Extended Experimental Procedures.
All procedures were approved by the Fondazione San Raffaele del Monte Tabor Ethical Committee. Human primary myoblasts were obtained from the Fields Center for FSHD of the Rochester Medical Center Dept. of Neurology, NY, USA and the Telethon BioBank of the C. Besta Neurological Institute, Milano, Italy. Muscle biopsies were obtained from the Telethon Neuromuscular Bank of the Department of Neurosciences, University of Padova, Italy. Details of the human samples used are listed in Table S1
Mammalian Cell Culture
HEK293T and CHO were obtained by ATCC; human chromosome 4/CHO hybrid (GM10115) was obtained from the Coriell Institute for Medical Research. Cells were maintained in DMEM-HIGH (Dulbecco's Modified Eagle's Medium, High Glucose with Sodium Pyruvate and L-Glutamine; EuroClone) supplemented with 10% FBS (Foetal Bovine Serum; EuroClone) and 1% Penicillin/Streptomycin (100 U/ml final concentration; EuroClone). Proline (final concentration 0.2 mM; Sigma) was added to CHO and human chr4/CHO cells. For the generation of stable cell lines infected with pLKO.1 or pTRIPZ lentiviruses, puromycin (6.5 μg/ml; InVivoGen) was added to the normal medium of human chr4/CHO cells in case of pLKO.1 lentiviruses, or to medium containing Tetracycline-negative serum (10%, EuroClone) in case of pTRIPZ. Resistant cells were maintained as a polyclonal population to avoid clone-to-clone variability. For the generation of stable cell lines transfected with either BAC RP11-462G22 or BAC CH16-291A23, CHO cells were cotransfected with pCDNA3.1 vector (Invitrogen) in a ratio 9:1, where BAC constructs where 9 times more concentrated than pCDNA3.1. Positively transfected cells were selected with Neomycin (1,000 μg/ml; InVivoGen) and maintained as a polyclonal population to avoid clone-to-clone variability. Human primary muscle cells from the Telethon BioBank were grown in DMEM-HIGH (Dulbecco's Modified Eagle's Medium, High Glucose with Sodium Pyruvate and L-Glutamine; EuroClone), supplemented with 20% FBS (Foetal Bovine Serum; EuroClone), 1% Penicillin/Streptomycin (100 U/ml final concentration; EuroClone), insulin (10 μg/ml final concentration; Sigma), human basic fibroblast growth factor (25 ng/ml; Peprotech) and epidermal growth factor (10 ng/ml; Peprotech). Human primary muscle cells from the Fields Center for FSHD were grown in F-10 Nutrient Media (Sigma), supplemented with 20% FBS (Foetal Bovine Serum; EuroClone), 1% Penicillin/Streptomycin (100 U/ml final concentration; EuroClone), human basic fibroblast growth factor (10 ng/ml; Peprotech), Dexamethasone (1 μM; Sigma). Muscle cells were induced to differentiate at 70% confluence by replacing the growth medium with a differentiation medium composed of DMEM-HIGH (Dulbecco's Modified Eagle's Medium, High Glucose with Sodium Pyruvate and L-Glutamine; EuroClone) supplemented with 5% Donor Horse Serum (EuroClone), 1% Penicillin/Streptomycin (100 U/ml final concentration; EuroClone), insulin (10 μg/ml final concentration; Sigma). Medium was replaced with fresh differentiation medium every 2 days.
For Doxycycline treatment, human chr4/CHO cells stably infected with pTRIPZ lentiviruses, were seeded at low confluence in tetracycline negative growth medium with Doxycycline (300 ng/ml, Sigma). Fresh medium containing Doxycycline was replaced every day. Cells were collected after 72h of doxycycline treatment. For 5-Aza-2′-deoxycytidine (AZA) and Trichostatin A (TSA) treatment, human chr4/CHO cells were seeded at low confluence in growth medium. The day after, AZA was added to the medium (final concentration 1 μM; Sigma). After 24 hr, the medium was replaced with fresh medium containing 1 μM AZA. After other 24 hr, fresh AZA (final concentration 1 μM) was added to the medium. After 12 hr, TSA was added to the medium (final concentration 1 μM; InVivoGen). Cells were collected after 12 hr to obtain a 72 hr AZA and 12 hr TSA treatment. In case of stable clones, puromycin (final concentration 6.5 μg/ml; InVivoGen) was added to all media.
