Wiesenfahrt Tobias, Duanmu Jingjie, Snider Frances, Moerman Don, Au Vinci, Li-Leger Erica, Flibotte Stephane, Parker Dylan M, Marshall Craig J, Nishimura Erin Osborne, Mains Paul E, McGhee James D
Department of Biochemistry and Molecular Biology, Alberta Children's Hospital Research Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1.
Department of Zoology and Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4.
G3 (Bethesda). 2018 May 4;8(5):1425-1437. doi: 10.1534/g3.118.200079.
The ELT-2 GATA factor normally functions in differentiation of the 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 :: 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 :: cDNA transgene to rescue the lethality of the double mutant. We performed this screen on a strain that has a single copy insertion of the transgene in an 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 gene was mutated in four independent strains. encodes the 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, , was mutated in two independent strains and encodes a glutamine-asparagine rich protein. and were verified as loss-of-function modifiers of the :: 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 and 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 <10 without mutagenesis and should be readily adapted to the general problem of identifying suppressors of lethal mutations.
ELT-2 GATA因子通常在内胚层特化下游的内胚层分化过程中发挥作用。我们之前已经表明，如果ELT-2表达足够早，它也能够特化内胚层并取代核心GATA因子转录级联反应的所有其他成员（END-1、END-3、ELT-7）。然而，这种拯救需要:: cDNA转基因的多个拷贝（大概是过表达）；转基因的单拷贝不能拯救。我们以这一观察结果为基础进行了遗传筛选，以寻找能够使:: cDNA转基因的单拷贝拯救双突变体致死性的遗传修饰因子。我们在一个在背景中有转基因单拷贝插入的菌株上进行了这个筛选。这些动物通过一个含有多个拯救转基因拷贝的游离型阵列而存活；该游离型阵列还含有一个在热休克控制下的毒素，用于对能够丢失拯救阵列的诱变存活者进行反选择。对约14,000个诱变单倍体基因组的筛选产生了17个独立的存活菌株。进行了全基因组测序以鉴定在多个存活菌株中发生独立突变的基因。基因在四个独立菌株中发生了突变。编码Taspase的同源物，Taspase是一种苏氨酸 - 天冬氨酸蛋白酶，在哺乳动物和昆虫中都已发现它能切割几种参与转录的蛋白质，特别是MLL1/三体同源盒蛋白和TFIIA。第二个基因在两个独立菌株中发生了突变，编码一种富含谷氨酰胺 - 天冬酰胺的蛋白质。通过RNA干扰和CRISPR/Cas9诱导的突变，验证了和是::转基因的功能丧失型修饰因子。在这两种情况下，基因缺失导致早期内胚层中ELT-2蛋白水平适度增加，尽管ELT-2水平与拯救并不严格相关。我们认为和代表了一类在早期胚胎中起作用的基因，可调节关键转录因子的水平或调节关键靶基因的反应性。该筛选的设计，即先用游离型转基因拯救致死性，然后进行反选择，在没有诱变的情况下背景存活率<10%，并且应该很容易适用于鉴定致死突变抑制因子的一般问题。
