Yeast culture
S. cerevisiae cells were cultured in either YPD medium or Synthetic media supplemented with dextrose. S. pombe cells were grown in 2×YES medium (Coolaber, cat. # DZPM4510). Both species of yeast were grown at 30°C.
Construction of plasmids and yeast strains
All plasmids and yeast strains were generated using established protocols and are listed in Tables S1 and S2, respectively. The delitto perfetto technique was used to construct Rco1 mutant knock-in strains 38. Briefly, a CORE cassette was integrated at the locus encoding amino acid residue 150 of Rco1 by a double-strand DNA break and subsequent homologous recombination. Subsequently, the CORE cassette was substituted with either wild-type or mutant Rco1 sequences, each carrying a 2×Flag-tag. The knock-in strain was confirmed via genotyping and western blotting.
To construct CPH1 or CPH2 deletion strains in S. pombe, PCR products containing the KANMX6 selective marker were transformed into wild-type S. pombe cells (a gift from Dr. Jilong Liu), utilizing the lithium acetate method 46,47. The transformants were selected on 2× YES G418+ solid medium, and the deletions were verified through both genotyping and RT-qPCR.
The STE11-HIS3 reporter system was described before 48. Viability assays were conducted by serial dilution of cells on media dropping out either LEU or LEU and HIS.
Yeast whole-cell extracts immunoblotting
Cells were grown in 10 ml YPD at 30°C until the OD600 reached 0.5. The cell pellets were washed twice with 1 ml of ice-cold wash buffer (20 mM Tris.HCl pH7.5, 150 mM NaCl and 1 mM EDTA) and then resuspended in 50–75 µl of WCE buffer (50 mM Tris. HCl pH8.0, 5 mM EDTA, 0.8 M NaCl, 15% glycerol and 0.01% IGEPAL CA-630) freshly supplemented with protease inhibitor cocktail tablets (Sigma, cat. # P8215), 10 mM DTT, 1 mM PMSF and 1 mM Benzamidine.HCl. After adding 100 µl of 0.5 mm glass beads, the samples were vortexed at high speed for 6 times of 15 seconds, with cooling intervals on ice. Subsequently, the samples were centrifuged at 14,000 rpm for 10 min, and the supernatant was carefully collected. Protein concentration of the whole cell extracts was determined using Bradford assay. Immunoblotting using anti-FLAG HRP antibody (Sigma, cat. # A8592) was performed according to the manufacturer's instructions.
Recombinant protein purification from E. coli.
To purify GST-tagged proteins, we expressed GST-CTD and GST-Rco1 N (1-260)-EGFP variants in BL21CodonPlus-RIL cells and followed a previously described protocol 48. Initially, cells were cultured in 2 L of LB media at 37°C until the OD600 reached a range of 0.4–0.6. Protein expression was induced by adding 0.2 mM IPTG, and the culture was incubated overnight at 16°C. For cell lysis, we used 2 M GST buffer composed of 2 M NaCl, 10 mM Tris.HCl (pH 7.5), 20 mM Na3PO4 (pH 6.8), 0.01% IGEPAL CA-630, and 1 mM β-ME. The lysate was obtained by sonication and subsequent centrifugation at 14,000 rpm for 10 minutes at 4°C. The resulting supernatant was collected and mixed with glutathione agarose beads (Shanghai Chuzhi Bio, cat. #SA008010) in 1 M GST buffer (1 M NaCl, 10 mM Tris.HCl pH 7.5, 20 mM Na3PO4 pH 6.8, 0.01% IGEPAL CA-630, and 1 mM β-ME). The mixture was incubated for 4 hours at 4°C on a SRT roller. After washing, the proteins were eluted using GST elution buffer (25 mM Tris.HCl pH 8.0, 50 mM NaCl, 10 mM β-ME, and 10% glycerol) supplemented with 100 mM Glutathione. Subsequently, the eluted proteins were dialyzed into GST elution buffer.
