VHL Synthetic Lethality Signatures Uncovered by Genotype-Specific CRISPR-Cas9 Screens.

Genome-wide CRISPR-Cas9 essentiality screening represents a powerful approach to identify genetic vulnerabilities in cancer cells. Here, we applied this technology and designed a strategy to identify target genes that are synthetic lethal (SL) with von Hippel-Lindau (VHL) tumor suppressor gene. Inactivation of VHL has been frequently found in clear cell renal cell carcinoma. Its SL partners serve as potential drug targets for the development of targeted cancer therapies. We performed parallel genome-wide CRISPR screens in two pairs of isogenic clear cell renal cell carcinoma cell lines that differ only in the VHL status. Comparative analyses of screening results not only confirmed a well-known role for mTOR signaling in renal carcinoma, but also identified DNA damage response and selenocysteine biosynthesis pathways as novel SL targets in VHL-inactivated cancer cells. Follow-up studies provided cellular and mechanistic insights into SL interactions of these pathway genes with the VHL gene. Our CRISPR and RNA-seq datasets provide a rich resource for future investigation of the function of the VHL tumor suppressor protein. Our work demonstrates the efficiency of CRISPR-based synthetic lethality screening in human isogenic cell pairs. Similar strategies could be employed to unveil SL partners with other oncogenic drivers.

BMP signaling components in embryonic transcriptomes of the hover fly Episyrphus balteatus (Syrphidae).

BACKGROUND: In animals, signaling of Bone Morphogenetic Proteins (BMPs) is essential for dorsoventral (DV) patterning of the embryo, but how BMP signaling evolved with changes in embryonic DV differentiation is largely unclear. Based on the extensive knowledge of BMP signaling in Drosophila melanogaster, the morphological diversity of extraembryonic tissues in different fly species provides a comparative system to address this question. The closest relatives of D. melanogaster with clearly distinct DV differentiation are hover flies (Diptera: Syrphidae). The syrphid Episyrphus balteatus is a commercial bio-agent against aphids and has been established as a model organism for developmental studies and chemical ecology. The dorsal blastoderm of E. balteatus gives rise to two extraembryonic tissues (serosa and amnion), whereas in D. melanogaster, the dorsal blastoderm differentiates into a single extraembryonic epithelium (amnioserosa). Recent studies indicate that several BMP signaling components of D. melanogaster, including the BMP ligand Screw (Scw) and other extracellular regulators, evolved in the dipteran lineage through gene duplication and functional divergence. These findings raise the question of whether the complement of BMP signaling components changed with the origin of the amnioserosa. RESULTS: To search for BMP signaling components in E. balteatus, we generated and analyzed transcriptomes of freshly laid eggs (0-30 minutes) and late blastoderm to early germband extension stages (3-6 hours) using Roche/454 sequencing. We identified putative E. balteatus orthologues of 43% of all annotated D. melanogaster genes, including the genes of all BMP ligands and other BMP signaling components. CONCLUSION: The diversification of several BMP signaling components in the dipteran linage of D. melanogaster preceded the origin of the amnioserosa.[Transcriptome sequence data from this study have been deposited at the NCBI Sequence Read Archive (SRP005289); individually assembled sequences have been deposited at GenBank (JN006969-JN006986).].

Maternal activation of gap genes in the hover fly Episyrphus.

The metameric organization of the insect body plan is initiated with the activation of gap genes, a set of transcription-factor-encoding genes that are zygotically expressed in broad and partially overlapping domains along the anteroposterior (AP) axis of the early embryo. The spatial pattern of gap gene expression domains along the AP axis is generally conserved, but the maternal genes that regulate their expression are not. Building on the comprehensive knowledge of maternal gap gene activation in Drosophila, we used loss- and gain-of-function experiments in the hover fly Episyrphus balteatus (Syrphidae) to address the question of how the maternal regulation of gap genes evolved. We find that, in Episyrphus, a highly diverged bicoid ortholog is solely responsible for the AP polarity of the embryo. Episyrphus bicoid represses anterior zygotic expression of caudal and activates the anterior and central gap genes orthodenticle, hunchback and Krüppel. In bicoid-deficient Episyrphus embryos, nanos is insufficient to generate morphological asymmetry along the AP axis. Furthermore, we find that torso transiently regulates anterior repression of caudal and is required for the activation of orthodenticle, whereas all posterior gap gene domains of knirps, giant, hunchback, tailless and huckebein depend on caudal. We conclude that all maternal coordinate genes have altered their specific functions during the radiation of higher flies (Cyclorrhapha).