If you think about it, one of the most fundamental jobs that cells have to perform is that of a wet, squishy Xerox machine. More specifically, a cell has to faithfully replicate its entire genome before dividing, ensuring each daughter cell gets its own complete genome copy—this concept forms the basis of biological inheritance and evolution as we know it. But genomes are big (if our genome were a book, it would be a large book!), and cells divide relatively quickly; thus, faithful genome replication is truly a race against time. Not unlike a busy office clamoring for access to a few overworked Xerox machines, cells have limited genome-replicating machinery which they must use wisely; and just like an office might address this challenge by sorting copy work into high- and low-priority piles, so too do cells replicate their genomes in systematic and highly stereotyped ways. In particular, eukaryotic cells tend to prioritize the replication of euchromatin (DNA which is generally considered ‘accessible’ for expression and enriched in protein-coding genes) over that of heterochromatin (DNA which is considered ‘inaccessible’ and enriched for more repetitive or noncoding regions of the genome). Messing with this order has implications for diseases like cancer and aging, but how exactly do cells manage this coordination in the first place?
This question drives researchers in the Bedalov lab in the Human Biology and Translational Science & Therapeutics Divisions at Fred Hutch, who use budding yeast as a model system to figure out the ins-and-outs of genome replication. In their recent study, published in eLife and led by Carmina Lichauco, they uncover important clues to how cells can precisely time the replication of different genomic regions. They focus on a particular portion of the yeast genome called the ribosomal DNA (rDNA), which in healthy yeast cells consists of roughly 150 identical repeats of the same modular sequence (cells need lots of rDNA to make enough protein-producing ribosomes to sustain their functions). As Dr. Eric Foss, a staff scientist in the Bedalov lab and co-author of the study, puts it, “the rDNA locus is so massive that it can’t rely on passive replication alone—for this reason, each rDNA repeat contains its own origin of replication. Out of roughly 500 highly active replication origins in the entire yeast genome, rDNA, with its 150 origins, poses a significant strain on the cell’s replicative machinery.” As rDNA is also generally heterochromatic, it’s normally replicated much later than the rest of the genome—classical studies have implicated a protein called SIR2 (a chromatin-modifying enzyme and founding member of a class of proteins called ‘sirtuins’) as the factor which normally suppresses rDNA replication.
If SIR2 is the office manager keeping cells from hogging the copiers with their lower-priority rDNA repeats, how exactly does it accomplish this task? “Classically, people assume that since SIR2 is a global chromatin-modifying enzyme, it regulates how accessible the rDNA locus is in a large-scale manner,’ notes Dr. Antonio Bedalov, “under normal conditions, SIR2 keeps rDNA in a heterochromatic, ‘closed’ state, preventing its replication until later in S phase.” Previous work from the Bedalov lab and others, however, challenged this notion: instead, it appeared that what mattered was not the overall accessibility of rDNA, but the specific positioning of nucleosomes (the protein complexes that DNA is normally wound around to form chromatin) in relation to the replicative helicase, MCM (a donut-shaped protein complex which initiates replication by unwinding DNA). But while SIR2 can do a lot, repositioning nucleosomes on DNA isn’t one of its skillsets—this hinted that SIR2 may have an accomplice in its regulation of rDNA replication timing.
Thus, the team began this current study with a scavenger hunt for a nucleosome-modifying enzyme which could perhaps reposition nucleosomes relative to MCM and thereby modify the timing of rDNA replication. Using sir2-mutant yeast (in which rDNA is replicated aberrantly early), Lichauco and colleagues systematically mutated known nucleosome-positioning enzymes and measured the relative timing of rDNA replication in these mutants. Before long, they found their suspect: FUN30, a chromatin remodeler whose loss significantly rescued rDNA replication timing in sir2-mutant yeast. A series of follow-up experiments revealed that FUN30 managed rDNA replication timing using its ATP-dependent nucleosome remodeling function, and that FUN30 loss had reciprocal effects on the replication of rDNA and non-rDNA regions of the genome. Furthermore, Lichauco and team found that SIR2 and FUN30 regulate rDNA replication not by altering the number of origins at which the Mcm helicase complex is loaded, but rather by regulating the propensity of those loaded helicases to be activated (or 'fire').
In sum, the team’s findings coalesced around a model in which, under wild-type conditions, the MCM replicative helicase on each rDNA repeat is loaded directly adjacent to a nucleosome, which delays origin firing relative to the rest of the origins in the genome. In sir2 mutants, this MCM is repositioned slightly downstream of this nucleosome—the net effect of this extra ‘breathing room’ upstream of MCM is an increased firing rate and early rDNA replication. The role of FUN30 is apparently to maintain the space between the original nucleosome and MCM—in the absence of FUN30, the DNA between the original nucleosome and the (now repositioned) MCM is filled by another nucleosome, effectively ‘closing up’ the space, reducing the origin firing rate, and once again stalling rDNA replication (see figure below).
What should we take away from this work? “On the highest level,” notes Bedalov, “we think this work challenges some basic assumptions about replication and overall DNA accessibility. While most people think of heterochromatin and ‘closed and inactive,’ there’s actually still a whole lot going on even in heterochromatic regions of the genome. What’s more, we show that rDNA replication is controlled not by regulating large-scale DNA accessibility, but instead by much more local and fine-grained changes in the rDNA landscape.” Foss is quick to add, “and while we studied most of these things in the context of sir2-mutant yeast, we have evidence to suggest that this mechanism of regulating MCM-nucleosome positioning also helps determine DNA replication timing under normal circumstances.” Thus, when it comes to regulating genome replication timing, the Bedalov lab urges us to think ‘small-scale.’
The spotlighted work was funded by the National Institutes of Health.
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium member Dr. Antonio Bedalov contributed to this study.
Lichauco, C., Foss, E. J., Gatbonton-Schwager, T., Athow, N. F., Lofts, B., Acob, R., Taylor, E., Marquez, J. J., Lao, U., Miles, S., & Bedalov, A. (2024). Sir2 and Fun30 regulate ribosomal DNA replication timing via MCM helicase positioning and nucleosome occupancy.
Science Spotlight writer David Sokolov is a graduate student in the Sullivan Lab at the Fred Hutch. He studies how cancer cells modify their metabolism to facilitate rapid proliferation and accommodate tumor-driving mitochondrial defects. He's originally from the east coast and has bachelors' and masters' degrees from West Virginia University. Outside of the lab, you'll find him enjoying the outdoors, playing music, or raising composting worms in his front yard.
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