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Preparation of mono-nucleosomal and poly-nucleosomal DNA ...

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Preparation of mono-nucleosomal and poly-nucleosomal DNA ...

    Birch et al. Supplementary Information

SUPPLEMENTARY INFORMATION

    Birch JL et al.

    FACT facilitates chromatin transcription by RNA polymerases I and III

RESULTS

    Displacement of histone H2A-H2B dimers from nucleosomal DNA by Pol I

    To determine whether there is H2A-H2B dimer displacement activity associated with Pol I?;;which would be consistent with FACT histone chaperone activity, we designed an experiment to evaluate H2A-H2B displacement during Pol I transcription in vitro. A

    biotinylated nucleosomal template containing Cy3-labelled H2A-H2B dimers (Figure S1A, the ‘donor’ template, D) was incubated with Pol I and NTPs in a transcription

    reaction mix together with an H3-H4 tetrasomal template (Figure S1A, the H2A-H2B ‘acceptor’ template, A). Following the transcription reaction, the ‘donor’ template was separated from the ‘acceptor’ template by binding the biotinylated ‘donor’ template to streptavidin-coated paramagnetic beads. The ‘donor’ template was then released from the beads by micrococcal nuclease digestion. To assess H2A-H2B displacement/transfer from the ‘donor’ template by Pol I, ‘donor’ and ‘acceptor’ template samples were

    analysed on a native polyacrylamide gel that was scanned for Cy3-fluorescence.

In the presence of Pol I and NTPs, the overall level of Cy3 signal was reduced

    compared to the control reaction lacking Pol I (Figure S1B, compare lane 1 to lane 5,

    and graph) - indicating H2A-H2B displacement from the donor template. A decrease in Cy3 level is also seen, though to a lesser extent, with Pol I in the absence of

    transcription (Figure S1B, compare lane 3 to lane 1 and 5, and graph), suggesting that binding of the Pol I-FACT complex to the nucleosomal template facilitates H2A-H2B dimer displacement. This depletion was specific to histone H2B as the total amount of donor DNA present in each lane was constant (data not shown). ATP-dependent chromatin remodeler RSC (Cairns et al., 1996), used here as a positive control for H2A-H2B displacement/transfer (Bruno et al., 2003), caused a complete remodeling of the ‘donor’ template and H2A-H2B (Cy3) displaced from the ‘donor’ template was, at least

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    in part, transferred to the ‘acceptor’ template (Figure S1B, lanes 7 and 8). The comparatively low efficiency of H2A-H2B displacement by Pol I (Figure S1B, lanes 1

    and 3, compared to lane 5) could reflect the low template usage. Notably, there was no detectable transfer of the Cy3 signal to the ‘acceptor’ templates (Figure S1B, lanes 2, 4 and 6), suggesting that the displaced H2A-H2B dimers are not free to associate with the ‘acceptor’ template after remodelling, perhaps because the H2A-H2B dimer maintains an

    association with FACT in Pol I. These preliminary studies suggest that Pol I possesses

    H2A-H2B dimer displacement activity through its association with the histone

    chaperone FACT.

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MATERIALS AND METHODS

    Preparation of mono-nucleosomal and poly-nucleosomal DNA templates

    For the mono-nucleosomal DNA template, forward primer 5’ AGA TAT AAA AGA

    GTG CTG ATT T 3’ and reverse 5’ Cy5-labelled ATC AAA ACT GTG CCG CAG were

    used to amplify a region of plasmid AB485 that contains the 147 bp Mouse Mammary Tumour Virus (MMTV) nucleosome positioning sequence A (NucA) plus an extra 31 bp 5’ of the positioning sequence (Flaus and Richmond, 1998). Large scale (5 ml) PCR was carried out using Taq polymerase (Bioline). A series of twelve 5S rDNA nucleosome-positioning sequences (NPS) in a DNA stretch of 2496 nt was used to create the poly-nucleosomal template. The DNA was purified from the PCR reactions using Pharmacia AKTA purifier 10 and a ResourceQ ion exchange column (GE Health). The DNA was salt-gradient eluted from the column and the eluted DNA was ethanol precipitated. Lyophilised E. coli expressed X. laevis recombinant histones (Luger et al., 1997) were

