Genome-Wide Profiling of Yeast DNA:RNA Hybrid Prone Sites with DRIP-Chip

<div><p>DNA:RNA hybrid formation is emerging as a significant cause of genome instability in biological systems ranging from bacteria to mammals. Here we describe the genome-wide distribution of DNA:RNA hybrid prone loci in <i>Saccharomyces cerevisiae</i> by DNA:RNA immunoprecipitation (DRIP) followed by hybridization on tiling microarray. These profiles show that DNA:RNA hybrids preferentially accumulated at rDNA, Ty1 and Ty2 transposons, telomeric repeat regions and a subset of open reading frames (ORFs). The latter are generally highly transcribed and have high GC content. Interestingly, significant DNA:RNA hybrid enrichment was also detected at genes associated with antisense transcripts. The expression of antisense-associated genes was also significantly altered upon overexpression of RNase H, which degrades the RNA in hybrids. Finally, we uncover mutant-specific differences in the DRIP profiles of a Sen1 helicase mutant, RNase H deletion mutant and Hpr1 THO complex mutant compared to wild type, suggesting different roles for these proteins in DNA:RNA hybrid biology. Our profiles of DNA:RNA hybrid prone loci provide a resource for understanding the properties of hybrid-forming regions <i>in vivo</i>, extend our knowledge of hybrid-mitigating enzymes, and contribute to models of antisense-mediated gene regulation. A summary of this paper was presented at the 26^th International Conference on Yeast Genetics and Molecular Biology, August 2013.</p></div

An Evolutionarily Conserved Synthetic Lethal Interaction Network Identifies FEN1 as a Broad-Spectrum Target for Anticancer Therapeutic Development

<div><p>Harnessing genetic differences between cancerous and noncancerous cells offers a strategy for the development of new therapies. Extrapolating from yeast genetic interaction data, we used cultured human cells and siRNA to construct and evaluate a synthetic lethal interaction network comprised of chromosome instability (CIN) genes that are frequently mutated in colorectal cancer. A small number of genes in this network were found to have synthetic lethal interactions with a large number of cancer CIN genes; these genes are thus attractive targets for anticancer therapeutic development. The protein product of one highly connected gene, the flap endonuclease <em>FEN1</em>, was used as a target for small-molecule inhibitor screening using a newly developed fluorescence-based assay for enzyme activity. Thirteen initial hits identified through <em>in vitro</em> biochemical screening were tested in cells, and it was found that two compounds could selectively inhibit the proliferation of cultured cancer cells carrying inactivating mutations in <em>CDC4</em>, a gene frequently mutated in a variety of cancers. Inhibition of flap endonuclease activity was also found to recapitulate a genetic interaction between <em>FEN1</em> and <em>MRE11A</em>, another gene frequently mutated in colorectal cancers, and to lead to increased endogenous DNA damage. These chemical-genetic interactions in mammalian cells validate evolutionarily conserved synthetic lethal interactions and demonstrate that a cross-species candidate gene approach is successful in identifying small-molecule inhibitors that prove effective in a cell-based cancer model.</p> </div

DNA:RNA hybrids are enriched at protein-encoding genes and retrotransposons of higher transcriptional frequency.

<p>(A) Average gene profile of DNA:RNA hybrids at ORFs enriched for DNA:RNA hybrids under wild type conditions. (B–D) CHROMATRA plots of DNA:RNA hybrid distribution along genes sorted by their length (B), grouped into five transcriptional frequency categories as per <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004288#pgen.1004288-Holstege1" target="_blank">[69]</a>) (C) or grouped into four GC content categories (D). Genes were aligned by their TSSs. (E) The average DNA:RNA hybrid score at Ty1, Ty2, Ty3, Ty4 and Ty5 retrotransposons in the left panel shows higher enrichment at Ty1 and Ty2 retrotransposons. The average profile of DNA:RNA hybrids at all retrotransposons under wild type conditions is shown in the right panel.</p

Genome-wide profile of DNA:RNA hybrids in wild type yeast revealed enrichment at rDNA, telomeres, retrotransposons and a subset of genes.

<p>DRIP-chip chromosome plot of DNA:RNA hybrids in the rDNA region and telomeric ends of chromosome XII. The black line represents the average of two wild type replicates. Bars indicate ORFs (grey), rDNA (purple), retrotransposons (green) or genes associated with an antisense transcript (red) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004288#pgen.1004288-Yassour1" target="_blank">[51]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004288#pgen.1004288-Xu1" target="_blank">[54]</a>). Grey boxes delineate telomeric repeat regions. Y-axis indicates relative occupancy of DNA:RNA hybrids. X-axis indicates chromosomal coordinates. P indicates probability of observing a number of enriched features by random chance below what was observed (P>0.99997).</p

Mutant specific trends in protein-coding genes prone to DNA:RNA hybrid formation.

<p>(A–C) CHROMATRA plots of DNA:RNA hybrid distribution along genes sorted by their length (A) grouped into five transcriptional frequency categories as per <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004288#pgen.1004288-Holstege1" target="_blank">[69]</a> (B) or grouped into four GC content categories (C). Genes were aligned by their TSSs.</p

Genome-wide profiles of DNA:RNA hybrids in revealed similar enrichment of rDNA, retrotransposons and telomeres in wild type and mutants.

