Strains and plasmids
The strains used in this study are listed in Table 5.
The rnh1 gene (RNase H1SPBC.336.06c) cDNA was amplified by PCR from genomic DNA and ligated between the XhoI and BamHI sites in pREP3X vector to obtain a thiamine repressible rnh1 expression construct. Similarly, the other construct nmt1-rnh201 (a gift of J-I. Nakayama34), encoding the subunit A of the three subunit enzyme RNase H2 (SPAC4G9.02), was PCR amplified from genomic DNA and cloned between the restriction sites BamHI and SalI upstream of the nmt1 promoter in the vector pREP1. All fission yeast media were prepared according to Moreno et.al.27
Sporulation was checked either microscopically or by staining the colonies with iodine vapours. Efficiently switching strains produce equal number of cells of opposite mating type, which mate to form zygotic asci that sporulate on minimal media. The spore cell wall contains a starchy compound that stains dark purple with iodine vapours. Thus, efficiently switching cells of an h90 strain grow into colonies that stain dark purple with iodine while colonies of cells that switch inefficiently give light yellowish staining27. Determining the percentage of dark colonies provided a semi-quantitative measure of the switching efficiency. After iodine staining the colonies were photographed under Olympus StereoZoom Microscope.
Silencing status of any reporter gene was assessed by monitoring its expression using different plate assays. Expression of the ura4 reporter was detected by dilution spotting, wherein 5μl of ten-fold serial dilutions of the overnight cultures of the required strains were spotted on non- selective plates, plates lacking urcail and those containing 5’FOA. FOA provided counte-r selection to ura- cells. Thus, ura+ strains that grow well on ura minus but grow poorly on FOA plates, while ura- cells grow poorly on ura minus but grow well on FOA plates. Cell densities of all the cultures were normalized before serial dilution.
Expression of heterochromatic ade6 was assessed by streaking the cells for single colonies on plates containing limited adenine. Cells expressing ade6 produced light pink or white colonies on these plates and cells failing to express ade6 gene produced dark red colonies. The fraction of colonies giving pink/white colour provide a qualitative measure of the silencing defect. Similarly, the extent of silencing of the his3 locus at subtelomeric locus (his3-telo)28 was measured qualitatively by growth on plates lacking histidine.
Cloning, site-directed mutagenesis, protein over-expression and purification
Lysine residues (KKK) at positions 242-244 in Swi6 were mutated to alanine (AAA) in the nmt1-GFP-swi6+ expression construct (kindly gifted by Dr. Alison Pidoux) using the QuickChange site-directed mutagenesis kit (Stratagene), as per the manufacturer’s instructiaonnds, confirmed by sequencing. The mutant is referred to as swi63K→3A. The wild type full length Swi6 and Swi63K→3A were tagged with GST at amino-terminus by cloning into pGEX-KG expression vector as BamHI-HindIII fragments.
Expression and purification of GST-tagged Swi6 protein
The recombinant constructs containing GST, GST-Swi6, GST-Swi63K→3A, GST-Swi6 -CD, GST-Swi6-CD-Hinge and GST-Swi6 (CSD) were transformed in to BL21(DE3) host cells. The transformed cells were cultured in the LB-Amp medium at 37°C. At mid-log phase, cells were induced with 1mM IPTG and incubated further at 25°C for 8hrs. For purification of GST-tagged proteins, 2-4 mg protein extract was added to 50 µl of Glutathione Sepharose beads (Cat. no. 17527901, GE healthcare Ltd.) equilibrated with 1XPBS and incubated for 10-14 hours at 4°C. Purified proteins were collected after subjecting the mixture to elution for 2hrs at 4°C with 250 µl elution buffer containing 10mM reduced glutathione. Purification was confirmed by SDS- PAGE.
Expression of the nmt1 promoter driven GFP tagged swi6+ as well as swi63K→-3A gene was induced by removing thiamine from the medium of an overnight grown culture by washing the cells thoroughly and growing them further in medium lacking thiamine. It took 17-18 hrs of induction to achieve maximum expression level. Swi6 protein was detected by Western blotting with either anti-Swi6 (in-house, 1:10000), anti-GFP [Santa Cruz (sc-9996) 1:1000] or anti-GST [Santa Cruz (sc-138), 1:1000] antibodies. Wherever required, anti-a-tubulin antibody (Sigma, cat# T-9026; 1:4000) was used to probe the loading control. Alkaline phosphatase conjugated anti-rabbit (Sigma, cat# A-3562; 1:20,000) antibody was used, as per instructions. Western blots were developed using nitroblue tetrazolium (NBT) and bromo-chloro-indolyl phosphate (BCIP) as chromogenic substrates.
