Author response: Rif1 inhibits replication fork progression and controls DNA copy number in Drosophila (2018)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Control of DNA copy number is essential to maintain genome stability and ensure proper cell and tissue function. In Drosophila polyploid cells, the SNF2-domain-containing SUUR protein inhibits replication fork progression within specific regions of the genome to promote DNA underreplication. While dissecting the function of SUUR’s SNF2 domain, we identified an interaction between SUUR and Rif1. Rif1 has many roles in DNA metabolism and regulates the replication timing program. We demonstrate that repression of DNA replication is dependent on Rif1. Rif1 localizes to active replication forks in a partially SUUR-dependent manner and directly regulates replication fork progression. Importantly, SUUR associates with replication forks in the absence of Rif1, indicating that Rif1 acts downstream of SUUR to inhibit fork progression. Our findings uncover an unrecognized function of the Rif1 protein as a regulator of replication fork progression. https://doi.org/10.7554/eLife.39140.001 Introduction Accurate duplication of a cell’s genetic information is essential to maintain genome stability. Proper regulation of DNA replication is necessary to prevent mutations and other chromosome aberrations that are associated with cancer and developmental abnormalities (Jackson et al., 2014). DNA replication begins at thousands of cis-acting sites termed origins of replication. The Origin Recognition Complex (ORC) binds to replication origins where, together with Cdt1 and Cdc6, it loads an inactive form of the MCM2-7 replicative helicase (Bell and Labib, 2016). Inactive helicases are phosphorylated by two key kinases, S-CDK and Dbf4-dependent kinase (DDK), which results in the activation of the helicase and recruitment of additional factors to form a pair of bi-directional replication forks emanating outward from the origin of replication (Siddiqui et al., 2013). Although many layers of regulation control the initiation of DNA replication, much less is known about how replication fork progression is regulated. In metazoans, replication origins are not sequence specific and are likely specified by a combination of epigenetic and structural features (Aggarwal and Calvi, 2004; Cayrou et al., 2011; Eaton et al., 2011; Mesner et al., 2011; Miotto et al., 2016; Remus et al., 2004). Furthermore, replication origins are not uniformly distributed throughout the genome. The result of non-uniform origin distribution is that, in origin-poor regions of the genome, a single replication fork must travel great distances to complete replication. If a replication fork encounters an impediment within a large origin-less region of the genome, then replication will be incomplete, resulting in genome instability (Newman et al., 2013). In fact, origin-poor regions of the genome are known to be associated with chromosome fragility and genome instability (Debatisse et al., 2012; Durkin and Glover, 2007; Letessier et al., 2011; Norio et al., 2005). This highlights the need to regulate both the initiation and elongation phases of DNA replication to maintain genome stability. DNA replication is also regulated in a temporal manner where specific DNA sequences replicate at precise times during S phase, a process known as the DNA replication timing program. While euchromatin replicates in the early part of S phase, heterochromatin and other repressive chromatin types replicate in the latter portion of S phase (Gilbert, 2002; Rhind and Gilbert, 2013). Although the process of replication timing has been appreciated for many years, the underlying molecular mechanisms controlling timing have remained elusive. The discovery of factors that regulate the DNA replication timing program, however, demonstrate that replication timing is an actively regulated process. One factor that regulates replication timing from yeast to humans is Rif1 (Rap1-interacting factor 1). Rif1 was initially identified as a regulator of telomere length in budding yeast (Hardy et al., 1992), but this function of Rif1 appears to be specific to yeast (Xu et al., 2004). Subsequently, Rif1 has been shown to regulate multiple aspects of DNA replication and repair. In mammalian cells, Rif1 has been shown to regulate DNA repair pathway choice by preventing resection of double-strand breaks and favoring non-homologous end joining (NHEJ) over homologous recombination (Chapman et al., 2013; Di Virgilio et al., 2013; Zimmermann et al., 