HeLa cells (Epitheloid carcinoma, cervix) and MEFs were cultivated in 4.5 g/l glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco) at 37°C and 5% CO2
. For SILAC labelling, HeLa cells were incubated in RPMI 1640 (–Arg, –Lys) medium containing 10% dialysed fetal bovine serum (Gibco) supplemented with 84 mg/l 13
arginine and 50 mg/l 13
lysine (Sigma Isotec or Euriso-top) or the corresponding nonlabelled amino acids, respectively. R1/E murine ES cells were also grown in 4.5 g/l GlutaMAX DMEM containing sodium pyruvate, but supplemented with 20% FBS, 50 μM 2-mercaptoethanol (Invitrogen), LIF (MPI-CBG protein expression facility), as well as 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco). For SILAC labelling of R1/E cells, DMEM (–Arg, –Lys) medium containing 20% dialysed FBS (Gibco) supplemented with 40 mg/l 13
arginine and 80 mg/l 13
lysine (Sigma Isotec or Euris-top) or the corresponding nonlabelled amino acids, respectively, was used. Cells were collected and nuclear extracts were prepared as described (Butter et al, 2010
Chemically synthesized oligonucleotides (; Metabion) were annealed and polymerized by T4 ligase (Fermentas) and biotinylated with biotin–dATP (Invitrogen) by Klenow fragment (Fermentas) following the manufacturer's instructions. Twenty-five microgram baits were immobilized on 750 μg paramagnetic streptavidin beads (Dynabeads MyOne C1, Invitrogen) and subsequently incubated with either 400 μg of SILAC-labelled nuclear extract or 20 μl of supernatants of crude E. coli
lysates (3 μg/ml) in PBB buffer (150 mM NaCl, 50 mM Tris–HCl pH 8.0, 10 mM MgCl2
, 0.5% NP-40, Complete Protease Inhibitor without EDTA (Roche)) for 2 h at 4°C on a rotation wheel. After three washes with PBB buffer bead fractions were pooled, bound proteins boiled at 80°C in 1 × LDS buffer (Invitrogen) and separated on a 4–12% gradient gel (Novex, Invitrogen; Butter et al, 2010
Oligonucleotides used for pull-down experiments
MS data acquisition
In-gel digestion and MS analysis was performed essentially as previously described by Butter et al, 2010
. Peptides were desalted on StageTips and analysed by nanoflow liquid chromatography on an EASY-nLC system from Proxeon Biosystems coupled to a LTQ-Orbitrap XL or a Orbitrap-Velos (Thermo Electron). Peptides were separated on a C18-reversed phase column (15 cm long, 75 μm inner diameter, packed in-house with ReproSil-Pur C18-AQ 3 μm resin (provided by Dr Maisch)) directly mounted on the electrospray ion source. We used a 107-min gradient from 2 to 60% acetonitrile in 0.5% acetic acid at a flow of 200 nl/min. The LTQ-Orbitrap XL was operated with a Top5 MS/MS spectra acquisition method in the linear ion trap per MS full scan in the Orbitrap, while for the Orbitrap-Velos a Top10 acquisition method with HCD fragmentation was used.
MS spectrum and data analysis
The raw files were processed with MaxQuant (Cox and Mann, 2008
) and searched with the Mascot search engine (Matrix Science) against IPI human v3.37 protein database concatenated with a decoy of the reversed sequences. Carbamidomethylation was set as fixed modification, while methionine oxidation and protein N-acetylation were considered as variable modifications. The search was performed with an initial mass tolerance of 7 p.p.m. mass accuracy for the precursor ion and 0.5 Da for the MS/MS spectra obtained with CID fragmentation and 20 p.p.m. for the MS/MS spectra in the HCD fragmentation mode. Search results were processed with MaxQuant filtered with a false-discovery rate of 0.01. Before statistical analysis, known contaminants and reverse hits were removed. The protein ratios of a ‘forward' experiment and the ‘reverse' experiment were plotted in R (prerelease version 2.8.0). Only proteins identified with at least two unique peptides and two quantitation events were plotted for the telomere pull-down (quality filter).
