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A novel forward genetic pipeline for the identification of genes required for transdifferentiation based on computational analysis of whole-genome sequences of large numbers of mutants


How cell fate is decided and maintained has been an ongoing question for decades. Early embryogenesis studies showed that embryonic stem cells are pluripotent and can differentiate into every cell type in vivo or in vitro by introducing the corresponding factors. Later, the cellular reprogramming discovered by Yamanaka opened a new area of generating induced pluripotent stem cell (iPSC) from a terminal differentiated post-mitotic cell. In this study, we aimed to develop a new forward genetic screening pipeline to illuminate the genetic requirements underlying developmental plasticity and to investigate how post-mitotic differentiated cells in an intact animal can be reprogrammed and remodeled into new cell types in the process of transdifferentiation (Td). In C. elegans, forced ubiquitous expression of the ELT-7 GATA-type transcription factor, which functions in intestinal differentiation, is capable of converting differentiated, post-mitotic cells of two organs, the pharynx and uterus, into cells with gene expression patterns and ultrastructural characteristics of normal intestine cells. The developmental arrest phenotype was also observed in ELT-7-mediated Td animals, plausibly due to the conversion of pharyngeal cells into intestine-like cells. Reverse genetic approaches have been applied to knock down or knock out hundreds of candidate genes to uncover the molecular mechanism of ELT-7-mediated Td; however, no positive result was found. This result suggests that none of the candidate genes was involved in Td, or the down-regulation of candidate genes approach was not a proper experimental design for identifying Td. For example, genes required for intestinal Td may also be crucial for normal animal development; thus, a regular gene knock-down approach is unsuitable. To address this problem and widen the search for candidate genes in Td, a large-scale forward genetic screening using the auxin-inducible degradation system to ubiquitously express ELT-7 was performed by selecting suppressors of development arrest. We hypothesize that genes required for ELT-7-mediated Td will more likely be selected in the forward genetic screening and show many unique mutations. Mutations in those Td-required genes would also have a high incidence of disrupting their protein function by affecting the conserved residues or the active domain. A total of 660 mutant lines were isolated that escaped developmental arrest after ELT-7 overexpression. Mutant lines were pooled into different group sizes for whole-genome sequencing and a hundred thousand SNPs were predicted and evaluated with the three analyses: 1) The mutation density in a gene, 2) The percentage of mutations in conserved amino acids, and 3) The distribution of mutations in a gene. This analysis identified the positive experimental control, elt-7 transgene, and among the top candidates identified, cdk-12 was confirmed as a causally associated gene required for ELT-7-mediated Td through complementation tests and rescue experiments. This novel pooled mutant sequencing strategy was highly efficient since it did not require procedures to pre-screen mutations that affect the system or clean up the background mutations before fine mapping. It also applies to non-viable phenotypes or genetic manipulation unavailable in the animal. Finally, pooled mutant sequencing was cost-effective, with 10x less expense compared to single animal sequencing. This method was ideal for organisms with large brood sizes, such as bacteria, yeast, worms, and flies.

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