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 …
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.
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 …
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 …
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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 …
Figure 2—figure supplement 2
Methylation by Sir3–M.EcoGII and sir3-bah∆-M.EcoGII at all 32 telomeres.
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 …
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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 …
Figure 3—figure supplement 2
Methylation upon overexpression of SIR3-M.ECOGII at all 32 telomeres.
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 …
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 …
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