Recombinant protein expression and deletion variant construction
The HOT1 clone was obtained from the ORFeome collection (IOH40784, Invitrogen). The sequence was subcloned into SLIC-compatible pETM44 or pETM14 vector via SLIC cloning (Li and Elledge, 2007
; Scholz et al, 2013
) and was expressed in E. coli
Rosetta at 18°C. E. coli
extracts with overexpressed recombinant proteins were used for binding studies on variant DNA motives. For crystallization, protein was purified under nondenaturing conditions using Ni2+
sepharose high-performance columns (GE Healthcare) followed by size-exclusion chromatography on Superdex 75 (GE Healthcare), using 150 mM NaCl and 20 mM Tris–HCl (pH 7.5) as a running buffer.
The HOT1 ORFeome clone was LR recombined into a gateway compatible pcDNA3.1 vector with N-terminal FLAG-tag (gift of Christian Brandts). A FLAG-tagged HOT1 homeobox deletion variant was constructed by PCR amplification of the pcDNA3.1-FLAG vector using primers with site-specific overhangs (; Metabion) following the QuickChange II Site-directed mutagenesis kit protocol (Stratagene; Zheng et al, 2004
). Point mutants were generated similarly using the QuickChange protocol. For expression in ES cells, constructs were subcloned into a pCAGGS vector. The constructs were sequence verified using an Applied Biosystems 3730 Genetic Analyzer (Applied Biosystems), according to the manufacturer's instructions.
Oligonucleotides used for QuickChange
Crystallization and structure determination
The complex of human HOT1 DBD and double-stranded telomeric DNA (5′-TTAGGGTTAGGGTTA-3′) was obtained as follows: synthetic single-strand complementary oligonucleotides (; Eurofins MWG) were dissolved in water and mixed to yield a 5-mM stock solution. For double-strand annealing, this solution was heated to 96°C for 10 min and allowed to cool down to room temperature (RT) in the switched-off heat block within 2–3 h. This solution was mixed in a 1–1.2 M excess with a solution of purified HOT1 DBD at 10 mg/ml and incubated on ice for 10 min. Crystals of the complex were grown at 4°C by sitting-drop vapour diffusion from drops formed by mixing equal volumes of the complex solution and the crystallization condition 24% PEG 3350, 100 mM Na Acetate (pH 4.5) and 100 mM KSO4
. For data collection, crystals were cryoprotected by soaking in a mother liquor supplemented with 20% glycerol and was flash-frozen in liquid nitrogen. Diffraction data were collected at the PXII beam line of the Swiss Light Source (SLS; Villigen, Switzerland) and processed using XDS (Kabsch, 1993
). The structure was solved by molecular replacement with the programme PHASER (McCoy et al, 2007
), using the NMR structure of the HOT1 DBD as a search model (PDB entry 2CUF). Model building and refinement was carried out with Phenix (Afonine et al, 2005
) and COOT (Emsley and Cowtan, 2004
), and models were validated with the programme Molprobity (Davis et al, 2007
Oligonucleotides used for crystallization
The alignment of the HOT1, TRF1 and TRF2 homeobox domain sequences was done in two steps. First, a secondary structure-based alignment was generated using the programme Chimera (www.cgl.ucsf.edu/chimera
) followed by feeding it into the web application Espript (espript.ibcp.fr) for a simple sequence alignment scoring and colouring.
His-MBP-tagged HOT1 (pETM44 vector construct) was expressed in E. coli Rosetta at 18°C in a fermentation tank. Cell pellet was lysed with Avestin and the soluble fraction subjected to affinity purification using Ni-sepharose. The elution fraction was concentrated with an Amicon Ultra 15 concentrator column and dialysed into buffer containing 50 mM K2PO3, 20 mM NaCl, 10% glycerol, 1 mM TCEP and protein inhibitors. Purified MBP-HOT1 was injected into rabbits for immunization and the rabbits were ultimately sacrificed. The antibody was affinity purified using His-MBP-HOT1 and MBP immobilized on HiTRAP desalting columns (GE Healthcare). First, the serum was applied to the His-MBP-HOT1 column, eluted and then applied to the MBP column. The flow trough was quantified and used for subsequent experiments.