2013). Rif1 from multiple organisms contains a Protein Phosphatase 1 (PP1) interaction motif and Rif1 is able to recruit PP1 to DDK-activated helicases to inactive them and prevent initiation of replication (Davé et al., 2014; Hiraga et al., 2014; Hiraga et al., 2017). In yeasts, flies and mammalian cells, Rif1 has been shown to regulate the replication timing program (Cornacchia et al., 2012; Hayano et al., 2012; Peace et al., 2014; Sreesankar et al., 2015; Yamazaki et al., 2012). The precise mechanism(s) through which Rif1 functions to control replication timing are not fully understood. For example, Rif1 has been show to interact with Lamin and is thought to tether specific regions of the genome to the nuclear periphery (Foti et al., 2016). How this activity is related to Rif1’s ability to inactivate helicases together with PP1 in controlling the timing program remains obscure. Studying DNA replication in the context of development provides a powerful method to understand how DNA replication is regulated both spatially and temporally. Although DNA replication is a highly ordered process, it must be flexible enough to accommodate the changes in S phase length and cell cycle parameters that occur as cells differentiate (Matson et al., 2017). For example, during Drosophila development the length of S phase can vary from ~8 hr in a differentiated mitotic cell to 3 – 4 min during early embryonic cell cycles (Blumenthal et al., 1974; Spradling and Orr-Weaver, 1987). Additionally, many tissues and cell types in Drosophila are polyploid, having multiple copies of the genome in a single cell (Edgar and Orr-Weaver, 2001; Lilly and Duronio, 2005; Zielke et al., 2013). In polyploid cells, copy number is not always uniform throughout the genome (Rudkin, 1969; Hua and Orr-Weaver, 2017; Spradling and Orr-Weaver, 1987). Both heterochromatin and several euchromatic regions of the genome have reduced DNA copy number relative to overall ploidy (Nordman et al., 2011). Underreplicated euchromatic regions of the genome share key features with common fragile sites in that they are devoid of replication origins, late replicating, display DNA damage and are tissue-specific (Andreyeva et al., 2008; Nordman et al., 2014; Sher et al., 2012; Yarosh and Spradling, 2014). The presence of underreplication is conserved in mammalian cells, but the mechanism(s) mammalian cells use to promote underreplication is unknown (Hannibal et al., 2014). In Drosophila, underreplication is an active process that is largely dependent on the distribution of ORC and on the Suppressor of Underreplication protein, SUUR (Hua et al., 2018; Makunin et al., 2002; Nordman and Orr-Weaver, 2015). Understanding how the SUUR protein functions will significantly increase our understanding of the developmental control of DNA replication. The SUUR protein has a recognizable SNF2-like chromatin remodeling domain at its N-terminus, but based on sequence analysis, this domain is predicted to be defective for ATP binding and hydrolysis (Makunin et al., 2002; Nordman and Orr-Weaver, 2015). Outside of the SNF2 domain, SUUR has no recognizable motifs or domains, which has hampered a mechanistic understanding of how SUUR promotes underreplication. Recently, however, SUUR was shown to control copy number by directly reducing replication fork progression (Nordman et al., 2014). SUUR associates with active replication forks and while loss of SUUR function results in increased replication fork progression, overexpression of SUUR drastically inhibits fork progression without affecting origin firing (Nordman et al., 2014; Sher et al., 2012). These findings, together with previous work showing that loss of SUUR function has no influence on ORC binding (Sher et al., 2012) and that SUUR associates with euchromatin in an S phase-dependent manner (Kolesnikova et al., 2013), further supports SUUR as a direct inhibitor of replication fork progression within specific regions of the genome. The mechanism through which SUUR is recruited to replication forks and how it inhibits their progression remains poorly understood. Here we investigate how SUUR is recruited to replication forks and how it inhibits fork progression. We show that localization of SUUR to replication forks, but not heterochromatin, is dependent on its SNF2 domain. We identify an interaction between SUUR and the conserved replication factor Rif1, indicating they are in the same protein complex. Importantly, we demonstrate that underreplication is dependent on Rif1. Critically, we have shown that Rif1 localizes