The cyclin-dependent kinase inhibitor Dacapo promotes replication licensing during Drosophila endocycles
2007; Springer Nature; Volume: 26; Issue: 8 Linguagem: Inglês
10.1038/sj.emboj.7601648
ISSN1460-2075
AutoresAmy Hong, Karine Narbonne-Reveau, Juan R. Riesgo‐Escovar, Haiqing Fu, Mirit I. Aladjem, Mary A. Lilly,
Tópico(s)Ubiquitin and proteasome pathways
ResumoArticle22 March 2007free access The cyclin-dependent kinase inhibitor Dacapo promotes replication licensing during Drosophila endocycles Amy Hong Amy Hong Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USAPresent address: Laboratory of Molecular Physiology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. Search for more papers by this author Karine Narbonne-Reveau Karine Narbonne-Reveau Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Juan Riesgo-Escovar Juan Riesgo-Escovar Departmento de Neurobiología del Desarrollo y Neurofisiología, Instituto de Neurobiología, Universidad Nacional Antonoma de Mexico, Queretaro, Mexico Search for more papers by this author Haiqing Fu Haiqing Fu Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Mirit I Aladjem Mirit I Aladjem Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Mary A Lilly Corresponding Author Mary A Lilly Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Amy Hong Amy Hong Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USAPresent address: Laboratory of Molecular Physiology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. Search for more papers by this author Karine Narbonne-Reveau Karine Narbonne-Reveau Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Juan Riesgo-Escovar Juan Riesgo-Escovar Departmento de Neurobiología del Desarrollo y Neurofisiología, Instituto de Neurobiología, Universidad Nacional Antonoma de Mexico, Queretaro, Mexico Search for more papers by this author Haiqing Fu Haiqing Fu Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Mirit I Aladjem Mirit I Aladjem Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Mary A Lilly Corresponding Author Mary A Lilly Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Author Information Amy Hong1,‡, Karine Narbonne-Reveau1,‡, Juan Riesgo-Escovar2, Haiqing Fu3, Mirit I Aladjem3 and Mary A Lilly 1 1Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA 2Departmento de Neurobiología del Desarrollo y Neurofisiología, Instituto de Neurobiología, Universidad Nacional Antonoma de Mexico, Queretaro, Mexico 3Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA ‡These authors contributed equally to this work *Corresponding author. Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA. Tel.: +1 301 435 8428; Fax: +1 301 402 0078; E-mail: [email protected] The EMBO Journal (2007)26:2071-2082https://doi.org/10.1038/sj.emboj.7601648 Present address: Laboratory of Molecular Physiology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The endocycle is a developmentally programmed variant cell cycle in which cells undergo repeated rounds of DNA replication with no intervening mitosis. In Drosophila, the endocycle is driven by the oscillations of Cyclin E/Cdk2 activity. How the periodicity of Cyclin E/Cdk2 activity is achieved during endocycles is poorly understood. Here, we demonstrate that the p21cip1/p27kip1/p57kip2-like cyclin-dependent kinase inhibitor (CKI), Dacapo (Dap), promotes replication licensing during Drosophila endocycles by reinforcing low Cdk activity during the endocycle Gap-phase. In dap mutants, cells in the endocycle have reduced levels of the licensing factor Double Parked/Cdt1 (Dup/Cdt1), as well as decreased levels of chromatin-bound minichromosome maintenance (MCM2–7) complex. Moreover, mutations in dup/cdt1 dominantly enhance the dap phenotype in several polyploid cell types. Consistent with a reduced ability to complete genomic replication, dap mutants accumulate increased levels of DNA damage during the endocycle S-phase. Finally, genetic interaction studies suggest that dap functions to promote replication licensing in a subset of Drosophila mitotic cycles. Introduction The