Finding targets of the retinoblastoma tumor suppressor pathway in Chlamydomonas reinhardtii
J. Matt Zones1, Su-Chiung Fang2, Brad Olson1, and James G. Umen1
1) Plant Biology Laboratory, Salk Institute, La Jolla 92037, USA
2) Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan
Size homeostasis is important to all proliferating cells and is thought to be mediated by checkpoints that couple growth and division. Size checkpoints are essential for regulating the multiple fission cell cycle of the unicellular alga Chlamydomonas reinhardtii in which a prolonged G1 phase is followed by a series of alternating S and M phases to produce daughter cells of uniform size. Deletion of the gene encoding the Chlamydomonas RB homolog MAT3 causes loss of cell size checkpoint control and excessive division resulting in tiny daughter cells. Mutations in DP1 and E2F1 that encode orthologs of the E2F and DP transcription factors suppressed the mat3 small cell mutant phenotype and caused a large-cell phenotype. We hypothesized that expression defects of E2F/DP target genes were the underlying cause of the cell size and cell cycle phenotypes that were observed in mat3 and dp1 or e2f1 mutant strains. However, we were unable to identify any cell cycle genes that were mis-expressed in the mutant strains, including some highly conserved targets of E2F/DP. Moreover, inhibition of transcription using the drug alpha-amanitin reduced cell cycle gene transcription but did not affect cell size control. Taken together these findings suggested that either (i) we had not identified the correct target genes or (ii), the RB pathway can regulate the cell cycle independently of transcription. Different approaches are used to gain further insights into the molecular defects of Chlamydomonas RB pathway mutants. Oligonucleotide-based microarrays are employed to assess differences in global gene expression between wild-type, mat3, and dp1 strains. Additionally, we have purified and reconstituted recombinant E2F1-DP1 heterodimers that will be used to identify a consensus binding site.
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