BIOLOGICAL OVERVIEW

In response to DNA damage, eukaryotic cells use a system of checkpoint controls to delay cell-cycle progression. Checkpoint delays provide time for repair of damaged DNA before its replication in S phase and before segregation of chromatids in M phase. The Chk2 tumor-suppressor protein has been implicated in certain checkpoint responses in mammalian cells. It directly phosphorylates and inactivates the mitosis-inducing phosphatase Cdc25 (see Drosophila String) in vitro and is required to maintain the G2 arrest that is observed in response to gamma-irradiation. Chk2 also directly phosphorylates p53 (see Drosophila p53) in vitro at a site that is implicated in its stabilization, and is required for stabilization of p53 and induction of p53-dependent transcripts in vivo upon gamma-ionizing radiation. Thus, Chk2 functions in both the G1 and G2 checkpoint responses. Like Chk2, the checkpoint protein kinase ATM (ataxia-telangiectasia-mutated) is required for correct operation of both the G1 and G2 damage checkpoints. ATM is necessary for phosphorylation and activation of Chk2 in vivo and can phosphorylate Chk2 in vitro (Xu, 2001; Abdu, 2002; Masrouha, 2003; and references therein).

The Drosophila serine/threonine kinase Loki (Entree Nucleotide record), here referred to alternatively as Dmnk (Oishi, 1998) or Drosophila Chk2, is the homolog of the yeast Mek1p, Rad53p, Dun1p, and Cds1 proteins as well as the human Chk2. Functional analyses led to the conclusion that, in flies, Chk2 is required for DNA damage-mediated cell cycle arrest and apoptosis (Xu, 2001). chk2 acts during early embryogenesis to monitor double-strand breaks (DSBs) caused by irradiation during S and G2 phases (Masrouha, 2003). Abdu (2002) presents convincing evidence that chk2 is part of a meiotic checkpoint functioning during oogenesis. The target of this signal is thought to be Vasa, which in turn regulates the translation of Gurken mRNA. Drosophila chk2 does not act at the same cell cycle phases as its yeast homologs, but seems rather to be involved in a pathway similar to the mammalian one, which involves signaling through the ATM/Chk2 pathway in response to genotoxic insults. Since mutations in human chk2 have been linked to several cancers, these similarities underscore the usefulness of the Drosophila model system (Masrouha, 2003).

In the mammalian system, Chk2 is a key player in maintaining the genome integrity. In the G1 checkpoint, ionizing radiation (IR) exposure can activate the ATM-Chk2 pathway. Activated Chk2 phosphorylates Ser123 in Cdc25A, targeting it for ubiquitin-dependent degradation (Falck, 2001). Since Cdc25A is downregulated, the activity of CyclinA-Cdk2 is inhibited and replication is slowed. In addition, downregulation of Cdc25A is thought to inhibit the activity of CyclinE-Cdk2, leading to the p53-independent initiation of the G1 checkpoint (Bartek, 2001a). In the G2/M checkpoint, Chk1 (Drosophila homolog: Grapes), Chk2, and p53 are three key transducers: the ATM and Rad 3-related (ATR)-Chk1 pathway is thought to be activated when cells are exposed to IR during G1 or S phase; the ATM-Chk2 pathway is thought to arrest cells in response to genotoxic insults during G2 phase (Abraham, 2001); recent findings indicate that p53 may play additional roles to help cells arrest at the G2/M transition (Taylor, 2001). Checkpoint signals relayed from Chk1, Chk2, and p53 arrest the cell cycle at the G2/M transition via downregulation of the kinase activity of CyclinB-Cdk1. Depending on the transducer, this downregulation step can involve one of the following intermediates: Cdc25C, p21, Gadd45, or 14-3-3 (Masrouha, 2003 and references therein).

