A Strategy To Isolate Modifiers of Caenorhabditis elegans Lethal Mutations: Investigating the Endoderm Specifying Ability of the Intestinal Differentiation GATA Factor ELT-2.
A Strategy To Isolate Modifiers of Caenorhabditis elegans Lethal Mutations: Investigating the Endoderm Specifying Ability of the Intestinal Differentiation GATA Factor ELT-2.
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DOI：
10.1534/g3.118.200079
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发表时间：
2018-05-04
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影响因子：
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通讯作者：
McGhee JD
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作者：
Wiesenfahrt T;Duanmu J;Snider F;Moerman D;Au V;Li-Leger E;Flibotte S;Parker DM;Marshall CJ;Nishimura EO;Mains PE;McGhee JD
ELT-2加塔因子在C.秀丽隐杆线虫内胚层，内胚层规格的下游。我们以前已经表明，如果ELT-2表达足够早，它也能够指定内胚层，并取代核心GATA因子转录级联的所有其他成员（END-1，END-3，ELT-7）。然而，这样的拯救需要end-1 p：：elt-2 cDNA转基因的多个拷贝（和可能的过表达）;转基因的单个拷贝不能拯救。我们已经将这一观察结果作为遗传筛选的基础，以寻找遗传修饰剂，该遗传修饰剂允许end-1 p：：elt-2 cDNA转基因的单拷贝挽救end-1 end-3双突变体的致死性。我们在一个菌株上进行了筛选，该菌株在末端-1末端-3背景中具有转基因的单拷贝插入。这些动物通过含有多拷贝拯救转基因的染色体外阵列保持存活;染色体外阵列还含有热休克控制下的毒素，以反选择已经能够失去拯救阵列的诱变幸存者。筛选14,000个诱变的单倍体基因组产生了17个独立的存活菌株。进行全基因组测序以鉴定在多于一种存活菌株中发生独立突变的基因。梭在四个独立的菌株中，线虫基因tasp-1发生突变。tasp-1编码C. Taspase是一种苏氨酸-天冬氨酸蛋白酶，在哺乳动物和昆虫中都发现它能切割几种参与转录的蛋白质，特别是MLL 1/三胸和TFIIA。第二个基因pqn-82在两个独立的菌株中突变，编码富含谷氨酰胺-天冬酰胺的蛋白质。通过RNAi和通过CRISPR/Cas9诱导的突变，tasp-1和pqn-82被验证为end-1 p：：elt-2转基因的功能丧失修饰剂。在这两种情况下，基因丢失导致早期内胚层中ELT-2蛋白水平的适度增加，尽管ELT-2水平与拯救并不严格相关。我们认为tasp-1和pqn-82代表了一类在早期胚胎中起作用的基因，以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，用染色体外转基因挽救致死性，然后进行反选择，在没有诱变的情况下，背景存活率<10−4，应该很容易适应识别C的抑制子的一般问题。elegans致命突变。
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-1end-3 double mutant. We performed this screen on a strain that has a single copy insertion of the transgene in an end-1end-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 <10−4 without mutagenesis and should be readily adapted to the general problem of identifying suppressors of C. elegans lethal mutations.