BackgroundSmaug is an RNA-binding protein that induces the degradation and represses the translation of mRNAs in the early Drosophila embryo. Smaug has two identified direct target mRNAs that it differentially regulates: nanos and Hsp83. Smaug represses the translation of nanos mRNA but has only a modest effect on its stability, whereas it destabilizes Hsp83 mRNA but has no detectable effect on Hsp83 translation. Smaug is required to destabilize more than one thousand mRNAs in the early embryo, but whether these transcripts represent direct targets of Smaug is unclear and the extent of Smaug-mediated translational repression is unknown.ResultsTo gain a panoramic view of Smaug function in the early embryo, we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug’s target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets, as well as many metabolic enzymes, including several glycolytic enzymes.ConclusionsSmaug plays a direct and global role in regulating the translation and stability of a large fraction of the mRNAs in the early Drosophila embryo, and has unanticipated functions in control of protein folding and degradation, lipid droplet function and metabolism.

R-loops are features of chromatin consisting of a strand of DNA hybridized to RNA, as well as the expelled complementary DNA strand. R-loops are enriched at promoters where they have recently been shown to have important roles in modifying gene expression. However, the location of promoter-associated R-loops and the genomic domains they perturb to modify gene expression remain unclear. To resolve this issue, we developed a bisulfite-based approach, bisDRIP-seq, to map R-loops across the genome at near-nucleotide resolution in MCF-7 cells. We found the location of promoter-associated R-loops is dependent on the presence of introns. In intron-containing genes, R-loops are bounded between the transcription start site and the first exon-intron junction. In intronless genes, the 3' boundary displays gene-specific heterogeneity. Moreover, intronless genes are often associated with promoter-associated R-loop formation. Together, these studies provide a high-resolution map of R-loops and identify gene structure as a critical determinant of R-loop formation.

Here, we have asked about post-transcriptional mechanisms regulating murine developmental neurogenesis, focusing upon the RNAbinding proteins Smaug2 and Nanos1. We identify, in embryonic neural precursors of the murine cortex, a Smaug2 protein/nanos1 mRNA complex that is present in cytoplasmic granules with the translational repression proteins Dcp1 and 4E-T. We show that Smaug2 inhibits and Nanos1 promotes neurogenesis, with Smaug2 knockdown enhancing neurogenesis and depleting precursors, and Nanos1 knockdown inhibiting neurogenesis and maintaining precursors. Moreover, we show that Smaug2 likely regulates neurogenesis by silencing nanos1 mRNA. Specifically, Smaug2 knockdown inappropriately increases Nanos1 protein, and the Smaug2 knockdownmediated neurogenesis is rescued by preventing this increase. Thus, Smaug2 and Nanos1 function as a bimodal translational repression switch to control neurogenesis, with Smaug2 acting in transcriptionally primed precursors to silence mRNAs important for neurogenesis, including nanos1 mRNA, and Nanos1 acting during the transition to neurons to repress the precursor state.

R-loops are features of chromatin consisting of a strand of DNA hybridized to RNA, as well as the expelled complementary DNA strand. R-loops are enriched at promoters where they have recently been shown to have important roles in modifying gene expression. However, the location of promoter-associated R-loops and the genomic domains they perturb to modify gene expression remain unclear. To resolve this issue, we developed a bisulfite-based approach, bisDRIPseq, to map R-loops across the genome at near-nucleotide resolution in MCF-7 cells. We found the location of promoter-associated R-loops is dependent on the presence of introns. In introncontaining genes, R-loops are bounded between the transcription start site and the first exonintron junction. In intronless genes, the 3' boundary displays gene-specific heterogeneity. Moreover, intronless genes are often associated with promoter-associated R-loop formation. Together, these studies provide a high-resolution map of R-loops and identify gene structure as a critical determinant of R-loop formation.

Recent studies have shown that mutations at non-coding elements, such as the TERT promoter and ESR1 enhancer, can act as cancer drivers. However, an important class of non-coding elements, namely CTCF/cohesin insulators, has been overlooked in the previous driver analyses. It is known that promoter and enhancer interactions are facilitated by partitioning of the human genome into DNA loops. These loops act as insulated neighborhoods preventing the interactions of enhancers and promoters across loops. They are held together by the binding of two CCCTCF binding factors (CTCF) and the cohesin complex, which consists of four core transcription factor subunits (SMC1, SMC3, RAD21 and either STAG2 or STAG1) at the loop ends. Disruption of the loop anchor regions, called CTCF/cohesin insulators, can lead to de novo enhancer-promoter interactions and subsequent dysregulation of associated genes. We used insulator annotations from cohesin ChIA-PET assays and analyzed somatic mutations in 1,962 whole-genomes from 21 cancer types. We find that the mutations predicted to disrupt DNA binding motifs of CTCF, RAD21 and SMC3 are enriched in many cancer types due to the sequence contexts of neutral mutational processes. Using a novel computational approach, CNCDriver (Cornell Non-Coding Driver), that balances the interplay between neutral mutational rate covariates of CTCF/RAD21/SMC3 motif loss and the stronger functional impact of motif-disrupting mutations, we identify the insulators that show signals of positive selection during tumor evolution. We find that the mutations in significantly mutated insulators are associated with differential expression of genes involved in multiple cancer pathways: TGF-β, Hedgehog and Wnt signaling. In particular, we find 16% of melanoma samples show mutations in an insulator that are associated with up-regulation of TGFB1, which is known to promote angiogenesis and tumor cell migration in melanoma. Thus, our study reveals insulators as a novel class of non-coding cancer drivers. Identification of significantly mutated insulators can complement the identification of other types of non-coding cancer drivers (promoters, enhancers and ncRNAs) to fully understand the role of non-coding alterations in tumorigenesis. Citation Format: Eric Minwei Liu, Alexander Martinez-Fundichely, Tawny Cuykendall, Jason G. Dumelie, Matthew MacKay, Priyanka Dhingra, Samie R. Jaffrey, Ekta Khurana. Pan-cancer analysis of 1,962 whole-genomes reveals significantly mutated CTCF/cohesin insulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3270.