The nucleosome DNA entry-exit site is important for transcription termination and prevention of pervasive transcription
- Department of Biological Sciences, University of Pittsburgh, United States
Figures
Figure 1
Histone residues in the DNA entry-exit site of the nucleosome are important for transcription termination.
(A) Yeast strains contain either an SNR47 transcription termination reporter (top, KY3220) or a control transcription cassette lacking the SNR47 terminator (bottom, KY3219) integrated at the LEU2 …
Figure 2 with 2 supplements
Mutations that alter the nucleosome DNA entry-exit site cause widespread 3’ extension of snoRNAs.
(A, B) 3’ extension index in (A) the H3 T45A mutant and (B) the H3 R52A mutant. The ratio (mutant/WT) of spike-in normalized RNA-seq read counts between mutant and wild-type strains produced by …
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Figure 2—source data 1
SNR gene 3' extension index RNA-seq data.
- https://cdn.elifesciences.org/articles/57757/elife-57757-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Agreement between biological replicates of RNA-seq datasets.
(A–C) Biplots showing agreement between biological replicates of RNA-seq data for (A) WT, (B) the H3 T45A mutant, and (C) the H3 R52A mutant. Log2-transformed, spike-in normalized RNA-seq read …
Figure 2—figure supplement 2
Levels of the Nrd1-unterminated transcripts (NUTs) change in DNA entry-exit site mutants.
(A, B) RNA-seq analysis of annotated NUTs (Schulz et al., 2013) in WT, H3 R52A and H3 T45A strains. Gray scale profiles show spike-in normalized data. Tri-color heatmaps show log2-fold change …
Figure 3 with 2 supplements
Transcriptional changes at protein-coding genes in DNA entry-exit site mutants.
(A) Heatmaps sorted by gene length and showing spike-in normalized RNA-seq and 4tU-seq read counts (gray scale) and log2-fold change between the H3 R52A or H3 T45A mutant and WT at protein-coding …
Figure 3—figure supplement 1
Pol II occupancy and antisense transcript levels at protein-coding genes are altered in DNA entry-exit site mutants.
(A) Box plots comparing log2-transformed spike-in normalized RNA-seq read counts in a window 150 bp downstream of the CPS. Box and whisker plots were generated in R Studio (RStudio Team, 2016) and …
Figure 3—figure supplement 2
Agreement between biological replicates of 4tU-seq and FLAG-Rpb3 ChIP-seq datasets.
(A–C) Biplots showing agreement between biological replicates of 4tU-seq results in the indicated strains. (D–E) Biplots showing agreement between biological replicates of FLAG-Rpb3 ChIP-seq results …
Figure 4 with 2 supplements
DNA entry-exit site mutants display aberrant transcription 5’ and 3’ to protein-coding genes.
(A) Heatmaps showing log2-fold change in spike-in normalized 4tU-seq read counts in H3 R52A or H3 T45A mutants relative to WT at divergent, convergent, and tandem genes. Heatmap rows represent 1186 …
Figure 4—figure supplement 1
Some genes in DNA entry-exit site mutants show little to no change in 5’ and 3’ expression.
(A, B) Browser tracks of 4tU-seq read density in WT, H3 T45A, and H3 R52A mutant cells visualized in IGV (Thorvaldsdóttir et al., 2013). Data displayed are spike-in normalized read density over the …
Figure 4—figure supplement 2
Transcriptional changes at ncRNA loci in DNA entry-exit site mutants.
(A–C) Heatmaps showing log2-fold change in spike-in normalized 4tU-seq read counts between H3 R52A or H3 T45A mutant and WT loci. Heatmap rows represent (A) 924 CUT (Xu et al., 2009), (B) 846 SUT (Xu…
Figure 5 with 2 supplements
H3 K36me3 and the DNA entry-exit site function through genetically distinct pathways.
(A) Left: western blot analysis of H3 K36me3 levels in H3 and H4 mutant strains. Library plasmids were transformed into KY812 for plasmid shuffling. Extract from a set2∆ strain was used to confirm …
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Figure 5—source data 1
H3 K36me3 western blot data.
