G1/S transcription factors assemble in increasing numbers of discrete clusters through G1 phase

Abstract

In budding yeast, the transcription factors SBF and MBF activate a large program of gene expression in late G1 phase that underlies commitment to cell division, termed Start. SBF/MBF are limiting with respect to target promoters in small G1 phase cells and accumulate as cells grow, raising the questions of how SBF/MBF are dynamically distributed across the G1/S regulon and how this impacts the Start transition. Super-resolution Photo-Activatable Localization Microscopy (PALM) mapping of the static positions of SBF/MBF subunits in fixed cells revealed each transcription factor was organized into discrete clusters containing approximately eight copies regardless of cell size and that the total number of clusters increased as cells grew through G1 phase. Stochastic modeling using reasonable biophysical parameters recapitulated growth-dependent SBF/MBF clustering and predicted TF dynamics that were confirmed in live cell PALM experiments. This spatio-temporal organization of SBF/MBF may help coordinate activation of G1/S regulon and the Start transition.

Growth-dependent signals drive an increase in early G1 cyclin concentration to link cell cycle entry with cell growth

“…We carried out a similar analysis for Whi5 and found that Whi5 concentration decreased by approximately 30% in rich carbon and 20% in poor carbon, consistent with the fact that Whi5 protein levels do not change substantially and there is only a small increase in cell size in G1 phase ( Figure 1—figure supplement 1F ). Our results are consistent with several previous studies that observed a relatively small increase in cell volume in G1 phase ( Ferrezuelo et al, 2012 ; Leitao and Kellogg, 2017 ) and little or no change in Whi5 concentration ( Dorsey et al, 2018 ; Litsios et al, 2019 ; Black et al, 2020 ). A previous study found that a small fraction of cells growing in complete synthetic media containing poor carbon show a 30–40% decrease in Whi5 concentration, but most cells showed a decrease of 20–30% or less ( Schmoller et al, 2015 ).…”

“…We used the same approach to calculate the fold change in Whi5 levels between the zero time point and bud emergence in multiple biological replicates ( Figure 1—figure supplement 1D ). Both plots suggest that levels of the Whi5 protein do not change substantially before bud emergence in rich or poor carbon, consistent with several previous studies that used microscopy to analyze fluorescently tagged versions of Whi5 ( Schmoller et al, 2015 ; Dorsey et al, 2018 ; Litsios et al, 2019 ; Black et al, 2020 ).…”

“…However, there are conflicting reports on the behaviors of Cln3 and Whi5 during growth in G1 phase (Tyers et al 1993;Zapata et al 2014;Liu et al 2015;Schmoller et al 2015;Lucena et al 2018;Dorsey et al 2018;Blank et al 2018;Litsios et al 2019;Barber et al 2020;Black et al 2020). Most previous studies Here, we used quantitative western blotting in synchronized cells to carry out a comprehensive analysis of the function and regulation of Cln3 and Whi5 during growth in G1…”

“…Distinguishing models will require a clear understanding of how changing concentrations of Cln3 and Whi5 influence cell cycle entry, and how the concentrations of Cln3 and Whi5 vary during growth and in response to changes in growth rate. However, there are conflicting reports on the behaviors of Cln3 and Whi5 during growth in G1 phase ( Tyers et al, 1993 ; Zapata et al, 2014 ; Liu et al, 2015 ; Schmoller et al, 2015 ; Lucena et al, 2018 ; Dorsey et al, 2018 ; Blank et al, 2018 ; Litsios et al, 2019 ; Barber et al, 2020 ; Black et al, 2020 ). Most previous studies used quantitative fluorescence microscopy to analyze the behavior of tagged proteins in living cells.…”

Single-molecule tracking (SMT) and localization of SRF and MRTF transcription factors during neuronal stimulation and differentiation

“…A third parameter quantified in cell differentiation besides TF bound-fraction ( figure 2 ) and residence time ( figure 3 ) was accumulation of SRF molecules in clusters by TALM ( figure 5 ). Others have already used TALM protocols for membrane and mitochondrial proteins [ 33 , 45 , 46 ] as well as nuclear proteins [ 65 , 66 ]. In the nucleus, previous studies argued that such TF aggregates or clusters might indicate presence of so-called transcriptional hot-spots or hubs where specific gene clusters are predominantly transcribed [ 7 , 43 ].…”