Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae
- Department of Molecular and Cell Biology, University of California, Berkeley, United States
Figures
Figure 1 with 3 supplements
The Sir3–M.EcoGII fusion protein strongly and specifically methylated HML and HMR.
(A) Sir3–M.EcoGII is a fusion protein that nonspecifically methylates adenines in regions that Sir3 binds. (B) RT-qPCR of HMLα2 and HMRa1 mRNA, normalized to ACT1 mRNA, in strains expressing no fusion proteins (wild type, JRY11699, JRY9316), sir3∆::M.ECOGII (JRY13029, JRY13030), SIR2-M.ECOGII (JRY13625, JRY13626), SIR3-M.ECOGII (JRY12840, JRY13027), and SIR4-M.ECOGII (JRY13021, JRY13022). Data are the average of three biological replicates, and bars mark one standard deviation. (C) ChIP-seq of Sir3-M.EcoGII-3xV5 (top row, JRY12839) and DNA m6A immunoprecipitation and sequencing (DIP-seq) of no EcoGII (row two, JRY11699), sir3∆::M.ECOGII (row three, JRY12838), and SIR3-M.ECOGII (row four, JRY12840). Shown are 10 kb regions centered at HML (left) and HMR (right). Input results are plotted but not visible due to the strong ChIP-seq and DIP-seq signals. (D) Aggregate results from long-read Nanopore sequencing of sir3∆::M.ECOGII (black line, JRY12838), SIR2-M.ECOGII (green line, JRY13625), SIR3-M.ECOGII (purple line, JRY13027), and SIR4-M.ECOGII (pink line, JRY13021). The y-axis represents the percentage of reads in each position called as methylated by the modified base-calling software Megalodon (see Materials and methods). Shown are 25 kb windows at a control region on chromosome III to show background methylation (top row), at HML (middle row), and at HMR (bottom row). (E) Single-read plots from long-read Nanopore sequencing of SIR3-M.ECOGII (JRY13027). Each row of the plots is a single read the spans the entire query region, ordered by lowest average methylation on the top to highest average methylation on the bottom. Methylated adenines are colored purple, and unmethylated adenines are colored gray. Shown are 5 kb windows at a control region on chromosome III to show background methylation (top row), at HML (middle row), and at HMR (bottom row). M.EcoGII nor a strain expressing unfused M.EcoGII from the SIR3 promoter showed appreciable DIP-seq signal (C, Figure 1—figure supplement 1).
Figure 1—figure supplement 1
Sir3–M.EcoGII strongly and specifically methylated HML and HMR.
Biological replicates of DIP-seq of no EcoGII (top row, JRY09316), sir3∆::M.ECOGII (middle row, JRY12838), and SIR3-M.ECOGII (bottom row, JRY13027).
Figure 1—figure supplement 2
Sir3–M. EcoGII strongly and specifically methylated HML and HMR.
(A) Aggregate results from long-read Nanopore sequencing of sir3∆::M.ECOGII (black line, JRY12838, same as Figure 1D), SIR3-M.ECOGII (blue line, JRY13027, same as Figure 1D), and a biological replicate of SIR3-M.ECOGII (purple line, JRY12840). Plots are as described in Figure 1D. (B) Single-read plots from a biological replicate of long-read Nanopore sequencing of SIR3-M.ECOGII (JRY13027). Plots are as described in Figure 1E.
Figure 1—figure supplement 3
Sir3–M.EcoGII preferentially methylated linker regions.
(A) Aggregate methylation signal by Nanopore sequencing (purple line) of SIR3-M.ECOGII (JRY13027) plotted against nucleosome occupancy (gray line, Chereji et al., 2018) over a 3 kb window at HML (top) and HMR (bottom). The y-axis represents the signal of both % of reads methylated and nucleosome occupancy scaled to fit in the same plot. Data were smoothed using a loess function. (B) The average % of methylated reads from SIR3-M.ECOGII (JRY13027) in linker regions at HML and HMR (nucleosome occupancy below a threshold of 0.4 from Chereji et al., 2018) compared to nucleosomal regions at HML and HMR (nucleosome occupancy above a threshold of 0.4 from Chereji et al., 2018). The center line of each box plot represents the median. The boxes represent the 25th and 75th percentiles. Whiskers represent the range of values within 1.5× the interquartile range. p value was calculated using a quasibinomial general linear model (glm, see Source code 5).