For His-tag purification, cells were lysed in His-tag binding buffer (0.5 M NaCl, 20 mM Tris.HCl pH8.0, 20 mM imidazole, 10% glycerol, and 5 mM β-ME). Following sonication, samples were centrifuged at 15,000 rpm at 4°C for 20 minutes. The supernatant was then incubated with pre-equilibrated Ni-agarose beads (QIAGEN, cat. # 30230) for 2 hours at 4°C. Subsequently, the beads were washed with His-tag binding buffer, followed by wash buffer (0.1 M NaCl, 20 mM Tris.HCl pH8.0, 40 mM imidazole, 10% glycerol, and 5 mM β-ME). Proteins were eluted with elution buffer (0.1 M NaCl, 20 mM Tris.HCl pH8.0, 300 mM imidazole, 10% glycerol, and 5 mM β-ME). After elution, proteins were dialyzed into the storage buffer (elution buffer without imidazole). Purified proteins were concentrated, aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C.
To purify the GST-cleaved Cph1 or Cph2 His-PHD1-MID/Alp13 heterodimers, we started with 1 mg of Cph1/2 His-PHD1-MID/GST-Alp13 dimers. These dimers were digested with TEV protease in TEV cleavage buffer (10 mM Tris pH 8.0, 150 mM KOAc, 1 mM DTT, and 0.5 mM EDTA) at 4°C overnight. After digestion, we used glutathione agarose beads (Shanghai Chuzhi Bio, cat. #SA008010) to remove any cleaved GST tags and undigested GST-Alp13 dimers. The resulting supernatant was then incubated with Ni-agarose beads (QIAGEN, cat. # 30230) supplemented with 20 mM imidazole for 2 hours at 4°C. Proteins were eluted using elution buffer and subsequently dialyzed into storage buffer.
Recombinant complexes purification from insect cells.
Recombinant Rpd3S, BUR and CTDK-I complexes were all purified from a Sf21 insect cell-based baculovirus expression system as previously described 48,49. Briefly, Sf21 cells (1×106 cells/ml) were co-infected with individual viruses encoding each subunit of Rpd3S, BUR or CTDK-I for 48 h. The cells were collected, washed with 10 ml of 1× PBS containing 1 mM PMSF, and lysed in BV-Lysis buffer (50 mM HEPES pH7.9, 300 mM NaCl, 10% glycerol, 0.5 mM EDTA, 2 mM MgCl2 and 0.2% Triton X-100) freshly supplemented with protease inhibitor cocktail tablets (Roche, cat. # 4693132001), 0.4 mM PMSF and 1 mM Benzamidine.HCl. The lysates were clarified by ultra-centrifugation (Beckman 50.2 Ti rotor) at 40,000 rpm for 30 min. Subsequently, 200 µl of anti-DYKDDDDK affinity agarose beads (Shanghai Chuzhi Bio, cat. # SA042005) were added to the supernatants and incubated at 4°C for at least 2 hours. The beads were washed 3 times with 10 ml of BV-Lysis buffer and eluted with 600 µl of 500 µg/ml of 3×FLAG peptides in BV elution buffer (50 mM HEPES pH7.9, 100 mM NaCl, 10% glycerol, 2 mM MgCl2 and 0.02% IGEPAL CA-630). For Flag-HA tandem purification, 5 volumes of BV-Lysis buffer were added to the Flag eluents, followed by the addition of 100 µl of anti-HA affinity agarose beads (Shanghai Chuzhi Bio, cat. # SA06850U). The mixture was then incubated at 4°C for 2 hours. After three times washing with BV-Lysis buffer, proteins were eluted using 600 µl BV elution buffer containing 500 µg/ml HA peptide (Beyotime, cat. # P9808).
TAP purification.