    resuspended in 0.5 ml of unfolding-buffer (6 M guanidium.HCl, 20 mM Na-acetate pH 5.2 or Tris.HCl pH7.5, 10 mM DTT) and left at room temperature for 1 hr, then aliquots of each histone were centrifuged at 12000xg for 5 min. An equimolar mixture of the histones was dialyzed at 4 ºC against 3 changes of refolding buffer (2 M NaCl; 10 mM Tris-Cl pH 7.5; 1 mM Na-EDTA; 5 mM β-mercaptoethanol) (each dialysis against 2

    litres for at least 3 hrs) using Spectrum 6-8 kDa MW cut-off dialysis tubing to refold the octamer. The contents of the dialysis tubing were spun at 12000xg for 15 min at 4ºC to remove any precipitate. The protein was concentrated to 1-2 ml in a Millipore Ultrafree 15 ml concentrator at 3000 rpm, 4ºC and the refolded octamer was purified by gel filtration using Superdex 200 HR (GE Health) at room temperature in filtered and degassed refolding-buffer (without β-mercaptoethanol) at a flow rate of 1 ml/min and a

    maximum back pressure of 40 psi. The octamer-containing fractions were pooled, concentrated to ~15-30 μM and stored on ice.

    The mono-nucleosomal template was produced by reconstitution of the 178 bp template DNA with octamer in a reaction mix consisting of 85 pmol DNA, 100 pmol octamer, 2 M NaCl and 10 mM Tris.HCl pH 7.5. The poly-nucleosomal template was produced by reconstitution of the poly-5S NPS DNA with octamer in an identical reaction, except that

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    the DNA component was 100 pmol NPS (8.3 pmol DNA). The reconstitution mixes were salt-dialyzed stepwise at 4ºC as follows: 0.85 M NaCl, 10 mM Tris.HCl pH 7.5 for 2 h; 0.65 M NaCl, 10 mM Tris.HCl pH 7.5 for 2 h; 0.5 M NaCl, 10 mM Tris.HCl pH 7.5 for 2 h; and, 0.1 M NaCl, 10mM Tris-Cl pH 7.5 overnight. The integrity of reconstitution of the mono-nucleosomal template was checked using 5% native polyacrylamide gel electrophoresis followed by Cy5 fluorescence scanning. The poly-nucleosomal template was checked on a polyacrylamide-agarose composite gel and by partial micrococcal nuclease digestion. To this end, 2.6 μg of reconstituted or unreconstituted DNA was

    digested in a 30 μl reaction volume using 0.8 U/μl MNase in dHO supplemented with 1 2

    mM CaCl for 2 min on ice. The reaction was stopped by the addition of 30 μl of a mix 2

    consisting of 0.4 M NaCl, 0.2% SDS and 20 mM EDTA. The mixture was phenol-chloroform extracted and ethanol precipitated, followed by analysis on a 1.2% agarose gel.

    Cross-linking of histones in the octamer of a mono-nucleosomal template The histones in the octamer of the reconstituted mono-nucleosomal template of 178 bp (5 g, in 40 mM NaCl and 10 mM HEPES pH7.0, rather than Tris) were cross-linked with 5

    3mM BS, a homo-bifunctional cross-linking reagent (Bis(sulfosuccinimidyl) suberate, Pierce) for 30 min at room temperature, and then the crosslinker was quenched with 160 mM glycine for 10 min at room temperature. To control for quenching (inactivation of 3BS), a sample was mixed into a transcription reaction with free DNA; transcription should not be inhibited if quenching was complete. Control reactions consisted of

    3nucleosomal template incubated with buffer only (mock treatment), incubated with BS

    inactivated with glycine prior to incubation with the nucleosomal template, or not treated. The integrity and concentration of the nucleosomal template were checked by electrophoresis on a 5% native polyacrylamide gel alongside non-reconstituted DNA of known concentration, scanning for Cy5 fluorescence and analysis of the signals using AIDA 1D software (Fuji imaging). The efficiency of crosslinking was checked by SDS-PAGE gel electrophoresis followed by staining with SYPRO Ruby (Molecular Probes). 0.45 μg of the templates were then used in the transcription assays.