endocycle, also known as the endoreplicative cycle, is a variant cycle used by both plants and animals to increase cell size and ploidy (Edgar and Orr-Weaver, 2001; Lilly and Duronio, 2005). During endocycle, cells undergo repeated rounds of DNA replication without intervening mitosis. Recently, considerable progress has been made in understanding how the core cell cycle machinery is modified to convert a mitotic cycle to an endocycle (Sigrist and Lehner, 1997; Shcherbata et al, 2004). Entry into the endocycle is contingent on the downregulation of the mitotic kinase Cdk1. Indeed, in Drosophila, removing the mitotic cyclins or the mitotic kinase Cdk1 shunts cells programmed to be in the mitotic cycle into a self-sustaining endocycle (Sauer et al, 1995; Hayashi, 1996; Weigmann et al, 1997). However, relative to the mitotic cycle, our basic understanding of the biochemical oscillator that drives the endocycle remains limited. Current data suggest that the Drosophila endocycle is driven by the oscillations of Cyclin E/Cdk2 activity (Edgar and Orr-Weaver, 2001; Lilly and Duronio, 2005). In Drosophila, Cyclin E/Cdk2 activity is required for DNA replication during endocycle and the mitotic cycle (Lane et al, 1996). Paradoxically, continuous overexpression of Cyclin E inhibits endocycle progression (Follette et al, 1998; Weiss et al, 1998). These data suggest that cells in the endocycle require a Gap-phase when overall Cyclin E/Cdk2 activity is low, in order to relicense DNA replication origins for successive rounds of DNA replication. An important factor controlling the periodicity of Cyclin E/Cdk2 activity is the regulated accumulation and destruction of the Cyclin E protein (Moberg et al, 2001). However, the exact nature of the regulator inputs that drive the oscillations of Cyclin E/Cdk2 activity during the endocycle is poorly defined. Perfect duplication of the genome depends on two sequential steps of DNA replication having opposite requirements for Cdk activity (Bell and Dutta, 2002; Diffley and Labib, 2002). Whereas the formation of pre-replication complexes (pre-RCs) or licensing of the DNA, requires low Cdk activity, the initiation of DNA replication is triggered by high Cdk activity. Pre-RCs are formed in late mitosis and G1 when the origin recognition complex recruits Cdt1 and Cdc6, which load the minichromosome maintenance (MCM2–7) complex onto the origin (reviewed in Bell and Dutta, 2002). In Drosophila, Cdt1 is referred to as Double Parked (Dup) (Whittaker et al, 2000). It has been shown in multiple organisms that increasing Cdk activity during the Gap-phase, by misexpressing G1 cyclins, inhibits pre-RC formation, resulting in genomic instability (Spruck et al, 1999; Lengronne and Schwob, 2002; Tanaka and Diffley, 2002; Ekholm-Reed et al, 2004b). Thus, precise regulation of Cdk activity is critical to the faithful duplication of the genome. The dacapo (dap) gene encodes a p21CIP/p27KIP1/p57KIP2-like cyclin-dependent kinase inhibitor that inhibits the activity of Cyclin E/Cdk2 complexes in Drosophila (de Nooij et al, 1996, 2000; Lane et al, 1996). In dap-null alleles, which are embryonic lethal, cells of the epidermis progress through an extra embryonic division cycle (de Nooij et al, 1996; Lane et al, 1996). Thus, as observed with other cyclin-dependent kinase inhibitors (CKIs), dap functions to coordinate cell cycle exit with terminal differentiation. However, a positive role has been proposed for dap in the regulation of the endocycle (de Nooij et al, 2000; Edgar and Orr-Weaver, 2001). Central to this proposal are the following two observations. First, Dap oscillations closely follow those of Cyclin E during the endocycle in the polyploid nurse cells of the ovary (de Nooij et al, 2000). Second, in many tissues of Drosophila, Cyclin E positively influences the accumulation of Dap (de Nooij et al, 2000). These two findings suggest a possible feedback loop in which increased Cyclin E promotes DNA replication, as well as accumulation of Dap. Ultimately, Dap levels may go high enough to