to replication forks in an SUUR-dependent manner, where it acts downstream of SUUR to control replication fork progression. Our findings provide mechanistic insight into the process of underreplication and define a new function for Rif1 in replication control. Results The SNF2 domain is essential for SUUR function and replication fork localization As a first step in understanding the mechanism of SUUR function, we wanted to define how it is localized to replication forks. SUUR has only one conserved domain: a SNF2-like domain in its N-terminal region that is predicted to be defective for ATP binding and hydrolysis (Makunin et al., 2002; Nordman and Orr-Weaver, 2015). To study the function of SUUR’s SNF2 domain, we generated a mutant in which the SNF2 domain was deleted and the resulting mutant protein was expressed under the control of the endogenous SuUR promoter. This mutant, SuUR∆SNF, was then crossed to an SuUR null mutant so that it was the only form of the the SUUR protein present (Figure 1A). We tested the function of the SUUR∆SNF mutant protein by assessing its ability to promote underreplication in the larval salivary gland. We purified genomic DNA from larval salivary glands isolated from wandering third instar larvae and generated genome-wide copy number profiles using Illumina-based sequencing. We compared the results we obtained from the SuUR∆SNF mutant to copy number profiles from wild-type (WT) and SuUR null mutant salivary glands. To identify underreplicated domains, we used CNVnator, which identifies copy number variants (CNVs) based on a statistical analysis of read depth (Abyzov et al., 2011). To be called as underreplicated, regions must not be called as underreplicated in 0 – 2 hr embryo samples that have uniform copy number and must be larger than 10 kb. Figure 1 with 2 supplements see all Download asset Open asset The SNF2 domain is essential for SUUR function and replication fork localization. (A) Schematic representation of the SUUR and SUURΔSNF proteins. (B) Illumina-based copy number profiles (Reads Per Million; RPM) of chr2L 1 – 20,000,000 from larval salivary glands. Black bars below each profile represent underreplicated regions identified by CNVnator. (C) Average read depth in regions of euchromatic underreplication domains called in wild-type salivary glands vs. the fully replicated regions of the genome. A Welch Two Sample t-test was used to determine p values. (D) Quantitative droplet-digital PCR (ddPCR) copy number assay for multiple underreplicated regions. Each bar is the average enrichment relative to a fully replicated control region for three biological replicates. Error bars are the SEM. (E) Localization of SUUR in wild-type and SuURΔSNF mutant follicle cells. A single representative stage 13 follicle cell nucleus is shown. Arrowheads indicate sites of amplification. Asterisk marks the chromocenter (heterochromatin). Scale bars are 2 μm. DAPI = blue, SUUR = green, EdU = red. The following source data, figure supplements and supplementary files are available for Figure 1: Figure 1—figure supplement 1; Figure 1—figure supplement 2; Supplementary file 1 - Underreplicated regions called by CNVnator; Figure 1—source data 1 – Raw data for 1D; Figure 1—figure supplements 2—source data 1 image intensity data. https://doi.org/10.7554/eLife.39140.002 Figure 1—source data 1 ddPCR data for Figure 1D. https://doi.org/10.7554/eLife.39140.006 Download elife-39140-fig1-data1-v2.xlsx The effect of deleting the SNF2 domain was qualitatively and quantitatively similar to the SuUR null mutant. Qualitatively, underreplication was suppressed in the SuUR∆SNF mutant and the copy number profile was similar to the SuUR null mutant (Figure 1B and Figure 1—figure supplement 1). Quantitatively, out of the 90 underreplicated sites identified in WT salivary glands, 59 were not detected in the SuUR∆SNF mutant (Supplementary file 1) and copy number was significantly increased in the euchromatic underreplicated domains similar to the SuUR null mutant (Figure 1C). We validated our deep-sequencing findings using quantitative droplet digital PCR (ddPCR) at four underreplicated domains (Figure 1D). Our findings show that the SNF2-like domain of SUUR is necessary to promote underreplication. To determine if the SUUR∆SNF protein was still able to associate with chromatin, we localized SUUR and the SUUR∆SNF mutant proteins in ovarian follicle cells. During follicle cell development, these cells undergo programmed changes in their cell cycle and DNA replication programs (Claycomb and Orr-Weaver, 