A gene-trap strategy identifies quiescence-induced genes in synchronized myoblasts
A gene-trap strategy identifies quiescence-induced genes in synchronized myoblasts
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DOI：
10.1007/s12038-008-0019-6
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发表时间：
2008-03-01
影响因子：
2.9
通讯作者：
Dhawan, Jyotsna
中科院分区：
文献类型：
--
作者：
Sambasivan, Ramkumar;Pavlath, Grace K.;Dhawan, Jyotsna
细胞静止的特征不仅在于减少有丝分裂和代谢活动，而且还在于改变基因表达。越来越多的证据表明，静止不仅仅是一种基础状态，而是受主动机制的调节。为了了解控制可逆细胞周期退出的分子程序，我们专注于肌源性细胞停滞和激活的培养模型中的静止相关基因表达。在这里，我们报告的沉默诱导基因的识别使用基因陷阱策略。使用逆转录病毒载体，我们在C2 C12成肌细胞中产生了一个基因陷阱库，通过活细胞分选（FACS-gal）筛选了该基因陷阱。几个独立的基因诱捕系揭示了β gal活性的抑制依赖性诱导，证实了FACS筛选的功效。在15个株系中鉴定了整合位点。在三个细胞系中，插入发生在先前参与静止控制的基因中，即EMSY -BRCA 2相互作用蛋白，p8/com 1-p300 HAT结合蛋白和MLL 5-SET结构域蛋白。我们的研究结果表明，染色质调节基因的表达诱导在G 0，提供支持的概念，这种可逆的逮捕状态是积极调节。
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 beta gal 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.