Purified MBP-HOT1 was injected into mice for immunization and the mice were ultimately sacrificed. Immortalized hybrid cells (hybridomas) were obtained by the fusion of B cells from the spleen of an immunized mouse with a myeloma cell line, which itself does not produce antibodies, using PEG and AH selection (Sigma Aldrich). Hybridoma clones were generated and screened using the Meso Scale Discovery platform (Meso Scale Diagnostics) by comparing affinity to His-MBP-HOT1 and His-MBP-Katanin as an unspecific negative control. Positive clones were subcloned by limiting dilution and retested using the MSD platform. Based on the subcloned hybridoma cell lines, antibodies were purified using HiTRAP protein G columns (GE Healthcare) followed by acid elution.
Cells were crosslinked with 1% formaldehyde (Thermo Scientific) for 10 min at 37°C, and the reaction was stopped by adding glycine to a final concentration of 0.125 M for 5 min at RT. Nuclei were prepared from fixed and washed cells by homogenization in cell lysis buffer (5 mM PIPES pH 8, 85 mM KCl, 0.5% NP-40) and centrifugation at 1800 g for 10 min. Finally, nuclei were lysed in 900 μl nuclei lysis buffer (50 mM Tris–HCl pH 8, 10 mM EDTA pH 8, 1% SDS) and lysates were sonicated for 30 min (30 s on/30 s off) in a Diagenode water-bath sonicator at speed 5. Following a centrifugation at 14 000 r.p.m. for 10 min, the cleared supernatants were snap-frozen in liquid nitrogen and stored at −80°C. Sonication efficiency was routinely monitored by DNA gel electrophoresis to ensure that the bulk of DNA fragments was between 100 and 500 bp.
Sonicated chromatin containing 50 μg DNA was diluted 10 times in ChIP dilution buffer (16.7 mM Tris–HCl pH 8, 167 mM NaCl, 1.2 mM EDTA pH 8, 1.1% Triton X-100, 0.01% SDS) and precleared for 2 h, rotating at 4°C, with 20 μl blocked beads (Dynabeads Protein A beads (Invitrogen) incubated for 2 h with 5 mg/ml BSA) before the overnight incubation with 5 μg of antibody. The following antibodies were used: mouse anti-HOT1 (MPI-CBG Antibody Facility), rabbit anti-TRF2 (NB110-57130, Novus), rabbit IgG (ab37415, Abcam) or mouse anti-GFP (ab1218, Abcam)). The bound material was recovered after a 2 h incubation, rotating at 4°C, with 30 μl blocked beads. The beads were washed for 10 min in each of the following wash buffers: low-salt buffer (20 mM Tris–HCl pH 8, 150 mM NaCl, 2 mM EDTA pH 8, 1% Triton X-100, 0.1% SDS), high-salt buffer (20 mM Tris–HCl pH 8, 500 mM NaCl, 2 mM EDTA pH 8, 1% Triton X-100, 0.1% SDS), LiCl buffer (10 mM Tris–HCl pH 8, 0.25 M LiCl, 1 mM EDTA pH 8, 1% NP-40, 1% Na deoxycholate) and twice, 5 min each, in TE. ChIPed material was eluted by two 15 min incubations at RT with 225 μl elution buffer (0.1 M NaHCO3, 1% SDS). Chromatin was reverse-crosslinked by adding 18 μl of 5 M NaCl and incubated overnight at 65°C, and DNA was submitted to RNase and proteinase K digestion and extracted by phenol–chloroform.
Purified DNA recovered by ChIP was denatured in 0.2 M NaOH by heating to 100°C for 10 min and spotted onto a positively charged Biodyne B nylon membrane (Pall, VWR). Membranes were hybridized at 42°C in 6 × SSC, 0.01% SDS, 0.1% milk with 20 pmol of DIG-labelled telomeric C-rich LNA probe (36 bp, Exiqon). Following hybridization washes (twice 5 min in 2 × SSC, 0.01% SDS, and once 2 min in 0.1 × SSC, 0.01% SDS), the signal was revealed using the anti-DIG-AP antibodies (Roche) and CDP-Star (Roche) following the manufacturer's instructions. Images were obtained using the Luminescent image analyser LAS-4000 mini (GE Healthcare). Following the detection of a telomeric signal, membranes were stripped twice for 15 min each in 0.5% SDS at 60°C and twice for 15 min each in 0.2 N NaOH, 0.1% SDS at 37°C. Stripped membranes were then hybridized at 65°C in 6 × SSC, 0.01% SDS, 0.1% milk with P32-random-primed labelled total genomic DNA from HeLa cells. Following hybridization washes (10 min in 2 × SSC, 0.01% SDS, 10 min in 0.5 × SSC, 0.01% SDS and 10 min in 0.1 × SSC, 0.01% SDS) the signal was revealed using the phosphorimager and an image obtained by the Typhoon scanner.