inhibit Cyclin E/Cdk2 activity, which either directly and/or indirectly brings S-phase to a halt, shunting cells into the Gap-phase. A similar role has been proposed for the mammalian Cdk inhibitor p57KIP2 during the endocycle of mammalian trophoblasts (Hattori et al, 2000). In the giant trophoblasts of the placenta, the levels of p57KIP2 oscillate with cell cycle kinetics similar to that of Dap in the polyploid nurse cells. Thus, the oscillation of one or more CKIs may be a common feature of endocycles in multiple species. In the Drosophila ovary, both germ-line derived nurse cells and somatic follicle cells enter the endocycle and become polyploid. Drosophila oogenesis takes place within a 16-cell interconnected cyst. However, only one of the 16 cells proceeds through meiosis and becomes a viable gamete, whereas the other 15 cells in the cyst enter the endocycle and develop as highly polyploid nurse cells. Individual egg chambers are produced when somatically derived follicle cells encapsulate the ovarian cysts. The follicle cells continue to divide mitotically until mid-oogenesis (stage 6), when they synchronously exit the mitotic cycle and enter the endocycle in a process requiring the Notch signaling pathway (Deng et al, 2001; Lopez-Schier and St Johnston, 2001). Because the complete process of polyploidization can be followed in two independent cell types, Drosophila oogenesis provides an excellent model system to examine mechanisms of endocycle regulation. Here, we present evidence that the CKI Dap promotes the accumulation of Dup/Cdt1 and licensing of DNA replication origins during Drosophila endocycles. In the absence of Dap, cells in the endocycle have low levels of Dup/Cdt1, as well as dramatically reduced levels of chromatin-bound MCM2–7 complex. Moreover, upon entering the endocycle, dap−/− cells accumulate DNA damage, consistent with the number of pre-RCs assembled during the endocycle Gap-phase being below the threshold required to complete genomic replication. Finally, reducing the dosage of dup/cdt1 in a dap−/− background reveals a possible role for Dap in replication licensing during the mitotic cycle. These studies represent one of the first reports of a CKI acting to promote replication licensing in a metazoan. Results Dap influences endoreplication in the polyploid nurse cells To explore the role of Dap in endoreplication in Drosophila, we used FLP/FRT-mediated mitotic recombination (Xu and Rubin, 1993) to generate dap−/− mutant clones using the dap4 allele. The dap4 allele is an amorph, which contains a deletion of the conserved Cdk binding domain (Lane et al, 1996). In developing wild-type egg chambers, the 15 polyploid nurse cells either have equal amounts of DNA, as measured by DAPI staining, or, in some stages of oogenesis, the posterior nurse cells are one or two endocycles ahead of the anterior nurse cells (Figure 1A; reviewed in Spradling, 1993). In Figure 1B, we used the FLP/FRT system to create an egg chamber in which all 16 cells within the germ-cyst are dap−/−. Note that the egg chamber containing the dap−/− germ-line clone has a posterior nurse cell, indicated by an arrowhead, with a dramatically smaller nuclear size than an anterior nurse cell, indicated by the arrow. Additionally, dap−/− nurse cells have a condensed chromatin structure (Figure 1C), similar to that observed in mutants that affect the oscillation of the Cyclin E protein (Lilly and Spradling, 1996; Doronkin et al, 2003). The smaller nuclear size often observed in dap−/− polyploid nurse cells suggests that in the absence of Dap, the nurse cell endocycle is compromised (Lilly and Spradling, 1996). Figure 1.dap−/− egg chambers have abnormal nurse cell nuclei. (A) Wild-type and (B, C) egg chamber containing a dap−/− germ-line clone stained with the DNA dye DAPI. Boxes in (A) and (C) are blow-ups of the indicated nurse cells. Note that dap−/− nurse cells have a condensed chromatin structure. Arrowhead in (B) denotes an example of a dap−/− nurse cell with an apparent DNA content lower than the anterior