|
Chromatin immunoprecipitation (ChIP)
Members of Pol III (Rpc82 and Rpc40), TFIIIB (Brf1) and TFIIIC
(Tfc1 and Tfc6) were tagged at their C-termini with three copies
of the hemagglutinin (HA) epitope at their genomic locus using
the Longtine method (1). Tagged components were precipitated
with monoclonal anti-HA antibody (clone 12CA5). The control ChIP
experiments (Fig. 1-3) were performed identically, using an isogenic
untagged strain (yHN3). Precipitation of the TBP component of
TFIIIB was performed using polyclonal TBP antisera (gift of Roger
Kornberg) with chromatin prepared from the Brf1-HA strain (YBC1988).
A control ChIP experiment for TBP with no antisera added yielded
similar results as the control above (data not shown).
The procedure used was adapted from one obtained from the Young
lab (http://web.wi.mit.edu/young/). Briefly, 50 ml of cells were
fixed in 1% formaldehyde overnight at 4°C. The fixed cells
were washed twice with ice-cold TBS and the cell pellets were frozen
and stored at -70°C. Pellets were thawed, resuspended in 500
ml lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCL, 1 mM EDTA,
1% Triton X-100, 0.1% sodium deoxycholate, 0.4mM DTT, 600 nM leupeptin,
2 mM pepstatin A, 2 mg/ml chymostatin, 500 mM PMSF, 2 mM benzamidine)
and transferred to 1.5 ml screwcap tubes. The tubes were topped
off with chilled glass beads (Biospec cat# 11079105) and processed
in a Minibeadbeater-8 (Biospec cat# 693) on homogenization setting
7x 1.5 min at 4°C with 2 min on ice in between sessions. The
lysates were collected from the beads in a refrigerated centrifuge.
Chromatin was sheared using a Sonicator XL (Misonix cat# XL-2020)
equipped with a microtip (Misonix cat# 418), sonicating 7x 25s,
setting of 3.5, 95% duty. This resulted in a peak fragment size
of 1-2 kb. The sonicated samples were centrifuged at 16,000x rcf
for 20 min. The supernatants were collected and Bradford assays
performed.
Either 50 ml (~2.0x107 beads) Pan Mouse IgG Dynabeads (Dynal Biotech
cat# 110.23) or 50 ml (~3.4x107 beads) Sheep anti-Rabbit IgG M-280
Dynabeads (Dynal Biotech cat# 112.03) per immunoprecipitation (IP)
were washed once in 20 volumes of PBS with 5mg/ml protease-free
BSA (Sigma cat# A3059) for 10 min. Beads were collected by magnet
and resuspended in 125 ml PBS with 5mg/ml protease-free BSA per
IP. 2.0 mg monoclonal anti-HA 12CA5 per IP was added to the Pan
Mouse IgG Dynabead suspensions. Either 1 ml rabbit TBP antisera
(gift of Roger Kornberg) per IP or no antibody was added to the
Sheep anti-Rabbit IgG M-280 Dynabeads suspensions. These suspensions
were rotated overnight at 4°C. Beads were collected by magnet,
washed once with 1.5 ml PBS with 5mg/ml protease-free BSA for 10
min, collected again and resuspended in 39 ml PBS with 5mg/ml protease-free
BSA per IP.
40 ml of the resuspended beads were added to 600 mg (protein) of
supernatant from the sheared chromatin above. In ChIP experiments
for ChIP-microarray analysis, 6.25 mg poly(dA)-poly(dT) (Amersham
cat# 27-7860-02) was added to each IP to reduce non-specific background,
but this was not added to IPs for the nutrient deprivation-reintroduction
analysis. The volume of each IP was brought to 400 ml with lysis
buffer (above). IPs were rotated for 4 hrs at 4°C.
Beads were collected by magnet and the supernatants discarded.
Beads were washed seven times for 5 min each with 900ml of the
following buffers: twice with lysis buffer (above), twice with
lysis buffer with an additional 360mM NaCl (500mM total) and no
DTT or protease inhibitors, twice with LiCL wash buffer (10mM Tris-HCl
pH 8.0, 250mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1mM EDTA),
once with TE (10mM Tris pH 7.5, 1mM EDTA). The bound material was
then eluted in 210 ml TE-SDS (10mM Tris pH 7.5, 1mM EDTA, 1% SDS)
for 10 min at 65°C. The supernatants were removed from the
beads. For a comparison of input DNA to these IP eluates, 30 mg
(protein) of the original supernatant from the sheared chromatin
above was added to 200 ml of TE-SDS; these input samples as well
as the IP eluates were processed as follows. 10 ml was removed
for Western analysis of the protein present (data not shown). The
remaining ~200 ml was incubated at 65°C overnight to reverse
the formaldehyde crosslinks.