Immunofluorescence and immunoFISH stainings on cycling cells
For immunofluorescence stainings, cells were seeded 24 h before the treatment on either glass coverslips (0.17 mm, assorted glass, Thermo Scientific) or in LabTek II chambered coverglass chambers (Labtek). After a brief wash with 1x PBS cells were fixed in 10% formalin solution (Sigma Aldrich) for 10 min at RT, followed by two washes with 1 × PBS+30 mM glycine. Cells were then permeabilized with 1 × PBS+0.5% Triton X-100 for 5 min at 4°C, followed by again two washes with 1 × PBS+30 mM glycine. Afterwards, cells were blocked in blocking solution (1 × PBS, 0.2% fish-skin gelatine (Sigma Aldrich)) for 15 min at RT. Primary antibodies were diluted in blocking solution and incubated for 1 h at RT. The following primary antibodies were used: mouse anti-HOT1 (MPI-CBG Antibody Facility, 1:1000), rabbit anti-Coilin (sc-32860, Santa Cruz, 1:500), and goat anti-GFP (MPI-CBG Antibody Facility, 1:2000). Cells were washed three times for 3 min each in blocking solution followed by a 30-min incubation at RT with secondary antibodies, which were diluted in blocking solution. As secondary antibodies, fluorescent-labelled donkey anti-rabbit-IgG or donkey anti-mouse-IgG antibodies with either Alexa488, Alexa555, Alexa594 or Alexa647 as fluorochromes (1:500, Invitrogen) were used. After three final washes for 3 min each in blocking solution, slides were briefly rinsed in distilled water and mounted using DAPI Prolong Gold Antifade Reagent (Invitrogen). In the case of Labtek chambers, samples were incubated for 5 min with blocking solution containing 1 μg/ml DAPI, followed by one wash in blocking solution.
If FISH stainings were combined with immunofluorescence stainings (immunoFISH), the FISH labelling was carried out directly after the IF protocol. Cells were post-fixed in 10% formalin solution (Sigma Aldrich) for 10 min at RT, followed by three washes in 70, 90 and 100% ethanol for 5 min each, followed by a standard telomeric FISH protocol as previously described.
All images were acquired with a DeltaVision Core Microscope (Applied Precision, Olympus IX71 microscope) using a 100 × /1.4 UPlanSApo oil-immersion objective. Z-stacks (0.2 μm optical sections) were collected and deconvolved using softWoRx (Applied Precision). Z-stacks were reconstructed in 3D using Imaris (Bitplane), and colocalization events were determined for signals above the background using the colocalization function.
The detailed protocol of esiRNA production has been previously published (Kittler et al, 2005
). Briefly, optimal regions for designing esiRNAs were chosen using the Deqor design algorithm (Henschel et al, 2004
) in order to fulfill two criteria: to obtain the most efficient silencing trigger in terms of silencing efficiency, and to get lowest chances to cross-silence other genes. The most favourable fragments were used to design gene-specific primers () using the Primer3 algorithm (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi
). Two esiRNAs for HOT1 and one each for TRF1 and TCAB1 were designed and synthesized. PCR products for the esiRNA production were sequenced using an Applied Biosystems 3730 Genetic Analyzer (Applied Biosystems) according to the manufacturer's instructions. All positions of sequence trace files were confirmed by manual inspection.
Oligonucleotides used for esiRNA production
esiRNA and plasmid transfection
For all transfections, HeLa cells were seeded in six-well plates (40 000 cells per well corresponding to 9.6 cm2) dish and incubated overnight before transfection. For esiRNA transfection, 30 μl Oligofectamine (Invitrogen) were diluted in 250 μl OptiMEM (Invitrogen) and incubated for 5 min at RT. In a separate tube, 2 μg esiRNA were diluted in 250 μl OptiMEM. Solutions were combined, mixed and incubated for 20 min at RT after which the transfection mix was evenly distributed over the dish.
