Systematic identification of cis-regulatory variants that cause gene expression differences in a yeast cross
- Department of Genetics, Cell Biology, & Development, University of Minnesota, United States
- Department of Chemistry & Biochemistry, University of California, Los Angeles, United States
- Department of Human Genetics, University of California, Los Angeles, United States
- Department of Biological Chemistry, University of California, Los Angeles, United States
- Howard Hughes Medical Institute, University of California, Los Angeles, United States
Figures
Figure 1 with 8 supplements
Schematic of MPRA design.
At the top, two native genes in the genome with multiple promoter variants (stars) are shown. Below the two genes, the TSS and Upstream MPRA library designs are illustrated. The TSS library tests …
Figure 1—figure supplement 1
Size distribution of indels in the MPRA design.
Figure 1—figure supplement 2
Distribution of the number of promoter variants per gene in the MPRA design.
Figure 1—figure supplement 3
Schematic of the library cloning procedure.
Figure 1—figure supplement 4
Distributions of barcodes.
(A) Number of barcodes tagging a given oligo in the TSS library. The inset shows the range from zero to 5000 barcodes, which contains the majority of the distribution. (B) as in (A), but for the …
Figure 1—figure supplement 5
Number of times a given designed oligo was observed in the TSS annotation sequencing run as a function of the first two nucleotides of the oligo.
Boxplots show the median as thick horizontal line, with the box showing the 25th and 75th percentiles. Whiskers show the largest value no further than 1.5 times the inter-quartile range; data points …
Figure 1—figure supplement 6
Barcode amplification.
The figure shows details of the molecular reactions used to make Illumina sequencing libraries for barcode counting. Primer names are given in quotes. For RNA, Protocol one is on the left and …
Figure 1—figure supplement 7
Reproducibility of oligo expression.
(A) Correlations of expression driven by oligos among replicates in the TSS library. (B) Average expression across TSS replicates driven by the 200 oligos from Sharon et al., 2012 compared to their …
Figure 1—figure supplement 8
Correlations of expression driven by 200 oligos common to the TSS and Upstream library.
Figure 2 with 1 supplement
Identification of single causal variants.
(A) A scatterplot showing the effect size and significance for each variant. The genome-wide significance threshold is shown as a dashed horizontal line. Variants with the most significant effects, …
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Figure 2—source data 1
Statistical tests for each variant.
Results for the TSS and Upstream libraries are in separate worksheets.
- https://cdn.elifesciences.org/articles/62669/elife-62669-fig2-data1-v2.xlsx
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Figure 2—source data 2
Statistical results for each variant after aggregating across the two sub libraries.
- https://cdn.elifesciences.org/articles/62669/elife-62669-fig2-data2-v2.xlsx
Figure 2—figure supplement 1
Reproducibility of variant effects.
(A) Variants measured in the same library (TSS or Upstream) but in opposite orientation (‘strand’) with respect to the reporter gene. Blue points: variants with significant (5% FDR) effects on the …
Figure 3
Comparison of variant effects to local eQTLs.
(A) Scatterplot showing the MPRA effect of the most significant causal variant per gene (y-axis) versus the effect of the local eQTL (x-axis). Red dots indicate local eQTLs with a LOD score of at …
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Figure 3—source data 1
Local eQTLs and variant results.
- https://cdn.elifesciences.org/articles/62669/elife-62669-fig3-data1-v2.xlsx
Figure 4 with 1 supplement
Epistasis among promoter variants.
Each panel shows, for one gene, MPRA expression driven by four oligos with the indicated combination of BY and RM alleles at the two variants. Each panel states the gene name in bold along with …
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Figure 4—source data 1
Results from the test for epistatic interactions.
- https://cdn.elifesciences.org/articles/62669/elife-62669-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Additional cases of significant epistasis between promoter variants.
Each panel shows, for one gene, MPRA expression driven by four oligos with the indicated combination of BY and RM alleles at the two variants. Each panel states the gene name in bold along with …
Figure 5 with 1 supplement
Association of features with variant causality.
(A) Non-TF features. The figure shows the strength of association between each feature and variant causality. Error bars show the standard error of the mean. Significant associations are indicated …
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Figure 5—source data 1
Single-feature regression analyses of causal variants.
