Regulation of the Chlamydomonas cell cycle by a stable, chromatin-associated retinoblastoma tumor suppressor complex.
We examined the cell cycle dynamics of the retinoblastoma (RB) protein complex in the unicellular alga Chlamydomonas reinhardtii that has single homologs for each subunit-RB, E2F, and DP. We found that Chlamydomonas RB (encoded by MAT3) is a cell cycle-regulated phosphoprotein, that E2F1-DP1 can bind to a consensus E2F site, and that all three proteins interact in vivo to form a complex that can be quantitatively immunopurified. Yeast two-hybrid assays revealed the formation of a ternary complex between MAT3, DP1, and E2F1 that requires a C-terminal motif in E2F1 analogous to the RB binding domain of plant and animal E2Fs. We examined the abundance of MAT3/RB and E2F1-DP1 in highly synchronous cultures and found that they are synthesized and remain stably associated throughout the cell cycle with no detectable fraction of free E2F1-DP1. Consistent with their stable association, MAT3/RB and DP1 are constitutively nuclear, and MAT3/RB does not require DP1-E2F1 for nuclear localization. In the nucleus, MAT3/RB remains bound to chromatin throughout the cell cycle, and its chromatin binding is mediated through E2F1-DP1. Together, our data show that E2F-DP complexes can regulate the cell cycle without dissociation of their RB-related subunit and that other changes may be sufficient to convert RB-E2F-DP from a cell cycle repressor to an activator.
Plant Molecular and Cellular Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
Bradley J S C
BJ
Olson
Michael
M
Oberholzer
Yubing
Y
Li
James M
JM
Zones
Harjivan S
HS
Kohli
Katerina
K
Bisova
Su-Chiung
SC
Fang
Jill
J
Meisenhelder
Tony
T
Hunter
James G
JG
Umen
D002453:Cell Cycle; D002696:Chlamydomonas / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism*; D050684:E2F Transcription Factors / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D010940:Plant Proteins / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D050680:Transcription Factor DP1 / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D002843:Chromatin; D050684:E2F Transcription Factors; D010750:Phosphoproteins; D010940:Plant Proteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D016160:Retinoblastoma Protein; D050680:Transcription Factor DP1; C432865:mat3 protein, Chlamydomonas