RESUMEN
Parkinson's disease (PD) is a neurodegenerative disorder caused by complex genetic and environmental factors. Genome-edited human pluripotent stem cells (hPSCs) offer the uniique potential to advance our understanding of PD etiology by providing disease-relevant cell-types carrying patient mutations along with isogenic control cells. To facilitate this experimental approach, we generated a collection of 55 cell lines genetically engineered to harbor mutations in genes associated with monogenic PD (SNCA A53T, SNCA A30P, PRKN Ex3del, PINK1 Q129X, DJ1/PARK7 Ex1-5del, LRRK2 G2019S, ATP13A2 FS, FBXO7 R498X/FS, DNAJC6 c.801 A>G+FS, SYNJ1 R258Q/FS, VPS13C A444P, VPS13C W395C, GBA1 IVS2+1). All mutations were generated in a fully characterized and sequenced female human embryonic stem cell (hESC) line (WIBR3; NIH approval number NIHhESC-10-0079) using CRISPR/Cas9 or prime editing-based approaches. We implemented rigorous quality controls, including high density genotyping to detect structural variants and confirm the genomic integrity of each cell line. This systematic approach ensures the high quality of our stem cell collection, highlights differences between conventional CRISPR/Cas9 and prime editing and provides a roadmap for how to generate gene-edited hPSCs collections at scale in an academic setting. We expect that our isogenic stem cell collection will become an accessible platform for the study of PD, which can be used by investigators to understand the molecular pathophysiology of PD in a human cellular setting.
RESUMEN
The MEK and extracellular signal-regulated kinase/mitogen-activated protein kinase proteins are established regulators of multicellular development and cell movement. By combining traditional genetic and biochemical assays with a statistical analysis of global gene expression profiles, we discerned a genetic interaction between Dictyostelium discoideum mek1, smkA (named for its role in the suppression of the mek1(-) mutation), and pppC (the protein phosphatase 4 catalytic subunit gene). We found that during development and chemotaxis, both mek1 and smkA regulate pppC function. In other organisms, the protein phosphatase 4 catalytic subunit, PP4C, functions in a complex with the regulatory subunits PP4R2 and PP4R3 to control recovery from DNA damage. Here, we show that catalytically active PP4C is also required for development, chemotaxis, and the expression of numerous genes. The product of smkA (SMEK) functions as the Dictyostelium PP4R3 homolog and positively regulates a subset of PP4C's functions: PP4C-mediated developmental progression, chemotaxis, and the expression of genes specifically involved in cell stress responses and cell movement. We also demonstrate that SMEK does not control the absolute level of PP4C activity and suggest that SMEK regulates PP4C by controlling its localization to the nucleus. These data define a novel genetic pathway in which mek1 functions upstream of pppC-smkA to control multicellular development and chemotaxis.
Asunto(s)
Quimiotaxis/fisiología , Dictyostelium/crecimiento & desarrollo , Dictyostelium/metabolismo , MAP Quinasa Quinasa 1/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Dictyostelium/citología , Dictyostelium/genética , Epistasis Genética , Perfilación de la Expresión Génica , MAP Quinasa Quinasa 1/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfoproteínas Fosfatasas/genética , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Protozoarias/genética , Transducción de Señal/fisiologíaRESUMEN
MOTIVATION: Coordinate regulation of gene expression can provide information on gene function. To begin a large-scale analysis of Dictyostelium gene function, we clustered genes based on their expression in wild-type and mutant strains and analyzed their functions. RESULTS: We found 17 modes of wild-type gene expression and refined them into 57 submodes considering mutant data. Annotation analyses revealed correlations between co-expression and function and an unexpected correlation between expression and function of genes involved in various aspects of chemotaxis. Co-regulation of chemotaxis genes was also found in published data from neutrophils. To test the predictive power of the analysis, we examined the phenotypes of mutations in seven co-regulated genes that had no published role in chemotaxis. Six mutants exhibited chemotaxis defects, supporting the idea that function can be inferred from co-expression. The clustering and annotation analyses provide a public resource for Dictyostelium functional genomics.
Asunto(s)
Quimiotaxis/genética , Dictyostelium/genética , Genes Protozoarios , Animales , Quimiotaxis/efectos de los fármacos , Análisis por Conglomerados , Biología Computacional , AMP Cíclico/farmacología , Citoesqueleto/genética , Dictyostelium/efectos de los fármacos , Dictyostelium/fisiología , Perfilación de la Expresión Génica/estadística & datos numéricos , Regulación de la Expresión Génica , Genómica/estadística & datos numéricos , Análisis de Secuencia por Matrices de Oligonucleótidos/estadística & datos numéricos , Fenotipo , Regulón , Ribosomas/genética , Transducción de SeñalRESUMEN
Classical epistasis analysis can determine the order of function of genes in pathways using morphological, biochemical and other phenotypes. It requires knowledge of the pathway's phenotypic output and a variety of experimental expertise and so is unsuitable for genome-scale analysis. Here we used microarray profiles of mutants as phenotypes for epistasis analysis. Considering genes that regulate activity of protein kinase A in Dictyostelium, we identified known and unknown epistatic relationships and reconstructed a genetic network with microarray phenotypes alone. This work shows that microarray data can provide a uniform, quantitative tool for large-scale genetic network analysis.
