A gene-trap strategy identifies quiescence-induced genes in synchronized myoblasts.
Cellular quiescence is characterized not only by reduced mitotic and metabolic activity but also by altered gene expression. Growing evidence suggests that quiescence is not merely a basal state but is regulated by active mechanisms. To understand the molecular programme that governs reversible cell cycle exit, we focused on quiescence-related gene expression in a culture model of myogenic cell arrest and activation. Here we report the identification of quiescence-induced genes using a gene-trap strategy. Using a retroviral vector, we generated a library of gene traps in C2C12 myoblasts that were screened for arrest-induced insertions by live cell sorting (FACS-gal). Several independent gene- trap lines revealed arrest-dependent induction of betagal activity, confirming the efficacy of the FACS screen. The locus of integration was identified in 15 lines. In three lines,insertion occurred in genes previously implicated in the control of quiescence, i.e. EMSY - a BRCA2--interacting protein, p8/com1 - a p300HAT -- binding protein and MLL5 - a SET domain protein. Our results demonstrate that expression of chromatin modulatory genes is induced in G0, providing support to the notion that this reversibly arrested state is actively regulated.
Sambasivan
Ramkumar
R
Centre for Cellular and Molecular Biology, Hyderabad, India.
Pavlath
Grace K
GK
Dhawan
Jyotsna
J
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D018929:Cell Culture Techniques; D002453:Cell Cycle; D002454:Cell Differentiation / Q000235:genetics; D049109:Cell Proliferation; D002478:Cells, Cultured; D003470:Culture Media; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000235:genetics; D015723:Gene Library; D005796:Genes / Q000502:physiology*; D005814:Genes, Viral; D005822:Genetic Vectors; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D016254:Mutagenesis, Insertional; D051788:Myeloid-Lymphoid Leukemia Protein / Q000235:genetics / Q000378:metabolism; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic; D001616:beta-Galactosidase / Q000032:analysis / Q000378:metabolism
D003470:Culture Media; D004268:DNA-Binding Proteins; C480344:EMSY protein, human; C530774:EMSY protein, mouse; C490632:Macf1 protein, mouse; D008840:Microfilament Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; C491644:Nupr1 protein, mouse; D012097:Repressor Proteins; D051788:Myeloid-Lymphoid Leukemia Protein; D001616:beta-Galactosidase