ATM activates human Chk2 (hChk2) by phosphorylating an amino terminal Thr residue (Thr 68) (Ahn, 2000; Melchionna, 2000), and hChk2, in turn, phosphorylates p53 on two sites, Serine 15 and Serine 20. The location of Ser 15 at the p53 amino terminus suggested that modification of this residue might trigger the dissociation of p53 from MDM2, a protein that targets p53 for ubiquitination, nuclear export, and proteosomal degradation. Therefore, if the model were correct, ATM/ATR-dependent phosphorylation of Ser 15 would free p53 from its destabilizing binding partner, thereby favoring p53 accumulation. It turns out, however, that Ser 15 phosphorylation is not sufficient to disrupt the p53-MDM2 interaction; rather, this modification stimulates the transactivating function of p53 by enhancing the binding of this protein to the transcriptional coactivator, p300. However, these results do not rule out the possibility that phosphorylation of p53 at Ser 15 sets this protein up for a secondary modification that does modify the binding of MDM2 to p53, thereby inhibiting p53 degradation. Indeed, Ser 15 phosphorylation greatly enhances the subsequent phosphorylation of p53 at Ser 18 by casein kinase I, at least under test-tube assay conditions with purified proteins. The presence of phosphates at Ser 15 and Ser 18 reduces the avidity of full-length p53 for MDM2 by approximately threefold. Further studies are required to determine whether, and under what conditions, the tandem modification of p53 by ATM/ATR and casein kinase I contributes to p53 accumulation in intact cells. Chk2 phosphorhylates yet another amino-terminal Ser residue (Ser 20) in p53 (Chehab, 2000; Hirao, 2000; Shieh, 2000). Unlike the Ser 15 modification of Chk2 by ATM, phosphorylation at Ser 20 interferes directly with the binding of p53 to MDM2, thereby favoring p53 accumulation in response to IR-induced DNA damage. The physiological relevance of hChk2 in the regulation of p53 is supported by the finding that loss-of-function mutations in hChk2 can give rise to a variant form of Li-Fraumeni syndrome (Bell, 1999), a heritable, cancer-prone disorder typically associated with germ-line mutations in p53 (Abraham, 2001 and references therein).

In Drosophila, Chk2 appears to act to regulate the apoptotic activity of p53 during genotoxic stress. The tumor suppressor function of p53 has been attributed to its ability to regulate apoptosis and the cell cycle. In mammals, DNA damage, aberrant growth signals, chemotherapeutic agents, and UV irradiation activate p53, a process that is regulated by several posttranslational modifications. Overexpression of Drosophila p53 (p53) in the eye induces apoptosis, resulting in a small eye phenotype. This phenotype is markedly enhanced by coexpression with Drosophila Chk2 and was almost fully rescued by coexpression with a dominant-negative (DN), kinase-dead form of Chk2. DN Chk2 also inhibits p53-mediated apoptosis in response to DNA damage, whereas overexpression of Grapes (Grp), the Drosophila Chk1-homolog, and its DN mutant has no effect on p53-induced phenotypes. Chk2 also activates the p53 transactivation activity in cultured cells. Mutagenesis of p53 amino terminal Ser residues reveals that Ser-4 is critical for its responsiveness toward Chk2. Chk2 activates the apoptotic activity of p53 and Ser-4 is required for this effect. Contrary to results in mammals, Grapes, the Drosophila Chk1-homolog, is not involved in regulating p53. Chk2 may be the ancestral regulator of p53 function (Peters, 2002).

Since maternal Chk2 protein expression persists into embryonic development, the gene may function during early embryogenesis; it was of interest to determine whether chk2 functions in DNA damage checkpoint activation in 3- to 4-hr-old embryos, which are in cycle 14. During cycle 14, cells are known to enter mitosis as stereotypical clusters called 'mitotic domains'. The timing of entry into mitosis of each one of these domains as well as the morphogenetic movements that comprise gastrulation are known to be invariant between different embryos. It was furthermore observed that DNA damage induced by irradiation or MMS treatment can delay entry into mitosis of cycle 14 (Su, 2000); this delay is primarily due to inhibitory phosphorylation of Cdk1, and nuclear exclusion of the Cdk1-Cyclin complex might also play a secondary role (Masrouha, 2003).

Embryos in interphase 14 (130- to 200-min-old embryos) were exposed to 600 rad of gamma-irradiation, which corresponds to the half-lethal dose. Irradiated embryos were allowed to recover for 45 min, after which they were fixed and stained for the mitotic-specific phospho-Histone3 (PH3) epitope. At the same time these embryos were stained for Vasa, a pole-cell-specific marker that shows the progression of the morphogenetic movements of gastrulation. In nonirradiated wild-type embryos, domain 1 initiates mitosis in stage 6. In wild-type embryos, irradiation causes a delay of entry into mitosis. For instance, domain 1 does not start mitosis until much later after irradiation (stage 8). In nonirradiated chk2^null mutant embryos, mitotic patterns in each specific gastrulation stage are the same as in the nonirradiated wild-type embryos. However, in irradiated chk2^null mutant embryos, each mitotic domain enters mitosis with the same timing as the nonirradiated control, indicating that the DNA damage checkpoint is defective. Similar defects in arresting the cell cycle were observed in irradiated chk2^null embryos that were allowed to recover for only 15 min after the gamma-ray exposure. Since S and G2 phases of cycle 14 last 50 and 20 min, respectively, this finding shows that chk2 is a damage checkpoint gene involved in mediating responses to DSBs induced during the S or G2 phases of the cell cycle. Thus, chk2 is required for the DNA damage checkpoint in somatic cells. It thus appears that low Chk2 levels are sufficient for this checkpoint to be active (Masrouha, 2003).