10ml cultures of cells expressing vector, nmt1-GFP-swi6+ or nmt1-GFP-swi63K→-3A in swi6Δ background were harvested by centrifugation at 5000 rpm at RT. Cells were fixed in chilled 70% ethanol, rehydrated sequentially in 50%, 30%, 20% ethanol and finally in water. The pellets were resuspended in 50μl of 1X PBS. 5μl of this suspension was then visualized unfldueorrescence microscope. Similar method was followed for visualizing subcellular localization of chromosomally GFP-tagged Swi6 under the influence of over-expressed rnh1 and rnh201.
Generation of [g-P32] labelled single stranded in vitro transcribed RNAs (ssRNA)
a) In vitro transcription
For synthesizing the single stranded RNA complementary to the heterochromatin-specific DNA sequences18,19, an in vitro transcription protocol was adapted from Wilusz lab protocol (http:// csu-cvmbs.colostate.edu/academics/mip/wilusz-lab/Pages/lab-protocols.aspx) with suitable modifications. The reaction was carried outby assembling nucleasefree water, 1μl of DNA template (1μg/μl conc.), 4 μl of ribonucleotidesmix (2.5mM each of ATP, GTP, CTP, and UTP) to a final concentration of 0.5mM, 2 μl of 10X RNA polymerase bufferand 1 μl of T7 RNA polymerase enzyme (Cat. no. AM2178, Ambion) were mixed and dilutedto 20 μl with nuclease- free water and incubatedat 37˚C for exactly 3 hour.s The reaction was subjected to Phenol (pH 5.2)/Chloroform extraction, followed by ethanol precipitation.
b) Polyacrylamide gel elution of in vitro transcribed RNAs
To obtain around 500 ng of in vitro transcribed RNA, about 10 reactions were set up and pooled. The pellet obtained after precipitation was resuspendedin 20 μl 1X formamide dye and loaded on 10% to 20% acrylamide/8M urea gels depending on the size of the required in vitro transcribed RNA. Subsequently, the gel was stained with Syber gold nucleic acid stain (Cat. no. S11494, Invitrogen Life technologies) for visualization under UV transilluminator. The RNA bands were located along the RNA marker (Cat. no. AM7778, Ambion) were excised and frozen at -20 ˚C for 20 min. Thereafter, the gel pieces were crushedand resuspended in 1 ml of Crush and Soak buffer. The tubes were incubated at 37˚C and 700 rpm for 12-14 hrs on a thermo mixer. The supernatant was then subjected to ethanol precipitation.
c) Dephosphorylation of 5’ends of RNA
The ssRNA generated by in vitro transcription was dephosphorylated before labeling. The dephosphorylation reaction was set up with 500ng of ssRNA, Alkaline phosphatase calf intestinal enzyme, CIP (Cat. no. M0290, New England BioLabs Inc., USA), 1X CIP buffer, RNase inhibitor (Cat. no. AM2696, Ambion) and nuclease-free water upto 25 μl. The reaction was incubated at 37˚C for 1 hour, phenol/chloroform extracted and ethanol precipitated.
d) 5-end labeling of RNA
To perform 5’-end labeling of dephosphorylated RNA, a reaction mix containing 500ng of dephosphorylated RNA, T4 Polynucleotide kinase (PNK) enzyme (Cat. no. M0201S, New England BioLabs Inc., USA), 1X T4 PNK buffer, RNase inhibitor, [g-32P]-dATP (>3300 Ci/ mmol) was obtained from Board of Radiation and Isotope Technology (BARC), Mumbai and nuclease-free water up to 50ml was prepared and incubated at 37˚C for 1hr. Unincorporated isotope was removed by passing through microspin G-50 columns (Cat. no. GE27-5330-01, GE Healthcare Ltd.). The eluent was stored at -70˚C and used for performing RNA-EMSA.
e) RNA-DNA hybrid formation
For producing RNA-DNA hybrids, the ssRNA was annealed with equimolar amount of complementary DNA oligo in presence of 1X transcription buffer. The reacting was incubated at 85oC for 5 minutes to remove the secondary structures and was subjected to gradual cooling to 55oC. At 55oC, the reaction was incubated overnight for annealing and checked on 10% native TBE gel before using for the EMSA experiments.
