Selection of a dominant negative retinoblastoma protein (RB) inhibiting satellite myoblast differentiation implies an indirect interaction between MyoD and RB

Citation
Fq. Li et al., Selection of a dominant negative retinoblastoma protein (RB) inhibiting satellite myoblast differentiation implies an indirect interaction between MyoD and RB, MOL CELL B, 20(14), 2000, pp. 5129-5139
Citations number
36
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Molecular Biology & Genetics
Journal title
MOLECULAR AND CELLULAR BIOLOGY
ISSN journal
0270-7306 → ACNP
Volume
20
Issue
14
Year of publication
2000
Pages
5129 - 5139
Database
ISI
SICI code
0270-7306(200007)20:14<5129:SOADNR>2.0.ZU;2-R
Abstract
Satellite myoblasts serve as stem cells in postnatal skeletal muscle, but t he genes responsible for choosing between growth versus differentiation are largely undefined. We have used a novel genetic approach to identify genes encoding proteins whose dominant negative inhibition is capable of interru pting the in vitro differentiation of C2C12 murine satellite myoblasts. The screen is based on fusion of a library of cDNA fragments with the lysosoma l protease cathepsin B (CB), such that the fusion protein intracellularly d iverts interacting factors to the lysosome. Among other gene fragments sele cted in this screen, including those of known and novel sequence, is the re tinoblastoma protein (RB) pocket domain. This unique dominant negative form of RB allows us to genetically determine if MyoD and RB associate in vivo. The dominant negative CB-RB fusion produces a cellular phenotype indisting uishable from recessive loss of function RB mutations. The fact that the do minant negative RB inhibits myogenic differentiation in the presence of non limiting concentrations of either RB or MyoD suggests that these two protei ns do not directly interact. We further show that the dominant negative RB inhibits E2F1 but cannot inhibit a forced E2F1-RB dimer. Therefore, E2F1 is a potential mediator of the dominant negative inhibition of MyoD by CB-RB during satellite cell differentiation. We propose this approach to be gener ally suited to the investigation of gene function, even when little is know n about the pathway being studied.