The DNA in the samples was purified using Qiaquick PCR cleanup
columns (Qiagen) exactly as described in the manufacturer protocol
and was eluted in 50 ml of 10mM Tris-HCl pH 8.0. 3 ml of 500 mg/ml
DNase-free RNase (Roche Diagnostics cat# 1119915) was added to
each 50 ml elution and incubated at 37°C for 2 hr to eliminate
any residual RNA contamination. This purified DNA was either used
directly in multiplex PCR (described below) and qPCR analysis for
the nutrient deprivation-reintroduction analysis presented in Fig.
4, 5 and 7 or was amplified and labeled (described below) for ChIP-microarray
analysis presented in Fig. 1-3.
Amplification and labeling of ChIP material
Our procedure is adapted from the amplification and labeling procedure
presented in Iyer et al. (2) and available at http://www.microarrays.org
as “Amplification and Labeling of DNA,” with the
following changes. After Round A, samples were diluted to a final
volume of 50 µl and 15 ml was used in the Round B PCR.
In the Round B PCR, 2.5 ml of 500 mM Primer B was added to each
100 ml reaction, instead of 1 ml of 100 mM. The extension cycle
(72°C) was for 1 min, instead of 2 min, and the total number
of cycles was 24. In Round C PCR, 15 ml of product from Round
B PCR was added to 4ml of 25 mM MgCl2, 5ml of 10x PCR Buffer
(500 mM KCl, 100 mM Tris pH 8.3), 0.5ml modified dNTP mix (25
mM dATP, 25 mM dGTP, 25 mM dCTP, 12.5 mM dCTP), 1 ml of 500 mM
Primer B, 0.5 ml of Taq DNA polymerase (5U/ml), 3 ml of Cy3-dCTP
or Cy5-dCTP (Amersham Biosciences cat# PA55321), and 21 ml water
for a total volume of 50 µl, instead of 100 ml. The PCR
cycles were performed as described for a total number of 24 cycles.
The DNA from the entire 50 ml PCR reaction was purified using
a Qiaquick PCR cleanup column (Qiagen) exactly as described in
the manufacturer protocol and was eluted in 50 ml water. The
appropriate Cy3-labeled input DNA (50 ml) and Cy5-labeled ChIP-enriched
DNA (50 ml) were combined for hybridization to microarray slides
(see below).
Microarray analysis
Labeled input and ChIP-enriched DNA were hybridized competitively
to glass slides containing the entire yeast genome arrayed in
~14,000 segments, parsed as open reading frames (ORF) and intergenic
regions (prepared from primers/ORFs from Research Genetics/Invitrogen).
Arrays were constructed using a Lucidea spotter (Amersham Biosciences),
scanned using a Genepix 4000s scanner (Axon Instruments), and
analyzed using Imagene analysis software (BioDiscovery)
After discarding data from spots of poor quality, Cy3 intensities
are normalized to Cy5 intensities such that the mean ratio of Cy5/Cy3
intensities is one. The normalized ratios of Cy5 (labeled ChIP
DNA) to Cy3 (labeled input DNA) were then calculated. A table (ChIP
data set 1) containing the Cy5/normalized Cy3 ratios for each individual
experiment, their percentile ranks, and the average percentile
ranks is included. Also included in the data set are the coordinates
of each segment and a number for each tRNA that corresponds to
its linear order with respect to all of the chromosomes. This number
marks the tRNA as well as segments both 5’ and 3’ to
the gene (when available). The high identity between tRNAs enables
cross-hybridization to other tRNA segments on the array, effectively
preventing occupancy designation on the physical map. Therefore,
we chose the 5’ proximal fragment as the primary identifier
of each putative Pol III transcribed locus as these fragments are
sufficiently unique in most cases. In the cases where this fragment
was not available on the array, the 3’ proximal fragment
was substituted. The MIPS naming convention for a given tRNA is
assigned to the fragment used for the primary determinations of
occupancy. In this manner, all of the 275 tRNAs were uniquely represented
on the array. The non-tRNA targets were sufficiently unique for
self-representation.
Northern blot analysis
Total RNA from the Rpc82-HA strain (YBC1846) was isolated throughout
the nutrient deprivation and reintroduction regimen by hot-phenol
extraction. Fifty micrograms was loaded into each lane and run
on an 8% polyacrylamide gel under denaturing conditions. RNA
was electro-blotted to Zeta-Probe GT (Bio-Rad) charged nylon
membrane and probed with 32P end-labeled oligonucleotides. Oligo
sequences: Table 3 (supporting information). Similar results
were obtained by preparing RNA from an untagged strain (FT4)
(data not shown).