In S. cerevisiae, the Chk2 family member Rad53p is required for the DNA damage and replication checkpoint and arrests the cell cycle at the G1/S transition, in S phase, or at the metaphase-anaphase transition in response to stresses. Nevertheless, Rad53p is not required for the meiotic pachytene checkpoint. Instead, a meiotic-specific version, Mek1p, is required for detecting DNA DSBs that arise as recombination occurs. In S. pombe, the Chk2 family member, Cds1, is mainly required for the S-phase DNA damage/replication checkpoint. Activated Cds1 arrests cells in S phase in response to unreplicated DNA or damaged DNA sensed during S phase. Whether Cds1 is required for the meiotic checkpoint is not yet known. Mammalian Chk2 is indispensable for G1/S, S, and G2/M checkpoint controls, but its role in the meiotic checkpoint is not clear. These functional and temporal divergences between the different CHK2 orthologs indicate that this protein kinase family displays an amazing degree of evolutionary plasticity (Meier, 2001). This plasticity is further supported when one compares C. elegans and Drosophila chk2. While the former has been shown to be required for meiotic chromosome pairing but is dispensable for typical DNA damage/replication checkpoint responses induced by gamma-irradiation or by HU, the results presented in the Masrouha study (2003) show that chk2 has no essential function in Drosophila meiosis. It is involved, however, in monitoring DSBs induced by gamma-rays, which places it closer to its vertebrate homologs and makes it an excellent invertebrate model for studying human chk2 function (Masrouha, 2003).

Calling some of these findings into doubt is a study by Abdu (2002), dealt with in more detail in the Effects of Mutation section. Abdu (2002) presents convincing evidence that chk2 transduces the DSB signal. The target of this signal is thought to be Vasa, which in turn regulates the translation of Gurken mRNA. Resolving this contradiction will require a number of follow-up experiments (Masrouha, 2003).

DNA damage-induced CHK2 activation compromises germline stem cell self-renewal and lineage differentiation

This study used germline stem cells (GSCs) in the Drosophila ovary to show that DNA damage retards stem cell self-renewal and lineage differentiation in a CHK2 kinase-dependent manner. Both heatshock-inducible endonuclease I-CreI expression and X-ray irradiation can efficiently introduce double-strand breaks in GSCs and their progeny, resulting in a rapid GSC loss and an accumulation of ill-differentiated GSC progeny. Elimination of CHK2 or its kinase activity can almost fully rescue the GSC loss and the progeny differentiation defect caused by DNA damage induced by I-CreI or X-ray. Surprisingly, checkpoint kinases ATM and ATR have distinct functions from CHK2 in GSCs in response to DNA damage. The reduction in BMP signaling and E-cadherin only makes limited contribution to DNA damage-induced GSC loss. Finally, DNA damage also decreases the expression of the master differentiation factor Bam in a CHK2-dependent manner, which helps explain the GSC progeny differentiation defect. Therefore, this study demonstrates, for the first time in vivo, that CHK2 kinase activation is required for the DNA damage-mediated disruption of adult stem cell self-renewal and lineage differentiation, and might also offer novel insight into how DNA damage causes tissue aging and cancer formation. It also demonstrates that inducible I-CreI is a convenient genetic system for studying DNA damage responses in stem cells (Ma, 2016).

Stem cells in adult tissues are responsible for generating new cells to combat against aging, and could also be cellular targets for tumor formation. Although aged stem cells have been shown to accumulate DNA damage, it remains largely unclear how DNA damage affects stem cell self-renewal and differentiation. A previous study has reported that upon weak irradiation apoptotic differentiated GSC progeny can prevent GSC loss by activating Tie-2 receptor tyrosine kinase signaling (Xing, 2015). This study shows that temporally introduced DNA double-stranded breaks cause premature GSC loss and slow down GSC progeny differentiation. Mechanistically, DNA damage causes GSC loss at least via two independent mechanisms, down-regulation of BMP signaling and E-cadherin-mediated GSC-niche adhesion as well as CHK2 activation- dependent GSC loss. In addition, CHK2 activation also decreases Bam protein expression by affecting its gene transcription and translation, slowing down CB differentiation into mitotic cysts and thus causing the accumulation of CB-like cells. Surprisingly, unlike in many somatic cell types, ATM, ATR, CHK1 and p53 do not work with CHK2 in DNA damage checkpoint control in Drosophila ovarian GSCs. Therefore, this study demonstrates that DNA damage-induced CHK2 activation causes premature GSC loss and also retards GSC progeny differentiation. The findings could also offer insight into how DNA damage affects stem cell-based tissue regeneration. In addition, this study also shows that the inducible I-CreI system is a convenient method for studying stem cell responses to transient DNA damage because it does not require any expensive irradiation equipment as the X-ray radiation does (Ma, 2016).

