Genomic Phenotyping by Barcode Sequencing Broadly Distinguishes between Alkylating Agents, Oxidizing Agents, and Non-Genotoxic Agents, and Reveals a Role for Aromatic Amino Acids in Cellular Recovery after Quinone Exposure

Toxicity screening of compounds provides a means to identify compounds harmful for human health and the environment. Here, we further develop the technique of genomic phenotyping to improve throughput while maintaining specificity. We exposed cells to eight different compounds that rely on different modes of action: four genotoxic alkylating (methyl methanesulfonate (MMS), N-Methyl-N-nitrosourea (MNU), N,N′-bis(2-chloroethyl)-N-nitroso-urea (BCNU), N-ethylnitrosourea (ENU)), two oxidizing (2-methylnaphthalene-1,4-dione (menadione, MEN), benzene-1,4-diol (hydroquinone, HYQ)), and two non-genotoxic (methyl carbamate (MC) and dimethyl sulfoxide (DMSO)) compounds. A library of S. cerevisiae 4,852 deletion strains, each identifiable by a unique genetic ‘barcode’, were grown in competition; at different time points the ratio between the strains was assessed by quantitative high throughput ‘barcode’ sequencing. The method was validated by comparison to previous genomic phenotyping studies and 90% of the strains identified as MMS-sensitive here were also identified as MMS-sensitive in a much lower throughput solid agar screen. The data provide profiles of proteins and pathways needed for recovery after both genotoxic and non-genotoxic compounds. In addition, a novel role for aromatic amino acids in the recovery after treatment with oxidizing agents was suggested. The role of aromatic acids was further validated; the quinone subgroup of oxidizing agents were extremely toxic in cells where tryptophan biosynthesis was compromised.Unilever (Firm)National Cancer Institute (U.S.) (R01-CA055042 (now R01-ES022872))Massachusetts Institute of Technology. Center for Environmental Health Sciences (Grant NIEHS P30-ES002109

Tryptophan biosynthesis rescues cells from ROS.

<p>A) A schematic of the tryptophan biosynthesis pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073736#pone.0073736-Braus1" target="_blank">[29]</a>. PRA: N-(5′-phospohribosyl)-anthsranilate, CDRP: 1-(o-carboxyphenylamino)-1-desoxyribuose-5-phosphate. B) Compound sensitivity of selected mutant strains were analyzed by spot assay. Strains were grown in liquid YPD+G418 overnight at 30°C and then diluted in YPD. Ten-fold serial dilutions of each yeast culture was spotted onto YPD plates in the absence (control) and presence of the different compounds: MMS (0.006%), MEN (40 µM), HYQ (3 mg ml⁻¹), and tBuOOH (0.75 mM). Plates were incubated at 30°C and growth was recorded after 48 h exposure.</p

Experimental workflow for barcoded genomic phenotyping.

<p>Schematic representation of A) experimental design (t is the time of cell harvest, which was at 10 or 20 generation times) and B) analysis and filtering of high-throughput sequencing data.</p

List of selected compounds and doses used in this study.

<p>List of selected compounds and doses used in this study.</p

The difference between alkylating and oxidizing agents can be explained by fitness profiles of the strains.

<p>A) Two-dimensional hierarchical clustering of fitness ratio (median log ratio of exposed/control) results using the strains sensitive after 10 and 20 generation times upon exposure to different chemicals. Compounds and doses are plotted across the horizontal axis. On the vertical axis, a subset of 508 strains with reduced fitness is shown. B) Protein-protein interaction networks with >5 toxicity-modulating proteins. The colors (explained in legend, same as labels in A) within the pie charts indicate the contribution of each of the eight compounds. Alkylating agents represented in shades of yellow-red, oxidizing agents in shades of blue and non-genotoxic compounds in green.</p

Functional enrichment reveals an alkylating agent-specific DNA repair and cell cycle dependency.

<p>Gene-annotation enrichment analysis heat map and clustering for sensitive strains to different compounds at early (10 generation times) or late (20 generation times) timepoints. Heat map colors correspond to the –log10 of the p-values.</p

Multiplexed DNA repair assays for multiple lesions and multiple doses via transcription inhibition and transcriptional mutagenesis

The capacity to repair different types of DNA damage varies among individuals, making them more or less susceptible to the detrimental health consequences of damage exposures. Current methods for measuring DNA repair capacity (DRC) are relatively labor intensive, often indirect, and usually limited to a single repair pathway. Here, we describe a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) that measures the ability of human cells to repair plasmid reporters, each bearing a different type of DNA damage or different doses of the same type of DNA damage. FM-HCR simultaneously measures repair capacity in any four of the following pathways: nucleotide excision repair, mismatch repair, base excision repair, nonhomologous end joining, homologous recombination, and methylguanine methyltransferase. We show that FM-HCR can measure interindividual DRC differences in a panel of 24 cell lines derived from genetically diverse, apparently healthy individuals, and we show that FM-HCR may be used to identify inhibitors or enhancers of DRC. We further develop a next-generation sequencing-based HCR assay (HCR-Seq) that detects rare transcriptional mutagenesis events due to lesion bypass by RNA polymerase, providing an added dimension to DRC measurements. FM-HCR and HCR-Seq provide powerful tools for exploring relationships among global DRC, disease susceptibility, and optimal treatment.American Cancer Society (Research Professor)National Institutes of Health (U.S.) (grant DP1-ES022576