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 tissues, 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.

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.

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

The fibroblast growth factor (FGF) and the transforming growth factor-β (TGF-β) 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-β inhibition in hESCs, identifying direct and indirect target genes.

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.

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.

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.

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).