DNA damage normally leads to cell apoptosis to eliminate potential cancer- forming cells. This study, shows that transient DNA damage causes GSC loss not through apoptosis based on twopieces of experimental evidence: first, DNA-damaged GSCs are not positive for the cleaved Caspase-3, a widely used apoptosis marker; Second, forced expression of a known apoptosis inhibitor p35 does not show any rescue effect on DNA damage-induced GSC loss. Thus, DNA damage-induced GSC loss is likely due to self-renewal defects though the possibility could not be ruled out that other forms of cell death are responsible. p53 is known to be required for DNA damage-induced apoptosis from flies to humans. This study, however, demonstrates that p53 prevents the DNA damage-induced GSC loss. Vacating DNA-damaged GSCs from the niche via differentiation might allow their timely replacement and restoration of normal stem cell function. Therefore, the findings argue strongly that DNA damage primarily compromises self-renewal, thus causing GSC loss. Both niche-activated BMP signaling and E-cadherin-mediated cell adhesion are essential for GSC self-renewal. Consistent with the idea that DNA damage compromises GSC self-renewal, it significantly decreases BMP signaling activity and apical accumulation of E-cadherin in GSCs. Since constitutively active BMP signaling alone or in combination with E-cadherin overexpression can only moderately rescue GSC loss caused by DNA damage, it is concluded that decreased BMP signaling and apical E-cadherin accumulation might partly contribute to the DNA damage-induced GSC loss. Therefore, the findings suggest that DNA damage-mediated down-regulation of BMP signaling and E-cadherin-mediated adhesion only moderately contributes to the GSC loss (Ma, 2016).

DNA damage leads to checkpoint activation and cell cycle slowdown, thus giving more time for repairing DNA damage. In various cell types, ATM-CHK2 and ATR-CHK1 kinase pathways are responsible for DNA damage-induced checkpoint activation. During Drosophila meiosis, ATR, but not ATM, is required for checkpoint activity, indicating that ATM and ATR could have different functions in germ cells. Both ATR and CHK2 have been shown to be required for DNA damage-evoked checkpoint control in Drosophilagerm cells and embryonic cells, while CHK1 can control the entry into the anaphase of cell cycle in response to DNA damage, the G2-M checkpoint activation as well as the Drosophila midblastula transition (Ma, 2016).

This study has shown that these four checkpoint kinases function differently in GSCs. First, CHK2 is required for DNA damage-induced GSC loss, but is dispensable for normal GSC maintenance. Particularly, inactivation of its kinase activity can almost fully rescue DNA damage-induced GSC loss. Interestingly, inactivation of CHK2 function can also rescue the female germ cell defect caused by DNA damage in the mouse ovary, indicating that CHK2 function in DNA damage checkpoint activation is conserved at least in female germ cells. However, it remains unclear if CHK2 behaves similarly in mammalian stem cells in response to DNA damage. Second, ATM promotes GSC maintenance in the absence and presence of DNA damage. This is consistent with the finding that ATM is required for the maintenance of mouse male germline stem cells and hematopoietic stem cells. It will be interesting to investigate if ATM also prevents the oxidative stress in Drosophila GSCs as in mouse hematopoietic stem cells. Third, ATR is dispensable for normal GSC maintenance, but it protects GSCs in the presence of DNA damage. Although CHK2 and ATR behave similarly in DNA damage checkpoint control during meiosis and late germ cell development, they behave in an opposite way in GSCs in response to DNA damage. Finally, CHK1 is dispensable for GSC self-renewal in the absence and presence of DNA damage. Consistent with the current findings, the females homozygous for grp, encoding CHK1 in Drosophila, can still normally lay eggs, but those eggs could not develop normally. It will be of great interest in the future to figure out how CHK2 inactivation prevents DNA damage-induced GSC loss and how ATM and ATR inactivation promotes DNA damage-induced GSC loss at the molecular level. A further understanding of the functions of CHK2, ATM and ATR in stem cell response to DNA damage will help preserve aged stem cells and prevent their transformation into CSCs. DNA damage-evoked CHK2 activation retards GSC progeny differentiation by decreasing Bam expression at least at two levels This study has also revealed a novel mechanism of how DNA damage affects stem cell differentiation. Bam is a master differentiation regulator controlling GSC- CB and CB-cyst switches in the Drosophila ovary: CB-like single germ cells accumulate in bam mutant ovaries, whereas forced Bam expression sufficiently drives GSC differentiation. This study shows that DNA damage causes the accumulation of CB-like cells in a CHK2- dependent manner because CHK2 inactivation can fully rescue the germ cell differentiation defect caused by DNA damage. In addition, a heterozygous bam mutation can drastically enhance, and forced bam expression can completely repress, the DNA damage-induced germ cell differentiation defect, indicating that DNA damage disrupts Bam-dependent differentiation pathways. Consistently, Bam protein expression is significantly decreased in DNA damaged mitotic cysts in comparison with control ones. Interestingly, CHK2 inactivation can also fully restore Bam protein expression levels in the DNA-damaged mitotic cysts. Taken together, CHK2 activation is largely responsible forBam down-regulation in DNA damaged mitotic cysts, which can mechanistically explain the DNA damage-induced germ cell differentiation defect. It was further shown that DNA damage decreases Bam protein expression at least at two different levels. First, the bam transcription reporter bam-gfp was used to show that DNA damage decreases bamtranscription in CBs and mitotic cysts. Second, the posttranscriptional reporter Pnos-GFP-bam 3'UTR was generated to show that DNA damage decreases Bam protein expression via its 3'UTR in CBs and mitotic cysts at the level of translation. Although the detailed molecular mechanisms underlying regulation of Bam protein expression by DNA damage await future investigation, these findings demonstrate that DNA damage causes the GSC progeny differentiation defect by decreasing Bam protein expression at transcriptional and translational levels (Ma, 2016).