The FLAG–HOT1 and the homeobox deletion variant construct were transfected using either Effectene (Qiagen) or Lipofectamine 2000 (Invitrogen) as the transfection reagent according to the manufacturers' instructions.
Telomeric quantitative FISH
For metaphase preparation, cells were incubated for 4 h with 200 nM nocodazole in order to induce mitotic arrest. A hypotonic shock was achieved in 0.03 M sodium citrate at 37°C for 40 min. Cells were fixed in an ethanol/acetic acid solution (3:1) and washed three times in this fixing reagent. Metaphase spreads were obtained by dropping suspensions of fixed cells onto clean glass slides.
The QFISH procedure was carried out as described, using an Alexa488-O-O-(CCCTAA)3
PNA probe (Panagene) (Londoño-Vallejo et al, 2001
Telomeric signals were quantified using the iVision software (Chromaphor). Telomere signals were segmented manually and average pixel intensities from every segment were quantified. For each metaphase, the average background intensity was determined and subtracted from individual telomere signals. Statistical analyses were done using Student's t-test.
The number of signal-free ends per metaphase was determined by manual inspection of the same metaphase images that were used for telomere signal-intensity quantification. Statistical analyses were done using Student's t-test.
One hundred picogram of Nde
I (NEB)-digested DNA were amplified according to the previously published Universal STELA PCR amplification protocol (Bendix et al, 2010
) with the following cycling conditions: 68°C for 5 min, 95°C for 2 min, 26 cycles of 95°C for 15 s, 58°C for 30 s and 72°C for 12 min, and 72°C for 15 min. In brief, 10 ng of DNA were digested with 0.4 units of Nsde
I and 0.4 units of Mse
I, annealed at 16°C overnight to 4.2 μM of both 42-mer (5′-TGTAGCGTGA-AGACGACAGAAAGGGCGTGGTGCGGACGCGGG-3′)and 11+2-mer (5′-TACCCGCGTCCGC-3′) panhandle-oligos with 1.3 units of T4 ligase (NEB) and ligated to 1 nM of telorette3 (5′-TGCTCCGTGCATCTGGCATCCCTAACC-3′) at 35°C over night. One hundred picogram of ligated DNA was then used for PCR amplification with 0.1 μM of Adapter (5′-TGTAGC-GTGAAGACGACAGAA-3′) and of Teltail (5′-TGCTCCGTGCATCTGGCATC-3′) primers by the Failsafe Enzyme with the FailSafe buffer premix H (Epicentre). The PCR products were resolved on a 1.2% agarose gel, blotted onto a nylon membrane by capillary osmosis and hybridized at 42°C over night to a DIG-labelled telomeric C-rich LNA probe (36 bp, Exiqon). The blots were then washed with Maleic Acid/Tween solution and DIG signal was revealed with anti-DIG-AP antibody (1:20 000, Roche) and CDP-star solution (Roche) according to the manufacturer's instructions. Statistical analyses were done using Student's t
Immunoprecipitations were carried out using the Dynabeads Protein G or A immunoprecipitation kit (Invitrogen). Fifty microlitre of beads were treated with 10 μg rabbit anti-HOT1, mouse anti-HOT1 (both MPI-CBG Antibody Facility), rabbit IgG (sc-66931, Santa Cruz; 2729s, Biolabs) or mouse IgG (ChromPure, Jackson ImmunoResearch) in PBB buffer (150 mM NaCl, 50 mM Tris–HCl pH 8.0, 10 mM MgCl2, 0.5% NP-40, Complete Protease Inhibitor–EDTA (Roche)) and subsequently incubated with 400 μg HeLa nuclear extract (SILAC-labelled if followed by MS analysis) for 2 h at 4°C on a rotation wheel, followed by three washes with PBB buffer. For MS, bead fractions were pooled, bound proteins were eluted and separated on a 4–12% gradient gel (Novex, Invitrogen). For co-IP experiments followed by western blot, bound proteins were simply eluted and subjected to western blot analysis.
