Correction of copy number induced false positives in CRISPR screens

<div><p>Cell autonomous cancer dependencies are now routinely identified using CRISPR loss-of-function viability screens. However, a bias exists that makes it difficult to assess the true essentiality of genes located in amplicons, since the entire amplified region can exhibit lethal scores. These false-positive hits can either be discarded from further analysis, which in cancer models can represent a significant number of hits, or methods can be developed to rescue the true-positives within amplified regions. We propose two methods to rescue true positive hits in amplified regions by correcting for this copy number artefact. The Local Drop Out (LDO) method uses the relative lethality scores within genomic regions to assess true essentiality and does not require additional orthogonal data (e.g. copy number value). LDO is meant to be used in screens covering a dense region of the genome (e.g. a whole chromosome or the whole genome). The General Additive Model (GAM) method models the screening data as a function of the known copy number values and removes the systematic effect from the measured lethality. GAM does not require the same density as LDO, but does require prior knowledge of the copy number values. Both methods have been developed with single sample experiments in mind so that the correction can be applied even in smaller screens. Here we demonstrate the efficacy of both methods at removing the copy number effect and rescuing hits from some of the amplified regions. We estimate a 70–80% decrease of false positive hits with either method in regions of high copy number compared to no correction.</p></div

MET Specific LDO correction in MKN45 in two different screens.

<p><b>A)</b> Sensitivity conferred by each guide (black dots) within the MET amplicon in MKN45 summarized by a boxplot for each gene in the amplicon. The red line displays the inverted copy number value scaled to the data. <b>B)</b> Sensitivity conferred by each guide (black dots) within the MET amplicon in MKN45 summarized by a boxplot for each gene in the amplicon. The red line displays the inverted copy number value scaled to the data.</p

LDO removes the copy number effect across samples and maintains sensitivity of essential genes.

<p><b>A)</b> Boxplot of dependency scores across copy number for uncorrected and LDO corrected data. <b>B)</b> The recall curve for essential, nonessential and amplified genes is shown before and after LDO copy number correction in the cell line DAN-G. <b>C)</b> The area under the recall curve is shown across samples for the essential, nonessential and amplified genes.</p

TEAD single and double motifs occur within most YAP1 binding sites.

<p>(A and B) Enrichment of (A) TEAD and (B) AP-1 motifs in YAP1 peaks. Full list provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005465#pgen.1005465.s017" target="_blank">S4 Table</a>. (C) YAP1 ChIP enrichment as determined by peak score in YAP1 peaks with/without TEAD and AP-1 motifs. (D) Number of TEAD motifs in YAP1 peaks. (E) Enrichment of TEAD double motif with several spacer lengths in YAP1 peaks. (F) Sequence conservation of YAP1 peak regions. (G) Sequence conservation of TEAD single and double motifs in YAP1 peak regions. (H) YAP1 ChIP enrichment as determined by peak score in YAP1 peaks with/without single/double TEAD motifs. (I) Luciferase reporter assay for two YAP1 binding regions with either intact double motif or with single or double mutations. Relative luciferase activity represents the ratio of Firefly and Renilla luciferase activity for each sample. The red line indicates the highest mean activity of the two negative control regions. Data are representative of at least three independent experiments. Error bars indicate the standard deviation of triplicate qPCR data.</p

YAP1 Exerts Its Transcriptional Control via TEAD-Mediated Activation of Enhancers

<div><p>YAP1 is a major effector of the Hippo pathway and a well-established oncogene. Elevated YAP1 activity due to mutations in Hippo pathway components or <i>YAP1</i> amplification is observed in several types of human cancers. Here we investigated its genomic binding landscape in YAP1-activated cancer cells, as well as in non-transformed cells. We demonstrate that TEAD transcription factors mediate YAP1 chromatin-binding genome-wide, further explaining their dominant role as primary mediators of YAP1-transcriptional activity. Moreover, we show that YAP1 largely exerts its transcriptional control via distal enhancers that are marked by H3K27 acetylation and that YAP1 is necessary for this chromatin mark at bound enhancers and the activity of the associated genes. This work establishes YAP1-mediated transcriptional regulation at distal enhancers and provides an expanded set of target genes resulting in a fundamental source to study YAP1 function in a normal and cancer setting.</p></div

Primers used for ChIP-qPCR.

<p>Primers used for ChIP-qPCR.</p

Primers used for RT-qPCR.

<p>Primers used for RT-qPCR.</p

Luciferase reporters (Fig 2I).

<p>Luciferase reporters (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005465#pgen.1005465.g002" target="_blank">Fig 2I</a>).</p

siRNAs (Fig 3A).

<p>siRNAs (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005465#pgen.1005465.g003" target="_blank">Fig 3A</a>).</p

Luciferase reporters (Fig 4E).

<p>Luciferase reporters (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005465#pgen.1005465.g004" target="_blank">Fig 4E</a>).</p