Biology subjects

Gresham, D.

Publications and source records attributed to Gresham, D..

4 recordsLinked to original sources

bioRxiv2018

Single-cell copy number variant detection reveals the dynamics and diversity of adaptation

Copy number variants (CNVs) are a pervasive, but understudied source of genetic variation and evolutionary potential. Long-term evolution experiments in chemostats provide an ideal system for studying the molecular processes underlying CNV formation and the temporal dynamics of de novo CNVs. Here, we developed a fluorescent reporter to monitor gene amplifications and deletions at a specific locus with single-cell resolution. Using a CNV reporter in nitrogen-limited chemostats, we find that GAP1 CNVs are repeatedly generated and selected during the early stages of adaptive evolution resulting in predictable dynamics of CNV selection. However, subsequent diversification of populations defines a second phase of evolutionary dynamics that cannot be predicted. Using whole genome sequencing, we identified a variety of GAP1 CNVs that vary in size and copy number. Despite GAP1s proximity to tandem repeats that facilitate intrachromosomal recombination, we find that non-allelic homologous recombination (NAHR) between flanking tandem repeats occurs infrequently. Rather, breakpoint characterization revealed that for at least 50% of GAP1 CNVs, origin-dependent inverted-repeat amplification (ODIRA), a DNA replication mediated process, is the likely mechanism. We also find evidence that ODIRA generates DUR3 CNVs, indicating that it may be a common mechanism of gene amplification. We combined the CNV reporter with barcode lineage tracking and found that 103-104 independent CNV-containing lineages initially compete within populations, which results in extreme clonal interference. Our study introduces a novel means of studying CNVs in heterogeneous cell populations and provides insight into the underlying dynamics of CNVs in evolution.

evolutionary biology↗

bioRxiv2018

Global analysis of gene expression dynamics identifies factors required for accelerated mRNA degradation

Cellular responses to changing environments frequently involve rapid reprogramming of the transcriptome. Regulated changes in mRNA degradation rates can accelerate reprogramming by clearing or stabilizing extant transcripts. Here, we measured mRNA stability using 4-thiouracil labeling in the budding yeast Saccharomyces cerevisiae during a nitrogen upshift and found that 78 mRNAs are subject to destabilization. These transcripts include Nitrogen Catabolite Repression (NCR) and carbon metabolism mRNAs, suggesting that mRNA destabilization is a mechanism for targeted reprogramming. To explore the molecular basis of destabilization we implemented a SortSeq approach to screen using the pooled deletion collection library for trans factors that mediate rapid GAP1 mRNA repression. We combined low-input multiplexed Barcode sequencing with branched-DNA single-molecule mRNA FISH and Fluorescence-activated cell sorting (BFF) to identify that the Lsm1-7p/Pat1p complex and general mRNA decay machinery are important for GAP1 mRNA clearance. We also find that the decapping modulator SCD6, translation factor eIF4G2, and the 5 UTR of GAP1 are important for this repression, suggesting that translational control may impact the post-transcriptional fate of mRNAs in response to environmental changes.

genetics↗

bioRxiv2017

Adaptive Evolution Of Transcription Factor Binding Affinities

Understanding the molecular basis of gene expression evolution is a central problem in evolutionary biology. However, connecting changes in gene expression to increased fitness, and identifying the functional basis of those changes, remains challenging. To study adaptive evolution of gene expression in real time, we performed long term experimental evolution (LTEE) of Saccharomyces cerevisiae (budding yeast) in ammonium-limited chemostats. Following several hundred generations of continuous selection we found significant divergence of nitrogen-responsive gene expression in lineages with increased fitness. In multiple independent lineages we found repeated selection for non-synonymous mutations in the zinc finger DNA binding domain of the activating transcription factor (TF), GAT1, that operates within incoherent feedforward loops to control expression of the nitrogen catabolite repression (NCR) regulon. Missense mutations in the DNA binding domain of GAT1 reduce its binding affinity for the GATAA consensus sequence in a promoter-specific manner, resulting in increased expression of ammonium permease genes via both direct and indirect effects, thereby conferring increased fitness. We find that altered transcriptional output of the NCR regulon results in antagonistic pleiotropy in alternate environments and that the DNA binding domain of GAT1 is subject to purifying selection in natural populations. Our study shows that adaptive evolution of gene expression can entail tuning expression output by quantitative changes in TF binding affinities while maintaining the overall topology of a gene regulatory network.

evolutionary biology↗

bioRxiv2017

Incorporation of unique molecular identifiers in TruSeq adapters improves the accuracy of quantitative sequencing

Quantitative analysis of next-generation sequencing data requires discriminating duplicate reads generated by PCR from identical molecules that are of unique origin. Typically, PCR duplicates are defined as sequence reads that align to the same genomic coordinates using reference-based alignment. However, identical molecules can be independently generated during library preparation. The false positive rate of coordinate-based deduplication has not been well characterized and may introduce unforeseen biases during analyses. We developed a cost-effective sequencing adapter design by modifying Illumina TruSeq adapters to incorporate a unique molecular identifier (UMI) while maintaining the capacity to undertake multiplexed sequencing. Incorporation of UMIs enables identification of bona fide PCR duplicates as identically mapped reads with identical UMIs. Using TruSeq adapters containing UMIs (TrUMIseq adapters), we find that accurate removal of PCR duplicates results in enhanced data quality for quantitative analysis of allele frequencies in heterogeneous populations and gene expression.\n\nMethod SummaryTrUMIseq adapters incorporate unique molecular identifiers in TruSeq adapters while maintaining the capacity to multiplex sequencing libraries using existing workflows. The use of UMIs increases the accuracy of quantitative sequencing assays, including RNAseq and allele frequency estimation, by enabling accurate detection of PCR duplicates.

genomics↗