| Popis: |
The G1/S transition governs cell proliferation in many systems, and involves essential transcription complexes such as SBF and MBF in Saccharomyces cerevisiae, or MBF in Schizosaccharomyces pombe. SBF and MBF are composed of the regulatory subunit Swi6p and the DNA-binding elements Swi4p (SBF) or Mbp1p (MBF). The fungal pathogen Candida albicans contains orthologues of SWI6, SWI4 and MBP1. Previous genetic, DNA expression and bioinformatic data suggest that Swi4p and Swi6p may be core components of a single MBF-like complex. However, direct evidence is lacking, and the role of Mbp1p is unclear. In order to determine the composition and mechanisms of action of the putative G1/S transcription complex in C. albicans, and identify other factors important for G1/S control, we determined physical interactions between Swi6p, Swi4p and Mbp1p using co-immunoprecipitation, systematically affinity-purified each protein and identified interacting factors through mass spectrometry, and investigated putative Swi4p targets using genome wide location analysis (ChIP-chip). We show that Swi6p physically interacts with Swi4p and Mbp1p, and Swi4p may bind Mbp1p. Affinity purifications did not identify many additional interacting proteins, suggesting that Swi6p, Swi4p and Mbp1p may be the core complex factors. Finally, ChIP-chip analysis identified putative Swi4p targets including G1 cyclins, cell wall-associated factors, and regulators of hyphal growth, consistent with the swi4/ phenotype. Unexpectedly, few putative Swi4p target promoters contained the conserved MBF binding site (ACGCGT). Rather, 8.6% contained a canonical SBF-binding (CNCGAAA) motif, and 43.0% contained a related motif, CACAAAA. Although the ChIP-chip data require confirmation by qPCR, the results suggest that Swi4p may be involved in regulating G1/S progression and hyphal development, but may not exclusively function through the conserved MBF-binding element. In summary, our results provide new insights on G1/S regulation in C. albicans, which have important implications for controlling cell proliferation and development in the pathogen. |