- https://cdn.elifesciences.org/articles/57757/elife-57757-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
SRAT expression is significantly increased in DNA entry-exit site mutants compared to wild-type.
Box and whisker plots showing log2-transformed spike-in normalized read counts for SRATs (Venkatesh et al., 2016) expressed in strains constructed by plasmid shuffling of WT, H3 T45A or H3 R52A …
Figure 5—figure supplement 2
Mutations to the DNA entry-exit site do not affect global levels of H3 K56ac.
Western blot analysis of H3 K56ac, total H3, and G6PDH levels in indicated mutants. Strains were constructed by plasmid shuffling of the histone mutant plasmids (TRP1-marked, CEN/ARS) into strain …
Figure 6 with 2 supplements
Mutation of the DNA entry-exit site alters nucleosomes genome-wide.
(A) Heatmap of the log2-fold change of MNase-seq read counts (spike-in normalized as described in Materials and methods) of the H3 R52A mutant relative to WT. Rows represent 6205 protein-coding …
Figure 6—figure supplement 1
Analysis of MNase digestion and MNase-seq data for the the H3 R52A mutant.
(A) Ethidium bromide stained agarose gel of MNase-treated chromatin with the indicated amounts of MNase. Input indicates untreated chromatin. Mono- and poly-nucleosome species are indicated. (B, C) …
Figure 6—figure supplement 2
DNA entry-exit site mutants exhibit chromatin- and transcription-related phenotypes.
(A) The Spt- phenotype was assessed at two Ty ∂ element insertion mutations, his4-912∂ and lys2-18∂, using a five-fold serial dilution series of a yeast culture starting at OD600 = 0.8. Histone …
Figure 7 with 1 supplement
Integration of a superbinder DNA sequence downstream of SNR48 suppresses the termination defect of the H3 R52A mutant.
(A) MNase-seq data from a 2.5 U digestion visualized in IGV (Thorvaldsdóttir et al., 2013) (top) compared to de novo transcript annotations generated from RNA-seq data from the same strains …
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Figure 7—source data 1
H2A ChIP data.
- https://cdn.elifesciences.org/articles/57757/elife-57757-fig7-data1-v1.xlsx
Figure 7—figure supplement 1
Detection of readthrough transcription of SNR48 by northern analysis in strains with and without the superbinder (SB) sequence.
(A) Location of the northern blot probe over the SNR48 locus (black line above SNR48) and the observed transcript types. (B) Northern blot analysis of transcripts associated with the SNR48 gene. A …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Saccharomyces cerevisiae) | S288C derivatives | This paper; Fred Winston (Harvard Medical School) Winston et al., 1995 | See Supplementary file 2. Yeast strains | |
| Strain, strain background (Saccharomyces cerevisiae) | 972 h- | Fred Winston (Harvard Medical School) | FWP10 | spike-in control |
| Strain, strain background (Saccharomyces cerevisiae) | NRRL Y-1140 | Nathan Clark (University of Utah) | spike-in control | |
| Genetic reagent, (Kluyveromyces lactis) | KLLA0D16170 g-3XFLAG:: NAT | Jin et al., 2017 | YSC193 | spike-in control |
| Recombinant DNA reagent | SHIMA plasmid library | Nakanishi et al., 2008 | recombinant histone mutant library | |
| Sequence-based reagent | PCR primers | This paper | See Supplementary file 1. Oligonucleo-tides | |
| Sequence-based reagent | histone superbinder (SB) sequence | Wang et al., 2011 | ||
| Recombinant DNA reagent | pMPY-3xHA plasmid | Schneider et al., 1995 | Plasmid vector used for SB-URA3-SB construct | |
| Recombinant DNA reagent | TOPO TA plasmid containing SB sequence | Hainer et al., 2015 | Source of SB DNA for construction of SB-URA3-SB construct | |