For western blot samples were boiled in Laemmli buffer (Sigma Aldrich) and subjected to SDS–PAGE (NuPage 4–12% Bis-Tris gels; Invitrogen). Gels were blotted to nitrocellulose (Protran; Schleicher & Schuell), blocked in 5% nonfat milk in PBST (PBS containing 0.1% Tween-20) for 1 h at RT and incubated over night at 4°C with primary antibody. The following primary antibodies were used: mouse anti-GFP (Roche Diagnostics, 1:4000 dilution), mouse anti-DM1alpha tubulin (MPI-CBG Antibody Facility, 1:50 000 dilution), mouse anti-Ku70 (sc17789, Santa Cruz, 1:1000 dilution), mouse anti-FLAG (M2, Sigma Aldrich, 1:5000) mouse anti-PCNA (sc-9847, Santa Cruz, 1:1000), mouse anti Histone3 (ab, 1791, Abcam, 1:10 000) and rabbit anti-TERT (gift from Madalena Tarsounas, 1:2000). The next day, membranes were washed three times for 10 min each in 5% milk PBST and were incubated for 1 h at RT with secondary antibody (goat anti-mouse antibody conjugated to horseradish peroxidase, Bio-Rad, 1:4000, or donkey anti-mouse IRDye 800CW or donkey anti-rabbit IRDye 800CW, both LI-COR Odyssey, 1:15 000). Membranes were washed three times for 10 min each in PBST followed by one PBS wash. Bands were visualized with enhanced chemiluminescence Western Blotting Detection Reagents (GE Healthcare) or with the LI-COR Odyssey imaging system. For detection of His-tagged HOT1, TRF1 and TBP, the Penta-His HRP Conjugate Kit (Qiagen) was used according to the manufacturer's instructions. As a molecular weight standard, Spectra Multicolor Broad Range Protein Ladder (Fermentas), Seablue 2 (Invitrogen) and MagicMark XP Western Protein Standard (Invitrogen) were used.
Immunoprecipitation of telomerase activity and quantitative TRAP assay
For the immunoprecipitation of telomerase activity HeLa cells were lysed in lysis buffer (50 mM Tris–HCl (pH 8.0), 150 mM NaCl and 1% NP-40 supplemented with Complete Protease Inhibitor–EDTA (Roche)) for 30 min on ice followed by a 30 min centrifugation step in a table-top centrifuge at 4°C and 20 000 g. Per IP assay, 25 μl of magnetic protein G beads (Invitrogen) were used. Beads were washed three times with 1 × PBS before use and incubated with 5 mg/ml BSA (in 1 × PBS) for 1 h at 4°C on a rotating wheel, while lysates were precleared with uncoated beads for 1 h at 4°C on a rotating wheel. Per IP assay, 1 mg lysate was incubated with 5 μg rabbit anti-HOT1 (MPI-CBG Antibody Facility), rabbit anti-DKC1 (ab64667, Abcam), rabbit anti-TRF1 (ab1423, Abcam), rabbit anti-TRF2 (NB110-57130, Novus), rabbit anti-TBP (sc-273, Santa Cruz), rabbit anti-YY1 (ab12132, Abcam), rabbit anti-STAT3 (9132, Cell Signaling), rabbit anti-Histone3K4tri-methylated (07-473, Upstate Antibodies), rabbit anti-CENP-B (ab25734, Abcam) or rabbit IgG (sc-66931, Santa Cruz) in PBS for 2 h at 4°C on a rotating wheel. BSA-coated beads were added, followed by a second incubation for 2 h at 4°C on a rotating wheel. Beads were then washed once with PBS, twice with lysis buffer and again once with PBS and finally recovered in 75 μl Chaps buffer (Chemicon).
The quantitative TRAP assay was carried out using GoTaq qPCR Master Mix (Promega) and both the TS (5′-AATCCGTCGAGCAGAGTT-3′) and ACX primer (5′-GCGCGGCTTACCCTTACCCTTACCCTAACC-3′) at 200 nM. The reaction was run on a Mx3000p real-time PCR system (Stratagene) with the following protocol: 25°C for 20 min, 95°C for 10 min and 32 cycles with 95°C for 30 s, 60°C for 30 s and 72°C for 1 min. Statistical analyses were done using Student's t-test.