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H2A-H2B dimer displacement assay

    Histone octamer folded with Cy3-labelled H2B was used to reconstitute the biotinylated 178 bp NucA template by salt dialysis (see above). A slightly reduced octamer: DNA ratio (1: 0.7) was used to maximise nucleosome formation and minimise free DNA as the labelled octamer is less readily reconstituted. This template formed the ‘donor’ (of Cy3-

    H2B) in the transcription reaction. A tetramer ‘acceptor’ template was produced by reconstitution of a 147 bp 601 sequence with unlabelled tetramer, using a similar salt dialysis.

    End-to-end transcription reactions contained Pol I and 0.5 mM NTPs (no radiolabelled

    CTP) with 0.5 μg of each template (‘donor’ and ‘acceptor’). Reactions were incubated for 45 mins at 37ºC. As a positive control, donor and acceptor templates (0.5 μg) were incubated with 3.6 μl of RSC in 50 mM Tris HCl pH7.9, 1 mM MgCl, 1 mM ATP in a 2

    total volume of 25 μl for 45 minutes at 30ºC. After incubation, the reactions were applied to 100 μg of streptavidin-coated paramagnetic beads (Dynabeads M-280, Dynal Biotech) that had been pre-equilibrated in TM10i. The biotinylated donor template was bound by rotation for 20 mins at 4ºC. The supernatant was removed and set aside. The bound material was washed three times in TM10i. To release the donor template, 0.05 U of MNase in 20 mM CaCl made up to 25 μl in TM10i was added to the beads and the 2

    samples incubated for 5 minutes at 37ºC. The released donor template was removed from the supernatant. Sucrose was added to each sample (bound and unbound) to a final 6% and the samples were loaded onto a 5% native polyacrylamide gel. After the run the gel was scanned for Cy3-fluorescence in a Fuji FLA-5100 phosphoimager.

Transcription assays

    Transcription reactions were performed at near physiological salt concentrations as follows: Pol I was incubated for 45 min at 37ºC with nucleosomal or non-nucleosomal templates with 500 M each of UTP, GTP and ATP (or AMP-PNP), 10 μM CTP, 5 μCi

    32of [α-P] CTP (3000 Ci/mmol), 4 U RNasin in TM10i (50 mM KCl, 25 mM Tris.HCl pH7.9, 12.5 mM MgCl10 % glycerol, 1 mM EDTA, 0.015 % NP40, 1 mM DTT, 1 mM 2,

    sodium-metabisulfite, 50-100 ng/μl Bovine Serum Albumin). The reactions were treated

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    with 10 U of DNase (RNase-free, Roche) for 5 min at 37ºC followed by a further 5 min incubation with 200 μl of transcription stop mix (0.2 M NaCl, 20 mM EDTA pH 8.0, 1 %

    SDS, 0.25 μg/μl E.coli tRNA and containing 20 μg of Proteinase K). The radiolabelled

    RNA was phenol-chloroform extracted, ethanol precipitated and analyzed by denaturing gel electrophoresis (7.5M urea, 8% polyacrylamide) with RNA size markers (T3 and T7 RNA polymerase body-labelled run-off transcripts). End-to-end transcript levels were quantified with the aid of a Fuji phosphorimager (and Aida software).

    Non-specific transcription assays with sheared calf thymus DNA templates were performed as follows: Pol I MonoS fractions (5 l), immunoprecipitated complex on

    Dynal paramagnetic beads, or fractions of the supernatants following immunoprecipitations were incubated for 1 hour at 37ºC in a 25 l reaction volume with