Logistic and linear regression (to predict log-fold change) are in separate worksheets.
- https://cdn.elifesciences.org/articles/62669/elife-62669-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Examples of predicted TFBS changes at individual variants.
(A) A variant in the promoter of the SFA1 gene alters a strong Msn2/4 motif (Yeastract consensus motif: CCCCT); as well as a strong Haa1 motif (SMGGSG). TFBSs detected by the Yeastract website in …
Figure 6
Prediction of causal variants and variant effects.
(A) Prediction results for 112 models. On the left, the plot shows the performance of binomial classifiers on the 10% test as black bars. On the right, the plot shows the performance of the linear …
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Figure 6—source data 1
Multiple-feature regression analyses of causal variants.
Logistic and linear regression (to predict log-fold change) are in separate worksheets.
- https://cdn.elifesciences.org/articles/62669/elife-62669-fig6-data1-v2.xlsx
Author response image 1
Tables
Table 1
Library design and representation in experiments.
| Library | TSS | Upstream |
|---|---|---|
| Designed oligos | 7211 | 9882 |
| Variants in design | 3645 | 4547 |
| Genes in design | 2172 | 1918 |
| Oligos in finished library | 6565 | 9646 |
| Barcodes | 9.2 million | 20 million |
| Median barcodes per oligo | 590 | 1008 |
| Variants with data | 2427 | 4467 |
| Genes with data | 1429 | 1824 |
| Number of replicates | 12 | 6 |
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Table 1—source data 1
Information on replicate samples.
- https://cdn.elifesciences.org/articles/62669/elife-62669-table1-data1-v2.xlsx
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Saccharomyces cerevisiae) | BY4741 | Albert et al., 2018 (doi:10.7554/eLife.35471) | ||
| Recombinant DNA reagent | RCP83 plasmid | This paper; (Addgene plasmid #163466) | Plasmid backbone | |
| Recombinant DNA reagent | SurePrint Oligonucleotide Libraries | Agilent | Custom DNA oligo library | |
| Gene (Aequorea victoria) | yEGFP | pKT0127 (Addgene plasmid #8728) | Reporter gene | |
| Software, algorithm | R version 3.5.0 | https://www.r-project.org | Data analysis |
Author response table 1
| Library | Fisher’s exact test (odds ratio) | Fisher’s exact test (p-value) | Correlation w/ number significant ASE datasets (rho) | Correlation w/ number significant ASE datasets (p-value) | Correlation with ASE magnitude (rho) | Correlation with ASE magnitude (p-value) |
|---|---|---|---|---|---|---|
| TSS | 3.1 | 3e-7 | 0.18 | 2e-8 | 0.11 | 0.0009 |
| Upstream | 2.8 | 6e-10 | 0.21 | 3e-12 | 0.13 | 0.00001 |
Additional files
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Supplementary file 1
Oligo design.
- https://cdn.elifesciences.org/articles/62669/elife-62669-supp1-v2.xlsx
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Supplementary file 2
Oligo counts per replicate.
- https://cdn.elifesciences.org/articles/62669/elife-62669-supp2-v2.xls
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Supplementary file 3
Non-TF features for each variant, along with statistical results for each variant.
- https://cdn.elifesciences.org/articles/62669/elife-62669-supp3-v2.xlsx
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Supplementary file 4
All features used in variant annotation, including TFBS.
(gzipped text file).
- https://cdn.elifesciences.org/articles/62669/elife-62669-supp4-v2.txt.gz
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Supplementary file 5
Primers sequences and sequences of various components of the reporter gene construct.
- https://cdn.elifesciences.org/articles/62669/elife-62669-supp5-v2.xlsx
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Supplementary file 6
BY/RM sequence variants used in MPRA design (gzipped vcf file).
- https://cdn.elifesciences.org/articles/62669/elife-62669-supp6-v2.vcf.gz
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Supplementary file 7
Sequence and map of a plasmid in the library after completed library construction.
In place of the library, the sequence contains an example promoter fragment.
- https://cdn.elifesciences.org/articles/62669/elife-62669-supp7-v2.gb.txt
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Transparent reporting form
- https://cdn.elifesciences.org/articles/62669/elife-62669-transrepform-v2.docx