Taken together, these findings from Drosophila ovarian GSCs could offer important insight into how DNA damage affects stem cell-based tissue regeneration, and have also established Drosophila ovarian GSCs as a new paradigm for studying how DNA damage affects stem cell behavior at the molecular level. Because many stem cell regulatory strategies are conserved from Drosophilato mammals, what has been learned from this study should help understand how mammalian adult stem cells respond to DNA damage (Ma, 2016).

A robust transposon-endogenizing response from germline stem cells

The heavy occupancy of transposons in the genome implies that existing organisms have survived from multiple, independent rounds of transposon invasions. However, how and which host cell types survive the initial wave of transposon invasion has remained unclear. This study shows that the germline stem cells can initiate a robust adaptive response that rapidly endogenizes invading P element transposons by activating the DNA damage checkpoint and piRNA production. Temperature modulates the P element activity in germline stem cells, establishing a powerful tool to trigger transposon hyper-activation. Facing vigorous invasion, Drosophila first shut down oogenesis and induce selective apoptosis. Interestingly, a robust adaptive response occurs in ovarian stem cells through activation of the DNA damage checkpoint. Within 4 days, the hosts amplify P element-silencing piRNAs, repair DNA damage, subdue the transposon, and reinitiate oogenesis. It is proposed that this robust adaptive response can bestow upon organisms the ability to survive recurrent transposon invasions throughout evolution (Moon, 2018).

Considered as 'selfish DNA sequences,' transposons have heavily accumulated in the genome of nearly all organisms during evolution. Although capable of fueling genomic divergence, the transposon invasion process is disruptive to host cells and often severely impacts host fertility or even survival. Therefore, taming invading transposons is an essential and endless task for the host organism. In this study, by using P element invasion as a model, temperature shifting was established as a powerful tool to adjust the intensity of transposon invasion. By investigating the response from the Drosophila adult ovaries, in which P element activity and germ cell development can be measured in detail, a robust transposon-endogenizing mechanism from the germline stem cells was uncovered. Centered on the key DNA damage checkpoint component, Chk2, this robust adaptive response renders hosts the ability to permanently silence invading transposons within just 4 days (Moon, 2018).

GFP::Vasa mobilization assay shows that the P element actively hops in germline stem cells. Does the P element also mobilize in other ovarian cells? Since nurse cells are polyploid and the developing oocytes are transcriptionally inactive, the current assay could not faithfully monitor P element mobilization in them. However, previous study shows that nurse cells express the protein P-element somatic inhibitor (PSI), which can block intron removal of P element transcripts and lead to the production of inactive transposases. Therefore, it is unlikely that P elements mobilize within developing egg chambers. As a type of DNA transposon, which employs the cut-and-paste mechanism for transposition, P elements cannot directly increase their copy number through mobilization. Instead, the propagation is likely achieved via homologous repair from the sister DNA strand during S-phase of the cell cycle. Hence, to amplify themselves during Drosophila oogenesis, perhaps P elements evolved to preferentially mobilize in the dividing germline stem cells but not in the developing oocytes, which are under cell cycle arrest (Moon, 2018).

By investigating adult oogenesis of Drosophila, this study uncovered the Chk2-mediated adaptive response from germline stem cells upon P element transposon invasion (Moon, 2018).