nurse cell indicated by an arrow. Download figure Download PowerPoint dap−/− mutants extend the endocycle S-phase To examine directly if the mitotic and/or endocycle S-phase is altered in dap−/− cells, we labeled ovaries containing dap−/− somatic and germ-line clones with the nucleotide analog, BrdU. When cells are in the mitotic cycle, the pattern of BrdU labeling in dap−/− germ-line and follicle cell clones is indistinguishable from wild type (data not shown). However, the pattern of BrdU incorporation diverges from wild type when the cells in dap−/− clones enter the endocycle. After a 1-h incubation, approximately 32±7% (n=81) of endocycling nurse cells from heterozygous (dap+/−) egg chambers were BrdU positive, indicating that approximately 1/3 of the nurse cells were in S-phase during the 1-h period of BrdU incorporation (Figure 2A and B). In contrast, in dap−/− homozygous germ-line clones almost twice as many cells, 62±7% (n=73), incorporate BrdU (Figure 2C and D). Similar to what is observed in dap−/− nurse cells, upon entry into the endocycle in stage 6 of oogenesis, an increased proportion of dap−/− follicle cells incorporate BrdU relative to adjacent heterozygous (dap+/−) cells (Figure 2E–G). Thus, in the absence of the CKI Dap, the endocycle is modified, such that cells spend a greater proportion of their cell cycle in S-phase. Figure 2.The endocycle S-phase is lengthened in dap−/− mutants. The FLP/FRT technique was used to generate dap−/− clones in a wild-type (dap+/−) background. (A–D) Egg chambers containing (A, B) wild-type and (C, D) dap−/− germ-line clones labeled with (A, C) DAPI and (B, D) the nucleotide analog BrdU. (E–G) dap−/− follicle cell clone from a stage 7 egg chamber labeled with (E) αBrdU and (F) αGFP. In (F) and (G), dap−/− clones are identified by the absence of αGFP staining. (G) An overlay of αBrdU (red), αGFP (green) and DAPI staining (blue). (H) Wild-type and (I) dap−/− nurse cell nuclei stained with DAPI. Arrows indicate the primarily heterochromatic fourth chromosome. Download figure Download PowerPoint During the Drosophila endocycle, S-phase is often truncated before the entire genome is replicated. This truncation, or premature entry into the Gap-phase, leads to underrepresentation of late-replicating heterochromatic sequences in many polyploid cell types (Gall et al, 1971; Spradling and Orr-Weaver, 1987). Several recently proposed models suggest that Dap oscillations may help bring down Cyclin E/Cdk2 activity at the end of each endocycle S-phase (de Nooij et al, 2000; Edgar and Orr-Weaver, 2001). One prediction from this model is that in dap−/− mutants, Cyclin E/Cdk2 kinase activity may stay above the threshold that will support DNA replication for an extended period of time. As an assay for the lengthening of the endocycle S-phase, we examined if nuclei from dap−/− polyploid nurse cells contain increased amounts of heterochromatin relative to wild-type nurse cells. One of the largest blocks of heterochromatin in the Drosophila genome corresponds to a stretch of 1.672 satellite DNA on the primarily heterochromatic fourth chromosome (Lohe and Brutlag, 1987). This block of heterochromatin can be identified as a bright blob of DAPI staining near the nuclear envelope (Figure 2H, arrow) (Dej and Spradling, 1999). Consistent with a global increase in the copy number of late-replicating heterochromatic sequences in the polyploid genome, in dap−/− nurse cells, the DAPI-stained fourth chromosome appears considerably larger and brighter than in similarly staged wild-type nurse cells (Figure 2I, arrow). This phenotype is nearly identical to that observed in the cycE01672 female sterile mutant, which has an increase in the proportion of nurse cells that are Cyclin E positive and is known to have a 2–3-fold increase in the copy number of several late-replicating sequences (Lilly and Spradling, 1996). To confirm that dap−/− mutants extend the endocycle S-phase, we compared the copy number of late-replicating sequences with the copy number of early replicating