Ron Ammar

Ron is a Postdoctoral Fellow in the Bader lab. He is currently developing tools for genome interpretation with MedSavant and The Sick Kids Genome Clinic.

Postdoctoral Project Descriptions

Under construction

Ph.D. Project Descriptions

Chromatin is an ancient innovation conserved between Archaea and Eukarya

The eukaryotic nucleosome is the fundamental unit of chromatin, comprising a protein octamer that wraps ∼147 bp of DNA and has essential roles in DNA compaction, replication and gene expression. Nucleosomes and chromatin have historically been considered to be unique to eukaryotes, yet studies of select archaea have identified homologs of histone proteins that assemble into tetrameric nucleosomes. Here we report the first archaeal genome-wide nucleosome occupancy map, as observed in the halophile Haloferax volcanii. Nucleosome occupancy was compared with gene expression by compiling a comprehensive transcriptome of Hfx. volcanii. We found that archaeal transcripts possess hallmarks of eukaryotic chromatin structure: nucleosome-depleted regions at transcriptional start sites and conserved −1 and +1 promoter nucleosomes. Our observations demonstrate that histones and chromatin architecture evolved before the divergence of Archaea and Eukarya, suggesting that the fundamental role of chromatin in the regulation of gene expression is ancient.

Our eLife paper

Genetic and Genomic Architecture of the Evolution of Resistance to Antifungal Drug Combinations

The evolution of drug resistance in fungal pathogens compromises the efficacy of the limited number of antifungal drugs. Drug combinations have emerged as a powerful strategy to enhance antifungal efficacy and abrogate drug resistance, but the impact on the evolution of drug resistance remains largely unexplored. Targeting the molecular chaperone Hsp90 or its downstream effector, the protein phosphatase calcineurin, abrogates resistance to the most widely deployed antifungals, the azoles, which inhibit ergosterol biosynthesis. Here, we evolved experimental populations of the model yeast Saccharomyces cerevisiae and the leading human fungal pathogen Candida albicans with azole and an inhibitor of Hsp90, geldanamycin, or calcineurin, FK506. To recapitulate a clinical context where Hsp90 or calcineurin inhibitors could be utilized in combination with azoles to render resistant pathogens responsive to treatment, the evolution experiment was initiated with strains that are resistant to azoles in a manner that depends on Hsp90 and calcineurin. Of the 290 lineages initiated, most went extinct, yet 14 evolved resistance to the drug combination. Drug target mutations that conferred resistance to geldanamycin or FK506 were identified and validated in five evolved lineages. Whole-genome sequencing identified mutations in a gene encoding a transcriptional activator of drug efflux pumps, PDR1, and a gene encoding a transcriptional repressor of ergosterol biosynthesis genes, MOT3, that transformed azole resistance of two lineages from dependent on calcineurin to independent of this regulator. Resistance also arose by mutation that truncated the catalytic subunit of calcineurin, and by mutation in LCB1, encoding a sphingolipid biosynthetic enzyme. Genome analysis revealed extensive aneuploidy in four of the C. albicans lineages. Thus, we identify molecular determinants of the transition of azole resistance from calcineurin dependence to independence and establish multiple mechanisms by which resistance to drug combinations evolves, providing a foundation for predicting and preventing the evolution of drug resistance.

Our PLoS Genetics paper

Tools for identifying human drug targets

Modern drug discovery methods have been developed for the challenging endeavor of identifying candidate drug targets and target pathways. Here I report a novel assay to identify human drug targets, called human Multi-copy Suppression Profiling (hMSP). This assay is optimized for use with a custom DNA microarray platform that was designed for high-throughput ORFeome studies. In hMSP, a collection of Saccharomyces cerevisiae strains, each expressing a different human protein, is exposed en masse to a drug at growth inhibitory concentrations. Strains that are resistant to drug inhibition are prioritized as candidate drug targets. I confirmed the utility of hMSP by identifying human dihydrofolate reductase as the target of methotrexate. hMSP was applied to study the β adrenergic receptor (βAR) antagonist propranolol, a drug used in the treatment of cardiovascular diseases. Propranolol has been shown to be an effective and safe therapeutic via a mechanism independent of its βAR activity. I identified yeast strains overexpressing the dual specificity phosphatases (DUSPs) 10 and 16 as resistant to propranolol treatment. These observations were confirmed in human embryonic kidney cells, where the overexpression of DUSP16 was able to rescue the cells from propranolol toxicity. Propranolol was subsequently shown to inhibit DUSP10 activity in vitro, suggesting that these dual-specificity phosphatases are bona fide targets of propranolol. This study emphasizes the value of in vivo chemical genomic assays in yeast for the purpose of identifying novel human drug targets in a rapid and unbiased manner.

As presented at GSA MOHB 2012 meeting. Manuscript in preparation.

Contact

Email: <ron DOT ammar AT mail DOT utoronto DOT ca>

Scripts

GC-based Nucleosome Prediction for Archaea and Eukarya

Other Resources/Pages

My Twitter Page: Science and other thoughts

http://elements.eaglegenomics.com/

Other/Past Affiliations

HIPHOP Lab

Collaborative Graduate Program in Genome Biology and Bioinformatics

Donnelly Centre for Cellular and Biomolecular Research University of Toronto 160 College St, Room 640, Toronto ON, M5S3E1, Canada