Figure 2 with 2 supplements
Nucleosome binding was required for spread, but not recruitment, of Sir3 to regions of heterochromatin.
(A) Schematic of Sir3 protein domains. (B) Protein immunoblotting in strains expressing Sir3 (no tag, JRY11699), Sir3-3xV5 (JRY12601), and sir3-bah∆–3xV5 (JRY13621). Top row are 3xV5-tagged Sir3 proteins, and bottom row is the loading control Hxk2. The unedited blot is in Figure 2—source data 1. (C) RT-qPCR of HMLα2 and HMRa1 mRNA, normalized to ACT1 mRNA, in strains expressing SIR3-M.ECOGII (JRY12840, JRY13027), sir3∆::M.ECOGII (JRY13029, JRY13030), and sir3-bah∆-M.ECOGII (JRY13438, JRY13439). Data are the average of three biological replicates, and bars mark one standard deviation. (D) Aggregate methylation results at HML (top) and HMR (bottom) from long-read Nanopore sequencing of sir3∆::M.ECOGII (JRY13029, JRY13030), SIR3-M.ECOGII (JRY12840, JRY13027), and sir3-bah∆-M.ECOGII (JRY13438). Plots are as described in Figure 1D. (E) Single-read plots from long-read Nanopore sequencing of sir3-bah∆-M.ECOGII (JRY13438) at HML (top) and HMR (bottom). Plots are as described in Figure 1E. (F) Aggregate methylation results at four representative telomeres (1 L, 2 L, 4 R, and 11 R) from long-read Nanopore sequencing of the same strains as D. Shown are 15 kb windows of each telomere. Plots are as described in Figure 1D. (G) Single-read plots from long-read Nanopore sequencing of SIR3-M.ECOGII (JRY13027) and sir3-bah∆-M.ECOGII (JRY13438) at two representative telomeres (1 L and 2 L). Shown are 10 kb windows of each telomere.
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Figure 2—source data 1
Uncropped protein immunoblot of Sir3 mutants.
- https://cdn.elifesciences.org/articles/75653/elife-75653-fig2-data1-v2.zip
Figure 2—figure supplement 1
DIP-seq of SIR3-M.ECOGII (top row, JRY13027), sir3∆::M.ECOGII (middle row, JRY13030), and sir3-bah∆-M.ECOGII (bottom row, JRY13438).
Input results are plotted but not visible due to the strong DIP-seq signals. (A) Shown are 10 kb regions centered at HML (left) and HMR (right). (B) Shown are 10 kb regions at two representative telomeres (1 L and 2 L).
Figure 2—figure supplement 2
Methylation by Sir3–M.EcoGII and sir3-bah∆-M.EcoGII at all 32 telomeres.
Aggregate methylation results from long-read Nanopore sequencing of the same strains as Figure 2D. Shown are 15 kb windows of each telomere. Plots are as described in Figure 1D. Highlighted in yellow are windows shorter than 15 kb due to discrepancies between the S288C and W303 genomes (see Ellahi et al., 2015).
Figure 3 with 2 supplements
Sir3 expression level was not limiting for its spread from recruitment sites.
