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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 H2AH2B 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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Birch et al. – Supplementary Information
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 nucleosomepositioning sequences (NPS) in a DNA stretch of 2496 nt was used to create the polynucleosomal 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 dH2O supplemented with 1
mM CaCl2 for 2 min on ice. The reaction was stopped by the addition of 30 μl of a mix
consisting of 0.4 M NaCl, 0.2% SDS and 20 mM EDTA. The mixture was phenolchloroform 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
mM BS3, 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
BS3), a sample was mixed into a transcription reaction with free DNA; transcription
should not be inhibited if quenching was complete. Control reactions consisted of
nucleosomal template incubated with buffer only (mock treatment), incubated with BS3
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 SDSPAGE 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 Cy3H2B) 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 MgCl2, 1 mM ATP in a
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 CaCl2 made up to 25 μl in TM10i was added to the beads and the
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
of [α-32P] CTP (3000 Ci/mmol), 4 U RNasin in TM10i (50 mM KCl, 25 mM Tris.HCl
pH7.9, 12.5 mM MgCl2, 10 % glycerol, 1 mM EDTA, 0.015 % NP40, 1 mM DTT, 1 mM
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
[-32P] CTP (3000 Ci/mmol), 0.1 mg/ml -amanitin (Sigma), 1.5 mM MnCl2, 0.03% NP40 in TM10 (25 mM Tris HCl pH 7.9, 12.5 mM MgCl2, 10 % glycerol, 1 mM EDTA)
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 sc28734, 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 immunoblotting 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 prerRNA and of tRNATyr and 7SL RNA levels
HeLa cells (in 24-well plates, cultured at 37ºC at 5% CO2 in Dulbecco’s modified Eagle’s
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,
2004). The Northern blot was probed with a
32
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) according to the manufacturer’s instructions. Pre-rRNA (5’ETS), tRNATyr
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, tRNATyr and 7SL RNA transcripts are as described in the ChIP
experiments. Transcript levels were normalized to GAPDH mRNA levels (GAPDH gene
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Birch et al. – Supplementary Information
accession no. 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 manufacturer’s
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 manufacturer’s 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 594conjugated 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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REFERENCES
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James MJ and Zomerdijk JC (2004) Phosphatidylinositol 3-kinase and mTOR signaling
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Leung AK, Gerlich D, Miller G, Lyon C, Lam YW, Lleres D, Daigle N, Zomerdijk J,
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Lowary PT and Widom J (1998) New DNA sequence rules for high affinity binding to
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Tan BC, Chien CT, Hirose S and Lee SC (2006) Functional cooperation between FACT
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LEGEND TO THE FIGURE
Figure S1. Displacement of histone H2A-H2B dimers from nucleosomal DNA by Pol
I.
A. Design of a procedure to investigate the fate of histones during Pol I transcription.
The ‘donor’ (D) template consisted of the 178-bp DNA reconstituted with a nucleosome,
which included Cy3-labelled histone H2B, and carried a biotin at the 5’ end. The
‘acceptor’ (A) template consisted of an H3-H4 tetramer assembled onto a ‘601’
nucleosome positioning sequence of 147 bp (Lowary and Widom, 1998; Schalch et al.,
2005).
B. Pol Idisplaces histone H2A-H2B dimers from nucleosomal DNA.
A mixture of ‘donor’ (D, nucleosomal) and ‘acceptor’ (A, tetrasomal) DNA templates
were incubated in a reaction mix containing: Pol I and NTPs (transcription conditions;
lanes 1 and 2); Pol I alone (lanes 3 and 4); NTPs alone (lanes 5 and 6); or RSC plus
ATP (positive control; lanes 7 and 8). Following the reactions, ‘donor’ and ‘acceptor’
templates were separated by binding of the ‘donor’ to streptavidin beads. The ‘donor’
template was then MNase-digested to release it from the beads and to yield a minimal
histone particle. Both ‘donor’ and ‘acceptor’ were analyzed on a native 5%
polyacrylamide gel and scanned for Cy3-fluorescence.. Cy3-fluorescence of the ‘donor’
template samples (combined signals from the upper and lower bands from lanes 1, 3 and
5) from three independent experiments were quantified and the means and standard
deviations are presented in a graph (y-axis in arbitrary units).
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