The native yeast Rpd3S complex was purified using tandem affinity purification, as previously described 24. Briefly, yeast strain expressing TAP-Rco1 were resuspended in extraction buffer (E buffer) (40 mM HEPES.KOH pH7.5, 350 mM NaCl, 10% glycerol and 0.1% tween 20), supplemented with complete proteinase inhibitors (Sigma, cat. #P8215) and lysed using a bead beater (Biospec). The extract was clarified by ultracentrifugation at 45,000 rpm for 1.5 hour before undergoing tandem purification by IgG Sepharose (Cytiva, cat. # 17096901) and Calmodulin resin (Cytiva, cat. # 17052901). Rpd3S complexes were eluted with calmodulin elution buffer (CEB) (10 mM Tris.HCl pH8.0, 150 mM NaCl, 1 mM Magnesium acetate, 1 mM imidazole, 2 mM EGTA pH8.0, 10 mM β-ME, 0.1% IGEPAL CA-630 and 10% glycerol). Pol II complex was purified by similar tandem affinity purification (Rpb9-TAP) as described with minor modification 24,50.
Purification of S. pombe Rpd3S (Pst2-Flag) complex.
Pst2-Flag complex was purified from Pst2-C3Flag-HphMX6 stain 43, which was kindly provided by Dr. Chengcheng Wang. Briefly, the cell pellet of 12-liter culture was resuspended in lysis buffer (40 mM HEPES.KOH pH7.5, 350 mM NaCl, and 15% glycerol) supplemented with complete sets of fresh proteinase inhibitors and lysed using a bead beater (Biospec). The resulting extracts were clarified twice at 12,000 rpm for 30 min and 12,000 rpm for 1 hour. The supernatant was incubated with anti-DYKDDDDK affinity agarose beads (Shanghai Chuzhi Bio, cat. # SA042005) and eluted with BV elution buffer containing 500 µg/ml of 3×Flag peptide.
CTD peptide pull-down assay
CTD peptide pull-down assays were carried out as previously described 17. Biotinylated CTD peptides containing three tandem heptads of YSPTSPS were immobilized to the Dyna M-280 streptavidin beads (Invitrogen, cat. # 11206D) in peptide coupling buffer containing 25 mM Tris.Cl pH8.0, 1 M NaCl, 1 mM DTT, 5% glycerol and 0.03% IGEPAL CA-630. For each assay, 8 µg of CTD peptide coupled beads were mixed with either 2 µg of Rpd3S or 1 µg of Rco1-Eaf3 dimers in a 50 µl volume of peptide binding buffer containing 25 mM Tris.Cl pH8.0, 50 mM NaCl, 1 mM DTT and 5% glycerol. After incubating for 2 hours at 4°C, the beads were washed twice with peptide binding buffer and eluted with 12 µl of 1×SDS loading buffer.
In vitro kinase assay
Kinase assays were performed in a 10 µl reaction system, containing 0.5 µg GST-CTD WT or GST-CTD28, the Bur1/Bur2 complex, 1×kinase buffer (50 mM Tris-HCl pH7.5, 10 mM MgCl2, 0.1 mM EDTA, 2 mM DTT, 0.01% IGEPAL CA-630), and 2 mM ATP. The reaction was incubated at 30°C for 1 hour and stopped by the addition of either SDS loading buffer or 1M GST buffer.
GST-CTD pull-down assay
0.5 µg of either GST-CTD or GST-CTD28 was phosphorylated by incubating it with 0.5 µg of the BUR or CTDK-I complex within 10 µl reaction systems containing 1× kinase buffer and 2 mM ATP at 30°C for 1 hour. Subsequently, 2 µl of pre-equilibrated Glutathione Sepharose beads and 30 µl of 1 M GST buffer (10 mM Tris-HCl pH7.5, 1 M NaCl, 0.01% IGEPAL CA-630, and 10 mM β-ME) were added to the reaction and incubated at 4°C for 2 hours. The beads were washed twice with 1M GST buffer and once with 0.1 M GST buffer. In each pull-down reaction, 1 µg of recombinant proteins and 100 µl 0.1 M GST buffer were added to the beads and incubated at 4°C for an additional 2 hours. Subsequently, the beads were washed three times with 0.1 M GST buffer before being eluted with 20 µl of SDS loading buffer for 5 minutes at 95°C. Majority of the experiments were conducted at 100 mM NaCl concentration with a few exceptions that employed 150 mM NaCl, as specified in the figure legend. The resulting reaction was resolved in 8% SDS-PAGE and subjected to western blotting analysis. To evaluate the pull-down efficiency of PHD1-MID/GST-Alp13 in binding to GST-CTD, we utilized a recombinant GST-CTD WT that featured a C-terminal HA-tag. In this case, instead of Glutathione Sepharose beads, HA affinity beads were used to perform the pull-down of PHD1-MID/GST-Alp13.