Multiplex PCR
Each lane is loaded with PCR product from a reaction that contained
as template: the input material for T=0 or ChIP material from
an untagged strain (FT4) or from the appropriately tagged strain
throughout the nutrient deprivation and readdition regimen. Multiplex
PCR was performed in reactions containing primer sets for two
amplicons: tRNAPhe(GAA)P2 and a control target (YNL102W ORF for
subunits other than TBP and TRA1 ORF for TBP). The PCR products
were resolved by electrophoresis in 2.5% Metaphor agarose (BioWhittaker
Molecular Applications) and visualized by ethidium bromide staining.
Primers and amplicon sizes: Table 3 (supporting information).
Supporting Methods References
1. Longtine, M. S., McKenzie, A., 3rd, Demarini, D. J., Shah, N.
G., Wach, A., Brachat, A., Philippsen, P. & Pringle, J. R.
(1998) Yeast 14, 953-61.
2. Iyer, V. R., Horak, C. E., Scafe C. S., Botstein, D., Snyder,
M. & Brown, P.O. (2001) Nature 409, 533-538.
Roberts et al., and B. Cairns
Supplemental Figure Legends, Methods, Figures and Tables
Figure Legends
Fig.
6 (below) Northern blot identifying SNR52 transcripts.
Northern blot of total RNA from strain FT4. Lane 1 Probe, SNR52
predicted mature form; Lane 2 probe, SNR52 A Box; Lane 3 probe,
SNR52 B Box. Each probe identifies a common band attributed to
the immature form of SNR52 migrating at ~250 nucleotides. The
probe to the predicted mature form, (lane 1) also identifies
a band of approximately 90-100 nucleotides accounting for ~95%
of the total signal in lane 1. The Asterisk (*) denotes a band
of unknown origin.
Fig.
7 (below) Occupancy analysis of tRNALys(CUU)Gl by qPCR in response
to changes in nutrient availability The primers for tRNALys(CUU)Gl
encompass the tRNA gene. The control primers amplify the CDC2 or
TRA1 ORFs. (A) The initial occupancy levels (T=0) were determined
as in Figs. 4 and 5. (B-C) The values from fig. 7A were set to
100% and the levels relative to T=0 for each timepoint determined.
Graphs derived from representative ChIP experiments are provided,
with the qPCR reactions in triplicate. One member of each of the
three complexes is shown in each graph. The asterisk (*) denotes
a value for TBP at this time point not obtained in isolated repeats,
which typically show it as unchanged; graphs represent entire time
courses with multiple factors.
Fig. 8 (below) A model for the regulation of Pol III transcription
in vivo during nutrient deprivation/re-addition.
Our whole-genome occupancy and qPCR analysis shows enrichment of
nearly all pol III genes by two members of each of the three complexes
TFIIIC, TFIIIB, and Pol III under optimal growth conditions. Our
work, together with others, suggests that the occupancy observed
by all three members of the Pol III machinery in rich medium reflects
an equilibrium between three states: 1) an initiation complex (containing
all three complexes), 2) a reinitiation complex (which recycles,
containing only TFIIIB and Pol III), and 3) a preinitiation complex
(containing only TFIIIC and TFIIIB). A commitment to transcription
by the initiation complex leads to the displacement of TFIIIC resulting
in one of two outcomes: TFIIIC ejection or tethering to Pol III
and TFIIIB, either of which lower its apparent occupancy during
the transcriptionally active state. We depict TFIIIC as semi-transparent
at active templates to reflect these two alternatives. Active transcription
of Pol III targets may involve a recycling TFIIIB-Pol III reinitiation
complex which our occupancy data suggests dominates the steady
state in nutrient replete conditions. However, this recycling reinitiation
complex may display a limited half-life, leading to passive loss
of Pol III at a slow rate. Nutrient deprivation may increase Pol
III loss and/or prevent reassociation. Loss of active Pol III allows
TFIIIC to re-occupy the template. TFIIIC then either recruits a
new TFIIIB or retains the TFIIIB resident on the template, alternatives
which are not distinguished by our data. We suggest that the nascent
TFIIIC-TFIIIB preinitition complex is either active (competent
to recruit Pol III) or inactive (not competent); the former dominates
during nutrient replete conditions and the latter during nutrient
deprivation/repression. TFIIIB semi-transparency in the preinitiation
complex reflects its high levels of occupancy during acute repression,
but reduced occupancy during prolonged repression. Nutrient readdition
enables recruitment of polymerase, forming a new initiation complex.
|