f) Synthesis of long RNAs
For in vitro synthesis of ‘Revcen’ RNA, a long centromeric sequence shown in Djupedal et al19 was PCR amplified and cloned in the Litmus-28i plasmid. The clone containing the DNA sequence complementary to ‘RevCen’ RNA was digested with HindIII to obtain a linear plasmid. The linearized plasmid was used as a template for in vitro transcription as described earlier. The reaction containing in vitro transcribed RNA was gel purified and subjected to alkaline phosphatase treatment followed by 5’end-labeling with [γ-32P] dATP (10 mCi/μl, specific activity 3300 Ci/mmol) and used for EMSA experiments. The ‘Cen100’ transcript, as reported by Keller et al (SPAC15E1.04)6, was also synthesized and labeled by the same protocol. The radio-labelled oligonucleotides were purified by passing through a BioGel P-6 column (Biorad Inc.).
Electrophoretic mobility shift assay (EMSA)
For EMSA, purified protein extract was mixed with ss siRNA or siRNA-DNA hybrid that was radiolabeled with [g-32P]ATP with polynucleotide kinase in the binding buffer containing 20mM HEPES (pH 7.5), 50mM KCl, 2mM EDTA, 0.01% NP40, 1mM DTT, 5 units RNase inhibitor, 1 μg BSA and 5% glycerol alongwith ~5 μg budding yeast total RNA or poly dI-dC as non- specific competitor, respectively), followed by incubation on ice. The samples were loaded on native TGE/ polyacrylamide (the tracking dyes Bromophenol blue and Xylene cyanol were not used in loading buffer as they interfere with the binding of proteins with nucleic acids). A negative control (without protein) was also loaded on the gel. 5X loading buffer containing both tracking dyes was added to negative control to track migration. For competition analysis, 0.1, 1.0 and 10 pmol of RNAs and DNAs were used. The gel was dried in gel dryer and exposed to X-ray film scanned in a phosphoimager.
The band intensities of the DNA-protein complex and free DNA for each lane were quantified using Scion Image software and the data were plotted according to a non-linear regression equation corresponding to one site specific-binding model as follows:
Y = Bmax.X /(Kd+X), where Y represents the amount of protein-bound radioactivity, X, the protein concentration, Bmax –the total change in radioactivity counts and Kd, the equilibtium binding constant23.
Chromatin Immunoprecipitation (ChIP) Assay
Cells of appropriate strains were grown to an OD600 of 0.6 and crosslinked for 30 min with 3% formaldehyde at 18oC. Immunoprecipitation was performed with the following antibodies: 1 μl anti-H3K9me2 (Upstate, 07-353) and 2 μl polyclonal anti-Swi6 per 400 μl reaction. Multiplex radioactive PCR amplifications of the immunoprecipitated chromatin samples, in the presence of α-P32ATP as the source of radiolabelled ATP in addition to non-radioactive dNTPs, were done using the following primers
dh For: 5’-GGAGTTGCGCAAACGAAGTT-3’
dh Rev: 5’-CTCACTCAAGTCCAATCGCA-3’
The products are resolved on 4% 1X TBE polyacrylamide gel, exposed to magnetic screen and scanned in Fuji ImageProcessor. The bands are quantified densitometrically from three independent sets of PCR using inbuilt MultiGauge Software. Data presented as mean SE. Data was analyzed through one-way ANOVA with Turkey’s post-hoc test where *** denotes p<0.001 when compared with vector and ns stands for non-significant.
Small RNA was isolated from immunoprecipitated samples from cells expressing GFP-Swi6+, GFP-Swi63K→3A and TAP-Tas3. Cells from 500 ml cultures were harvested at log-phase (OD595= 0.5-0.7) were washed once with 10 ml ice-cold water and once with ice-cold STOP buffer (0.5% SDS, 5mM EDTA pH 8, 100 µg proteinase K) at 4°C. Cells were resuspended in (one-fifth the culture volume) HB buffer (25mM MOPS pH 7.2, 15mM MgCl2, 15mM EGTA, 1mM DTT, 1% Triton-X100, 10% glycerol) supplemented with PMSF (to a final concentration of 1mM) and protease inhibitor cocktail (PIC) (at 1:100 dilution) and dispensed as 1 ml aliquots into bead beater tubes. Ice-cold zirconium or acid-washed RNase-free glass beads were added upto 500μl mark, given 10-12 pulses of 1 min each alternated with 5 min incubations on ice. The lysate was centrifuged at 13500 rpm at 4°C for 1 hr. The clarified supernatants were removed to fresh tubes and mixed with 100% glycerol to a final concentration of 10%. The expression of the protein of interest can be checked at this point by Western analysis (as described previously).