EMSA assay (nucleosome binding assay)
Reconstituted unmethylated and H3K36me3 mono-nucleosomes were prepared and purified as described previously 51, utilizing a Cy5.5-labeled 216 bp DNA template that included the 601-positioning sequence. The binding reactions were carried out in 15 µl EMSA buffer (10 mM HEPES pH7.9, 50 mM KCl, 5% Glycerol, 4 mM MgCl2, 5 mM DTT, 0.05 mg/ml BSA and 0.1 mM PMSF). Wild-type and mutant Rpd3S complexes were incubated with either unmethylated or H3K36me3 mono-nucleosomes at 30°C for 45 minutes. The mixtures were then directly loaded onto a 3.5% native polyacrylamide gel (37.5:1) in 0.3×TBE. The gels were scanned with Typhoon 5 (Amersham) after three hours of electrophoresis at 4°C.
Droplet formation assay
Protein samples were added into reaction buffer composed of 50 mM Tris.HCl pH 7.0, 1 mM DTT, and 0.5 mM EDTA. The mixture also included varying concentrations of NaCl and either dextran (Sangon Biotech, cat. # A600375) or PEG-8000 (Fisher Scientific, cat. # BP233), as specified. The samples were immediately analyzed using a Nikon microscope for imaging. Three images of each condition were captured from different fields of view. The images were analyzed using OpenCFU software (version_3.9.0) to perform statistical analysis. The droplet area was set between 100 and 500 pixels as the threshold.
Droplet sedimentation assay
Proteins were mixed with droplet formation buffer containing TEV proteinase and incubated at 30°C for 1 hour, followed by another hour of incubation at 4°C. After collecting input fractions, the samples were centrifuged for 30 minutes at 13,300 rpm at room temperature. Subsequently, equal proportions of both the supernatant and pellet fractions were subjected to Coomassie blue staining and quantified by Image J (version_2.1.0/1.53c) software.
Yeast RNA isolation and RT-qPCR
Yeast cells from 10 ml OD600 0.5 culture was collected and washed with 1 ml of ice-cold STE buffer before being resuspended in 400 µl of AE buffer (50 mM NaAc pH5.2 and 10 mM EDTA), 40 µl of 10% SDS and 400 µl of acidic phenol (Sigma, cat. # P4682). The suspensions were incubated at 65°C for 10 minutes while being vortexed every minute to ensure uniform heating, and then centrifuged at 12000 rpm for 15 minutes at 4°C. The supernatant was incubated on ice for 5 min and mixed with 400 µl of chloroform by vertexing. After centrifugation at 12000 rpm for 10 min, the upper layer was carefully removed and isopropanol was added. The RNAs were finally resuspended in 30 µl of RNase-free water and stored at -80˚C.
cDNA was reverse-transcribed from 1 µg of total RNA using HiScript III RT SuperMix (Vazyme Biotech, cat. # R312-01) according to the manufacturer’s protocol. RT-qPCR reactions were performed in triplicate in 384-well plates using SYBR Green qPCR Master Mix (EZBioscience, cat. # A0001-R2) with gene-specific forward and reverse primers (Table S3). cDNA was amplified for 40 cycles using a Roche LC480 Real-time PCR system.