(A) RT-qPCR of SIR3 and M.ECOGII mRNA normalized to ACT1 mRNA in SIR3-M.ECOGII strains carrying an empty multicopy 2μ vector (JRY13670, JRY13671) and strains carrying a multicopy 2μ SIR3-M.ECOGII plasmid (JRY13672, JRY13673). Data are the average of four biological replicates, and bars mark one standard deviation. (B) Protein immunoblotting in the same strains as A (JRY13670-JRY13671) as well as SIR3-M.ECOGII (no plasmid, JRY13027) and sir3∆::M.ECOGII (JRY13029). Top row is Sir3, and bottom row is the loading control Pgk1. The unedited blot is in Figure 3—source data 1. (C) RT-qPCR of HMLα2 and HMRa1 mRNA normalized to ACT1 mRNA in the same strains as A as well as sir3∆::M.ECOGII (JRY13029, JRY13030). Data are the average of four biological replicates, and bars mark one standard deviation. (D) Aggregate methylation results at a control region on chromosome III to show background levels of methylation (top row), at HML (middle row) and HMR (bottom row) from long-read Nanopore sequencing of the same strains in A. The two colors for each genotype correspond to two biological replicates. Plots are as described in D. (E) RT-qPCR of CHA1 and OCA4 mRNA normalized to ACT1 mRNA in the same strains as C. (F) Aggregate methylation results at three representative telomeres (6 R, 15 R, and 10 R) from long-read Nanopore sequencing of the same strains as A. The two colors for each genotype correspond to two biological replicates. Shown are 15 kb windows of each telomere. Plots are as described in Figure 1D.
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Figure 3—source data 1
Uncropped Protein Immunoblot of Sir3-M.EcoGII overexpression strains.
- https://cdn.elifesciences.org/articles/75653/elife-75653-fig3-data1-v2.zip
Figure 3—figure supplement 1
Binding of Sir3–M.EcoGII measured by ChIP-seq upon overexpression of SIR3-M.ECOGII.
ChIP-seq of two biological replicates of Sir3–M.EcoGII strains carrying an empty 2μ plasmid (top two rows of each panel, JRY13670 and JRY13671) and two biological replicates of Sir3–M.EcoGII strains carrying a SIR3-M.ECOGII 2μ plasmid (bottom two rows of each panel, JRY13672 and JRY13673). Input signal is black, and immunoprecipitated signal is green. Note that each row is scaled to the maximum signal in the window shown to compare the relative levels of Sir3–M.EcoGII at recruitment and spreading sites across samples. (A) ChIP-seq signal at HML (left) and HMR (right). (B) ChIP-seq signal at telomere 6 R (left), telomere 15 R (center), and telomere 10 R (right).
Figure 3—figure supplement 2
Methylation upon overexpression of SIR3-M.ECOGII at all 32 telomeres.
Aggregate methylation results from long-read Nanopore sequencing of the same strains as Figure 3B. Shown are 15 kb windows of each telomere. Plots are as described in Figure 1D. Highlighted in yellow are windows shorter than 15 kb due to discrepancies between the S288C and W303 genomes (see Ellahi et al., 2015).
Figure 4 with 2 supplements
Repression of HML and HMR preceded heterochromatin maturation.
(A) Schematic of temperature-shift time course with sir3-8-M.ECOGII. (B) Protein immunoblotting in a strain expressing sir3-8-3xV5 (JRY13467) constitutively at 25°C (first lane), constitutively at 37°C (second lane), and at 30, 60, 90, 120, and 150 min after a shift to 25°C. Top row is 3xV5-tagged sir3-8 protein, the middle row is the same as the top row but at a higher exposure, and the bottom row is the loading control Hxk2. The unedited blot is in . (C) Aggregate methylation results at HML (top) and HMR (bottom) from long-read Nanopore sequencing of strains expressing sir3-8-M.ECOGII (JRY13114) grown constitutively at 25 or 37°C. Plots are as described in Figure 1D. (D) Aggregate methylation results at HML (top) and HMR (bottom) from long-read Nanopore sequencing of a strain expressing sir3-8-M.ECOGII (JRY13134) grown constitutively at 25°C (dotted gray line) and collected at 0, 15, 45, and 90 min after a temperature switch from 37 to 25°C. (E) RT-qPCR of HMLα2 (left) and HMRa1 (right) mRNA in strains expressing SIR3-M.ECOGII (black, JRY13027, JRY12840), sir3∆::M.ECOGII (green, JRY13029, JRY13030), or sir3-8-M.ECOGII (purple, JRY13114, JRY13134) collected at 0, 30, 60, 90, 120, and 150 min after a temperature switch from 37 to 25°C. Points are the average of three biological replicates and bars mark one standard deviation.