For Actinomycin D treatment (final concentration 5 μg/ml; Sigma), the drug was added to the medium 15′ before harvesting the cells.
For cell transfection Lipofectamine LTX (Invitrogen) was used. Human chromosome 4/CHO hybrid cells were transfected with pcDNA3 empty vector or with nt 1280-7755 of AF117653 and collected after 48 hr for gene expression analysis. CHO cells were transfected with pGEM42 (Kowaljow et al., 2007
) or pGEM-T as control and collected after 24 hr for RNA extraction.
Nonsilencing and Ash1L shRNAs cloned in pLKO.1 or nonsilencing and Suz12 shRNAs cloned in pTRIPZ were directly obtained by Open Biosystems.
DBE and NDE siRNAs were designed by using a program (siRNA Designer, previously available on Promega website) based on parameters specified in the scientific literature (contact Promega for details). SiRNA sequences were cloned into lentiviral pLKO.1 vector to express them as shRNAs driven by the human U6 promoter according to the instructions of the RNAi TRC consortium (http://www.broadinstitute.org/rnai/trc
). Briefly, each siRNA sequence was used to generate two complementary oligonucleotides: 5′-CCGG-sense-CTCGAG-antisense-TTTTTG-3′ and the complementary 5′-AATTCAAAAA-sense-CTCGAG-antisense-3′. Oligo pairs were mixed at a final concentration of 1 μg/μl in 1X NEB buffer 2 (New England Biolabs Restriction Endonuclease Reaction Buffer 2;
New England Biolabs), denatured 4′ at 95°C and annealed for 10′ at 70°C followed by a slow cool down to RT. The obtained dsDNA fragment has AgeI and EcoRI sticky ends and is ready for cloning into digested pLKO.1 plasmid.
- DBE: GCTCACCGCCATTCATGAAGG
- NDE: AACGTCACGGACAAGGCCAGA
- Nonsilencing: TCTCGCTTGGGCGAGAGTAAG
- Ash1L: GCTGGTCATTTATTGCTCAAT
- Suz12: TTCTACAAACAGCATACAG
Lentiviruses Production and Transduction
HEK293T cells were seeded in DMEM-HIGH supplemented with 10% FBS without antibiotics in T25 tissue culture flasks (1 flask per infection). The day after, cells at 60%–70% confluence were transfected (Lipofectamine LTX with Plus reagent; Invitrogen) by using these quantities of DNA: 2.925 μg of packaging plasmid (pCMV-dR8.91; http://www.broadinstitute.org/rnai/trc
), 325 ng of envelope plasmid (VSV-G/pMD2G; http://www.broadinstitute.org/rnai/trc
) and 3.25 μg of hairpin-pLKO.1 or hairpin-pTRIPZ vector. After 5 hr, the medium was replaced with regular growth medium. After 18 hr from transfection, the medium was replaced by a high serum growth medium DMEM-HIGH supplemented with 30% FBS and 1% Penicillin/Streptomycin. Cells were incubated for 24 hr and the medium containing the lentiviral particles was harvested, filtered by using a 0.22 μm filter unit and placed at 4°C for short time storage or −80°C for long-term storage. Fresh high serum growth medium was added to cells. After 24 hr the viral harvesting was repeated and pooled with the previous one.