sequences in endocycling wild-type versus dap−/− nurse cells. We used the R1 (Endow and Glover, 1979) and the Bari-1 (Marsano et al, 2003) transposable elements as markers for late-replicating heterochromatic DNA and euchromatic sequences from the mio and CCR4 genes as markers for early replicating DNA (Iida and Lilly, 2004; Morris et al, 2005). Diploid nuclei, as well as all nuclei from 64C to 128C, were collected by FACS analysis from wild-type and dap−/− ovaries (Lilly and Spradling, 1996). The dap−/− ovaries were obtained from the small percentage of homozygous dap−/− females that develop to adulthood. The proportion of nuclei contributed by the 64C peak versus 128C peak was approximately equal in both mutant and wild-type samples. DNA was extracted from the samples and real-time PCR was performed to determine the copy number of the heterochromatic and euchromatic regions. DNA from the 2C peak, representing DNA from diploid cells, was assumed to be 100% represented for all heterochromatic sequences and was used for normalization. Real-time PCR using two probes from the R1 element (R1 and R1′) confirm that whereas the R1 element is underrepresented approximately four-fold in wild-type nurse cells from the 64–128C samples, it is nearly fully represented in nuclei from the dap−/− 64–128C sample (Table I). Similar to what is observed with the R1 element, the Bari-1 element is nearly fully represented in dap−/− nurse cells but is underrepresented nearly two-fold in 64–128C nuclei from wild-type nurse cells (Table I). Taken together, these data support the model that the endocycle S-phase is lengthened in dap−/− mutants to include late replication. Table 1. dap−/− nurse cells have an increased copy number of two heterochromatic sequences Bari-1 R1 R1′ mio CCR4 Wild type 0.49±0.12 0.16±0.20 0.36±0.16 0.93±0.38 1.00±0.22 dap−/− 0.96±0.13 1.05±0.06 0.87±0.17 1.14±0.17 1.00±0.24 Data are presented as the ratio of the >64C versus 2C for mutant versus wild-type cell lines, normalized for CCR4, +s.e.m. mio is a euchromatic sequence. To further explore if the levels of Cyclin E/Cdk2 activity are increased in dap−/− nurse cells, as is suggested by the lengthening of S-phase, we stained ovaries that contained dap−/− clones with the αMPM2 antibody (Davis et al, 1983). Although traditionally used to follow mitotic phosphoepitopes, the αMPM2 antibody is a useful marker for monitoring Cyclin E/Cdk2 activity in Drosophila, with the presence of one or more αMPM2-positive subnuclear spheres correlating with high levels of Cyclin E/Cdk2 activity (Calvi et al, 1998; Royzman et al, 1999). In endocycling nurse cells, Cyclin E levels oscillate (Lilly and Spradling, 1996). αMPM2 staining levels also oscillate, with approximately 30% of wild-type nurse cells being αMPM2 positive at any given time (Supplementary Figure 1A). In dap−/− clones, the proportion of nurse cells that are αMPM2 positive is dramatically increased, with greater than 95% of the mutant nurse cells containing αMPM2 subnuclear spheres (Supplementary Figure 1B). These data suggest that in dap−/− nurse cells, the baseline level of Cyclin E/Cdk2 activity is increased. Similarly, dap−/− follicle cell clones contain a higher proportion of cells that are αMPM2 positive (Supplementary Figure 1C–E). Thus, Dap may influence the dynamics of Cyclin E/Cdk2 kinase activity in multiple cell types. These data support the model that it is increased Cyclin E/Cdk2 activity that drives the lengthening of the endocycle S-phase in dap−/− mutants. Dap promotes the accumulation of Dup/Cdt1 during the endocycle In human cells, Cyclin E overexpression inhibits pre-RC assembly (Ekholm-Reed et al, 2004a). Experiments described above suggest that Cyclin E/Cdk2 activity is increased in dap−/− mutants. To explore the possibility that high Cyclin E/Cdk2 kinase activity in dap−/− mutant cells inhibits the licensing of DNA replication origins, we compared the behavior of the licensing factor Dup/Cdt1 in wild-type versus dap−/− clones. The Dup/Cdt1 protein accumulates during the Gap-phase