Yeast Strains, Plasmids, and Proteins
All in vivo experiments were performed with S. cerevisiae
strains congenic to W303 (Thomas and Rothstein, 1989
) and include elc1Δ::URA3
(Harreman et al., 2009
). Untransformed cells were grown in YPD. For activation of the GAL
genes, cells transformed with pRS314-GAL10-GAL7
were grown at 30°C overnight in synthetic complete media (lacking tryptophan, with raffinose as the only sugar source) to stationary phase before being diluted and grown overnight to mid-log phase. Galactose or glucose was added to cells (as indicated) at a final concentration of 2% for the indicated time.
The plasmid GAL10-GAL7
used throughout this study was constructed by obtaining a GAL10
PCR product using primers targeted 343 bp upstream of the ORF to include all promoter elements (5′AGAGAAAGCTCGAG CTTTATTGTTCGGAGCAGTGCGG3′) and 47 bp upstream of the stop codon, to ensure no terminator elements were present (5′AGAGAGAGATCGAT TCAAGGTTACACAATCTTTCCAGTTCTC3′). This product was cloned between the XhoI and ClaI sites of pRS314. A GAL7
PCR product (or GAL7ΔTATA
PCR product for the control) was obtained with primers hybridizing 446 bp upstream of the ORF (5′AGAGAGAGGAGCTC ATATCACTCACAACTATTGCGAAGCG3′) and 38 bp upstream of the GAL7
stop codon (5′AGAGAGAGACTAG TTCTTAGTTTTTCAGCAGCTTGTTCCG3′). The fragment was cloned into the SacI and SpeI sites in a convergent orientation to the GAL10
gene. A 100 bp G-less cassette was PCR amplified from pGAL4CG (Lue et al., 1989
) and cloned into an EcoRI site at the 5′ end of the GAL10
ORF to form the promoter proximal G-less cassette (forward 5′AGAGAGAGGAATTCACTCACCCAATACTCCCTACTC3′; reverse 5′AGAGAGAGGAATTCGGGAGTGGAATGAGAAATG3′). Finally, a 371 bp G-less cassette obtained from the 365 bp G-less cassette of pGAL4CG (forward 5′AGAGAGAGATCGATCCTCCATACCCTTCCTCC3′; reverse 5′AGAGAGAGACTAGTGGGAGTGGAATGAGAAATG3′) was cloned into the SpeI and ClaI sites between the 3′ ends of the GAL7
) and GAL10
ORFs. The plasmids pYC10-7Fus and pYC10-7Fus-Δ7 (Prescott and Proudfoot, 2002
) used as templates for the PCR amplifications above were kindly provided by Nick Proudfoot.
FLAG-tagged RNAPII and 3HA-tagged RNAPII were purified from 100 l of yeast culture using previously published techniques (heparin column, ammonium sulfate precipitation, 8WG16 affinity purification, and MonoQ anion-exchange chromatography) (Cramer et al., 2001
), using strains with the endogenous Rpb3 or Rpb1 subunits (respectively) tagged at their genomic locus.
RNA Extraction and Northern Blotting
Cells were harvested following addition of 2% glucose or galactose for 75 min at 30°C, and RNA was extracted using the QIAGEN RNeasy kit and standard protocol.
Equal amounts of RNA were treated with 200 U of RNase T1 (Roche) for 1 hr at 37°C, prior to phenol-chloroform extraction. Formamide gel loading buffer (95% deionized formamide, 5 mM EDTA, 0.004% bromophenol blue, 0.004% xylene cyanol) was then added to the RNA, which was heated to 65°C for 10 min and separated by 7% denaturing (8.3 M urea) PAGE. RNA was transferred to Hybond-N+ nylon membrane (GE Healthcare) using semidry transfer blotting at 400 mA for 1 hr and UV crosslinked. Northern membranes were incubated with a random-primed 32P-labeled double-stranded DNA probe (corresponding to the long G-less cassette) for 1 hr at 65°C. This was followed by four washes with WB1 (2× SSC, 0.05% SDS) for 10 min at room temperature and two washes with WB2 (0.1× SSC, 0.1% SDS), each for 15 min at 50°C. Probed membranes were exposed to phosphor imager screens or Kodak BioMax MR film. “% Distal Cassette Transcribed” was calculated using data from the phosphor imager. The proximal cassette signals were equalized for “+/− Convergent Transcription.” The “+ Convergent Transcription” distal cassette signal was then calculated as a percentage of the “− Convergent Transcription” distal cassette signal (= 100%). The mean value and standard error were calculated from two biological replicates.