For viral transduction, human chr4/CHO cells were seeded in T25 tissue culture flasks and infected at 70% confluence. Cells were incubated overnight with the viral supernatant diluted 1:2 with fresh growth medium, supplemented with 0.2 mM proline and polybrene (final concentration 8 μg/ml; Sigma). The day after, cells were passed 1:7. At 24–48 hr postinfection puromycin selection was started and it was continued until all noninfected control cells died (typically, 5 days).
RNA Extraction, RT-PCR, and qRT-PCR Analysis
RNA extraction was performed with Trizol reagent (Invitrogen) followed by purification with RNA spin columns (PureLink RNA MiniKit; Invitrogen) and digestion with DNaseI following the manufacturer's instructions.
For reverse transcription, equal amounts of DNA-free RNA (100 ng-1 μg) were retro-transcribed with SuperScript III First-Strand Synthesis SuperMix (Invitrogen) following the suggested conditions.
RT-PCR assays for the different 8 regions encompassing NDE-DBE and for Gapdh (Gapdh F2-Gapdh R2) were performed by retrotranscribing 2.5 μg of total RNA derived from human chr4/CHO cells treated or untreated with AZA-TSA. For PCR reactions Go Taq Flexi (Promega) or Accuprime (Invitrogen) were used. Conditions varied according to length and base composition. More detailed information on each region is available upon request.
To amplify the single NDE-DBE transcript (primers NDE F+DBE R), 1 μg of total RNA extracted from CHO cells transfected with pGEM42 or pGEM-T as control, was retrotranscribed as previously described. cDNA was PCR amplified by using Expand Long Range dNTPack (Roche). For PCR conditions and amplification steps the manufacturer's instructions were followed, with addition of 6% DMSO in the reaction.
For gene expression analysis, real-time PCR with Sybr GreenER qPCR kit (Invitrogen) was used. GAPDH or β-actin were used as housekeeping genes for sample normalization.
The specificity of the amplified products was monitored by performing melting curves at the end of each amplification reaction.
PCR products obtained from human samples were cloned into pGEM-T vector and sequenced to make sure that they were selectively from the human 4q35 region.
The primers used in qPCR are listed below. In order to allow expression analysis with the ΔΔCT method, all primers were tested and selected for amplification efficiencies ranging between 90%–110%.
Sequences of the primers used are listed in Table S2
All experiments were repeated at least three times.
5′ RACE (Invitrogen) was carried out by using 1.5 μg of total RNA extracted from CHO cells transfected with pGEM42. NDE R primer was used in the retrotranscription reaction. A first PCR reaction (Go Taq Flexi, Promega) was performed by using 5RACE1 and Abridged Anchor primer (provided by the kit) with the following conditions: 1 mM MgCl2, 0.4 mM each dNTP and 0.2 μM of each primer (final concentrations). Amplification was performed as follows: initial denaturation 1′ at 95°C, denaturation 30″ at 95°C, annealing 30″ at 60°C, extension 4′ at 72°C. Repeated for 35 cycles. The obtained band was gel purified (QIAGEN) and PCR amplified with 5RACE2 and Abridged Universal Amplification Primer (provided by the kit) with the following conditions: 1.5 mM MgCl2, 0.2 mM each dNTP and 0.2 μM of each primer (final concentrations). Amplification was performed as follows: initial denaturation 1′ at 95°C, denaturation 30″ at 95°C, annealing 30″ at 56°C, extension 1′ at 72°C. Repeated for 35 cycles. The obtained band was sequenced.
