Rif1 Functions in a Tissue-Specific Manner To Control Replication Timing Through Its PP1-Binding Motif

Abstract

Replication initiation in eukaryotic cells occurs asynchronously throughout S phase, yielding early- and late-replicating regions of the genome, a process known as replication timing (RT). RT changes during development to ensure accurate genome duplication and maintain genome stability. To understand the relative contributions that cell lineage, cell cycle, and replication initiation regulators have on RT, we utilized the powerful developmental systems available in Drosophila melanogaster. We generated and compared RT profiles from mitotic cells of different tissues and from mitotic and endocycling cells of the same tissue. Our results demonstrate that cell lineage has the largest effect on RT, whereas switching from a mitotic to an endoreplicative cell cycle has little to no effect on RT. Additionally, we demonstrate that the RT differences we observed in all cases are largely independent of transcriptional differences. We also employed a genetic approach in these same cell types to understand the relative contribution the eukaryotic RT control factor, Rif1, has on RT control. Our results demonstrate that Rif1 can function in a tissue-specific manner to control RT. Importantly, the Protein Phosphatase 1 (PP1) binding motif of Rif1 is essential for Rif1 to regulate RT. Together, our data support a model in which the RT program is primarily driven by cell lineage and is further refined by Rif1/PP1 to ultimately generate tissue-specific RT programs.

Replication timing analysis in polyploid cells reveals Rif1 uses multiple mechanisms to promote underreplication in Drosophila

“…Additionally, while characterizing the variant RT regions, we noted that majority of the variant regions, 64.45% and 78.28% respectively (Figure 2A-B), fall within known underreplicated regions of salivary glands. The differences in RT measured by TIGER and the G1/S method are consistent with expected differences due to cell-type specific changes in the RT program (Armstrong et al, 2020). Taken together, we conclude that TIGER is an effective method to generate genome-wide RT profiles from polyploid cells.…”

“…In contrast, nearly 50% of the genome is dependent on Rif1 for proper RT. The fraction of the salivary gland genome that is dependent on Rif1 for RT is significantly greater than what was observed for Drosophila follicle cells or wing discs (Armstrong et al, 2020). While differences in the statistical methods used to perform variant calling could be responsible for subset of these differences, it is clear that Rif1 differentially affects RT in these three cell types.…”

“…RT profiles generated with TIGER correlate with RT profiles generated by the G1/S method While the data presented in Figure 1 strongly suggests that the profiles generated by TIGER represent RT in the larval salivary gland, we wanted to compare these TIGER-generated profiles to RT profiles generated by conventional methods. Therefore, we compared directly TIGER-generated RT profiles in the larval salivary gland to RT profiles of larval wing discs and follicle cells of the adult ovary that our lab profiled using the G1/S method (Armstrong et al, 2020). If the TIGER-generated profiles truly reflect RT, then we would expect the RT profiles produced by TIGER to be comparable to these previously published.…”

“…Of all the regions that displayed differential RT in the Rif1 mutant relative to wild type, only 29.88% fell within known UR regions (Figure 4B). This indicates that, in contrast to SUUR, Rif1 functions as a global regulator of RT in the polyploid larval salivary gland similar to its function in other diploid cell types (Armstrong et al, 2020;Cornacchia et al, 2012;Foti et al, 2016;Hayano et al, 2012;Peace et al, 2014;Yamazaki et al, 2012).…”

“…One major regulator of RT is the trans-acting factor Rif1 (Rap1-interacting factor 1). Rif1 controls genome-wide RT from yeast to humans (Armstrong et al, 2020;Cornacchia et al, 2012;Hayano et al, 2012;Peace et al, 2014;Seller & O'Farrell, 2018;Sreesankar et al, 2015;Yamazaki et al, 2012). The prevalent model for how Rif1 controls the RT program is based upon its conserved Protein Phosphatase 1 (PP1)-interaction motif.…”