For verification that signals were due to the presence of the characteristic TRAP ladder, samples were run on a 20% precast TBE gel (Invitrogen) for 2 h at 200 V. For size reference, the GeneRuler Ultra Low Range DNA Ladder (Fermentas) was used. The gel was stained with EtBr for visualization.
Quantitative real-time PCR
For quantification of TCAB1 mRNA levels RNA was extracted with the RNeasy kit (Qiagen), including DNaseI digestion and from the eluted RNA cDNA was synthesized using the SuperScript III first-strand synthesis kit with oligodT according to manufacturer's instructions. qPCR primers () were used at 70 nM concentration together with the Absolute qPCR SYBR green mix (Abgene) on a Mx3000p real-time PCR system (Stratagene). Target gene mRNA levels were normalized against quantification of GAPDH mRNA levels for housekeeping.
Oligonucleotides used for quantitative PCRs
Statistical analyses were done using student's t-test.
The following BACs (bacterial artificial chromosomes) were used in this study: human HOT1 RP11-789B24 (Invitrogen), DKC1 RP11-107C18, Coilin RP23-375L19 and POT1 CTD-3053M7 and mouse TRF1 RP24-402F23 (BACPAC Resource Center). A LAP (localization and affinity purification) cassette was inserted as a C-terminal fusion using recombineering. Isolated BAC DNA was transfected and selected for stable integration as described (Poser et al, 2008
). The BAC RP11-789B24 does not cover the entire HOT1
gene and was complemented by insertion of a cDNA fragment covering the missing coding and 3′-UTR sequence ().
Oligonucleotides used for cloning and BAC engineering
Immunofluorescence stainings on testes chromosome spreads and testes sections
Testes were isolated from wild-type 129T2/SvEms male mice, between 4–6 weeks old. To prepare chromosome spreads, the tunica albuginea was detached and the seminiferous tubules were incubated in 500 μl of 1 mg/ml Collagenase Type I (Gibco) in PBS for 10 min at 32°C. The tubules were agitated slightly, and the liquid was removed in order to remove a proportion of interstitial cells. A fresh 500 μl aliquot of 1 mg/ml collagenase was added and incubated at 32°C for 30 min, disaggregating by pipetting every 10 min. The cells were spun at 2000, r.p.m. for 5 min at 4°C. The pellet was resuspended in 1 ml cold PBS and filtered through a 40 μm nylon membrane to create a single-cell suspension.
Fifteen microlitre of fixation and permeabilization solution (1% PFA, 5 mM sodium borate pH 8.5, 0.2% Triton X-100) were added to each well (Ø 7 μm) on a 10-well glass slide (StarFrost coating, Engelbrecht). An ImmEdge pen (Vector Laboratories) was used to circle the wells and prevent leakage. The cells were diluted 1:5 into cold 100 mM sucrose in 1 × PBS for 2–3 min to allow hypertonic swelling. One to two microlitre of cell suspension were added to each well. The slides were incubated in a humidified chamber for 30 min, followed by 2–3 h of drying in a laminar hood or on the bench at 22°C. The slides were washed briefly three times in 0.5% Photo-Flo (Kodak) then once in distilled water.
To prepare testes tissue sections, whole testes were immersed in O.C.T Compound (Tissue-Tek 4583) in specimen molds (Tissue-Tek 4566 Cyromold 15 mm × 15 mm × 5 mm) and frozen at −80°C. Seven-micrometer sections were cut using a Leica CM1900 and placed on microscope slides (StarFrost K078; 76 × 26 mm). Sections were fixed using 4% formaldehyde (Sigma F8775) in 1 × PBS for 15 min at 22°C and permeabilized using 0.15% Triton X-100 for 10 min at 22°C and washed twice in 1 × PBS.
