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A gene-trap strategy identifies quiescence-induced genes in synchronized myoblasts

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Abstract

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 genetrap lines revealed arrest-dependent induction of βgal 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-interactin proteinm, 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.

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Abbreviations

gBgal:

gBgalactosidase

CFU:

colony forming units

ENT:

EMSY N-terminus

FACS:

fluorescent activated cell sorting

FDG:

fluorescein di-β-D-galactopyranoside

LTR:

long terminal repeat

MLL5 :

mixed lineage leukaemia gene 5

MOI:

multiplicity of infection

MRF:

myogenic regulatory factor

PETG:

phenyl ethyl thiogalactoside

PHD:

plant homoeo domain

PR:

polymerase chain reaction

RNAi:

RNA interference

RT:

reverse transcriptase

SA:

splice acceptor

SET:

conserved in Su(var), Enhancer of Zeste, Trithorax

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Author notes

  1. Ramkumar Sambasivan

    Present address: Department of Developmental Biology, Pasteur Institute, 75724, Cedex 15 Paris, France

Authors and Affiliations

  1. Centre for Cellular and Molecular Biology, Hyderabad, 500 007, India

    Ramkumar Sambasivan & Jyotsna Dhawan

  2. Department of Pharmacology, Emory University, Atlanta, GA, 30322, USA

    Grace K. Pavlath

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  1. Ramkumar Sambasivan
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  2. Grace K. Pavlath
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  3. Jyotsna Dhawan
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Correspondence to Jyotsna Dhawan.

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Sambasivan, R., Pavlath, G.K. & Dhawan, J. A gene-trap strategy identifies quiescence-induced genes in synchronized myoblasts. J Biosci 33, 27–44 (2008). https://doi.org/10.1007/s12038-008-0019-6

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  • DOI: https://doi.org/10.1007/s12038-008-0019-6

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