AZA+TSA treated human chr4/CHO hybrid cells were detached by treating with 1X Trypsin, counted and centrifuged at RT 168 g for 5′. The pellet was lysed with 175 μl/106 cells of cold RLN1 solution (50 mM Tris HCl pH 8.0; 140 mM NaCl; 1.5 mM MgCl2; 0,5% NP-40; 2mM Vanadyl Ribonucleoside Complex; Sigma; reagent stocks were prepared in RNase-free H20) and incubated 5′ in ice. Next, the suspension was centrifuged at 4°C and 300 g for 2′ and the supernatant, corresponding to the cytoplasmic fraction, was transferred into a new tube and stored in ice. The pellet containing nuclei was extracted with 175 μl/106 cells of cold RLN2 solution (50 mM Tris HCl pH 8.0; 500 mM NaCl; 1.5 mM MgCl2; 0,5% NP-40; 2mM Vanadyl Ribonucleoside Complex; stocks were made in RNase-free H20) and 5′ incubated in ice. The suspension was centrifuged at 4°C and 16360 g for 2′ and the supernatant, corresponding to the nuclear-soluble fraction, was transferred into a new tube and stored in ice. The remaining pellet corresponds to the chromatin-associated fraction.
Total RNA was extracted by using PureLink RNA MiniKit (Invitrogen) following the manufacturer's instructions for the extraction from aqueous solutions for the cytoplasmic and nuclear-soluble fractions, whereas the chromatin-associated fraction was considered as a pellet. The samples were treated with on column DNaseI, washed and then eluted in 30 μl RNase-free water.
Preparation of FISH Probes. For D4Z4 DNA and β-actin transcript detection, 4 different DNA aminoallyl-modified oligonucleotides were used (the Midland Certified Reagent Company). Each oligo was resuspendend in 100 μl of H20 and purified with chloroform extraction (1vol). DNA was then precipitated with 1/10 vol of 3 M NaAc and 2.5 vol of 100% EtOH for 30′ at −80°C. After centrifuging 15′ at 16,300 g, the pellet was washed with 70% EtOH, centrifuged again 5′ at 16300 g, air-dried and resuspended in 150 μl of H20. After DNA quantification, 5 μg of each of the 4 oligos were pooled together and H20 was added to a final volume of 20 μl. To this, 12 μl of NaHCO3 25 mg/ml and 8 μl of Alexa Fluor 555 (resuspended at 30 μg/μl in DMSO; Invitrogen) were added. After a brief vortex, the samples were let stand in the dark at RT overnight to allow the coupling of the fluorochrome. The day after, the oligos were purified by using QIAquick Nucleotide Removal kit (QIAGEN) and eluted in 40 μl of Rnase free H20. This corresponds to the stock solution. We calculated the relative efficiency of labeling measuring the base/dye ratio as described by Invitrogen's protocol of labeling with amine-reactive reagent. For the D4Z4 probe we obtained a base/dye ratio of 18.22, whereas for the β-actin RNA probe we obtained a base/dye ratio of 15.20.
For DBE transcript detection, 3 FAM-labeled LNA oligonucleotides were used (Exiqon). Each LNA was resuspended in RNase-free H20 at 2 μg/μl. To prepare a stock solution, an aliquot of each was pooled together and RNase-free H20 was added to reach a dilution of 1:4 for each LNA.
To perform FISH, the oligo and LNA probes were diluted 1:100 and 1:500, respectively, in hybridization mix (50% deionized formamide [Sigma], 2X SSC [recipe of 20X SSC from Sambrook and Russel (2001)
], 30 mM phosphate buffer, 10% dextran sulfate; [pH 7.0]) containing salmon sperm ssDNA (final concentration 700 ng/μl; Sigma), human Cot1 DNA (final concentration 300 ng/μl; Invitrogen) and Vanadyl Ribonucleoside Complex (final concentration 2 mM; Sigma), denatured 5′ at 80°C and incubated at 37°C for at least 20′ before use.
Hybridization. Hybridization was performed essentially as described in Custódio et al., 2006.