but rapidly disappears once cells enter the S-phase (Whittaker et al, 2000; Thomer et al, 2004). In Drosophila, Cyclin E/Cdk2 activity promotes the destruction of Dup/Cdt1 (Thomer et al, 2004). In wild-type nurse cells, the levels of Dup/Cdt1 oscillate during the endocycle, with nurse cells with low, medium and high levels of Dup/Cdt1 found within a single egg chamber (Figure 3C, asterisk). In contrast, nurse cells in dap−/− germ-line clones, marked by the absence of anti-β-gal staining, invariably have low levels of Dup/Cdt1 (Figure 3B and C, arrowhead). The decrease in Dup/Cdt1 levels observed in dap−/− germ-line clones in region 3 of the germarium is temporally coincident with entry into the endocycle. Earlier in oogenesis, dap−/− germ-line clones undergoing the mitotic cysts divisions in region 1 of the germarium have wild-type levels of Dup/Cdt1 (Figure 3D–F, arrowhead). Dap is also differentially required for the accumulation of Dup/Cdt1 during the endocycles in the somatic follicle cells. Before stage 6 of oogenesis, when the cells are in the mitotic cycle, dap−/− follicle cell clones have wild-type levels of Dup/Cdt1 (data not shown). However, upon entry into the endocycle in stage 6, dap−/− follicle cells exhibit decreased levels of Dup/Cdt1 relative to adjacent wild-type cells (Figure 3G–I). Additionally, consistent with a role for Dap in promoting the accumulation of Dup/Cdt1, the overexpression of Dap in endocycling follicle cells results in the increased accumulation of the Dup/Cdt1 protein (Figure 3J–L). Figure 3.Dap differentially affects the accumulation of Dup/Cdt1 in endocycling versus mitotic cells. The FLP/FRT technique was used to generate dap−/− clones in a wild-type (dap+/−) background. (A–I) Ovaries containing dap−/− clones were stained with (A, D and I) DAPI, (B) α-β-gal, (E, F, G, I) αGFP and (C, F, H, I) αDup/Cdt1 antibodies. In (B), dap−/− clones are identified by the absence of α-β-gal staining, whereas in (E, F, G, and I), dap−/− clones are identified by the absence of αGFP staining. (A–C) An egg chamber containing a dap−/− germ-line clone, arrowhead, flanked by two wild-type egg chambers. Note the low levels of Dup/Cdt1 in the polyploid nurse cells of the dap−/− cyst. (D–F) In contrast, Dup/Cdt1 is present in dap−/− germ-line clones undergoing the mitotic cyst division in region 1 of the germarium (F, arrowhead). (G–I) dap−/− follicle cell clones, marked by the absence of (G) αGFP, have reduced levels of (H) αDup/Cdt1 staining compared with adjacent wild-type cells. (J–L) The FLP/Gal4 system was used to clonally express Dap with β-gal. Follicle cells overexpressing Dap, marked by (J) α-β-gal staining, have higher levels of (K) α-Dup/Cdt1 staining than neighboring wild-type cells. Download figure Download PowerPoint dap−/− nurse cells have reduced levels of the chromatin-bound MCM2–7 complex The loading of the MCM2–7 complex onto chromatin requires Dup/Cdt1 (Bell and Dutta, 2002). Therefore, one might predict that in dap−/− nurse cells, where Dup levels are low, the loading of the MCM2–7 complex onto chromatin may be compromised. To explore this possibility, we stained dap−/− ovaries with an antibody that recognizes a conserved epitope present in all six Drosophila MCM2–7 subunits (Claycomb et al, 2002). Immunostainings were performed under two conditions: first, using a low-salt wash, we examined the total nucleoplasmic pool of MCM2–7 proteins in wild-type versus dap−/− nurse cell nuclei (Figure 4A–D). From this experiment, we determined that dap−/− nurse cells have overall levels of MCM2–7 proteins similar to that observed in wild-type egg chambers. However, when we used high-salt wash to remove non-chromatin-bound MCM proteins from the nuclei in order to reveal what fraction of MCM proteins are loaded onto chromatin, we saw a striking difference between dap−/− and wild-type nurse cells (Figure 4E–H). Wild-type egg chambers contain a fraction of nurse cells with chromatin-bound MCM2–7, as well as a fraction of nurse cells with no obvious chromatin-bound