| Recombinant DNA reagent | SB-URA3-SB plasmid | This study | KB1479 | Used to generate yeast strains with integrated SB |
| Gene (Saccharomyces cerevisiae) | hht2-HHF2 or HHT2-hhf2 | Nakanishi et al., 2008 | ||
| Antibody | α-H3 (rabbit polyclonal) | Tomson et al., 2011 | western analysis (1:15,000) | |
| Antibody | α-H3 K36me3(rabbit polyclonal) | Abcam | ab9050 | western analysis (1:1000) |
| Antibody | α-HA (mouse monoclonal) | Roche | 12CA5 | western analysis (1:3000) |
| Antibody | α-G6PDH (rabbit) | Sigma-Aldrich | A9521 | western analysis (1:20,000) |
| Antibody | α-H3 K56ac (rabbit) | Paul Kaufman (UMass Medical School) | western analysis (1:5000) | |
| Antibody | α-rabbit IgG-HRP | GE Healthcare | NA934 | western analysis (1:5000) |
| Antibody | α-mouse IgG-HRP | GE Healthcare | NA931 | western analysis (1:5000) |
| Commercial assay or kit | Pico Plus chemilumine-scence substrate | Thermo Fisher | #34580 | |
| Antibody | α-FLAG M2 affinity gel (mouse monoclonal) | Sigma-Aldrich | A2220 | 30 µL α-FLAG beads per 700 µL chromatin (ChIP) |
| Antibody | α-H2A (rabbit polyclonal) | ActiveMotif | #39235 | 1 µL α-H2A per 700 µL chromatin (ChIP) |
| Ather | Protein A conjugated to sepharose beads | GE Healthcare | GE17-5280-01 | 30 µL per chromatin IP |
| Commercial assay or kit | QIAquick PCR purification kit | Qiagen | #28106 | |
| Commercial assay or kit | NEBNext Ultra II library kit for Illumina | New England Biolabs | E7645 | |
| Commercial assay or kit | NEBNext Ultra II sequencing indexes | New England Biolabs | E7335, E7500, E7710 | |
| Commercial assay or kit | SoLo RNA-seq library preparation kit | TECAN | 0516–32 | |
| Commercial assay or kit | Maxima 2X SYBR Master Mix | Thermo Fisher | K0221 | |
| Commercial assay or kit | TURBO DNA-free kit | Invitrogen | AM1907 | |
| Commercial assay or kit | RiboPure Yeast RNA extraction kit | Ambion | AM1926 | |
| Commercial assay or kit | MTSEA Biotin-XX | Biotium | #90066 | |
| Commercial assay or kit | Streptavidin beads | Invitrogen | #65001 | |
| Commercial assay or kit | MinElute columns | Qiagen | #74204 | |
| Commercial assay or kit | QuikChange II kit | Agilent | #200523 | |
| Peptide, recombinant protein | Micrococcal nuclease | Thermo Fisher | #88216 | |
| Sequence-based reagent | [α-32P]dATP and [α-32P]dTTP | PerkinElmer | used to generate Northern probes | |
| Other | S. cerevisiae reference genome | Ensembl | R64-1-1 | |
| Other | S. pombe reference genome | Ensembl | EF2 | |
| Other | K. lactis reference genome | Ensembl | ASM251v1 | |
| Software, algorithm | HISAT2 | Kim et al., 2015 | ||
| Software, algorithm | SAMtools | Li et al., 2009 | ||
| Software, algorithm | RSubread featureCounts | Liao et al., 2014 | ||
| Software, algorithm | deepTools2 | Ramírez et al., 2014; Ramírez et al., 2016 | ||
| Software, algorithm | R Studio | RStudio Team, 2016 | ||
| Software, algorithm | DANPOS2 | Chen et al., 2013 | ||
| Software, algorithm | wigToBigWig UCSC utility | Kent et al., 2010 | ||
| Software, algorithm | BEDtools | Quinlan, 2014; Quinlan and Hall, 2010 |
Additional files
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Supplementary file 1
Oligonucleotides.
- https://cdn.elifesciences.org/articles/57757/elife-57757-supp1-v1.docx
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Supplementary file 2
Yeast strains.
- https://cdn.elifesciences.org/articles/57757/elife-57757-supp2-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/57757/elife-57757-transrepform-v1.docx
