title: Forward genetic screening for regulators involved in cholesterol synthesis using validation-based insertional mutagenesis.
abstract: Somatic cell genetics is a powerful approach for unraveling the regulatory mechanism of cholesterol metabolism. However, it is difficult to identify the mutant gene(s) due to cells are usually mutagenized chemically or physically. To identify important genes controlling cholesterol biosynthesis, an unbiased forward genetics approach named validation-based insertional mutagenesis (VBIM) system was used to isolate and characterize the 25-hydroxycholesterol (25-HC)-resistant and SR-12813-resistant mutants. Here we report that five mutant cell lines were isolated. Among which, four sterol-resistant mutants either contain a truncated NH2-terminal domain of sterol regulatory element-binding protein (SREBP)-2 terminating at amino acids (aa) 400, or harbor an overexpressed SREBP cleavage-activating protein (SCAP). Besides, one SR-12813 resistant mutant was identified to contain a truncated COOH-terminal catalytic domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase). This study demonstrates that the VBIM system can be a powerful tool to screen novel regulatory genes in cholesterol biosynthesis.
authors.affiliation: Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Jing-Jie
authors.initials: JJ
authors.lastname: Tang
authors.affiliation: Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Hong-Hua
authors.initials: HH
authors.lastname: Miao
authors.affiliation: Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Yu-Xiu
authors.initials: YX
authors.lastname: Qu
authors.affiliation: Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Qin
authors.affiliation: Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Xu
authors.affiliation: School of Life Sciences, Lanzhou University, Lanzhou, China.
authors.forename: Jinbo
authors.initials: J
authors.lastname: Yang
authors.affiliation: Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Bo-Liang
authors.initials: BL
authors.lastname: Li
authors.affiliation: Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China; College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Bao-Liang
authors.initials: BL
authors.lastname: Song
mesh_terms: D000818:Animals; D016466:CHO Cells; D002784:Cholesterol / Q000096:biosynthesis*; D003412:Cricetulus; D004164:Diphosphonates / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D005820:Genetic Testing / Q000379:methods; D005822:Genetic Vectors; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D006888:Hydroxycholesterols / Q000494:pharmacology; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016254:Mutagenesis, Insertional / Q000379:methods*; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000187:drug effects; D051782:Sterol Regulatory Element Binding Protein 2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D004164:Diphosphonates; D006888:Hydroxycholesterols; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C100355:SR 12813; C102196:SREBP cleavage-activating protein; D051782:Sterol Regulatory Element Binding Protein 2; C007997:25-hydroxycholesterol; D002784:Cholesterol; D006903:Hydroxymethylglutaryl CoA Reductases