Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Element cis-régulateur et facteurs en trans contrôlant l'expression de Krox20 lors de la segmentation du rhombencéphale

During development, the posterior brain undergoes a transient segmentation process along the anteroposterior axis leading to its subdivision into 7 transversal units called rhombomeres (r) that form developmental and genetic expression units. The zinc finger factor plays a key role in this process by coupling the formation and the specification of the rhombomeres 3 and 5. Three regulatory elements controlling Krox20 expression have been characterized previously. Elements B and C are active in r5 and in r3-r5, respectively, and are involved in the onset of Krox20 expression whose amplification and upkeep are carried out by the autoregulatory element A. Thus, element C is a key tool to investigate the mechanisms responsible for the regionalized expression of Krox20 in r3. In order to deepen the characterization of Krox20 transcriptional control in r3, I undertook a functional analysis of the sequences of element C by mutagenesis and identified several conserved blocks required for its activity in r3. Furthermore, I established the complete quantitative and differential transcriptomic map of the mouse early hindbrain by RNA-Seq and I characterized several regulators of element C activity. Finally, I analyzed the mouse mutant where element C is deleted and I revealed new properties of Krox20 transcriptional control, notably a cis-cooperation between elements A and C. As a conclusion, this work shows how the combination of different approaches allows the elucidation of a complex transcriptional regulation mechanism that plays an essential role in cell specificationLa segmentation du rhombencéphale désigne le processus de subdivision rostro-caudale du cerveau postérieur embryonnaire en 7 compartiments cellulaires, appelés rhombomères (r), qui constituent des unités de développement et d'expression génique. Le facteur de transcription à doigts à zinc Krox20 joue un rôle central dans ce processus en couplant la formation et la spécification des rhombomères 3 et 5. Trois éléments régulateurs contrôlant l'expression de Krox20 ont été caractérisés. Les éléments B et C, actifs respectivement dans r5 et r3-r5, sont impliqués dans le démarrage de l'expression de Krox20 dont l'amplification et le maintien sont ensuite assurés par l'élément autorégulateur A. L'élément C constitue donc une clé d'investigation pour élucider les mécanismes responsables de l'expression régionalisée de Krox20 dans r3. Afin de poursuivre la caractérisation du contrôle transcriptionnel de Krox20 dans r3, j'ai entrepris une analyse fonctionnelle des séquences de l'élément C par mutagénèse et mis en évidence plusieurs blocs nécessaires à son activité. J'ai également établi le répertoire transcriptomique exhaustif, quantitatif et différentiel du rhombencéphale murin en début de segmentation par RNA-Seq et j'ai pu ainsi identifier plusieurs régulateurs de l'activité de l'élément C. Enfin, j'ai réalisé l'analyse du mutant murin d'excision de l'élément C et révélé de nouvelles propriétés du contrôle transcriptionnel de Krox20, dont la coopération en cis entre les éléments A et C. Ces travaux illustrent comment la combinaison de différentes approches permet d'élucider un mécanisme de régulation transcriptionnel complexe jouant un rôle essentiel dans la spécification cellulair

Krox20 hindbrain regulation incorporates multiple modes of cooperation between cis-acting elements

International audienceDevelopmental genes can harbour multiple transcriptional enhancers that act simultaneously or in succession to achieve robust and precise spatiotemporal expression. However, the mechanisms underlying cooperation between cis-acting elements are poorly documented, notably in vertebrates. The mouse gene Krox20 encodes a transcription factor required for the specification of two segments (rhombomeres) of the developing hindbrain. In rhombomere 3, Krox20 is subject to direct positive feedback governed by an autoregulatory enhancer, element A. In contrast, a second enhancer, element C, distant by 70 kb, is active from the initiation of transcription independent of the presence of the KROX20 protein. Here, using both enhancer knock-outs and investigations of chromatin organisation, we show that element C possesses a dual activity: besides its classical enhancer function, it is also permanently required in cis to potentiate the autoregulatory activity of element A, by increasing its chromatin accessibility. This work uncovers a novel, asymmetrical, long-range mode of cooperation between cis-acting elements that might be essential to avoid promiscuous activation of positive autoregulatory elements

Physical interactions within the <i>Krox20</i> locus.

