The Investigation of disease causing genes in C. elegans

线虫致病基因的研究

基本信息

批准号：
9356216
负责人：
Andy Golden
金额：
$ 62.25万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9356216
关键词：
Alleles Amino Acids Animal Model Animals BHLH Protein Binding Biological Assay Biological Process CRISPR/Cas technology Caenorhabditis elegans Cells Chotzen Syndrome Clinical DNA Binding Data Defecation Disease Dominant-Negative Mutation Drosophila genus Enhancers Enteral Gene Targeting Generations Genes Genetic Glutamic Acid Human Investigation Larva Lead Learning Modeling Molecular Muscle Mutate Mutation Nature Orthologous Gene Pathway interactions Phenotype Reporter Site Suppressor Mutations Syndrome System Tail Technology Testing Therapeutic Variant Work Zebrafish craniofacial disease phenotype disease-causing mutation egg gene function gene replacement therapy genetic analysis genome editing human disease insight interest loss of function mutation male meetings mutant promoter sex standard of care stem cell therapy therapeutic target tool

项目摘要

For human autosomal recessive diseases in which the responsible gene is known, we are using C. elegans to study the function of that gene and to genetically identify other factors that act in the same pathway. There are a number of criteria that must be met in order for this strategy to work. First, there must be a convincing and clear C. elegans ortholog. Second, there would have to be a mutation or deletion in this gene that already exists. Towards this end, we are using CRISPR technology to generate mutant alleles analogous to those found in human diseases. Third, there would have to be a scorable phenotype. The more penetrant the phenotype, the better. If these criteria are met, genetic suppressor and enhancer screens could be performed to identify interacting factors that function with any given gene and the biological process in which it functions. In the past year, we have identified a number of C. elegans orthologs of human disease-causing genes. We have determined that many of these candidates satisfy all of the above criteria- there are mutations in these genes and they reveal very penetrant and scorable phenotypes. We have initiated a project to model human craniofacial syndromes in C. elegans. We were approached by colleagues to determine whether mutations in the sole C. elegans orthologs of the Twist basic helix-loop-helix (bHLH) transcription factor results in distinct phenotypes in C. elegans. There are two Twist genes in humans, Twist1 and Twist2. Twist mutations have already been implicated in other craniofacial disorders such as Saethre-Chotzen Syndrome. Interesting, our clinical colleagues have recently shown that mutations in a conserved glutamic acid residue in the conserved DNA-binding basic domain of Twist1 and Twist2 are implicated in three other distinct craniofacial syndromes, all of which are autosomal dominant and hypothesized to result in dominant-negative variants of Twist. In each case, this conserved glutamic acid is altered to one of five other amino acid residues. Using CRISPR/Cas9 genome-editing technology, we have made the orthologous changes in this conserved glutamic acid in the C. elegans hlh-8 gene, the sole ortholog of the Twist genes in humans. We were able to screen for our mutations by PCR, restriction digests, and sequencing and were able to generate all of the desired mutations. Each of our mutations resulted in a very visible phenotype; homozygous animals were egg-laying defective (Egl). Interestingly, only some of the mutations resulted in a constipated (Con) phenotype and displayed a very deformed tail. We have characterized these strains quantitatively to determine how penetrant each of these phenotypes are. We have also crossed these strains with GFP reporter strains to look at the expression of known HLH-8 targets. We have also examined the expression of an hlh-8::gfp reporter to learn more about the M lineage in the developing larvae. The M lineage is responsible for the generation of the muscles that make up the egg-laying and defecation systems in C. elegans. Using this marker, we can determine when the M descendants divide and migrate. This marker also allows us to assay the generation of the sex muscles (SMs). With all these markers, we have been able to characterize the mutant phenotypes of these hlh-8 alleles at the cellular and molecular level and better group our alleles into distinct phenotypic classes. Interestingly, these mutants also display diverse male tail phenotypes, further allowing us to put these mutants into distinct classes. To date, our data suggest that the vulval muscles are most sensitive to each of these mutant alleles, while the enteric muscles responsible for defecation are more variable in their sensitivity. And each of these alleles, like the human diseases, is also semi-dominant in nature. We suspect that they are also acting in a dominant-negative fashion, interfering with the proper expression of target genes. We are eager to determine the molecular mechanism of these phenotypes: do these mutants bind promoters and inappropriately turn on or turn off genes that lead to these phenotypes? We also plan to test the weakest alleles in genetic suppressor screens to determine whether we can isolate suppressor mutants that reverse the mutant phenotypes and restore them to a more wild-type-like condition.

