Chromatin structure restricts origin utilization when quiescent cells re-enter the cell cycle

Quiescent cells reside in G0 phase, which is characterized by the absence of cell growth and proliferation. These cells remain viable and re-enter the cell cycle when prompted by appropriate signals. Using a budding yeast model of cellular quiescence, we investigated the program that initiated DNA replication when these G0 cells resumed growth. We performed BrdU IP-Seq to examine the DNA replication profile genome-wide. These data revealed that the initiation of replication was delayed and fewer origins were active when G0 cells entered the cell cycle compared to the entry of G1 cells into S phase. We found that both the transcript and protein levels of these replication factors were significantly diminished during the development of proliferating cells into quiescence. The levels of these factors, including MCM2-7 helicase, increased as G0 cells re-entered the cell cycle, suggesting that the replication program is re-established de novo. Consistent with these results, Mcm4 ChIP-seq analysis showed fewer and reduced binding at a number of origins during the re-entry of G0 cells into the cell cycle. In support of this evidence, the chromatin context surrounding inactive origins of G0 cells exhibits greater increased nucleosome occupancy and reduced periodicity of nucleosome positioning. Altogether, these observations provide insights into the important role of chromatin context that determines the ability of replication origin to accrue limiting replication factors during the the re-entry of quiescent cells into the cell cycle. For BrdU IP-seq, G1 or Go cells were released into YPD+0.2M HU and incubated with 800 μg/ml of BrdU for overlapping periods of 20 min prior to harvesting. G1 released cells were collected at 25, 40, 55, and 70 min and G0 released cells were collected at 85, 100, 115, 130, 145 and 165 min after BrdU pulse-labeling. For Mcm4 ChIP-seq, G0 cells were released into YPD+0.2 M HU and collected at 90 and 105 min. G1 cells were collected after incubation with alpha-factor for 135 min. The ChIP and input DNA from collected samples were amplified separately using an Ion Xpress Plus Fragment Library kit and sequenced with the Ion S5 System (ThermoFisher).