Figure 4—figure supplement 1
DIP-seq of sir3-8-M.ECOGII (JRY13114).
Shown are 10 kb regions centered at HML (left) and HMR (right). Cells were grown constitutively at either 25 or 37°C. Input results are plotted but not visible due to the strong DIP-seq signals.
Figure 4—figure supplement 2
Nanopore sequencing over temperature switch time course (biological replicate).
Aggregate methylation results at HML (top) and HMR (bottom) from long-read Nanopore sequencing of a strain expressing sir3-8-M.ECOGII (JRY13114) grown constitutively at 25°C (dotted gray line) and collected at 0, 15, 45, and 90 min after a temperature switch from 37 to 25°C.
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Saccharomyces cerevisiae) | Various | This paper | NCBITaxon:4932 | W303 Background; see Supplementary file 1 |
| Antibody | Anti-V5 (mouse monoclonal) | Thermo Fisher Scientific | Cat # R960-25 | (5 µl for ChIP, 1:5000 for Western) |
| Antibody | Anti-Hexokinase (rabbit polyclonal) | Rockland | Cat # 100-4159 | (1:20,000) |
| Antibody | Anti-m6A (rabbit polyclonal) | Synaptic Systems | Cat # 202-003 | 300 ng per 1 μg IP DNA |
| Antibody | Anti-Pgk1 (mouse monoclonal) | Invitrogen | Cat # 459,250 | (1:40,000) |
| Antibody | Anti-Sir3 (rabbit polyclonal) | N. Dhillon & R. Kamakaka | (10 μl for ChIP, 1:1000 for Western) | |
| Recombinant DNA reagent | SIR2-M.ECOGII | This paper | Fusion of SIR2 and bacterial adenine methyltransferase M.ECOGII | |
| Recombinant DNA reagent | SIR3-M.ECOGII | This paper | Fusion of SIR3 and bacterial adenine methyltransferase M.ECOGII | |
| Recombinant DNA reagent | SIR4-M.ECOGII | This paper | Fusion of SIR4 and bacterial adenine methyltransferase M.ECOGII | |
| Recombinant DNA reagent | YEp24 | doi:https://doi.org/10.1016/0378-1119(79)90004-0 | Empty 2 μm overexpression plasmid; see Supplementary file 2 | |
| Recombinant DNA reagent | pMB34 | This paper | M.ECOGII inserted into pFA6a-natMX6; see Supplementary file 2 | |
| Recombinant DNA reagent | pMB54 | This paper | SIR3-M.ECOGII inserted into YEp24; see Supplementary file 2 | |
| Recombinant DNA reagent | pFA6a-natMX6 | doi:10.1002/yea.1291 | natMX6 marker integration cassette; see Supplementary file 2 | |
| Sequence-based reagent | Various oligonucleotides | This paper | qPCR and cloning primers | See Supplementary file 3 |
| Commercial assay or kit | NEBNext Ultra II Library Prep Kit for Illumina | New England Biolabs | Cat # E7645 | |
| Commercial assay or kit | NEBNext Multiplex Oligos for Illumina | New England Biolabs | Cat # E7335/E7500 | |
| Commercial assay or kit | Qubit dsDNA HS | Invitrogen | Cat # Q32854 | |
| Commercial assay or kit | Qiaquick PCR Purification Kit | Qiagen | Cat # 28,104 | |
| Commercial assay or kit | Accel-NGS 1S Plus DNA Library Kit | Swift Biosciences | Cat # 10,024 | |
| Commercial assay or kit | Swift Single Indexing Primers Set A | Swift Biosciences | Cat # X6024 | |
| Commercial assay or kit | YeaStar Genomic DNA Kit | Genesee Scientific | Cat # 11-323 | |
| Commercial assay or kit | Covaris g-TUBE | Covaris | Cat # 520,079 | |
| Commercial assay or kit | NEB Oxford Nanopore Companion | New England Biolabs | Cat # E7180S | |
| Commercial assay or kit | NEBNext Quick Ligation Reaction Master Mix | New England Biolabs | Cat # B6058 | |