Interestingly, it appears that arrested germ cells are not equally capable of taming transposons, and Chk2 activation promotes adaptation by eliminating the cells with lower competency. Several lines of evidence support the occurrence of selective cell elimination. First, a significant increase in cell death was detected once P elements became hyperactive after the temperature shift. Second, although GFP-negative egg chambers directly connected to germaria at 25°C were occasionally observed from the GFP::Vasa mobilization assay, no GFP-negative cells were detected in later stage egg chambers at any time points. This suggests that the germ cells that maintained high P element activity, and were presumably less competent to adapt, were eliminated at early stages of oogenesis. Third, the number of new P element insertion events declined to 44% in recovered ovaries after adaptation. This dramatic decline indicates that only the stem cells that had lower transposition rates survived the selection. Therefore, it is tempting to speculate that not all germ cells are created equal and that in addition to germarial arrest, the Chk2-mediated DNA break checkpoint also has a role in selecting the survivors from P element invasion and promoting adaptation (Moon, 2018).

In the surviving ovarian stem cells, Chk2-mediated oogenesis arrest provides a critical time window to propel piRNA generation from the paternally inherited clusters, initiating the amplification cycles for piRNA biogenesis. With at least two piRNA clusters containing P element sequences in the paternally inherited genome, invaded progeny are capable of generating P element-silencing piRNAs de novo. Although it is still unclear when the clusters become active during pre-adult development, it has been shown that the primordial germ cells in larval ovaries can already initiate de novo piRNA production. Consistently, low levels of piRNAs were detected corresponding to P element before adaptation. However, it appears that the amount of piRNAs produced at this stage is too scarce to silence invading P elements. Their activation results in sterility and triggers the Chk2-dependent acute adaptive response from germline stem cells. Subsequently, the Chk2-mediated arrest blocks differentiation, which would allow the newly produced P element-silencing piRNAs to quickly reach a concentration sufficient for Ping-Pong amplification. Finally, these newly produced piRNAs silence transposons at the post transcriptional level and also initiate transcriptional silencing (Moon, 2018).

Besides promoting piRNA production, the arrest period also allows germ cells to repair DNA lesions before reinitiating oogenesis, thereby preventing the proliferation of cells with DNA damage and defective differentiation. Having the ability to repair damage and endogenize invading transposons in germline stem cells ensures permanent restoration of robust oogenesis and protection of all daughter cells from transposon activation (Moon, 2018).

Transposon silencing in the Drosophila female germline is essential for genome stability in progeny embryos

The Piwi-interacting RNA pathway functions in transposon control in the germline of metazoans. The conserved RNA helicase Vasa is an essential Piwi-interacting RNA pathway component, but has additional important developmental functions. This study addresses the importance of Vasa-dependent transposon control in the Drosophila female germline and early embryos. Transient loss of vasa expression during early oogenesis leads to transposon up-regulation in supporting nurse cells of the fly egg-chamber. Elevated transposon levels have dramatic consequences, as de-repressed transposons accumulate in the oocyte where they cause DNA damage. Suppression of Chk2-mediated DNA damage signaling in vasa mutant females restores oogenesis and egg production. Damaged DNA and up-regulated transposons are transmitted from the mother to the embryos, which sustain severe nuclear defects and arrest development. These findings reveal that the Vasa-dependent protection against selfish genetic elements in the nuage of nurse cell is essential to prevent DNA damage-induced arrest of embryonic development (Durdevic, 2018).

This study shows that a transient loss of vas expression during early oogenesis leads to up-regulation of transposon levels and compromised viability of progeny embryos. The observed embryonic lethality is because of DNA DSBs and nuclear damage that arise as a consequence of the elevated levels of transposon mRNAs and proteins, which are transmitted from the mother to the progeny. This study thus demonstrates that transposon silencing in the nurse cells is essential to prevent maternal transmission of transposons and DNA damage, protecting the progeny from harmful transposon-mediated mutagenic effects (Durdevic, 2018).

The finding that suppression of Chk2-mediated DNA damage signaling in loss-of-function vas mutant flies restores oogenesis, and egg production demonstrates that Chk2 is epistatic to vas. However, hatching is severely impaired, because of the DNA damage sustained by the embryos. The defects displayed by vas, mnk double mutant embryos resembled those of PIWI (piwi, aub, and ago3) single and mnk; PIWI double mutant embryos. Earlier observation that inactivation of DNA damage signaling does not rescue the development of PIWI mutant embryos led to the assumption that PIWI proteins might have an essential role in early somatic development, independent of cell cycle checkpoint signaling. By tracing transposon protein and RNA levels and localization from the mother to the early embryos, it is suggested that, independent of Chk2 signaling, de-repressed transposons are responsible for nuclear damage and embryonic lethality. This study indicates that transposon insertions occur in the maternal genome where they cause DNA DSBs that together with transposon RNAs and proteins are passed on to the progeny embryos. Transposon activity and consequent DNA damage in the early syncytial embryo cause aberrant chromosome segregation, resulting in unequal distribution of the genetic material, nuclear damage and ultimately embryonic lethality. This study shows that early Drosophila embryos are defenseless against transposons and will succumb to their mobilization if the first line of protection against selfish genetic elements in the nuage of nurse cell fails (Durdevic, 2018).