Human chr4/CHO or CHO cells were seeded on glass coverslips and, following treatment with AZA+TSA, were briefly washed twice with PBS and fixed with 4% PFA (Electron Microscopy Sciences) for 10′ at RT. Next, three washes of 5′ with PBS and a rinse with 70% ice cold EtOH were performed. Slides were stored in 70% ice-cold EtOH at −20°C. After at least an overnight incubation, slides were rehydrated in PBS for 5′ twice. Next, cells were permeabilized with a 10′ incubation with gentle swirl in 0.5% Triton X-100, 2 mM Vanadyl Ribonucleoside Complex (not used if RNase treatment was following) in PBS. Then, three washes of 5′ in PBS and two washes of 5′ in 2X SSC, 0.05% Tween 20 (National Diagnostics) were performed. In control experiments requiring an RNase treatment at this step, cells were incubated with RNase A+T1 (final concentrations 20 μg/ml RNase A and 50 U/ml RNase T1; Fermentas) in 10 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5 mM EDTA (pH 7.5) for 1 hr at 37°C in humid chamber. At the end of the treatment, cells were washed twice with 2X SSC, 0.05% Tween 20. Before hybridization, cells were incubated for 30′ at RT in 2X SSC, 0.05% Tween 20, supplemented with 1% BSA (Sigma), 1 μg/μl tRNA (Sigma) and 2 mM Vanadyl Ribonucleoside Complex in a humid chamber. The blocking solution was carefully removed and the denatured LNA probe (see previous section) 1:500 was added. Samples were incubated in the dark in a humid chamber at 37°C overnight. The day after, two washes of 30′ at 37°C with 1X SSC, 0.025% Tween 20, 50% deionized formamide, pH 7.0 were performed. Next, after 3 × 5′ washes with 2X SSC, 0.05% Tween 20 (2 performed at 37°C and 1 performed at RT), slides were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen) if simply RNA FISH was performed. Alternatively, for sequential RNA/DNA FISH experiments, cells were denatured at 80°C for 30′ in prewarmed 1X SSC, 0.025% Tween 20, 50% deionized formamide pH 7.0. Next, cells were hybridized with denatured oligonucleotides to D4Z4 1:100 for 3 hr. Next, the same washes described previously after hybridization to RNA were performed. Slides were mounted with ProLong Gold antifade reagent with DAPI. Analyses were performed on single Z stacks acquired with an Olympus IX70 DeltaVision RT Deconvolution System microscope. All experiments were repeated at least three times. Probe sequences are listed in Table S3
Human chr4/CHO cells were seeded in 5–15 15 cm dishes, whereas human primary myoblasts were seeded in 2–3 10 cm dishes. In order to collect chromatin, cells were briefly washed once in PBS and immediately fixed for 10′ at RT in 1% formaldehyde in PBS (from a 37.5% formaldehyde/10% methanol stock; Sigma). After formaldehyde quenching with Glycine (final concentration 125 mM) for 2′, cells were washed three times for 5′ in PBS with gentle swirl. Cells were harvested by using a silicon scraper and cold PBS. Cells were collected in 50 ml tubes and centrifuged at 1350 g for 5′ at 4°C. The resulting pellet was resuspended in 3 ml of PBS every 3 × 15 cm dishes of starting material, and aliquoted in 15 ml tubes. Human primary myoblasts were kept in a single 15 ml tube. Cells were centrifuged at 1,350 g for 5′ at 4°C. Each pellet was then lysed in 5 ml of LB1 solution (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100) and incubated 10′ in ice. Next, the samples were centrifuged at 1350 g for 5′ at 4°C. The resulting pellet was washed in 5 ml of LB2 solution (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) with