    2.5 g sheared calf thymus DNA, 500 M ATP, GTP and UTP, 10 M CTP and 2.5 Ci

    32[-P] CTP (3000 Ci/mmol), 0.1 mg/ml -amanitin (Sigma), 1.5 mM MnCl, 0.03% NP-2

    40 in TM10 (25 mM Tris HCl pH 7.9, 12.5 mM MgCl, 10 % glycerol, 1 mM EDTA) 2

    with 0.05 M KCl. The reaction was stopped by the addition of 200 l of 50 mM sodium

    pyrophosphate, 50 mM EDTA, 0.5 mg/ml calf thymus DNA and then nucleic acid was precipitated with 800 l of 12.5% ice-cold Trichloroacetic acid for at least 1 hour on ice. Precipitated nucleic acids were recovered on Whatman GF/C filters, which were then washed with 10 ml of ice-cold 0.1 M sodium pyrophosphate and 1 mM HCl, followed by a rinse in 100% ethanol. Filters were air-died and incorporation of radiolabel was determined by Cherenkov counting.

Co-immunoprecipitation of Pol I and FACT

    Pol Iα was incubated with 5 μg of either SSRP1, Spt16 antibodies (sc-25382 and sc-

    28734, respectively) or control rabbit IgGs in TM10/0.1 M KCl for 2 h at 4ºC. The antibodies were captured with 20 μl of Protein A paramagnetic beads (Dynal, Invitrogen) (pre-equilibrated in TM10/0.1, 0.015% NP-40 and 1 mg/ml BSA). The supernatant was removed and the bound material washed twice in TM10/0.1 and once in TM10/0.05 M KCl. Supernatant and immunoprecipitated material were analysed by non-specific

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    transcription assay or western blotting using antibodies specific for Pol I subunit PAF53 (P95220).

    Immunoprecipitation of FACT from HeLa cell nuclear extract was performed as follows: HeLa nuclei were isolated and lysed in nuclear extraction buffer (10 mM HEPES with pH7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% Triton X-100, 0.2 M NaCl, 10% glycerol, 1 mM DTT, 10 mM PMSF, 1 g/ml leupeptin and 1 g/ml pepstatin A).

    Immunoprecipitation was performed with the SSRP1 (10D1) antibody prebound to protein G-Sepharose (GE Health) for 3 h at 4 ?, and washed in the lysis buffer. To test

    whether proteins immunoprecipitated independently of DNA, ethidium bromide or DNase I were included during the immunoprecipitation, as described previously (Tan et al., 2006). Immunocomplexes were boiled in SDS sample buffer and subsequently analyzed by SDS-PAGE and immunoblotting using antibodies specific for FACT subunits, CAST/PAF49 (Bethyl Laboratories), RNA pol II (CTD4H8, Santa Cruz Biotechnology), and RPC5 (Abgen).

    HeLa cells were transfected with an expression vector for Flag-tagged CAST/hPAF49 (Panov et al., 2006) and harvested 48 h post-transfection. Nuclear extracts from these cells were incubated with Flag-specific antibodies (M2-agarose, Sigma) for 2 h at 4ºC. Immunoprecipitated material and the resulting supernatant were analyzed by immuno- blotting with antibodies specific for FACT subunit Spt16 (sc-28734) and PAF53 (P95220).

Indirect immunofluorescence and confocal microscopy

    All steps of the immunostaining procedures were done at room temperature. HeLa cells grown on coverslips were first washed twice, followed by fixation with fresh 2% paraformaldehyde for 15 min. After a brief rinse in PBS, cells were permeabilized with 0.5% Triton X-100 in PBS (5 min), blocked with 1% bovine serum albumin in PBS, and probed with the indicated primary antibodies (SSRP1: monoclonal antibody 10D1; CAST/PAF49: rabbit polyclonal antibodies from Bethyl Laboratories, A301-294A). Secondary antibody incubation was performed for 1 h using Alexa 488-conjugated goat anti-rabbit IgG and Alexa Fluor 594-conjugated goat anti-mouse IgG (Molecular Probes

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    Inc.). To visualize DNA, cells were stained with DAPI. Cells were analyzed with the Zeiss LSM-510 inverted confocal laser-scanning microscope, using a 63×/NA 1.4 oil

    immersion objective lens.