MCM2–7 (Figure 4E). This pattern reflects the asynchrony of nurse cell endocycles within a single egg chamber, with some cells in the S-phase and others in the Gap-phase. In contrast, dap−/− nurse cells have uniformly low levels of chromatin-bound MCM2–7 (Figure 4G). Thus, consistent with the low levels of Dup/Cdt1, endocycling dap−/− nurse cells have decreased levels of the chromatin-bound MCM2–7 complex. Figure 4.dap−/− nurse cells have reduced levels of the chromatin-bound MCM2–7 complex. (A, B) Wild-type and (C, D) dap−/− egg chambers stained with (A, C) αMCM2–7 antibody and (B, D) DAPI, and washed with low salt to reveal the nucleoplasmic pool of MCM2–7. (E, F) Wild-type and (G–H) dap−/− egg chambers stained with (E, G) αMCM2–7 antibody and (F, H) DAPI and washed with high salt to reveal chromatin-bound MCM2–7. Download figure Download PowerPoint dap−/− mutants accumulate increased γ-H2Av staining upon endocycle entry In budding yeast and mammals, overexpression of G1 cyclins results in genomic instability owing to inhibition of pre-RC formation (Tanaka and Diffley, 2002). The genomic instability is thought to arise, at least in part, because a lower density of licensed origins results in the production of stalled replication forks during the S-phase. Stalled replication forks often collapse and break, resulting in DNA damage (Zhou and Elledge, 2000). Thus, we wanted to determine if the apparent decrease in pre-RC formation observed in endocycling cells in dap−/− mutants results in a corresponding increase in DNA damage. In order to address this question, we used an antibody against the phosphorylated form of the variant histone H2Av (Madigan et al, 2002). In Drosophila, the variant histone H2Av is phosphorylated on its C-terminal tail in response to the presence of a double-stranded break (Madigan et al, 2002). H2Av is the only H2A histone variant present in Drosophila and is thought to be the functional homolog of both H2Az and H2Ax (Madigan et al, 2002). Phosphorylated H2Av is referred to as γ-H2Av. In wild-type ovaries, coincident with entry into the first endocycle, γ-H2Av staining abruptly appears in stage 6 follicle cells (Figure 5, compare panel D with B). The staining is not uniform throughout the nucleus, but is found as a series of small intranuclear dots or clumps, often near the nuclear envelope. During the very first endocycle S-phase, follicle cells fail to replicate approximately 25% of their genome and thus are predicted to contain numerous stalled replication forks that in subsequent rounds of S-phase would produce truncated DNAs (Lilly and Spradling, 1996; Leach et al, 2000). The truncated DNAs are predicted to form between the junctions of the fully replicated euchromatin and the underreplicated heterochromatin (Leach et al, 2000). Consistent with this model, γ-H2Av staining in wild-type endocycling follicle cell nuclei is often present near the DAPI bright chromocenter, which contains the centric heterochromatin as well as the fourth chromosome (Figure 5C and D, arrows). In mammals, stalled DNA replication forks often collapse, resulting in DNA breaks (Kurose et al, 2006). Thus, the intranuclear foci of γ-H2Av staining likely mark the sites of accumulated stalled replication forks and/or truncated DNAs that accumulate during the incomplete endocycle S-phase. Figure 5.dap−/− mutants accumulate increased α-γ-H2Av staining upon entry into the endocycle. Wild-type (A, B) mitotic and (C, D) endocycling follicle cells stained with (A, C, white; B, D, blue) DAPI and (B, D, red) α-γ-H2Av antibody. (A, B) Wild-type mitotic follicle cells have little α-γ-H2Av staining, whereas (C, D) α-γ-H2Av staining accumulates near the chromocenter upon entry into the endocycle (C, D, arrows). (E, F) Two dap−/− germ-line cysts marked by the absence of (F) α-GFP staining have increased levels of (E, F) γ-H2Av staining compared with a neighboring older wild-type cyst. (G–I) A stage 9 egg chamber containing a dap−/− clone of follicle cells t
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