gentle swirl 10′ at RT. Next, samples were centrifuged at 1350 g for 5′ at 4°C and the resulting pellet was lysed in 3 ml of LB3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-laurylsarcosine). LB1, LB2 and LB3 were supplemented with protease inhibitor (Complete EDTA-free Protease Inhibitor Cocktail Tablets; Roche). Lysates were sonicated with Bioruptor (Diagenode). Briefly, 1 ml aliquots of LB3 lysates were put in 15 polystirene tubes and were sonicated for 10′ (medium intensity 30″ on 30″ off). An aliquot (55 μl) of the sonicated material was collected to determine the quality of the chromatin by adding 0.1M NaHCO3
, 1% SDS (100 μl), 5 μl of Proteinase K (20 mg/ml in 50 mM Tris-HCl pH 8.0, 10 mM CaCl2
, 50% glycerol; Promega) and incubate 1 hr at 55°C for cross-link reversal. Next, samples were precipitated with 5M LiCl (3.2 μl) and 1 ml 100% EtOH by centrifuging for 30′ at 4°C at 16,360 g. The pellet was washed in 70% EtOH and centrifuged for 10′ at 4°C at 16,360 g. After air dry, the pellet was resuspended in H2
O and loaded on 1% agarose gel for electrophoresis. We considered good a chromatin enriched in fragments of 500-300 bp. The samples were quantified with Nanodrop spectrophotometer to determine the concentration of chromatin. Before ChIP, Triton X-100 was added to chromatin samples at a final concentration of 1% and a clarification step of 10′ at 16,360 g at 4°C followed. Supernatants were transferred into a new tube and were precleared with 1/50 vol of Dynabeads protein G (Invitrogen) rotating for 2 hr at 4°C. Fifty to one hundred micrograms of chromatin were used for each ChIP to a protein target, whereas 5–10 μg of chromatin were used for each ChIP to histones. For each ChIP, 100 μl of Dynabeads protein G (50 μl in ChIPs to histones) were washed three times with 0.5% BSA in PBS and incubated with 10 μg of antibody (5 μg in ChIPs to histones) in 250 μl of 0.5% BSA in PBS for 2–3 hr on rotation at 4°C. Next, the beads-antibody complex was washed three times with 0.5% BSA in PBS and resuspended in 50 μl of 0.5% BSA in PBS. Precleared chromatin and beads-antibody complexes were incubated on rotation overnight at 4°C. The day after, before starting the washes, 5% of the total ChIP volume was taken from the control IgG supernatant as input fraction. Next, six washes of 5′ on rotation at 4°C in RIPA buffer were performed (50 mM HEPES-KOH pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate). An additional wash of 5′ on rotation at 4°C in TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) with 50 mM NaCl was performed. Next, samples were centrifuged for 3′ at 1,000 g at 4°C. The supernatant was discarded and to the beads-antibody-chromatin complex were added 240 μl of elution buffer (TE buffer with 2% SDS). Samples were incubated in a thermo mixer for 15′ at 65°C, shaking, and then centrifuged for 1′ at RT at 16,360 g. The elute supernatant was transferred to a new tube and samples, together with the input fraction to which 3 vol of elution buffer were added, were cross-linked reverted overnight at 65°C. For samples purification, the QIAquick PCR purification kit was used (QIAGEN), following the manufacturer recommendations. DNA was eluted in 70 μl of TE buffer and 1–2 μl were used in qPCR with Sybr GreenER qPCR kit (Invitrogen). Chromatin Immunoprecipitation for H2Aub1 and IgM as control was performed according Stock et al. (2007)
with 20 μg of chromatin produced and quantified as previously described. All experiments were repeated at least three times.
