selectively amplify fragments cut by PvuRts1I, the ligated products were amplified by PCR with Phusion HF DNA Polymerase using a Linker- specific Forward Primer (For 5´CTC GTA GAC TGC GTA CCA TG 3´) and gene- specific Revers Primers (PP1 Rev:
5´ GAG TCA GAC CTT GCT GCC AAA 3´, and PP2 Rev 5´ GCC GTC TAA GCA ATG GAA GAA 3´), resulting in different fragment sizes depending on the cutting site. The PCR pattern was analyzed on a 2 % agarose gel and a library of the 10 % 5hmC fragments was generated using the Zero Blunt® PCR Cloning Kit. Randomly selected clones were sequenced and analyzed for potential cutting sites.
2.2.2.13 DNA methylation analysis
Bisulfite conversion of genomic DNA (0.5 - 1.5 µg) was carried out using the EZ DNA Methylation-Gold Kit according to the manufacturer´s instructions. For amplification we used Qiagen Hot Start Polymerase in 1x Qiagen Hot Start Polymerase buffer supplemented with 0.2 mM dNTPs, 0.2 µM forward primer, 0.2 µM reverse primer, 1.3 mM betaine and 60 mM tetramethylammonium-chloride. Primer sequences (listed in 2.1.5.4 and 2.1.5.5) were designed using MethPrimer (Li and Dahiya, 2002) and CpG islands were identified with the CpG Island Searcher using default settings (Takai and Jones, 2002). DNA methylation analysis by pyrosequencing was carried out by Varionostic GmbH (Ulm, Germany). For bisulfite sequencing, PCR products were subcloned using the StrataCloneTM PCR Cloning Kit and StrataConeTM SoloPack® Competent Cells according to the manufacturer´s instructions.
Sequences were analyzed with BISMA software using default settings (Rohde et al., 2010).
2.2.2.14 Cloning of oct4 dTALEs
Cloning of oct4 dTALEs was performed by our collaboration partner Robert Morbitzer (group of Thomas Lahaye, LMU Munich). A Gateway cassette from pGWB5 (Nakagawa et al., 2007b) was amplified (Forward primer 5´GGG GCG ATC GCA CAA GTT TGT ACA AAA AAG CTG AAC GAG 3´; reverse primer 5´ GGG GCG GCC GCA ACC ACT TTG TAC AAG AAA GCT GAA CG 3´), thereby adding AsiSI and NotI restriction sites and subcloned into pCAG_mCH (Niwa et al., 1991) using AsiSI and NotI, generating pCAG_mCH_GW. The VP16AD was amplified from RSV E2F1-VP16 using the following primers: Forward primer 5´
GGG GGT CTC TCA CCA TGG ATC CTG CCC CCC CGA CCG ATG TCA GC 3, Reverse primer 5´ GGG GGT CTC CCT TCT ACC CAC CGT ACT CGT CAA TTC CAA GG 3´ (Johnson et al., 1994), thereby adding a BamH1 restriction site to the 5' end and
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cloned into pENTR-D-TOPO generating pENTR-D-BamHI_VP16AD. TALE repeat arrays were generated via multi-fragment cut-ligation using golden gate cloning (Engler et al., 2009) and ligated either into pENTR-D-TALE-∆rep-BpiI-AC or pENTR-D-TALE-∆rep-BpiI-AC-VP16AD. All entry clones were transferred by LR recombination into the expression vector pCAG_mCh_GW.
2.2.2.15 Cloning of oct4 reporter contruct
Cloning of the oct4 reporter construct was performed by Sebastian Bultmann. The vector GOF-18 (Yeom et al., 1996) was cut by XhoI/ AvrII, resulting in the basepairs -1 to -4716 upstream of the transcriptional start site of oct4, which was ligated into pGL-3 basic together with a linker oligo (5´ CCT AGG TGA GCC GTC TTT CCA CCA GGC CCC CGG CTC GGG GTG CGA TCG CCG CCC ATG G 3´) using XhoI/ NcoI. Subsequently, the luciferase ORF was removed by with KasI/ FseI and replaced by the eGFP ORF (Forward primer 5´ AAA GGC GCC AGT GAG CAA GGG CG 3´, reverse primer 5´. AAA GGC CGG CCT TAC TTG TAC AGC TCG TCC 3´).