Journal of Biosciences

, Volume 33, Issue 1, pp 27–44

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

Authors

  • Ramkumar Sambasivan
    • Centre for Cellular and Molecular Biology
  • Grace K. Pavlath
    • Department of PharmacologyEmory University
    • Centre for Cellular and Molecular Biology
Article

DOI: 10.1007/s12038-008-0019-6

Cite this article as:
Sambasivan, R., Pavlath, G.K. & Dhawan, J. J Biosci (2008) 33: 27. doi:10.1007/s12038-008-0019-6

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.

Keywords

Gene-trap MLL5 myoblast p8 quiescence

Abbreviations used

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

Copyright information

© Indian Academy of Sciences 2008