ChIP q-PCR Analysis
To determine the enrichment obtained by using nonhistone antibodies, we normalized ChIP-qPCR data for input chromatin (reported as % input in the figures). For histone modification antibodies, we expressed the enrichment as percentage of total histone (reported as % H3 or % H2A in the figures). To normalize for the highly variable amount of D4Z4 repeats present in human samples, the EZH2 ChIP-qPCR signal shown in was normalized for the input signal of five different single copy regions. Primers used are listed in Table S2
This assay was carried out mainly as described in Jeon and Lee, 2011
. Briefly, cells were seeded in 15 cm dishes (1 for each RNA IP). After treatment with AZA+TSA, cells were UV cross-linked with 100,000 μJ/cm2
twice (interval of 1′ between the two irradiations) on ice. Lysed in 0.5% NP40, 0.5% Na Deoxycholate, 300 U/ml Superase Inhibitor (Ambion), Protease inhibitor in PBS pH 7.9 and put on rotation for 25′ at 4°C. Samples were treated with 30 U of Turbo DNase (Ambion) and incubated 15′ at 37°C. After centrifuging 5′ at 1,350 g at 4°C, the supernatant was used in RNA IP. For each RNA IP 100 μl of Dynabeads Protein G, with 10 μg of anti Ash1L antibody (Sigma) or IgG (Jackson lab) as control, were used. Before performing the RNA IP, 10% of the supernatant was saved as input. The antibody-conjugated beads were added to the samples and put on rotation at 4°C over night. Beads were washed three times (5′ at 4°C) with PBS supplemented with 1% NP40, 0.5% Na Deoxycholate, additional 150 mM NaCl (final 300 mM), and 1:200 Superase inhibitor (Ambion). Beads were resuspended in 100 μl of PBS + 10X DNase buffer and 3 U (1.5 μl) of Turbo DNase (Ambion) was added. Samples were incubated 30′ on rotation at 37°C. Beads were washed three times (5′ at 4°C) with PBS supplemented with 1% NP40, 0.5% Na Deoxycholate, 10 mM EDTA, additional 150 mM NaCl (total 300 mM), and 1:200 Superase inhibitor (Ambion). RNA was eluted with 100 μl of 100 mM Tris HCl (pH 7.5), 50 mM NaCl, 10 Mm EDTA, 100 μg Proteinase K, 0.5% SDS for 30′ at 55°C, with shaking. Eluate was centrifuged at 16100 g at RT and the supernatant was collected. Samples were purified with Trizol LS following the manufacturer's instructions. After RNA resuspension in 21.5 μl of RNase-free water, samples were treated with DNase (Turbo DNase Ambion) and 5 μl of DNA-free RNA were used in RT reactions with Superscript III supermix (Life technologies). All experiments were repeated at least three times.
In Vitro RNA Pulldown Assay
Recombinant GST-fusion proteins were prepared as described previously (Tanaka et al., 2008
). Briefly, Rosetta host cells (Novagen) were transformed with pGEX-6P vectors (Amersham) with or without Ash1L SET domain (including pre-SET and post-SET regions, as described in Tanaka et al., 2007
) and induced with 1 mM isopropyl b-D-1-thiogalactopyranoside. Next, bacteria were resuspended in a buffer containing 30 mM Tris-HCl pH 8.0, 1 mM EDTA, 20% sucrose for 10 min and in ice-cold water for 30 min. Subsequently, cells were lysed in 20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 0.1 mg/ml lysozyme, 1 × Protease Inhibitor Cocktail (Roche), and 0.001% b-mercaptoethanol for 30 min on ice. After one freeze-thawing cycle at −80°C, cell lysates were cleared by ultracentrifugation at 20,000 rpm for 90′ by using Avanti HP-25 (Beckman). Supernatants were incubated with Glutathione Agarose beads (Sigma) for 2 hr at 4°C on rotation. Protein/beads complexes were quantified by SDS-PAGE by using a BSA standard curve.
To produce in vitro transcribed DBE-T, a fragment containing nt 4317–7463 of AF117653 was cloned into pGEM-T (Promega). The construct was linearized and DBE-T was transcribed in vitro by using T7 RNA polymerase (Promega). Transcripts were treated with RNase-free DNase (Promega) for 1 hr at 37°C, TRIzol (Invitrogen) purified, treated with TURBO DNase (Ambion) and renatured by heating 2′ at 90°C and slow-cooling at room temperature. Next, 300 ng of RNA per pull-down were precleared by a 30′ incubation at room temperature with Glutathione Agarose beads. 1/10 of this material was saved and TRIzol purified as input. RNA was incubated with two micrograms of GST-Ash1L SET domain or GST protein-beads complexes at room temperature for 1 hr in PBS containing 2 mM MgCl2, 0.2 mM ZnCl2, 1mM DTT, 100 U/ml RNase Inhibitor, 0.1 mg/ml yeast tRNA (Sigma), 0.05% BSA, and 0.2% NP40. Beads were washed with the same incubation buffer supplemented with additional 150 mM NaCl (total 300 mM NaCl) five times with 4′ washes at room temperature. The recovered RNA was TRIzol purified and 1/3 was analyzed by qRT-PCR. All experiments were repeated at least three times.