Reconstitution of Elongation Complexes
ECs were reconstituted using 150 nt DNA oligonucleotides from DNA technology, the sequences of which are as follows:
The DNA oligonucleotides were received HPLC purified, but subjected to further purification by 5.2% denaturing PAGE (8.3 M urea).
The sequences of RNA oligonucleotides were as follows: RNA Oligo1 (CCAGGAUAC) and Oligo2 (AUGGAGAGG); these were purchased from Dharmacon and purified via 20% denaturing PAGE.
Mono-ECs were assembled in Reconstitution Buffer (20 mM Tris [pH 7.9], 40 mM KCl, 20 μM ZnCl2, 5 mM DTT, 0.2 mM MgCl2, 0.75 μg/μl BSA) by incubating 1 pmol of DNA Oligo1 with 2 pmol of RNA Oligo1 (including 400 counts per second [CPS] of 32P end-labeled RNA Oligo1) at 65°C for 5 min followed by step-wise cooling to 25°C over a 40 min period. Next, 3 pmol of FLAG-tagged RNAPII was added for 25 min at 25°C, followed by the addition of 6 pmol of DNA Oligo2 (which was placed at 65°C for 5 min then stored on ice before addition) at 37°C for 10 min.
Di-ECs were formed by incubation of 1 pmol of DNA Oligo1 with 2 pmol of RNA Oligo1 (including 400 CPS 32P end-labeled RNA1), while in a separate tube 6 pmol of DNA Oligo2 was incubated with 12 pmol of unlabelled RNA Oligo2. Next, 3 pmol of FLAG-tagged RNAPII was added to the Oligo1-RNA1 mixture, and 18 pmol of HA-tagged RNAPII was added to the DNA Oligo2-RNA Oligo2 mixture. Following incubation for 25 min at 25°C, the DNA-RNA-RNAPII mixtures from each tube were mixed and allowed to hybridize for 10 min at 37°C.
Purification of Elongation Complexes
Mono- and di-ECs were incubated with Anti-FLAG M2 Affinity Gel (Sigma) for 1 hr at 4°C with shaking, prior to washing with EC-WB (50 mM Tris [pH 7.5], 500 mM NaCl, 0.5 mM EDTA, 10 μM ZnCl2, 0.05% NP-40, 10% glycerol) and 20 mM Tris-Cl (pH 7.5), followed by elution with 300 μg/ml FLAG peptide in TB (20 mM Tris [pH 7.9], 40 mM KCl, 20 μM ZnCl2, 5 mM DTT, 7 mM MgCl2, 0.75 μg/μl BSA). Semipurified ECs were then incubated with Anti-HA Affinity Matrix (Roche) for 1 hr at 4°C with shaking to isolate di-ECs, followed by repeated washes with EC-WB and 20 mM Tris-Cl (pH 7.5).
Ubiquitylation of Elongation Complexes
Unpurified ECs were incubated with yeast Uba1, Ubc5, and Rsp5 ± ubiquitin (with all lysine residues mutated) for 90 min at 30°C prior to transcription. Ubiquitylation efficiency was assayed by western blot with 4H8 Rpb1-CTD antibody.
ECs in TB were incubated with 600 μM NTPs (either AUC or AUCG) for 5 min (or as specified). Transcription was stopped with STOP buffer (20 mM Tris [pH 7.9], 40 mM KCl, 20 μM ZnCl2, 5 mM DTT, 20 mM EDTA [pH 8.0], ± 1 mg/ml proteinase K) and, if proteinase K was added, incubated at 37°C for 30 min. Samples were either phenol-chloroform extracted and the RNA ethanol precipitated for analysis of transcripts by 8.3 M urea denaturing PAGE (6% polyacrylamide), or resuspended in loading buffer (3% glycerol, 2.5 mM 2-mercaptoethanol, 0.75 μg/μl BSA) for native agarose electrophoresis on a 0.7% agarose gel (with 5 mM 2-mercaptoethanol, 0.1 mM MgCl2, 10 μM ZnCl2).
Purification of RNA from the agarose gel was performed by staining in a 1:10,000 dilution of Sybr Gold, allowing visualization and excision of the band corresponding to the mono- or di-ECs, followed by maceration, phenol-chloroform extraction, butanol extraction, and ethanol precipitation (Saeki and Svejstrup, 2009
). Transcripts were then analyzed by denaturing PAGE and exposure to phosphor imager screens or Kodak BioMax MR film.