    Down-regulation of SSRP1 expression in cells through RNAi and analysis of pre-

    TyrrRNA and of tRNA and 7SL RNA levels

    HeLa cells (in 24-well plates, cultured at 37ºC at 5% CO in Dulbeccos modified Eagles 2

    medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin and streptomycin) were transfected (INTERFERin, PolyPlus) with synthetic SSRP1-siRNA (0.1, 1, 10 and 20 nM final concentration; Santa Cruz Biotechnology, sc-37877) or with control siRNA (20 nM final concentration; Santa Cruz Biotechnology, sc-36869). After 24 h, transfection was repeated and cells were re-seeded into 6-well plates. Cells were harvested 24 h or 48 h later and whole cell extracts were analyzed by immunoblotting. Total RNA was isolated from the siRNA-treated cells (Qiagen RNeasy kit) to assess the 47S pre-rRNA levels by Northern blotting and to determine the levels of synthesis of the first 40 nt of the pre-rRNA by S1 nuclease protection as described (James and Zomerdijk,

    322004). The Northern blot was probed with a P end-labelled oligonucleotide

    complementary to 5`-end of rRNA gene (81 to 125 relative to transcription start site at +1; human rDNA sequence U13369). 47S pre-rRNA and S1 assay signals were normalized to the 28S rRNA signal from the ethidium bromide-stained agarose gel.

    Spt16-targeting siRNA: 5’-GGAATTAAGACATGGTGTG-3’ (nucleotides 942-960 in

    the cDNA). Cellular RNA was isolated from the transfected HeLa cells with Trizol Reagent (Invitrogen). Following isolation, RNA was subjected to DNase I (Roche) treatment to ensure complete elimination of the DNA from the sample. First-strand cDNA synthesis was performed with the SuperScriptII Reverse Transcriptase (Invitrogen)

    Tyraccording to the manufacturer’s instructions. Pre-rRNA (5’ETS), tRNA and 7SL RNA

    transcripts levels were determined by quantitative real-time PCR as described in the chromatin immunoprecipitation section. Triplicate PCRs were performed for each sample. Sequences of the primers used to PCR-amplify the 47S pre-rRNA (5-ETS), 18S rRNA,

    TyrtRNA and 7SL RNA transcripts are as described in the ChIP experiments. Transcript levels were normalized to GAPDH mRNA levels (GAPDH gene accession no.

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AF261085): 5’-GGTATCGTGGAAGGACTCATGAC-3’ (sense), 5’-

    ATGCCAGTGAGCTTCCCGTTCAGC-3’ (antisense).

    BrUTP incorporation in cells in which SSRP1 expression is downregulated by a plasmid-based dsRNAi

    To establish a plasmid-based dsRNAi system targeting endogenous SSPR1 or hSpt16, annealed oligonucleotides corresponding to a specific sequence of SSRP1 (5-

    TGGCAAGACCTTTGACTAC-3; nucleotides 677-695 in the cDNA) or Spt16 (5’-

    GGAATTAAGACATGGTGTG-3’; nucleotides 942-960 in the cDNA) were designed

    and ligated to the pSuper-neo+GFP (OligoEngine) according to the manufacturers

    instructions. The same sequence in the inverted orientation was used as the nonspecific dsRNAi control. The RNAi expression vectors were transfected in HeLa or HeK293 cells with Lipofectamine 2000 according to the manufacturers instructions (Invitrogen).

    SSRP1-siRNA or control- transfected HeLa cells were permeabilized for 6 min on ice with 1 mg/ml saponin in PBS, and incubated with 10 mM BrUTP for 10 min at 37ºC; in this short period BrUTP incorporation is primarily detected in transcriptionally active nucleoli (Leung et al., 2004). Cells were subsequently treated with fresh 4% paraformaldehyde for 20 min at 4ºC and 0.5% Triton X-100 (10 min, room temperature). To simultaneously visualize BrU-labelled RNA and FACT, staining was performed first with the SSRP1 monoclonal antibody 10D1 (Tan and Lee, 2004) and an Alexa Fluor 594-conjugated goat anti-mouse IgG, and subsequently with an Alexa Fluor 488-conjugated monoclonal anti-BrdU antibody (1:20 dilution; BD Biosciences Pharmingen). Stained cells were then analyzed by confocal microscopy.

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