SDS-PAGE and Immunoblotting
After SDS-PAGE electrophoresis, transfers were performed by using the iBlot dry blotting system following the manufacturer recommended conditions (Invitrogen). After transfer, the filters were prehybridized in 5% nonfat milk (Regilait) in TBS-Tween 0.1% solution (Tris Buffered Saline: 10 mM TrisHCl pH 7.4, 140 mM NaCl, Tween-20; National Diagnostics) for Tubulin and Suz12 or in 5% BSA (Sigma) in PBS 0.2% Tween-20 for Ash1L western, for 1h at RT or overnight at 4°C. Incubation with the primary antibody was performed overnight at 4°C in 5% nonfat milk TBS-Tween 0.1% solution for Tubulin (1:20,000; Sigma T9026) and Suz12 (1:300; Abcam) or in 2% BSA in PBS 0.2% Tween-20 for Ash1L (1:2,000; Bethyl Laboratories A301-749A). Filters were then washed three times at RT (10′ each) with TBS-Tween 0.1% for Tubulin and Suz12, or PBS 0.2% Tween-20 for Ash1L. HRP-conjugated secondary antibody (1:20,000; Jackson ImmunoResearch Laboratories) was incubated for 1-2 hr at RT in 5% nonfat milk TBS-Tween 0.1% solution for Tubulin and Suz12 or in 2% BSA in PBS 0.2% Tween-20 for Ash1L. Filters were washed again as described, and for detection they were incubated for 5 min with horseradish peroxidase (HRP) chemiluminescent substrate (SuperSignal West Pico Chemiluminescent Substrate; Thermo scientific).
Chromosome Conformation Capture
3C was performed essentially as described in Bodega et al., 2009
. Briefly, a total of 50 × 106
human chr4/CHO cells for each treatment were lysed and nuclei were cross-linked with 1% formaldehyde for 10′ at room temperature. Digestion was performed with 600–800 U of PvuII
(50U/ml, NEB, R0151M) at 37°C overnight with constant agitation.
An appropriate amount of DNA that would amplify within the linear range was subsequently used for the experiments. A total of 33 rounds of PCR amplification were used. PCR products were run on 2% agarose gels and quantified by Typhoon (GE Healthcare).
One microgram of polyA+ RNA from control or AZA+TSA treated human chr4/CHO cells was analyzed by northern blotting by using the Northern Max-Gly kit (Ambion) following manufacturer's instructions. Filters were hybridized with probes to NDE, DBE, DUX4 homeobox region and Gapdh.
q-PCR Quantification of Relative Amount of D4Z4 Repeats
Chromatin DNA deriving from 5 independent control and AZA+TSA treatment experiments on human chr4/CHO cells were analyzed by qPCR to DBE in order to detect signal from D4Z4 repeats and normalized to a single copy region located on the human chromosome 4, namely SSLP. The graph is expressed as relative amount of D4Z4 repeats. The error bars represent standard error of the mean (SEM).
Characterization of BAC CH16-291A23
The BAC clone 291A23 containing D4Z4 repeats was isolated by using D4Z4 as probe to screen the CHORI-16 BAC library generated from sheared DNA (Osoegawa et al., 2007
). The BAC extremities were sequenced with SP6 and T7 oligonucleotides. The number of D4Z4 repeats was inferred based on restriction digestion with EcoRI and XhoI enzymes and by q-PCR normalized to a single copy region, as described above.