A recent study showed that in p53 mutants, transposon RNAs are up-regulated and accumulate at the posterior pole of the oocyte, without deleterious effects on oogenesis or embryogenesis. It is possible that the absence of pole plasm in vas mutants results in the release of the transposon products and their ectopic accumulation in the oocyte. Localization of transposons to the germ plasm may restrict their activity to the future germline and protect the embryo soma from transposon activity. Transposon-mediated mutagenesis in the germline would produce genetic variability, a phenomenon thought to play a role in the environmental adaptation and evolution of species. It would therefore be of interest to determine the role of pole plasm in transposon control in the future (Durdevic, 2018).

Transposon up-regulation in the Drosophila female germline triggers a DNA damage-signaling cascade. In aub mutants, before their oogenesis arrest occurs, Chk2-mediated signaling leads to phosphorylation of Vasa, leading to impaired grk mRNA translation and embryonic axis specification. Considering the genetic interaction of vas and mnk (Chk2) and the fact that Vasa is phosphorylated in Chk2-dependent manner, it is tempting to speculate that phosphorylation of Vasa might stimulate piRNA biogenesis, reinforcing transposon silencing and thus minimizing transposon-induced DNA damage. The arrest of embryonic development as a first, and arrest of oogenesis as an ultimate response to DNA damage, thus, prevents the spreading of detrimental transposon-induced mutations to the next generation (Durdevic, 2018).

Nuclear lamina dysfunction triggers a germline stem cell checkpoint

LEM domain (LEM-D) proteins are conserved components of the nuclear lamina (NL) that contribute to stem cell maintenance through poorly understood mechanisms. The Drosophila emerin homolog Otefin (Ote) is required for maintenance of germline stem cells (GSCs) and gametogenesis. This study shows that ote mutants carry germ cell-specific changes in nuclear architecture that are linked to GSC loss. Strikingly, both GSC death and gametogenesis are rescued by inactivation of the DNA damage response (DDR) kinases, ATR and Chk2. Whereas the germline checkpoint draws from components of the DDR pathway, genetic and cytological features of the GSC checkpoint differ from the canonical pathway. Instead, structural deformation of the NL correlates with checkpoint activation. Despite remarkably normal oogenesis, rescued oocytes do not support embryogenesis. Taken together, these data suggest that NL dysfunction caused by Otefin loss triggers a GSC-specific checkpoint that contributes to maintenance of gamete quality (Barton, 2018).

The Drosophila emerin homolog Ote has an essential requirement for GSC survival and germ cell differentiation. This study shows that Ote loss causes GSC-specific nuclear defects that include a thickened and irregular NL and aggregation of heterochromatin. Strikingly, inactivation of two DDR kinases, ATR, and Chk2, rescues oogenesis in ote-/- females, a rescue that is cell-type specific. Genetic and cytological features of the checkpoint pathway present in ote mutant GSCs differ from those found in canonical DDR pathways. In addition, although heterochromatin coalesce is present, such defects by themselves do not trigger the checkpoint. Instead, the data correlate Chk2 activity with defects in NL structure, indicating that NL dysfunction is responsible for the activation of a checkpoint pathway in GSCs. Despite remarkably normal oogenesis, rescued oocytes do not support embryogenesis. It is suggested that this NL checkpoint pathway functions in GSCs to ensure that only healthy gametes are passed on to the next generation (Barton, 2018).

These studies identify ATR as the critical responder kinase and Chk2 as the critical transducer kinase in the NL checkpoint. This signaling axis differs from the canonical ATM-to-Chk2 or ATR-to-Chk1 axes. Several factors might contribute to the choice of responder and transducer kinase. First, species-specific constraints might exist. ATR, but not ATM is essential in mammals, whereas ATM, but not ATR, is essential in Drosophila. Second, cell-type specific distinctions are apparent. In both the fly and mouse germline, persistent meiotic double-strand breaks activate ATR and Chk2, implying that the ATR-Chk2 axis might be dominant in germ cells. Third, the nature of the trigger might influence which proteins are involved in signaling. For example in Drosophila, ATR and Chk2 are both required for the patterning defects caused by a failure to repair meiotic double-strand breaks. However, in DNA-damaged GSCs, ATR protects against GSC death, whereas Chk2 promotes it. These data suggest that in the case of the NL checkpoint, both ATR and Chk2 promote germ cell death. These studies add to growing evidence that the DDR pathway is modular, with selective use of pathway components in response to various cellular stresses (Barton, 2018).

