WDR5, BRCA1, and BARD1 Co-regulate the DNA Damage Response and Modulate the Mesenchymal-to-Epithelial Transition during Early Reprogramming.

Differentiated cells are epigenetically stable, but can be reprogrammed to pluripotency by expression of the OSKM transcription factors. Despite significant effort, relatively little is known about the cellular requirements for reprogramming and how they affect the properties of induced pluripotent stem cells. We have performed high-content screening with small interfering RNAs targeting 300 chromatin-associated factors and extracted colony-level quantitative features. This revealed five morphological phenotypes in early reprogramming, including one displaying large round colonies exhibiting an early block of reprogramming. Using RNA sequencing, we identified transcriptional changes associated with these phenotypes. Furthermore, double knockdown epistasis experiments revealed that BRCA1, BARD1, and WDR5 functionally interact and are required for the DNA damage response. In addition, the mesenchymal-to-epithelial transition is affected in Brca1, Bard1, and Wdr5 knockdowns. Our data provide a resource of chromatin-associated factors in early reprogramming and underline colony morphology as an important high-dimensional readout for reprogramming quality.V.B. and C.B. are funded by the Stand Up to Cancer campaign for Cancer Research UK, and Cancer Research UK Program Foundation Award to C.B. (C37275/1A20146). K.M. was supported by an NWO-VIDI grant (864.12.010)

Quantitative imaging of single-cell phenotypes in cancer cells cultured on hydrogel surfaces

Summary: Small interfering RNA (siRNA) screening approaches used with quantitative single-cell analysis can uncover the roles of genes in cell morphogenesis. Here, we present a high-throughput automated phenotypic screening technique to quantify a single cell shape in cancer cells cultured on top of soft 3D hydrogels. We describe reverse transfection of cells with siRNAs and seeding of these cells on top of collagen, followed by image analysis to quantify morphology of a single cell and population levels in low-elasticity matrices.For complete details on the use and execution of this protocol, please refer to Bousgouni et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

A high-content RNAi screen reveals multiple roles for long noncoding RNAs in cell division.

Genome stability relies on proper coordination of mitosis and cytokinesis, where dynamic microtubules capture and faithfully segregate chromosomes into daughter cells. With a high-content RNAi imaging screen targeting more than 2,000 human lncRNAs, we identify numerous lncRNAs involved in key steps of cell division such as chromosome segregation, mitotic duration and cytokinesis. Here, we provide evidence that the chromatin-associated lncRNA, linc00899, leads to robust mitotic delay upon its depletion in multiple cell types. We perform transcriptome analysis of linc00899-depleted cells and identify the neuronal microtubule-binding protein, TPPP/p25, as a target of linc00899. We further show that linc00899 binds TPPP/p25 and suppresses its transcription. In cells depleted of linc00899, upregulation of TPPP/p25 alters microtubule dynamics and delays mitosis. Overall, our comprehensive screen uncovers several lncRNAs involved in genome stability and reveals a lncRNA that controls microtubule behaviour with functional implications beyond cell division

ARHGAP9 siGENOME MDA-MB-231, high YAP/TAZ ratio (2).bmp

ARHGAP9 siGENOME MDA-MB-231, hit for high YAP/TAZ rati

MDA-MB-231_siGENOME_GEFGGAP screen (Data Record 3).xlsx

Raw data for the siGENOME RhoGEF and RhoGAP RNAi screen on MDA-MB-231 cell

ARHGAP26 siGENOME MDA-MB-231, no change in YAP/TAZ ratio.bmp

ARHGAP26 siGENOME MDA-MB-231, no change in YAP/TAZ rati

Signaling Networks Converge on TORC1-SREBP Activity to Promote Endoplasmic Reticulum Homeostasis

<div><p>The function and capacity of the endoplasmic reticulum (ER) is determined by multiple processes ranging from the local regulation of peptide translation, translocation, and folding, to global changes in lipid composition. ER homeostasis thus requires complex interactions amongst numerous cellular components. However, describing the networks that maintain ER function during changes in cell behavior and environmental fluctuations has, to date, proven difficult. Here we perform a systems-level analysis of ER homeostasis, and find that although signaling networks that regulate ER function have a largely modular architecture, the TORC1-SREBP signaling axis is a central node that integrates signals emanating from different sub-networks. TORC1-SREBP promotes ER homeostasis by regulating phospholipid biosynthesis and driving changes in ER morphology. In particular, our network model shows TORC1-SREBP serves to integrate signals promoting growth and G1-S progression in order to maintain ER function during cell proliferation.</p></div

LM2_GEFGAP_siGENOME (Data Record 1).xlsx

Raw data for the siGENOME RhoGEF and RhoGAP RNAi screen on LM2 cells

Oligonucleotides used in the study.

<p>Oligonucleotides used in the study.</p