The extracts were kept at -80°C until further use. For immunoprecipitating TAP tagged proteins, commercially available TAP antibody conjugated agarose beads (Santa Cruz, sc-32319 AC) were used and no coupling was needed. These beads were directly used after equilibration with IP buffer. 2μl of anti-Swi6 antibody was conjugated to 50 µl protein-A sepharose beads (Sigma, P9424-1ml) in a 500 μl 1X PBS containing coupling reaction for 16hrs at 4°C with continuous mixing. The antibody bound beads are then washed twice with 500μl of 0.2M borate buffer (pH 9) and subsequently incubated in 500μl of 0.2M respective borate buffer containing dimethylpimelimidate (DMP) at a concentration of 5mg/ml for 30 minutes at RT with mixing.
DMP-mediated coupling was stopped by washing the beads once with 500μl 0.2M of ethanoleamine (pH 8.0). The residual DMP was quenched by incubating the beads in 500μl 0.2M of ethanoleamine (pH 8.0) for 2 hrs at RT. The beads were then washed once with 1X IP buffer [50mM Tris-Cl (pH 7.5), 150mM NaCl, 0.5% NP40 and 0.5mg/ml BSA). Approximately 3 mg of crude extract was bound in the presence of 1X IP buffer for 16hrs at 4°C with mixing. Beads were then washed once with IP wash buffer [50mM Tris-Cl (pH 7.5), 150mM NaCl, 0.1% NP40 (or 0.1% Tween-20) and 1mM EDTA (pH 8.0)]. To each 50 μl beads loaded with immunoprecipitate, 200μl each of LETS buffer [100mM LiCl, 10mM EDTA (pH8.0), 10mM Tris (pH 7.4), 0.2% SDS] and phenol (pH 5.2) were added. Samples were vortexed gently with intermittent ice incubation (30 sec on, 30sec off) followed by 1 hr at 37°C. The beads are then centrifuged at RT for 5min at 12000rpm. The collected supernatants were extracted once with equal volumes (~400μl) of phenol (pH 5.2): chloroform (1:1). RNA in the upper aqueous phase was precipitated with 20μg glycogen, one-tenth volume of 5M LiCl (40μl) and 2.5 volume of chilled absolute ethanol (1100μl) at -80oC for 16hrs. Pellet was washed with 300μl chilled 80% ethanol and dissolved in 250μl of RNase-free water. To the dissolved RNA, 250μl PEG precipitation solution (20% PEG (MW8000) in 2M NaCl) was added. Contents were incubated on ice for 30 min and centrifuged at 12000rpm for 20 min at 4oC. The supernatants were transferred to fresh tubes while the pellets were first dissolved in TE (pH 8.0), ethanol precipitated, washed with 80% ethanol, dried and resuspended in RNase-free water. This contains the population of long RNA while the supernatant isolated after PEG precipitation contains the small RNAs. Supernatant obtained after PEG precipitation was extracted once with phenol (pH 5.2): chloroform and small RNA present in the aqueous layer was precipitated using LiCl-ethanol system as earlier. The isolated small RNA was checked by running on a 15% 7M denaturing urea gel. This RNA was subjected to Northern blotting with probes complementary to dh sequences of the S. pombe centromeric repeat regions. Alternatively, they were used as riboprobes for Southern blots of PCR products generated from the centromeres and the mating type loci of fission yeast, like, dh, dhk and dg; act1 was taken as a negative control. Finally, one set of the isolate RNA was subjected to next generation sequencing.
The NEBNext® Multiplex Small RNA Library Prep Set for Illumina based system was used for 3’ adaptor ligation, cDNA synthesis, 5’ adaptor ligation and PCR, followed by size selection and sequencing using the Illumina platform was performed by Bionivid Technology Pvt. Ltd., Bangalore.
The sequencing data was analyzed using bowtie2 and R-packages as detailed in the following section.