2005; Hua and Orr-Weaver, 2017). At a precise time in their differentiation program, follicle cells cease genomic replication and amplify six defined sites of their genome through a re-replication-based mechanism. Early in this gene amplification process, both initiation and elongation phases of replication are coupled. Later in the process, however, initiation no longer occurs and active replication forks can be visualized by pulsing amplifying follicle cells with 5-ethynyl-2’-deoxyuridine (EdU) (Claycomb et al., 2002). Active replication forks resolve into a double-bar structure, where each bar represents a series of active replication forks travelling away from the origin of replication (Claycomb and Orr-Weaver, 2005). By monitoring SUUR localization in amplifying follicle cells, we can unambiguously determine if SUUR associates with active replication forks. SUUR has two distinct modes of chromatin association during the endo cycle. It constitutively localizes to heterochromatin and dynamically associates with replication forks (Kolesnikova et al., 2013; Nordman et al., 2014; Swenson et al., 2016). In agreement with previous studies, SUUR localized to both replication forks and heterochromatin in amplifying follicle cells (Figure 1E) (Nordman et al., 2014). In contrast, the SUUR∆SNF mutant localized to heterochromatin, but its recruitment to active replication forks was severely reduced (Figure 1E; Figure 1—figure supplement 2). Together, these results demonstrate that the SNF2 domain is important for SUUR recruitment to replication forks and is essential for SUUR-mediated underreplication. SUUR associates with Rif1 Interestingly, overexpression of the SNF2 domain and C-terminal portion of SUUR have different underreplication phenotypes. Whereas overexpression of the C-terminal two-thirds of SUUR promotes underreplication (Kolesnikova et al., 2005), overexpression of the SNF2 domain suppresses underreplication in the presence of endogenous SUUR (Kolesnikova et al., 2005). The C-terminal region of SUUR, however, has no detectable homology or conserved domains (Makunin et al., 2002). These observations, together with our own results demonstrating that the SNF2 domain of SUUR is responsible for its localization to replication forks, led us to hypothesize that SUUR is recruited to replication forks through its SNF2 domain where it could recruit an additional factor(s) through its C-terminus to inhibit replication fork progression. To test the hypothesis that a critical factor interacts with the C-terminal region of SUUR to promote underreplication, we used immunoprecipitation mass spectrometry studies to identify SUUR-interacting proteins. We generated flies that expressed FLAG-tagged full-length SUUR or the SNF2 domain of SUUR under control of the hsp70 promoter, induced and immunoprecipitated these constructs and identified associated proteins through mass spectrometry. We verified that both full-length SUUR and the SNF2 domain were expressed equally (Figure 2—figure supplement 1B). If SUUR recruits a factor to replication forks outside of its SNF2 domain, then we would expect this factor to be present only in full-length purifications and not in the SNF2 domain purification. A single protein fulfilled this criteria: Rif1 (Figure 2A). This result raises the possibility Rif1 works together with SUUR to inhibit replication fork progression. Figure 2 with 2 supplements see all Download asset Open asset SUUR associates with Rif1. (A) Total spectrum counts of FLAG-SUUR, FLAG-SNF2 and Oregon R (no FLAG control) for three independent IP-mass spectrometry experiments (biological replicates). A Fisher’s Exact test of spectrum counts was used to determine significance. (B) Immunoprecipitation of FLAG-SUUR and no FLAG control (wild-type) from 0 to 24 hr embryos extracted with NP40 lysis buffer with or without Benzonase treatment. Membranes were probed with anti-Rif1 and anti-FLAG antibodies to monitor Rif1 and SUUR, respectively. The following source data and figure supplement are available for Figure 2: Figure 2—figure supplement 1; Figure 2—figure supplement 2; Figure 2—source data 1 – SUUR mass spectrometry total spectrum counts; Figure 2—figure supplements 2—source data 1 – embryo hatch rata data. https://doi.org/10.7554/eLife.39140.007 Figure 2—source data 1 Results of SUUR IP-mass spec screen. https://doi.org/10.7554/eLife.39140.011 Download elife-39140-fig2-data1-v2.xlsx To ensure that the association between SUUR and Rif1 was not bridged by chromatin, we used NP40 to extract chromatin proteins and