In vivo single-cell CRISPR uncovers distinct TNF programmes in tumour evolution.

The tumour evolution model posits that malignant transformation is preceded by randomly distributed driver mutations in cancer genes, which cause clonal expansions in phenotypically normal tissues. Although clonal expansions can remodel entire tissues1-3, the mechanisms that result in only a small number of clones transforming into malignant tumours remain unknown. Here we develop an in vivo single-cell CRISPR strategy to systematically investigate tissue-wide clonal dynamics of the 150 most frequently mutated squamous cell carcinoma genes. We couple ultrasound-guided in utero lentiviral microinjections, single-cell RNA sequencing and guide capture to longitudinally monitor clonal expansions and document their underlying gene programmes at single-cell transcriptomic resolution. We uncover a tumour necrosis factor (TNF) signalling module, which is dependent on TNF receptor 1 and involving macrophages, that acts as a generalizable driver of clonal expansions in epithelial tissues. Conversely, during tumorigenesis, the TNF signalling module is downregulated. Instead, we identify a subpopulation of invasive cancer cells that switch to an autocrine TNF gene programme associated with epithelial-mesenchymal transition. Finally, we provide in vivo evidence that the autocrine TNF gene programme is sufficient to mediate invasive properties and show that the TNF signature correlates with shorter overall survival of patients with squamous cell carcinoma. Collectively, our study demonstrates the power of applying in vivo single-cell CRISPR screening to mammalian tissues, unveils distinct TNF programmes in tumour evolution and highlights the importance of understanding the relationship between clonal expansions in epithelia and tumorigenesis.

Monitoring the 5'UTR landscape reveals isoform switches to drive translational efficiencies in cancer.

Transcriptional and translational control are key determinants of gene expression, however, to what extent these two processes can be collectively coordinated is still poorly understood. Here, we use Nanopore long-read sequencing and cap analysis of gene expression (CAGE-seq) to document the landscape of 5' and 3' untranslated region (UTR) isoforms and transcription start sites of epidermal stem cells, wild-type keratinocytes and squamous cell carcinomas. Focusing on squamous cell carcinomas, we show that a small cohort of genes with alternative 5'UTR isoforms exhibit overall increased translational efficiencies and are enriched in ribosomal proteins and splicing factors. By combining polysome fractionations and CAGE-seq, we further characterize two of these UTR isoform genes with identical coding sequences and demonstrate that the underlying transcription start site heterogeneity frequently results in 5' terminal oligopyrimidine (TOP) and pyrimidine-rich translational element (PRTE) motif switches to drive mTORC1-dependent translation of the mRNA. Genome-wide, we show that highly translated squamous cell carcinoma transcripts switch towards increased use of 5'TOP and PRTE motifs, have generally shorter 5'UTRs and expose decreased RNA secondary structures. Notably, we found that the two 5'TOP motif-containing, but not the TOP-less, RPL21 transcript isoforms strongly correlated with overall survival in human head and neck squamous cell carcinoma patients. Our findings warrant isoform-specific analyses in human cancer datasets and suggest that switching between 5'UTR isoforms is an elegant and simple way to alter protein synthesis rates, set their sensitivity to the mTORC1-dependent nutrient-sensing pathway and direct the translational potential of an mRNA by the precise 5'UTR sequence.

Inhibition of FGF and TGF-ß Pathways in hESCs Identify STOX2 as a Novel SMAD2/4 Cofactor.

The fibroblast growth factor (FGF) and the transforming growth factor-ß (TGF-ß) pathways are both involved in the maintenance of human embryonic stem cells (hESCs) and regulate the onset of their differentiation. Their converging functions have suggested that these pathways might share a wide range of overlapping targets. Published studies have focused on the long-term effects (24-48 h) of FGF and TGF-ß inhibition in hESCs, identifying direct and indirect target genes. In this study, we focused on the earliest transcriptome changes occurring between 3 and 9 h after FGF and TGF-ß inhibition to identify direct target genes only. Our analysis clearly shows that only a handful of target transcripts are common to both pathways. This is surprising in light of the previous literature, and has implications for models of cell signaling in human pluripotent cells. In addition, we identified STOX2 as a novel primary target of the TGF-ß signaling pathway. We show that STOX2 might act as a novel SMAD2/4 cofactor. Taken together, our results provide insights into the effect of cell signaling on the transcription profile of human pluripotent cells.

Some like it translated: small ORFs in the 5'UTR.

The 5' untranslated region (5'UTR) is critical in determining post-transcriptional control, which is partly mediated by short upstream open reading frames (uORFs) present in half of mammalian transcripts. uORFs are generally considered to provide functionally important repression of the main-ORF by engaging initiating ribosomes, but under specific environmental conditions such as cellular stress, uORFs can become essential to activate the translation of the main coding sequence. In addition, a growing number of uORF-encoded bioactive microproteins have been described, which have the potential to significantly increase cellular protein diversity. Here we review the diverse cellular contexts in which uORFs play a critical role and discuss the molecular mechanisms underlying their function and regulation. The progress over the last decades in dissecting uORF function suggests that the 5'UTR remains an exciting frontier towards understanding how the cellular proteome is shaped in health and disease.

Gaining Insights into the Function of Post-Translational Protein Modification Using Genome Engineering and Molecular Cell Biology.

Modifications by kinases are a fast and reversible mechanism to diversify the function of the targeted proteins. The OCT4 transcription factor is essential for preimplantation development and pluripotency of embryonic stem cells (ESC), and its activity is tightly regulated by post-transcriptional modifications. Several phosphorylation sites have been identified by systemic approaches and their functions proposed. Here, we combined molecular and cellular biology with CRISPR/Cas9-mediated genome engineering to pinpoint the function of serine 12 of OCT4 in ESCs. Using chemical inhibitors and an antibody specific to OCT4 phosphorylated on S12, we identified cyclin-dependent kinase (CDK) 7 as upstream kinase. Surprisingly, generation of isogenic mESCs that endogenously ablate S12 revealed no effects on pluripotency and self-renewal, potentially due to compensation by other phosphorylation events. Our approach reveals that modification of distinct amino acids by precise genome engineering can help to clarify the functions of post-translational modifications on proteins encoded by essential gene in an endogenous context.

Comparative analysis of differential gene expression tools for RNA sequencing time course data.

RNA sequencing (RNA-seq) has become a standard procedure to investigate transcriptional changes between conditions and is routinely used in research and clinics. While standard differential expression (DE) analysis between two conditions has been extensively studied, and improved over the past decades, RNA-seq time course (TC) DE analysis algorithms are still in their early stages. In this study, we compare, for the first time, existing TC RNA-seq tools on an extensive simulation data set and validated the best performing tools on published data. Surprisingly, TC tools were outperformed by the classical pairwise comparison approach on short time series (<8 time points) in terms of overall performance and robustness to noise, mostly because of high number of false positives, with the exception of ImpulseDE2. Overlapping of candidate lists between tools improved this shortcoming, as the majority of false-positive, but not true-positive, candidates were unique for each method. On longer time series, pairwise approach was less efficient on the overall performance compared with splineTC and maSigPro, which did not identify any false-positive candidate.

A Concise Protocol for siRNA-Mediated Gene Suppression in Human Embryonic Stem Cells.

Human embryonic stem cells hold great promise for future biomedical applications such as disease modeling and regenerative medicine. However, these cells are notoriously difficult to culture and are refractory to common means of genetic manipulation, thereby limiting their range of applications. In this protocol, we present an easy and robust method of gene repression in human embryonic stem cells using lipofection of small interfering RNA (siRNA).