RIP-seq data processing
Gene annotations and reference genome of fission yeast were downloaded from https://www.pombase.org/. Reference genome was indexed using ‘bowtie2-build’ *
http://bowtie-bio.sourceforge.net/bowtie2/index.shtml). We trimmed the 5’ and 3’ Illumina small RNA-seq adapters sequences (GUUCAGAGUUCUACAGUCCGACGAUC and TGGAATTCTCGGGTGCCAAGG, respectively) using ‘cutadapt’ (https://cutadapt.readthedocs.io/). Duplicates were removed using ‘rmdup’ utility of SeqKit package (https://bioinf.shenwei.me/seqkit). The clean reads were mapped onto the indexed reference genome using bowtie2 with ‘–sensitive’ setting. The quality of the reads was assessed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) at each step. We binned the raw reads at 5kb (for boxplot) and 200 bp (for line plot) resolutions and quantile normalized the read counts across samples using the function ‘normalize.quantiles’ of ‘preprocessCore’ R-package (https://www.bioconductor.org/packages//2.11/bioc/html/preprocessCore.html). The centromere annotations were obtained from https://www.pombase.org/status/centromeres. The telomeric and sub-telomeric regions were defined as 0-50Kb and 50-100Kb regions from the chromosome ends. The read-counts for swi6+ and swi63K→3A samples were divided by those of control (‘vector’) sample. We calculated the log2 ratio of read counts of swi63K→3A to swi6+ for plotting purpose. Sequence and annotations for mating-type region were obtained from https://www.ncbi.nlm.nih.gov/nuccore/FP565355.1. The reads were independently mapped onto mating-type sequence following the same procedure as earlier and processed at 200 bp resolution. We further identified the 5kb bins that had at-least one of the sequence motifs known to interact with Swi6 (Table X) using in-house script. P-values were calculated using two-tailed Mann-Whitney U tests on R.
The quality scores of RIP-Seq data is shown in Extended Data Table 6.
ChIP-seq data processing
We downloaded the ‘sra’ files of the ChIP-seq experiments, such as H3K4me3, H3K36me3, H3K9me2, H3K9me3, RNA PolII and Swi6 from GSE83495. We converted the files to ‘fastq’ format using ‘fastq-dump’ of NCBI SRAtoolkit. The reads were then mapped on the indexed genome reference using bowtie2 with default parameters. We converted the ‘sam’ files into ‘bam’ files using ‘samtools’ and ‘bam’ to ‘bed’ using ‘bedtools’ with default parameters. We binned the raw reads at 5kb resolution and labeled bins appropriately when their coordinates mapped into telomeres, centromeres and mating region. We plotted the density of read counts for the bins with RIP-seq log2 ratio (mutant to wild-type) > +1 or < -1. The p-values were calculated using Mann Whitney U tests.
Gene expression analysis
FPKM values for WT pombe strain were downloaded from GSE104546 and the average gene expression was calculated for each 5kb bin. We plotted the distribution of average gene expression values for the bins with RIP-seq log2 ratio (mutant to wild-type) > +1 or < -1. The p- value was calculated using Mann Whitney U test.
DNA-RNA hybrid Immunoprecipitation (DRIP) Analysis
Cells harvested from 500 ml log phase culture were washed with 1X PBS, resuspended in 2 ml PEMS (100mM PIPES, 1mM MgCl2, 1mM EDTA, 1.2 M sorbitol) supplemented with 20ul lyticase/zymolase and keep at 37°C for 30 min for spheroplasting. Spheroplasts were resuspended in 1.6ml TE containing 42ul of 20% SDS and 10ul of 10mg/ml Proteinase K and overnight at 37oC. Supernatant was extracted once with phenol:CIA and genomic DNA in the aqueous phase ethanol precipitated. 10ug of this DNA was digested overnight with Sau3AI at 37°C in 150 µl reaction. Following a phenol clean-up ethanol precipitated Sau3AI digested DNA was dissolved in TE without RNase A. One aliquot of the digested DNA was subjected to overnight RNase H (NEB cat#M0297) treatment at 37°C. Subsequently, RNAse H treated samples were purified through phenol extraction and ethanol precipitation and pellet dissolved in 50 µl TE mixed with binding buffer [10mM NaPO4 (pH 7.0), 0.14M NaCl, 0.05% TritonX-100]. 50 µl of both RNase H treated and untreated samples were saved as non-immunoprecipitated samples (NIPs). The DNA was immunoprecipitated overnight with 2 µl of anti-DNA-RNA hybrid mouse mono clonal antibody30 (S9.6; ATCC HB8730) at 4°C with constant mixing.
DNA-RNA hybrid/anti-body complex was harvested with protein G Sepharose beads and eluted in a buffer containing 50mM Tris (pH 8.0), 10mM EDTA, 0.5% SDS and 1.5 µg/ml Proteinase K at 55°C for 45 min. DNA from this complex was retrieved through phenol:CIA clean-up followed by ethanol precipitation. 2-3ng of this DNA was used for each RT-qPCR reaction set up with primers specific to the centromeric dh repeat region using SYBR Master Mix (ThernoFisher) in QuantStudio3 (ThermoFisher). Fold enrichment was calculated using ΔΔCt method39.