A functional genome-wide genetic screening identifies new pathways controlling the G1/S transcriptional wave

Abstract

The Schizosaccharomyces pombe MBF complex activates the transcription of genes required for DNA synthesis and S phase. The MBF complex contains several proteins, including the core components Cdc10, Res1 and Res2, the co-repressor proteins Yox1 and Nrm1 and the co-activator Rep2. It has recently been shown how MBF is regulated when either the DNA damage or the DNA synthesis checkpoints are activated. However, how MBF is regulated in a normal unperturbed cell cycle is still not well understood. We have set up a genome-wide genomic screen searching for global regulators of MBF. We have crossed our knock-out collection library with a reporter strain that allows the measurement of MBF activity in live cells by flow cytometry. We confirm previously known regulators of MBF and show that COP9/ signalosome and tRNA methyltransferases also regulate MBF activity.

Genetic-interaction screens uncover novel biological roles and regulators of transcription factors in fission yeast

“…There were 7 previously characterized interaction pairs generated in the screens and 4 that we captured as SDL interactions: scr1 + overexpression with Δ sds23 , as well as yox1 + overexpression with Δ cds1 , Δ cdt2 , and Δ gad8 ( Gómez-Escoda et al 2011 ; Stewart et al 2011 ; Matsuzawa et al 2012 ; Saitoh et al 2015 ; Cohen et al 2016 ; Gaspa et al 2016 ). Both Sds23 and Cds1 are kinases that directly negatively regulate their respective transcription factors.…”

“…No interaction was detected between scr1 + overexpression and the loss of another previously confirmed upstream regulator of Scr1, ssp2 + , despite it having a similar regulatory role on Scr1 nuclear localization as observed in the Δsds23 strain ( Matsuzawa et al 2012 ). The deletion of cdt2 + or gad8 + has been shown to have a role in the regulation of the MBF complex and Yox1 through indirect and less well-characterized methods ( Cohen et al 2016 ; Gaspa et al 2016 ). However, the Cul4-RING E3 complex subunit Ddb1, from the same complex as Cdt2, did not have a synthetic lethal interaction with yox1 + overexpression when the gene was deleted ( Gaspa et al 2016 ).…”

“…The deletion of cdt2 + or gad8 + has been shown to have a role in the regulation of the MBF complex and Yox1 through indirect and less well-characterized methods ( Cohen et al 2016 ; Gaspa et al 2016 ). However, the Cul4-RING E3 complex subunit Ddb1, from the same complex as Cdt2, did not have a synthetic lethal interaction with yox1 + overexpression when the gene was deleted ( Gaspa et al 2016 ). Finally, the activation of the SREBP transcription factor Sre2 by the ubiquitin ligase Dsc1 was not captured by the screen because it is not a SDL interaction and, therefore, not expected to be detected in this screen ( Stewart et al 2011 ).…”

Nutritional cell cycle reprogramming reveals that inhibition of Cdk1 is required for proper MBF-dependent transcription

“…It has been reported that nrm1Δ cells exhibit upregulated MBF activity (Gaspa et al, 2016;Caetano et al, 2014). We constructed the triple mutant nrm1Δ rum1Δ ste9Δ to test whether upregulation of MBF by deleting nrm1 + could rescue the DNA damage phenotype of rum1Δ ste9Δ mutant cells.…”

“…As a control, we determined the mRNA levels of cdc18 + and cdc22 + in cells lacking Rep2 (rep2Δ), a positive regulator of MBF that shows reduced MBFdependent transcription (Fig. 7A, right panel) (Baum et al, 1997;Gaspa et al, 2016;Tournier and Millar, 2000).…”