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1. The microprotein Nrs1 rewires the G1/S transcriptional machinery during nitrogen limitation in budding yeast

   https://doi.org/10.1371/journal.pbio.3001548  

   Tollis, Sylvain; Singh, Jaspal; Palou, Roger; Thattikota, Yogitha; Ghazal, Ghada; Coulombe-Huntington, Jasmin; Tang, Xiaojing; Moore, Susan; Blake, Deborah; Bonneil, Eric; et al (March 2022, PLOS Biology)

   Martin, Sophie G. (Ed.)

   Commitment to cell division at the end of G1 phase, termed Start in the budding yeast Saccharomyces cerevisiae , is strongly influenced by nutrient availability. To identify new dominant activators of Start that might operate under different nutrient conditions, we screened a genome-wide ORF overexpression library for genes that bypass a Start arrest caused by absence of the G1 cyclin Cln3 and the transcriptional activator Bck2. We recovered a hypothetical gene YLR053c , renamed NRS1 for Nitrogen-Responsive Start regulator 1, which encodes a poorly characterized 108 amino acid microprotein. Endogenous Nrs1 was nuclear-localized, restricted to poor nitrogen conditions, induced upon TORC1 inhibition, and cell cycle-regulated with a peak at Start. NRS1 interacted genetically with SWI4 and SWI6 , which encode subunits of the main G1/S transcription factor complex SBF. Correspondingly, Nrs1 physically interacted with Swi4 and Swi6 and was localized to G1/S promoter DNA. Nrs1 exhibited inherent transactivation activity, and fusion of Nrs1 to the SBF inhibitor Whi5 was sufficient to suppress other Start defects. Nrs1 appears to be a recently evolved microprotein that rewires the G1/S transcriptional machinery under poor nitrogen conditions. 

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2. The consequences of differential origin licensing dynamics in distinct chromatin environments

   https://doi.org/10.1093/nar/gkac003  

   Mei, Liu; Kedziora, Katarzyna_M; Song, Eun-Ah; Purvis, Jeremy_E; Cook, Jeanette Gowen (January 2022, Nucleic Acids Research)

   Abstract Eukaryotic chromosomes contain regions of varying accessibility, yet DNA replication factors must access all regions. The first replication step is loading MCM complexes to license replication origins during the G1 cell cycle phase. It is not yet known how mammalian MCM complexes are adequately distributed to both accessible euchromatin regions and less accessible heterochromatin regions. To address this question, we combined time-lapse live-cell imaging with immunofluorescence imaging of single human cells to quantify the relative rates of MCM loading in euchromatin and heterochromatin throughout G1. We report here that MCM loading in euchromatin is faster than that in heterochromatin in early G1, but surprisingly, heterochromatin loading accelerates relative to euchromatin loading in middle and late G1. This differential acceleration allows both chromatin types to begin S phase with similar concentrations of loaded MCM. The different loading dynamics require ORCA-dependent differences in origin recognition complex distribution. A consequence of heterochromatin licensing dynamics is that cells experiencing a truncated G1 phase from premature cyclin E expression enter S phase with underlicensed heterochromatin, and DNA damage accumulates preferentially in heterochromatin in the subsequent S/G2 phase. Thus, G1 length is critical for sufficient MCM loading, particularly in heterochromatin, to ensure complete genome duplication and to maintain genome stability. 

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3. RB dependent transcriptional regulation at mitotic centromeres preserves genome stability

   https://doi.org/10.26508/lsa.202503433  

   Crowley, Elizabeth A; Manning, Amity L (December 2025, Life Science Alliance)

   Transcripts derived from centromere repeats play a critical role in the localization and activity of kinetochore components during mitosis such that disruption of RNA polymerase II-dependent transcription compromises the fidelity of chromosome segregation. Here, we show that the retinoblastoma tumor suppressor protein (RB), a critical regulator of the G1/S cell cycle transition, additionally plays an important role in the regulation of centromere transcription during mitosis. We find that cells lacking RB experience increased RNA Polymerase II activity at mitotic centromeres and a corresponding increase in nascent RNA transcripts derived from centromere sequences. Together with high levels of centromere transcription and corresponding R-loop formation, RB-deficient cells exhibit centromere DNA breaks and local activation of ATR that correspond with increased centromere localization of Aurora B, destabilization of kinetochore-microtubule attachments, and an increase in anaphase defects. Importantly, reduction of DNA damage, ATR activity, and mitotic defects following inhibition of RNA Pol II, or targeted repression of centromere transcription through centromere tethering of Suv420h2, support that mitotic defects in RB-deficient cells are linked to centromere transcription. 

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4. Cell-size regulation in budding yeast does not depend on linear accumulation of Whi5

   https://doi.org/10.1073/pnas.2001255117  

   Barber, Felix; Amir, Ariel; Murray, Andrew W. (June 2020, Proceedings of the National Academy of Sciences)

   Cells must couple cell-cycle progress to their growth rate to restrict the spread of cell sizes present throughout a population. Linear, rather than exponential, accumulation of Whi5, was proposed to provide this coordination by causing a higher Whi5 concentration in cells born at a smaller size. We tested this model using the inducibleGAL1promoter to make the Whi5 concentration independent of cell size. At an expression level that equalizes the mean cell size with that of wild-type cells, the size distributions of cells with galactose-induced Whi5 expression and wild-type cells are indistinguishable. Fluorescence microscopy confirms that the endogenous andGAL1promoters produce different relationships between Whi5 concentration and cell volume without diminishing size control in the G1 phase. We also expressed Cln3 from the GAL1 promoter, finding that the spread in cell sizes for an asynchronous population is unaffected by this perturbation. Our findings indicate that size control in budding yeast does not fundamentally originate from the linear accumulation of Whi5, contradicting a previous claim and demonstrating the need for further models of cell-cycle regulation to explain how cell size controls passage through Start. 

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5. The timing of Start is determined primarily by increased synthesis of the Cln3 activator rather than dilution of the Whi5 inhibitor

   https://doi.org/10.1091/mbc.E21-07-0349  

   Litsios, A.; Goaswami, P.; Terpstra, Coffin; H.M; Vuillemeot, L.-A.; Rovetta, M.; Ghazal, G.; Guerra, P.; Buczak, K.; Schmidt, A.; et al (May 2022, Molecular biology of the cell)

   Sophie Martin (Ed.)

   In their Letter to Molecular Biology of the Cell, Schmoller et al. (2022) raise questions about the results and conclusions presented in our published studies (Dorsey et al., 2018; Litsios et al., 2019). Here, we respond to the criticisms of Schmoller et al. and demonstrate how wide-field fluorescence microscopy experiments to determine nuclear Whi5 concentration dynamics can be confounded by uncontrolled effects, which include photobleaching, partial confocal effects, and nuclear-to-cytoplasmic volume scaling. Further, we provide additional experimental evidence demonstrating that nuclear Whi5 concentration is essentially constant as cells grow in G1 phase and that Cln3 and protein synthesis dynamics occur as reported in Litsios et al. (2019). These results suggest that instead of being triggered by dilution of the stable inhibitor Whi5, Start is rather primarily controlled by the increase in protein synthesis rate in G1 and the concomitant production of the unstable activator Cln3. 

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