| Commercial assay or kit | NEB Blunt/TA Ligase Master Mix | New England Biolabs | Cat # M0367 | |
| Commercial assay or kit | Oxford Ligation Sequencing Kit | Oxford Nanopore Technologies | Cat # SQK-LSK109 | |
| Commercial assay or kit | Oxford Native Barcoding Expansion 1–12 | Oxford Nanopore Technologies | Cat # EXP-NBD104 | |
| Commercial assay or kit | Qiagen RNeasy kit | Qiagen | Cat # 74,104 | |
| Commercial assay or kit | SuperScript III First-Strand Synthesis System | Invitrogen | Cat # 18080051 | |
| Commercial assay or kit | DyNAmo HS SYBR Green kit | Thermo Fisher | Cat # F410L | |
| Commercial assay or kit | RNase-free DNase set | Qiagen | Cat # 79,254 | |
| Software, algorithm | SAMtools | doi:10.1093/bioinformatics/btp352 | ||
| Software, algorithm | Bowtie2 | doi:10.1038/nmeth.1923 | ||
| Software, algorithm | IGV | doi:10.1093/bib/bbs017 | ||
| Software, algorithm | Guppy Basecaller | Oxford Nanopore Technologies | ||
| Software, algorithm | Megalodon | Oxford Nanopore Technologies | https://github.com/nanoporetech/megalodon | |
| Software, algorithm | All-context rerio model | Oxford Nanopore Technologies | res_dna_r941_ min_modbases-all-context_v001.cfg | For Megalodon modified base-calling; https://github.com/nanoporetech/rerio |
| Peptide, recombinant protein | Protein A Dynabeads | Thermo Fisher | Cat # 10,002D | |
| Peptide, recombinant protein | Proteinase K | New England Biolabs | Cat # P8107S | |
| Peptide, recombinant protein | RNase A | Thermo Fisher | Cat # EN0531 |
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/75653/elife-75653-transrepform1-v2.docx
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Supplementary file 1
Strains used in this study.
- https://cdn.elifesciences.org/articles/75653/elife-75653-supp1-v2.xlsx
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Supplementary file 2
Plasmids used in this study.
- https://cdn.elifesciences.org/articles/75653/elife-75653-supp2-v2.xlsx
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Supplementary file 3
Oligonucleotides used in this study.
- https://cdn.elifesciences.org/articles/75653/elife-75653-supp3-v2.xlsx
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Source code 1
Creation of bedgraph files from fastq files.
- https://cdn.elifesciences.org/articles/75653/elife-75653-code1-v2.zip
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Source code 2
Normalization of bedgraph files to genome-wide median.
- https://cdn.elifesciences.org/articles/75653/elife-75653-code2-v2.zip
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Source code 3
Extract nanopore read IDs corresponding to each barcode from sequencing summary file.
- https://cdn.elifesciences.org/articles/75653/elife-75653-code3-v2.zip
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Source code 4
Extract single read data corresponding to a given chromosome.
- https://cdn.elifesciences.org/articles/75653/elife-75653-code4-v2.zip
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Source code 5
R scripts used to construct nanopore data into figures.
- https://cdn.elifesciences.org/articles/75653/elife-75653-code5-v2.zip
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Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae
eLife 11:e75653.
https://doi.org/10.7554/eLife.75653
