Positive feedback of G1 cyclins ensures coherent cell cycle entry

Abstract

In budding yeast, the Start checkpoint integrates multiple internal and external signals into an allor-none decision to enter the cell cycle. Here, we show that Start behaves like a switch due to systems-level feedback in the regulatory network. In contrast to current models proposing a linear cascade of Start activation, transcriptional positive feedback of the G1 cyclins Cln1,2 induces the near-simultaneous expression of the ~200-gene G1/S regulon. Nuclear Cln2 drives coherent regulon expression, while cytoplasmic Cln2 drives efficient budding. cln1,2-deleted cells frequently arrest as unbudded cells, incurring a large fluctuation-induced fitness penalty due to both the lack of cytoplasmic Cln2 and insufficient G1/S regulon expression. Thus, positivefeedback-amplified expression of Cln1,2 simultaneously drives robust budding and rapid, coherent regulon expression. A similar G1/S regulatory network in mammalian cells, comprised of nonorthologous genes, suggests either the conservation of regulatory architecture or convergent evolution.Positive feedback in genetic control networks can ensure that cells do not slip back and forth between either cell cycle phases or developmental fates. For example, commitment to sporulation in budding yeast is driven by transcriptional positive feedback of the meiotic inducer IME1 [1][2][3] . In Xenopus laevis, positive feedback underlies the all-or-none characteristics of oocyte maturation 4, 5 and mitotic entry 6,7 , suggesting the frequent use of positive feedback to regulate cellular transitions.Absent from this list of examples is the well-studied Start checkpoint controlling cell cycle commitment in budding yeast. Nutrient limitation and pheromone exposure arrests cells prior to DNA replication, while size control extends G1 in small daughter cells [8][9][10][11] . Beyond Start, cells proceed through division almost independently of size and environment 9 . Previous experiments suggested that Start represents a feedback-free cascade of events 12 (see schematic in Fig. 1a; omitting red arrows). The transition is initiated by the G1-cyclin Cln3 [13][14][15] , which in complex with Cdc28 activates the transcription of about 200 genes 16 by phosphorylating promoter-bound protein complexes that include the transcription factors SBF and MBF 17 and the transcriptional inhibitor Whi5 18,19 . Phosphorylation and inactivation of Whi5 is rate-limiting, and phosphorylated Whi5 rapidly exits the nucleus. The G1/S regulon, which includes two additional G1-cyclins, CLN1,2, contributes to the activation of B-type cyclins, DNA replication, spindle pole body duplication, and bud emergence. Mitotic B-type cyclins then inactivate SBF 20 and, with NRM1, inactivate MBF 21 , thus turning off the G1/S regulon.Correspondence and requests for materials should be addressed to J.M.S. (e-mail: jskotheim@rockefeller.edu). Any one of the three G1-cyclins suffices to activate the regulon, suggesting the potential for transcriptional positive feedback of CLN1,2 on their own expression 22,23 . Howev...

The critical size is set at a single-cell level by growth rate to attain homeostasis and adaptation

“…4d), and similar results were obtained with cells lacking both Cln1 and Cln2 (Fig. 4f), the two G1 cyclins that contribute to Start in a positive feedback loop [25][26][27] . Thus, rather than being mediators, individual G1 cyclins would be target effectors of the sizing mechanism.…”

“…Perhaps, indirectly, Ydj1 could also be required for proper sBF and mBF function (dashed arrow). Cln1 and Cln2 cyclins generate a positive feedback loop essential for coherent and robust sBF and mBF activation [25][26][27] , and stb1 is a transcriptional modulator that contributes to inhibit sBF-and mBF-dependent transcription during G1 48 . sfp1 acts as a negative regulator of start (continuous arrow) and, as it is a transcription activator in ribosome biogenesis, sfp1 would also upregulate Cln3 levels (dashed arrow) as a result of increased translation 4 .…”

“…YDJ1 overexpression (oYDJ1) was assessed in daughter cells elutriated 3 h after adding galactose to a culture grown in raffinose. As they contained a MET3p-CLN2 construct to avoid accumulation of aberrantly shaped cells, cln1,2 cells were grown in SC medium without methionine, which was added immediately before the time-lapse experiment 27 . Newly born cells were obtained by elutriation 19 .…”

Hybrid modeling and simulation of stochastic effects on progression through the eukaryotic cell cycle

“…13,35 Stochastic models of cell cycle regulation are necessary to understand these experimental results in quantitative terms. Kar et al successfully converted Tyson and Novak's three-variable, deterministic, cell cycle model into a stochastic version by unpacking the phenomenological rate laws into detailed elementary reactions.…”

Déplétion des nucléosomes dans les régions promotrices

“…Dans la plupart des études précédentes, les données transcriptionnelles étaient exprimées comme la moyenne de mesures faites dans des populations de cellules. Au contraire, nous avons travaillé sur des cellules individuelles de levures et mesuré in vivo, dans des expériences de vidéomi-croscopie de fluorescence, la transcription de gènes spécifiques en fonction du temps [12][13][14]. Deux promoteurs de gènes régulés au cours du cycle cellulaire, CLN2 (une cycline active lors de la transition G1/ S) et HO 1 , ont été sélectionnés pour étudier l'influence des NDR sur l'activation transcriptionnelle.…”