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Differential effects of Cdc68 on cell cycle-regulated promoters in Saccharomyces cerevisiae
D Lycan1, G Mikesell, M Bunger
1Lewis and Clark College, Portland, Oregon 97219.
Abstract:
Swi4 and Swi6 form a complex which is required for Start-dependent activation of HO and for high-level expression of G1 cyclin genes CLN1 and CLN2. To identify other regulators of this pathway, we screened for dominant, recessive, conditional, and allele-specific suppressors of swi4 mutants. We isolated 16 recessive suppressors that define three genes, SSF1, SSF5, and SSF9 (suppressor of swi four). Mutations in all three genes bypass the requirement for both Swi4 and Swi6 for HO transcription and activate transcription from reporter genes lacking upstream activating sequences (UASs). SSF5 is allelic with SIN4 (TSF3), a gene implicated in global repression of transcription and chromatin structure, and SSF9 is likely to be a new global repressor of transcription. SSF1 is allelic with CDC68 (SPT16). cdc68 mutations have been shown to increase expression from defective promoters, while preventing transcription from other intact promoters, including CLN1 and CLN2. We find that CDC68 is a required activator of both SWI4 and SWI6, suggesting that CDC68's role at the CLN promoters may be indirect. The target of CDC68 within the SWI4 promoter is complex in that known activating elements (MluI cell cycle boxes) in the SWI4 promoter are required for CDC68 dependence but only within the context of the full-length promoter. This result suggests that there may be both a chromatin structure and a UAS-specific component to Cdc68 function at SWI4. We suggest that Cdc68 functions both in the assembly of repressive complexes that form on many intact promoters in vivo and in the relief of this repression during gene activation.
Insights
Researchers identified new genes (SSF1, SSF5, SSF9) that regulate the cell cycle by suppressing swi4 mutations. These genes bypass the need for Swi4 and Swi6, impacting HO and CLN gene expression, with CDC68 playing a key activator role.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Cell Cycle Regulation
Background:
- The Swi4-Swi6 complex is crucial for initiating cell cycle progression by activating HO and CLN1/CLN2 genes.
- Understanding regulators of this pathway is key to deciphering cell cycle control mechanisms.
Purpose of the Study:
- To identify novel genes that regulate the Swi4-Swi6 pathway.
- To elucidate the function of these novel regulators in transcription and cell cycle control.
Main Methods:
- Genetic screening for suppressors of swi4 mutants.
- Gene mapping and allelism tests.
- Reporter gene assays to assess transcriptional activation.
- Analysis of gene interactions and promoter elements.
Main Results:
- Identified three recessive suppressor genes: SSF1, SSF5, and SSF9.
- Mutations in SSF1, SSF5, and SSF9 bypass the requirement for Swi4/Swi6 in HO transcription and activate UAS-lacking reporters.
- SSF5 is allelic to SIN4 (global transcription repressor), and SSF9 is a potential new global repressor.
- SSF1 is allelic to CDC68 (SPT16), a known regulator of transcription and chromatin.
- CDC68 acts as a required activator for both SWI4 and SWI6, suggesting an indirect role at CLN promoters.
- CDC68's function at the SWI4 promoter involves both chromatin structure and UAS-dependent elements.
Conclusions:
- CDC68 is a critical activator upstream of Swi4 and Swi6, influencing cell cycle gene expression.
- Cdc68 likely functions in relieving transcriptional repression at promoters, involving both chromatin remodeling and specific DNA elements.
- The identified SSF genes offer new insights into the complex regulatory network controlling yeast cell cycle initiation.