A Strategy To Isolate Modifiers of Caenorhabditis elegans Lethal Mutations: Investigating the Endoderm Specifying Ability of the Intestinal Differentiation GATA Factor ELT-2
| Autor: | James D. McGhee, Vinci Au, Tobias Wiesenfahrt, Craig J. Marshall, Dylan M. Parker, Paul E Mains, Erica Li-Leger, Frances Snider, Don Moerman, Stephane Flibotte, Erin Osborne Nishimura, Jingjie Duanmu |
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| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Embryo Nonmammalian Genotype Zygote Transgene Mutant QH426-470 GATA Transcription Factors 03 medical and health sciences C . elegans Extrachromosomal DNA medicine Genetics Animals Amino Acid Sequence Genetic Testing Caenorhabditis elegans Caenorhabditis elegans Proteins Molecular Biology Gene intestine Genetics (clinical) tasp-1 Genes Modifier pqn-82 Whole Genome Sequencing biology Endoderm Mutant Screen Report Reproducibility of Results Cell Differentiation Extrachromosomal array biology.organism_classification Survival Analysis ELT-2 GATA factor Cell biology Intestines 030104 developmental biology medicine.anatomical_structure endoderm specification taspase Mutation C. elegans Genetic screen |
| Zdroj: | G3: Genes, Genomes, Genetics, Vol 8, Iss 5, Pp 1425-1437 (2018) G3: Genes|Genomes|Genetics |
| ISSN: | 2160-1836 |
| DOI: | 10.1534/g3.118.200079 |
| Popis: | The ELT-2 GATA factor normally functions in differentiation of the C. elegans endoderm, downstream of endoderm specification. We have previously shown that, if ELT-2 is expressed sufficiently early, it is also able to specify the endoderm and to replace all other members of the core GATA-factor transcriptional cascade (END-1, END-3, ELT-7). However, such rescue requires multiple copies (and presumably overexpression) of the end-1p::elt-2 cDNA transgene; a single copy of the transgene does not rescue. We have made this observation the basis of a genetic screen to search for genetic modifiers that allow a single copy of the end-1p::elt-2 cDNA transgene to rescue the lethality of the end-1 end-3 double mutant. We performed this screen on a strain that has a single copy insertion of the transgene in an end-1 end-3 background. These animals are kept alive by virtue of an extrachromosomal array containing multiple copies of the rescuing transgene; the extrachromosomal array also contains a toxin under heat shock control to counterselect for mutagenized survivors that have been able to lose the rescuing array. A screen of ∼14,000 mutagenized haploid genomes produced 17 independent surviving strains. Whole genome sequencing was performed to identify genes that incurred independent mutations in more than one surviving strain. The C. elegans gene tasp-1 was mutated in four independent strains. tasp-1 encodes the C. elegans homolog of Taspase, a threonine-aspartic acid protease that has been found, in both mammals and insects, to cleave several proteins involved in transcription, in particular MLL1/trithorax and TFIIA. A second gene, pqn-82, was mutated in two independent strains and encodes a glutamine-asparagine rich protein. tasp-1 and pqn-82 were verified as loss-of-function modifiers of the end-1p::elt-2 transgene by RNAi and by CRISPR/Cas9-induced mutations. In both cases, gene loss leads to modest increases in the level of ELT-2 protein in the early endoderm although ELT-2 levels do not strictly correlate with rescue. We suggest that tasp-1 and pqn-82 represent a class of genes acting in the early embryo to modulate levels of critical transcription factors or to modulate the responsiveness of critical target genes. The screen’s design, rescuing lethality with an extrachromosomal transgene followed by counterselection, has a background survival rate of |
| Databáze: | OpenAIRE |
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