ChIP-seq analysis
ChIP was performed as previously reported 23. Briefly, 200 ml of OD600 = 1 yeast cells were collected and cross-linked by 1% formaldehyde (Thermo Scientific, cat. # 28908). The cells were then resuspended in 0.8 ml of FA-lysis SDS buffer (50 mM HEPES.KOH pH7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.2% SDS freshly added protease inhibitor cocktails, 0.6 mM PMSF and 1 mM Benzamidine.HCl) and vigorously vortexed with equal volume of glass beads, then rotationally incubated at 4°C for 40 mins. Cell lysates were passed through a 21G needle and sonicated with a duty cycle of 90% (0.9 sec ON; 0.1 sec OFF) for 12 pulses for 5 times, followed by clarification by centrifugation. An additional 10 cycles of sonication (12 pulses each time) were carried out to yield DNA fragments of approximately 300–500 bp. For immunoprecipitation, 30 µg of chromatin solution was mixed with 1 µl of H4Ac antibody (Millipore, cat. # 06-866) and incubate at 4°C for 2 hours in 300 µl FA-lysis buffer. The immunoprecipitated complexes were eluted twice at 65°C for 30 min with 200 µl of elution buffer, followed by addition of 2 µl of proteinase K (NEB, cat. # P8107S) and incubation at 55°C for more than 1 hour. The samples were then reverse-crosslinked overnight at 65°C. The ChIP-seq library was constructed using VAHTS® Universal DNA Library Prep Kit for Illumina V3 (Vazyme Biotech, cat. # ND607), and the resulting library was sequenced on an Illumina NovaSeq 6000 platform.
QUANTIFICATION AND STATISTICAL ANALYSIS
ChIP-seq data processing
Clean reads were mapped to S. cerevisiae S288C R64-2-1 and S. pombe ASM294v2 using Bowtie2 (version bowtie2_2.2.5), respectively. Subsequently, multiple-mapped reads were filtered using SAMtools (version SAMtools _1.16.1), and PCR-duplicated reads were removed with Picard (version Pichard_3.1.0). Index of the bam files was generated using SAMtools. BigWig files were generated for visualizing in Integrative Genomics Viewer (IGV) using BamCoverage of deepTools (version deeptools_3.5.1). BamCompare was applied to calculate the log2 fold change of mutant versus wild-type. ComputeMatrix, plotProfile and plotHeatmap functions from deepTools were also used to plot the gene body and flanking region heatmap graphs using the normalized signal intensity. The reproducibility of two biological replicates or correlation between ChIP-seq was assessed using Pearson correlation coefficient calculated by multiBamSummary (the read coverage of 1kb-binned matrices) and plotCorrelation function of deepTools.
RNA-seq and data processing
S. pombe cells were cultured in 2×YES medium at 30°C until OD600 reached 0.6–1.2. Total RNA was extracted with hot acidic phenol method, followed by cleanup with the RNeasy Mini kit (Qiagen, cat. # 74004). RNA-Seq libraries were prepared from total RNA using poly(A) enrichment of the mRNA to remove ribosomal RNA, and subsequently sequenced on an Illumina NovaSeq 6000 platform.
Clean reads were aligned to the Schizosaccharomyces pombe genome (ASM294v2) using STAR (version STAR_2.7.10a). Low mapping quality reads and PCR duplicates were removed using SAMtools (version SAMtools_1.16.1) and Picard (version Pichard_3.1.0). Counts per genes were generated using featureCounts tools (version subread_2.0.1). The DESeq2 R package (version DESeq2_1.36.0) was used for principal component analysis (PCA). For differential expression analysis, the edgeR package (version edgeR_3.38.4) was applied to evaluate changes. The threshold log2 fold change > 1 or < -1 and BH adjusted p-value from DESeq2 < 0.01 was used to determine statistical significance.
Analysis of the features of amino acids
R studio (version_4.2.2) was used to represent the distribution of the specified amino acid. To assess the conservation of the Rco1 protein, PhylomeDB in conjunction with Jalview software was applied to analysis the conservation among yeasts. For predicting the intrinsically disorder regions within the Rco1 protein, the PONDR-VSL2 tool (predictor of natural disordered regions based on various training data for short and long disordered sequences) was employed.
Quantification and data analysis
The statistical analyses were performed using the GraphPad Prism 9 software and statistical details are provided in figure legends. Significance was defined by p values determined by Two-tailed Student’s t-test.