<p><b>(A)</b> Alignment of data in the <i>Krox20</i> and adjacent loci from Hi-C in ES cells [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006903#pgen.1006903.ref011" target="_blank">11</a>], 4C-seq in E9.5 whole mouse embryos, using the <i>Krox20</i> and <i>Nrbf2</i> promoters as viewpoints (this work, 2 biological replicates) and CTCF ChIP-seq in E14.5 mouse brain (ENCODE, [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006903#pgen.1006903.ref058" target="_blank">58</a>]). <b>(B)</b> Zoom in on the <i>Krox20</i> locus, showing 4C-seq data from the <i>Krox20</i> promoter, element A, element B and element C as viewpoints. CTCF ChIP-seq data in E14.5 mouse brain (ENCODE) are indicated below. Signals from simultaneously processed E9.5 whole embryo (dark blue) and E8.5 embryo head (light blue) samples are shown. On the right, normalized distributions of the 4C-seq signals in different genomic regions are indicated. TADs as defined in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006903#pgen.1006903.ref007" target="_blank">7</a>] or by our additional analysis (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006903#pgen.1006903.s002" target="_blank">S2 Fig</a>) are indicated above, with dashed lines in the graphs demarcating TAD boundaries. Genes (black/red), <i>cis</i>-regulatory elements (orange) and genomic coordinates are indicated below each set of data. Arrowheads above each 4C track pinpoint viewpoints.</p

A model for <i>Krox20</i> regulation and the dual function of element C.

<p>(<b>A</b>) Schematic representation of the regulation of <i>Krox20</i> in r3. Three situations are envisaged in wild type embryos. Left: silent locus. If both element C and the new enhancer (NE) are inactive, no expression occurs. Middle: early expression phase. At this stage, elements C and NE have been bound by their respective transcription factors and have initiated the expression of <i>Krox20</i> via their classical enhancer functions. Nevertheless, element C has not yet been unlocked (decompacted) element A and/or the concentration of the KROX20 protein has not reached high enough levels to allow the establishment of a stable feedback loop with a significant probability. Right: late expression phase. Via its potentiator function, element C has unlocked element A, which can bind the KROX20 protein, which has now accumulated at a high enough concentration. Activation of enhancer A establishes the autoregulatory loop. <b>(B)</b> Three mutations that disrupt the positive feedback loop are presented at late expression phase. Left: mutation of the KROX20 protein preventing binding to element A. Middle: mutation of element A, preventing the binding of the KROX20 protein. Right: mutation of element C, preventing unlocking of element A.</p

Genetic analysis of element C function.

<p><b>(A)</b> Strategy for the construction of conditional and null alleles of element C. The targeting vector was introduced into the locus in ES cells by homologous recombination and one of the ES clones subsequently allowed germ line transmission in the mouse. The floxed allele, <i>Krox20</i>^<i>Cflox</i>, was obtained by crossing the founder mouse line with a <i>Flp</i> (targeting FRT sites) deletor line. The null allele, <i>Krox20</i>^<i>ΔC</i>, was obtained by crossing the <i>Krox20</i>^<i>Cflox</i> line with a <i>Cre</i> (targeting loxP sites) deletor line, PGK-Cre. <b>(B)</b> In situ hybridization for <i>Krox20</i> mRNA performed on <i>Krox20</i>^<i>+/ΔC</i> and <i>Krox20</i>^{<i>ΔC/ΔC</i>} embryos at the indicated somite stages. Embryos were flat-mounted with anterior toward the top. Rhombomere positions are indicated on the left.</p

<i>Krox20</i> hindbrain regulation incorporates multiple modes of cooperation between <i>cis</i>-acting elements - Fig 2

<p><b>Cooperation in <i>cis</i> between elements A and C. (A)</b> In situ hybridization for <i>Krox20</i> mRNA was performed on wild type (WT), <i>Krox20</i>^<i>+/Cre</i>, <i>Krox20</i>^{<i>ΔA/ΔA</i>}, <i>Krox20</i>^{<i>ΔC/ΔC</i>} and composite heterozygous <i>Krox20</i>^{<i>ΔA/ΔC</i>} embryos at the indicated somite stages. <b>(B)</b> In situ hybridization for <i>Krox20</i> mRNA was performed on <i>Krox20</i>^<i>+/Cre</i> and <i>Krox20</i>^{<i>Cflox/Cre</i>} embryos at the indicated somite stages. In (A) and (B) embryos were flat-mounted with anterior toward the top.</p