treated the extract with Benzonase to digest DNA. We then immunoprecipitated FLAG-SUUR and used Western blotting to determine if Rif1 could co-IP using a highly specific anti-Rif1 antibody (Figure 2—figure supplement 2). Even in these conditions, SUUR was able to co-IP Rif1 (Figure 2B; Figure 2—figure supplement 1A). We conclude that SUUR and Rif1 exist in the same protein complex and the interaction between SUUR and Rif1 is independent of chromatin bridging. Underreplication is dependent on Rif1 If SUUR recruits Rif1 to replication forks to promote underreplication, then underreplication should be dependent on Rif1. To test this hypothesis, we used CRISPR-based mutagenesis to generate Rif1 null mutants in Drosophila (Bassett et al., 2013; Gratz et al., 2013) (Figure 3A). Western blot analysis of ovary extracts from two deletion mutants, Rif11 and Rif12, show no detectable Rif1 protein (Figure 2—figure supplement 2A). Also, no signal was detected in the Rif11/Rif12 mutant by immunofluorescence (Figure 2—figure supplement 2B). The Rif11/Rif12 null mutant was viable and fertile showing only a modest defect in embryonic hatch rate relative to wild-type flies with a 92% hatch rate for wild-type embryos vs. 88% for the Rif11/Rif2 mutant embryos (Figure 2—figure supplement 2C). This is in contrast to a previous study reporting Rif1 is essential in Drosophila (Sreesankar et al., 2015) and consistent with a recent study that generated an independent Rif1 null mutant using CRISPR-based mutagenesis (Seller and O'Farrell, 2018). Rif1’s essentiality, however, was based on RNAi and not a mutation of the Rif1 gene (Sreesankar et al., 2015). The most likely explanation for this discrepancy is that the lethality in the RNAi experiments was due to an off-target effect. Figure 3 with 3 supplements see all Download asset Open asset Rif1 is required for underreplication. (A) Schematic representation of the Rif1 gene and CRISPR-induced Rif1 mutants. Lightning bolts represent the 5’ and 3’ gRNA positions. (B) Illumina-based copy number profiles of the chr2L from larval salivary glands. Black bars below each profile represent underreplicated regions identified by CNVnator. The wild-type and SuUR profiles are the same as in Figure 1b. (C) Average read depth in regions of euchromatic underreplication domains called in wild-type salivary glands vs. the fully replicated regions of the genome. A Welch Two Sample t-test was used to determine p values. (D) Quantitative droplet-digital PCR (ddPCR) copy number assay for multiple underreplicated regions. Each bar is the average enrichment relative to a fully replicated control region for three biological replicates. Error bars are the SEM. The following source data and figure supplement are available for Figure 3: Figure 3—figure supplement 1; Figure 3—figure supplement 2; Figure 3—figure supplement 2; Figure 3—source data 1; Figure 3—figure supplement 2—source data 1 - Raw data for 2B. https://doi.org/10.7554/eLife.39140.012 Figure 3—source data 1 ddPCR data for Figure 3D. https://doi.org/10.7554/eLife.39140.017 Download elife-39140-fig3-data1-v2.xlsx To determine if Rif1 is necessary for underreplication, we dissected salivary glands from Rif11/Rif12 (herein referred to as Rif1-) heterozygous larvae and extracted genomic DNA for Illumina-based sequencing to measure changes in DNA copy number. Strikingly, underreplication is abolished upon loss of Rif1 function (Figure 3B and C; Figure 3—figure supplement 1). We validated our sequence-based copy number assays with quantitative PCR at a subset of underreplicated regions using ddPCR (Figure 3D). Furthermore, we determined the read density at all euchromatic sites of underreplication called in our wild-type samples, which quantitatively demonstrates that Rif1 is essential for underreplication (Figure 3C). These results demonstrate that underreplication is dependent on Rif1. It is possible that the Rif1 mutant indirectly influences underreplication through changes in replication timing. Underreplicated domains, both euchromatic and heterochromatic, tend to be late replicating regions of the genome (Belyaeva et al., 2012; Makunin et al., 2002). Therefore, if these regions replicated earlier in S phase in a Rif1 mutant, then this change could prevent their underreplication. In fact, SUUR associates with late replicating regions of the genome (Filion et al., et al., to their large polyploid salivary cells be to genome-wide replication timing heterochromatin replicates in late S phase, however, late replication can be visualized EdU is in