<p>DRIP-chip chromosome plot of DNA:RNA hybrids in wild type, <i>rnh1</i><b><i>Δ</i></b><i>rnh201</i><b><i>Δ</i></b>, <i>hpr1</i><b><i>Δ</i></b> and <i>sen1-1</i> at chromosome XII. The average of two replicates per strain is shown. Bars indicate ORFs (grey), rDNA (purple), retrotransposons (green) or genes associated with an antisense transcript (red) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004288#pgen.1004288-Yassour1" target="_blank">[51]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004288#pgen.1004288-Xu1" target="_blank">[54]</a>). Grey boxes delineate telomeric repeat regions. Y-axis indicates relative occupancy of DNA:RNA hybrids. X-axis indicates chromosomal coordinates. P indicates probability of observing a number of enriched features below what was observed (P>0.99997).</p

Screening for FEN1 inhibitors <i>in vitro</i>.

<p>Schematic representation of the fluorescence-based assay employed to identify FEN1 inhibitors. In the absence of inhibitor, FEN1 cleaves the 5′ flap to which the 6-FAM fluorophore is attached, allowing it to diffuse away from the BHQ-1 quencher and fluoresce. Activity is read as increasing fluorescence over time.</p

Cell-based assays for flap endonuclease inhibitor activity reveal two compounds that selectively inhibit the proliferation of cells deficient in <i>CDC4</i>.

<p>(A) siRNA-mediated knockdown of FEN1 selectively inhibits the proliferation of <i>CDC4</i>-knockout HCT116 cells. siRNA transfections were carried out as described in Materials and Methods. Cells were fixed and imaged four days following siRNA transfection. Data were analyzed by one-way ANOVA followed by a Tukey test. Shown is mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001. (B) Some flap endonuclease inhibitors recapitulate the genetic interaction between <i>FEN1</i> and <i>CDC4</i> in HCT116 cells. Cells were incubated with compound at the indicated concentration for 72 hours in optically clear 96-well plates prior to fixation and imaging as described in Materials and Methods. Data were analyzed by one-way ANOVA followed by a Tukey test. Shown is mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001. (C) RF00974 and NSC645851 recapitulate the genetic interaction between <i>FEN1</i> and <i>CDC4</i> in DLD-1 cells. Experiments were carried out as in (B). Data were analyzed by one-way ANOVA followed by a Tukey test. Shown is mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.</p

Genes associated with DNA:RNA hybrids were significantly associated with antisense transcripts.

<p>(A) Antisense association of DNA:RNA hybrid-enriched genes in wild type. The p-value indicates significant enrichment (Fisher's exact test) of antisense-associated genes among DNA:RNA hybrid-enriched genes compared to the Yassour et al. 2010 antisense-annotated dataset (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004288#pgen.1004288-Yassour1" target="_blank">[51]</a>). (B) CHROMATRA plots of DNA:RNA hybrid distribution along genes sorted by their length and separated by whether they are antisense associated or not. Genes were aligned by their TSSs. (C) Average gene profile of DNA:RNA hybrids at genes associated with antisense transcripts. (D) Genes with increased mRNA levels upon RNase H overexpression were significantly associated with antisense transcripts compared to all transcripts represented by the microarray. (E) Antisense-associated DNA:RNA hybrid-enriched genes in wild type have lower transcription frequency compared to non-antisense-associated DNA:RNA hybrid-enriched genes. Genes up-regulated at the transcript level by RNase H overexpression have lower transcription frequency compared to all genes on the expression microarray. Intervals indicate range of the 95% of genes closest to the average in each sample. Averages stated above each bar. P values indicate significant decrease in transcriptional frequency (Wilcoxon rank sum test). (F) Overlap between DNA:RNA hybrid-enriched genes and RNase H-modulated transcripts sorted by antisense association according to the Yassour et al. 2010 database. For genes that are both hybrid-enriched and modulated at the transcript level by RNase H overexpression, the antisense association (100%) is significantly higher (Fisher's exact test p<2.2e⁻¹⁶) than those of the parent datasets (37.4% for DNA:RNA hybrid-enriched genes, 43.9% for RNase H-modulated genes).</p

Evolutionary conservation of synthetic lethal interactions in HCT116 cells.

<p>(A) A yeast cancer-ortholog synthetic lethal network. Lines indicate synthetic lethal genetic interactions. Yeast genes and human orthologs are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003254#pgen-1003254-t001" target="_blank">Table 1</a>. Red circles represent <i>S. cerevisiae</i> orthologs of genes mutated in cancer; blue circles indicate common interacting partners, which are referred to as “central” genes. (B–D) Representative data depicting mean percentage of remaining HCT116 cells (± SEM) treated with pooled siRNAs targeting central genes (top) and cancer genes (along <i>x</i>-axis) relative to GAPDH-silenced controls. Blue circles, siRNA targeting central gene alone. Red circles, siRNA targeting cancer gene alone. Yellow triangles, predicted viability of double siRNA treatment. Green circles, observed viability of double siRNA treatment. For raw data, please refer to . GAPDH siRNA does not significantly reduce viability relative to a non-silencing siRNA (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003254#pgen.1003254.s002" target="_blank">Figure S2A</a>). (E) Representative data depicting mean percentage viability of hTERT cells (± SEM) treated with pooled siRNAs targeting FEN1 and cancer genes (along <i>x</i>-axis) relative to GAPDH-silenced controls. Symbols are as in B. (F) Mammalian genetic interaction network. Solid grey line, interaction observed in both <i>S. cerevisiae</i> and HCT116 cells; green dashed line, interaction observed only in <i>S. cerevisiae</i>; orange dotted line, interaction observed only in HCT116 cells.</p