To perform immunofluorescence for both chromosome spreads and tissue sections, blocking solution (0.2% fish gelatin (Sigma) in PBS−0.1% Tween-20) was added for 1 h at RT. Primary antibodies were added for 16 h at RT. The following primary antibodies were used: rabbit anti-HOT1 (MPI-CBG Antibody Facility, 1:1000), rabbit anti-TRF2 (sc-9143, Santa Cruz, 1:100) and mouse anti-SYCP3 (as previously described (Adelfalk et al, 2009
)). The cells were washed 3 × in blocking solution for 10 min each and secondary antibodies (goat anti-rabbit Alexa555 or goat anti-mouse Alexa488 at 1:500, Invitrogen) were added for 1 h at RT. After 3 × washing in blocking solution for 10 min each, Vectashield (Vecta Laboratories) containing 1 μg/ml DAPI was added and a glass cover slip was placed on top of the wells and sealed closed. Slides were stored at −20°C.
Images were acquired with a DeltaVision Core Microscope (Applied Precision, Olympus IX71 microscope) using a 100 × /1.4 UPlanSApo oil-immersion objective. Z-stacks (0.2–0.5 μm optical sections) were collected and deconvolved using softWoRx (Applied Precision). Z-stacks were reconstructed in 3D using Imaris (Bitplane) and colocalization events were determined for signals above the background using the colocalization function. For imaging of entire tubules, separate images were acquired as one panel and stitched together using the stitch function in softWoRx for visualization.
mice and generation of MEFs
embryos were retrieved from the Center for Animal Resources and Development and are based on the Exchangeable Gene Trap Clones system using the exchangeable pU21 trap vector (Araki et al, 2009
). Live animals were retrieved by embryo transfer and were outcrossed for several generations against C57BL/6 wild-type animals. MEFs were generated from littermate embryos at E13.5 by crossing C57BL/6-Hot1Gt(pU-21T)346Card/+
animals with each other.
For genotyping of Hot1Gt(pU-21T)346Card/(Gt(pU-21T)346Card mice, primers were designed to amplify the wild-type and mutant allele in one PCR reaction, both sharing the same forward primer. The PCR was carried out with a reaction containing 2.5 μl 10 × PCR buffer, 0.8 μl 50 mM MgCl2, 0.2 μl 25 mM dNTP mix, 2 μl 10 μM WT-F primer, each 1 μl 10 μM WT-R and MUT-R primers, 1 μl TaqRed polymerase (Bioloine) and 2 μl crude genomic DNA extracts, filled up with HPLC-grade H2O to a total volume of 25 μl (for primer sequences see ). PCR conditions were as follows: initial hotstart denaturation at 94°C for 3 min followed by 10 cycles with 94°C for 30 s, 62°C for 30 s with a 0.5°C touchdown decline per cycle and 72°C for 60 s, followed by 25 cycles with 94°C for 30 s, 57°C for 30 s and 72°C for 60 s, finished by a final elongation at 72°C for 5 min. The reaction was run on a DNA Engine Thermocycler (Bio-Rad). After the reaction the PCR products were checked by standard gel electrophoresis
Oligonucleotides used for genotyping PCRs
Cell fractionation analysis for TERT binding to chromatin was carried out as previously described, with minor modifications (Tejera et al, 2010
). Per sample one million cells were washed once in cold PBS and then resuspended in 200 μl ice-cold buffer A+ (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2
, 0.34 M sucrose, 10% glycerol, Complete Protease Inhibitor–EDTA (Roche)). Samples were incubated for 5 min on ice and centrifuged for 5 min at 1300 g
and 4°C. The supernatant was further cleaned by centrifugation for 25 min at 20 000 g
and 4°C, and represents the cytoplasmic fraction. The pellet was washed twice in 500 μl buffer A+ and resuspended in 100 μl ice-cold buffer B (3 mM EDTA, 0.2 mM EGTA, Complete Protease Inhibitor–EDTA (Roche)). Samples were incubated for 30 min on ice and centrifuged for 5 min at 1700 g
and 4°C. The supernatant was further cleaned by centrifugation for 25 min at 20 000 g
and 4°C, and represents the nuclear soluble fraction. The pellet was washed twice in 500 μl buffer B, resuspended in 100 μl Laemmli buffer (Sigma Aldrich) and sonicated twice for 10 min each in a water-bath sonicator. This sample represents the chromatin fraction. The other fractions were equally mixed with Laemmli buffer, and all samples were boiled at 95°C for 5 min and separated on a 4–12% gradient gel (Novex, Invitrogen).