Activated Chk2 is commonly associated with phosphorylation and activation of p53. Canonically, p53 activation leads to cell cycle arrest and apoptosis. These studies demonstrate that GSC loss persists in ote-/-; p53-/- females, suggesting that classical apoptosis is not responsible for GSC death. These findings are consistent with the absence of classic markers of apoptosis in ote mutants and observations that the p53 regulatory network differs in GSCs. Recently, an alternative cell death pathway was identified in spermatogonia of Drosophila testes. This pathway is responsible for spontaneous elimination of spermatogonia, using activated lysosomal and mitochondrial-associated factors. Additional studies are needed to determine whether ATR/Chk2-dependent GSC loss in ote mutants targets a similar pathway (Barton, 2018).

The data suggest that NL dysfunction is the primary cause of the ATR/Chk2 checkpoint in GSCs. Notably, NL defects are found only in affected cells and persist in rescued chk2-/-, ote-/- double mutants. Multiple mechanisms might connect nuclear architecture changes to ATR/Chk2 activation. First, altered NL structure might change genomic contacts needed for appropriate transcriptional regulation, with resulting gene expression changes prompting activation of the checkpoint. While global transcriptional changes during oogenesis were not observed, identification of transcriptional changes specific to GSCs or early germ cells would have been masked in these studies. Second, disruptions in the NL might affect trafficking of products between the nucleus and cytoplasm. Notably, a recent study identified large ribonucleoparticles (megaRNPs) that exit the nucleus by egress or budding through the inner and outer nuclear membranes, a process disrupted by defects in the NL. As such, it remains possible that the thickened NL in ote-/- GSCs disrupts large ribonucleoprotein (megaRNP) egress, leading to cellular stress and ATR/Chk2 activation. Third, defects in the NL structure might alter scaffolding of components of the DDR pathway, leading to checkpoint activation. Indeed, proteomic studies from Drosophila cultured somatic cells found that Ote interacted with proteins involved in DNA replication and repair, implying that Ote might assemble responder and transducer kinases complexes at the NL. However, observations that the ATR/Chk2-dependent checkpoint is GSC-specific, coupled with findings that meiotic double-strand breaks are repaired appropriately in chk2-/-, ote-/- germaria, argue against this model. Fourth, structural alteration in the nuclear envelope itself might trigger ATR/Chk2 activation. Indeed, emerging evidence implicates ATR as a general sensor of the structural integrity of cellular components. Further studies are needed to identify how NL dysfunction triggers the GSC-specific checkpoint (Barton, 2018).

Mutations in NL LEM-D proteins cause dystrophic diseases. Much evidence suggests that these diseases result from compromised stem cell populations that underlie the defects in tissue homeostasis. Indeed, a wealth of evidence links NL defects to increased DNA damage. The data are consistent with these reports, as it was shown that elevated accumulation of the commonly used DNA damage marker. However, this study found that phosphorylation of the H2A variant occurs downstream of Chk2, suggesting that accumulation of DNA damage in cells with a dysfunctional NL might be a consequence of cells dying, not the primary cause. These unexpected results suggest that caution is needed in linking causation of γH2Av/H2X accumulation to DNA damage and a failure in DNA repair. Indeed, recent studies of progerin-expressing cells indicated that the cellular defect in Hutchinson-Gilford progeria cells does not lie in defective DNA repair and DNA damage, even though these cells accumulate phosphorylated H2AX. These findings establish a new context for consideration of mechanisms of laminopathic diseases, suggesting that detrimental effects of NL dysfunction are primary events that are linked to checkpoint activation and stem cell loss (Barton, 2018).

GENE STRUCTURE

cDNA clone length - 2059 (short form)

Bases in 5' UTR - 270

Exons - 6

Bases in 3' UTR - 409

PROTEIN STRUCTURE

Amino Acids - 459 and 476 (long form)

Structural Domains

A phylogenetic analysis was performed with Loki and its most similar sequences. The Loki polypeptide sequence identified 48 sequences with considerable identity (>25%) in the NCBI databases using the BLAST algorithm. The most conserved sequence, the kinase domain, was then used to perform a multiple alignment that served to generate the phylogenetic tree. The neighbor-joining phylogeny produced a high percentage (94%) branched clade containing Loki, Chk2, Mek1p, Rad53p, Cds1, and Dun1p. In addition, Loki contains a FHA domain (52-112 aa) followed by a kinase domain (157-424 aa), which is the distinguishing feature of Rad53p, Mek1p, Dun1p, Cds1, and Chk2. It is thus likely to have a similar checkpoint function in flies as its homologs in their respective organisms (Masrouha, 2003).

date revised: 25 October 2023

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