ChIP was performed with WT or elc1Δ
strains containing GAL10-GAL7
. For the steady-state ChIP (), cells were crosslinked directly for 20 min at room temperature with 1% formaldehyde. For the ChIP in , the 0 min time point was taken after 2 hr in galactose and crosslinked (using conditions stated above). Glucose was added (2% final concentration) and time points taken every 2 min (up to 10 min) and crosslinked. Crosslinking was quenched with 200 mM glycine prior to resuspension and cell lysis in FA Lysis buffer (50 mM Tris [pH 7.5], 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate, 1× protease inhibitors [Otero et al., 1999
]). The chromatin was sonicated to a fragment length of 200–500 bp and then incubated with 2.2 μg of 4H8 (anti-CTD antibody) or 2.2 μg mouse IgG (where appropriate) for 2 hr prior to incubation with Protein G Agarose (Pierce) for 2 hr. Beads were washed three times for 3 min at room temperature in FA Lysis, once in FA500 (FA lysis with 500 mM NaCl), once in ChIP wash buffer (10 mM Tris [pH 8.0], 250 mM LiCl, 0.5% NP-40, 0.5% Na-Deoxycholate, 1 mM EDTA), and once in TES (10 mM Tris [pH 7.5], 1 mM EDTA, 100 mM NaCl). Finally, 100 μl ChIP elution buffer (50 mM Tris [pH 8.0], 10 mM EDTA, 1% SDS) was added and the samples were incubated at 65°C for 10 min. Thirty-five micrograms of RNase A (Sigma) was added to the eluate for 30 min at 37°C, followed by addition of 20 μg Proteinase K (Roche) for 2 hr at 42°C. DNA-protein crosslinks were reversed by incubating at 65°C for 6 hr, and the DNA was isolated using a PCR Purification Kit (QIAGEN).
Quantitative PCR was performed using 18 μl reaction buffer (0.2 μM forward primer, 0.2 μM reverse primer, 1× iQ Custom SYBR Green SuperMix [BioRad]) and 2 μl DNA. Primers targeted to the distal cassette of the GAL10-GAL7 construct were used (forward 5′GAGGGGATATGGAAAGGGAA3′; reverse 5′CCGGTGATTTCTTGTCTGCT3′) as well as control primers directed to the telomeres of chromosome 6 (forward 5′TAACAAGCGGCTGGACTACTTT3′; reverse 5′GATAACTCTGAACTGTGCATCC3′) and primers targeted to endogenous GAL1 (forward 5′ACGAGTCTCAAGCTTCTTGC3′; reverse 5′TATAGACAGCTGCCCAATGC3′).
The steady-state ChIP () values were divided by the input, telomere, and IgG control values and normalized to the signal for GAL10-GAL7ΔTATA (= 1). Values obtained for the ChIP in were divided by the input and telomere signal and normalized to the 0 min time point (= 100%). Columns on the graphs represent the mean value and bars show standard error, calculated from three biological replicates.
WT and elc1Δ cells were grown in YPD to mid-log phase and crosslinked as indicated earlier; however, cells were resuspended in FA500 before lysis. Sonicated chromatin was incubated for 2 hr with 2.2 μg 4H8 or mouse IgG prior to incubation with Protein G Agarose (Pierce) for 1 hr. Beads were washed three times for 3 min with FA500, twice with FA lysis, twice with ChIP wash buffer, and twice with TES. Elution, RNase treatment, and reverse crosslinking were performed as described for standard ChIP. DNA was purified by two rounds of phenol-chloroform extraction followed by ethanol precipitation before being subjected to standard library preparation techniques (Illumina) and Advanced Sequencing on an Illumina GAIIx DNA sequencer.
WT and elc1Δ cells were grown in YPD to mid-log phase prior to total RNA extraction (QIAGEN RNeasy Kit). RNA was subjected to standard library preparation techniques (Illumina), including Ribo-Zero hybrid-selection (Epicenter Biotechnologies), and Advanced Sequencing on an Illumina GAIIx sequencer.
Short read sequences from ChIP-Seq and RNA-Seq were aligned to the S288c reference genome (Version 20110326 downloaded from the Saccharomyce
s Genome Database [SGD] [Cherry et al., 1998
]) using the Novoalign (http://www.novocraft.com
) software. Reads at each position along the genome were extracted. In ChIP-Seq data analysis, ChIP signals (Log2 values) were divided by the relative IgG control for each genomic position excluding the ones with < 5 reads coverage. Quantile normalization between samples was then performed. Normalized ChIP-Seq values for the regions flanking the middle positions of intergenic regions (± 500 bp) between convergent or divergent gene pairs were extracted, and the median values of the signal in each position in these gene pairs were calculated and plotted.
RNA-Seq coverage at each position along the genome was divided by the total number of reads mapped in each sample. Regions flanking the end positions of every gene (± 500 bp) were taken, and normalized RNA-Seq signals were extracted for every gene. The mean values of RNA-Seq signals at each position in all genes were calculated and plotted.