对于已知负责基因的人类常染色体隐性疾病，我们正在使用秀丽隐杆线虫研究该基因的功能，并在遗传上鉴定在同一途径中起作用的其他因素。为了使该策略起作用，必须满足许多标准。首先，必须有一个令人信服且清晰的秀丽隐杆线虫直系同源物。其次，该基因中必须存在突变或缺失。为此，我们正在使用CRISPR技术来产生类似于人类疾病的突变等位基因。第三，必须有一个值得注意的表型。表型的渗透越多，越好。如果满足这些标准，则可以执行遗传抑制和增强子筛选，以识别与任何给定基因作用的相互作用因子及其功能的生物学过程。在过去的一年中，我们已经确定了许多致病基因的秀丽隐杆线虫直系同源物。我们已经确定许多这些候选者都满足上述所有标准 - 这些基因中有突变，它们揭示了非常渗透和可记忆的表型。我们发起了一个项目，以对秀丽隐杆线虫中的人类颅面综合征进行建模。同事们与我们接触，以确定曲折基本螺旋 - 环螺旋（BHLH）转录因子的唯一秀丽隐杆线虫直系同源物中的突变是否导致秀丽隐杆线虫的明显表型。人类有两个扭曲基因，Twist1和Twist2。扭曲突变已经与其他颅面疾病有关，例如塞斯雷 - chotzen综合征。有趣的是，我们的临床同事最近表明，保守的DNA结合基本结构型Twist1和Twist2中保守的谷氨酸残基中的突变与其他三种不同的颅面综合征有关，所有这些都具有常染色体优势，并假设导致扭曲的主导变体。在每种情况下，这种保守的谷氨酸都会改变为其他五个氨基酸残基之一。使用CRISPR/CAS9基因组编辑技术，我们在秀丽隐杆线虫HLH-8基因中进行了这种保守的谷氨酸的直系同源，这是人类扭曲基因的唯一直系同源物。我们能够通过PCR，限制消化和测序来筛选我们的突变，并能够生成所有所需的突变。我们的每个突变导致表型非常明显。纯合的动物是卵子有缺陷（EGL）。有趣的是，只有某些突变导致了便秘的（con）表型，并显示出非常变形的尾巴。我们已经定量地表征了这些菌株，以确定每种表型的渗透率。我们还将这些菌株与GFP报告基因菌株越过这些菌株，以查看已知的HLH-8靶标的表达。我们还研究了HLH-8 :: GFP记者的表达，以了解有关发展幼虫中M谱系的更多信息。 M谱系负责构成秀丽隐杆线虫中卵形和排便系统的肌肉的产生。使用此标记，我们可以确定M后代何时分裂和迁移。该标记还使我们能够测定性肌肉的产生（SMS）。使用所有这些标记，我们能够表征这些HLH-8等位基因在细胞和分子水平上的突变表型，并更好地将等位基因分为不同的表型类别。有趣的是，这些突变体还显示出不同的男性尾巴表型，进一步使我们能够将这些突变体置于不同的类别。迄今为止，我们的数据表明，外阴肌肉对这些突变等位基因中的每个等位基因最敏感，而负责排便的肠肌的敏感性更具变化。这些等位基因（如人类疾病）本质上也是半主导的。我们怀疑它们还以主导的阴性方式起作用，干扰了靶基因的适当表达。我们渴望确定这些表型的分子机制：这些突变体是否结合启动子并不当打开或关闭导致这些表型的基因？我们还计划测试遗传抑制筛网中最弱的等位基因，以确定我们是否可以隔离抑制突变体，以逆转突变体表型并将其恢复到更野生型的状态。