regions of To if Rif1 mutants have a of late replication in larval salivary glands, we isolated salivary glands from early instar which are actively endo We these salivary glands with EdU to sites of replication and with an antibody to In wild-type salivary glands, only of we EdU in regions of heterochromatin (Figure 3—figure supplement 2). This is consistent with the of heterochromatin replication due to underreplication. In contrast, in both SuUR and Rif1 mutants, we could cells that were EdU within regions of heterochromatin of for SuUR and of for (Figure 3—figure supplement 2). Therefore, we conclude that Rif1 mutants still have a of late replication. that heterochromatin underreplication is suppressed in a Rif1 mutant, it is still late replicating, that replication timing the of underreplication associated with loss of Rif1 function. While Rif1’s in underreplication and of DNA replication in endo cells, we in the regions of SuUR and Rif1 mutants. underreplication is suppressed in both mutants (Figure 3 and Figure 3—figure supplement the were large in the Rif1 mutant relative to an SuUR mutant as by DAPI consistent with the in Rif1 mutant cells (Figure 3—figure supplement (Cornacchia et al., 2012). this was present in all endo cells, it was in the ovarian cells (Figure 3—figure supplement Illumina-based copy number profiles an increase in copy number in heterochromatin regions in the Rif1 mutant relative to the SuUR mutant (Figure 3—figure supplement 1). these results that heterochromatin is but not fully replicated in SuUR mutant endo cells, consistent with previous analysis et al., In contrast, loss of Rif1 function appears to replication in endo cells. Rif1 replication fork progression SUUR-mediated underreplication occurs through of replication fork progression (Nordman et al., 2014; Sher et al., 2012). If SUUR acts together with Rif1 to promote underreplication, then Rif1 is to control replication fork progression. DNA assays in and cells from multiple have to different as to Rif1 replication fork progression et al., 2017; et al., 2012; Hiraga et al., 2017; Yamazaki et al., 2012). Rif1, however, has been shown to be associated with replication forks through chromatin an used to proteins associated with active replication forks et al., 2014). To determine directly if Rif1 replication fork progression, we copy number assays on amplifying follicle cells. amplification in ovarian follicle cells occurs at six sites in the genome through a based mechanism. number of these domains provides a quantitative of the number of of origin firing and the replication forks have during the amplification process, us to the initiation and elongation phases of DNA replication. To determine if Rif1 origin firing replication fork progression, we isolated wild-type and Rif1 mutant stage 13 which represent the end of the amplification process, and quantitative DNA copy number of Rif1 function in an increase in replication fork progression without significantly affecting copy number at the origin of replication at all sites of amplification (Figure The increase in fork progression in the Rif1 mutant was not due to a of the developmental time for gene as was no in distribution between wild-type and Rif1 mutant (Figure supplement 1). Figure 4 with 1 supplement see all Download asset Open asset Rif1 regulates replication fork progression. (A) Illumina-based copy number profile of sites of follicle cell gene amplification. DNA was extracted from and Rif1 mutant stage 13 and compared to DNA extracted from 0 to 2 hr The resulting are the of relative to embryonic DNA. below the represent the between the copy number on each of the replication (B) of cells that display amplification in each stage of gene amplification. Average of two biological replicates in which two from each stage were used biological – follicle cells were Error bars are the SEM. The following source data, supplementary file and figure supplement are available for Figure Figure supplement 1; Supplementary file 2 – of for all Figure data 1 – data for Figure supplements 1—source data data for distribution of biological replicates. Figure data 1 Data for Figure Download To the changes in fork progression we at sites of we determined the of amplification and the region on each of the domain that represents one of the copy number at the of the (Nordman et al., 2014). This quantitative analysis of origin firing and replication fork progression that origin firing was not in the Rif1 mutant, as no change in copy number was detected at the origin of replication