WDR5, BRCA1, and BARD1 Co-regulate the DNA Damage Response and Modulate the Mesenchymal-to-Epithelial Transition during Early Reprogramming
| Autor: | Klaas W. Mulder, Susan Waarlo, Georgina Peñalosa-Ruiz, Vicky Bousgouni, Simon J. van Heeringen, Joris V. van de Ven, Chris Bakal, Jan P. Gerlach, José C. R. Silva, Gert Jan C. Veenstra, Tim E. Veenstra |
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| Přispěvatelé: | Rebelo Da Silva, Jose [0000-0001-5487-1117], Apollo - University of Cambridge Repository |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
WDR5 DNA Repair Cellular differentiation Biochemistry functional interactions Mice 0302 clinical medicine mesenchymal-to-epithelial transition DNA damage repair Induced pluripotent stem cell lcsh:QH301-705.5 lcsh:R5-920 Gene knockdown BRCA1 Protein Intracellular Signaling Peptides and Proteins Cellular Reprogramming Chromatin Cell biology Phenotype Molecular Developmental Biology lcsh:Medicine (General) Reprogramming Signal Transduction Epithelial-Mesenchymal Transition DNA damage DNA repair Ubiquitin-Protein Ligases iPSCs chromatin factors Biology Article 03 medical and health sciences Genetics BARD1 Animals Humans Transcription factor Gene Expression Profiling Tumor Suppressor Proteins reprogramming Cell Biology BRCA1 030104 developmental biology lcsh:Biology (General) 030217 neurology & neurosurgery Developmental Biology DNA Damage Transcription Factors |
| Zdroj: | Stem Cell Reports Stem Cell Reports, 12, 743-756 Stem Cell Reports, 12, 4, pp. 743-756 Stem Cell Reports, Vol 12, Iss 4, Pp 743-756 (2019) |
| ISSN: | 2213-6711 |
| Popis: | Summary 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. Graphical Abstract Highlights • High-content imaging screen of chromatin factors in reprogramming MEFs to iPSCs • Colony level phenotypes followed up with secondary screen using RNA sequencing • Wdr5 functionally interacts with Brca1 and Bard1 in early reprogramming processes • WDR5, BRCA1, and BARD1 affect DNA repair and mesenchymal-to-epithelial transition To assess the role of chromatin-associated factors in reprogramming of fibroblasts to induced pluripotency, high-content imaging screening with siRNAs targeting chromatin-associated factors was performed to identify colony level phenotypes. RNA sequencing was used as a secondary screen. It was found that WDR5 functionally interacts with BRCA1 and BARD1, and that these factors are important for DNA repair and the mesenchymal-to-epithelial transition. |
| Databáze: | OpenAIRE |
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