grapes: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - grapes

Synonyms -

Cytological map position - 36A10--36A14

Function - cell cycle checkpoint protein

Keywords - cell cycle, response to DNA damage

Symbol - grp

FlyBase ID:FBgn0261278

Genetic map position -

Classification - Chk1 homolog - kinase

Cellular location - presumably nuclear



NCBI links: | Entrez Gene
Recent literature
Deneke, V.E., Melbinger, A., Vergassola, M. and Di Talia, S. (2016). Waves of Cdk1 activity in S phase synchronize the cell cycle in Drosophila embryos. Dev Cell 38: 399-412. PubMed ID: 27554859
Summary:
Embryos of most metazoans undergo rapid and synchronous cell cycles following fertilization. Using biosensors of Cdk1 and Chk1 activities, this study dissected the regulation of Cdk1 waves in the Drosophila syncytial blastoderm. Cdk1 waves were shown not to be controlled by the mitotic switch but by a double-negative feedback between Cdk1 and Chk1. S phase Cdk1 waves were shown to be fundamentally distinct and propagate as active trigger waves in an excitable medium, while mitotic Cdk1 waves propagate as passive phase waves. These findings show that in Drosophila embryos, Cdk1 positive feedback serves primarily to ensure the rapid onset of mitosis, while wave propagation is regulated by S phase events. 

Lefebvre, F. A., Benoit Bouvrette, L. P., Bergalet, J. and Lecuyer, E. (2017). Biochemical fractionation of time-resolved Drosophila embryos reveals similar transcriptomic alterations in replication checkpoint and histone mRNA processing mutants. J Mol Biol [Epub ahead of print]. PubMed ID: 28167048
Summary:
In higher eukaryotes, maternally provided gene products drive the initial stages of embryogenesis until the zygotic transcriptional program takes over, a developmental process called the midblastula transition (MBT). In addition to zygotic genome activation, the MBT involves alterations in cell-cycle length and the implementation of DNA damage/replication checkpoints that serve to monitor genome integrity. Previous work has shown that mutations affecting histone mRNA metabolism or DNA replication checkpoint factors severely impact developmental progression through the MBT, prompting this study to characterize and contrast the transcriptomic impact of these genetic perturbations. This study defined gene expression profiles that mark early embryogenesis in Drosophila through transcriptomic analyses of developmentally staged (early syncytial vs late blastoderm) and biochemically fractionated (nuclear vs cytoplasmic) wild type embryos. The transcriptomic profiles were compared of loss-of-function mutants of the Chk1/Grapes replication checkpoint kinase and the Stem Loop Binding Protein (SLBP), a key regulator of replication-dependent histone mRNAs. This analysis of RNA spatial and temporal distribution during embryogenesis offers new insights into the dynamics of early embryogenesis. In addition, it was found that grp and Slbp mutant embryos display profound and highly similar defects in gene expression, most strikingly in zygotic gene expression, compromising the transition from a maternal to a zygotic regulation of development.
Kizhedathu, A., Bagul, A. V. and Guha, A. (2018). Negative regulation of G2-M by ATR (mei-41)/Chk1(Grapes) facilitates tracheoblast growth and tracheal hypertrophy in Drosophila. Elife 7. PubMed ID: 29658881
Summary:
Imaginal progenitors in Drosophila are known to arrest in G2 during larval stages and proliferate thereafter. This study investigated the mechanism and implications of G2 arrest in progenitors of the adult thoracic tracheal epithelium (tracheoblasts). Tracheoblasts were shown to pause in G2 for ~48-56 h and grow in size over this period. Surprisingly, tracheoblasts arrested in G2 express drivers of G2-M like Cdc25/String (Stg). Mechanisms that prevent G2-M are also in place in this interval. Tracheoblasts activate Checkpoint Kinase 1/Grapes (Chk1/Grp) in an ATR/mei-41-dependent manner. Loss of ATR/Chk1 led to precocious mitotic entry ~24-32 h earlier. These divisions were apparently normal as there was no evidence of increased DNA damage or cell death. However, induction of precocious mitoses impaired growth of tracheoblasts and the tracheae they comprise. It is proposed that ATR/Chk1 negatively regulate G2-M in developing tracheoblasts and that G2 arrest facilitates cellular and hypertrophic organ growth.
BIOLOGICAL OVERVIEW

Treatment of cells with radiation causes the cell cycle to pause in the G2 phase, thus stopping cells from proceeding through mitosis. In addition, when the G2 phase is shortened for any reason, cells become sensitive to radiation. These observations have led to the concept of "checkpoints" - specific times within the cell cycle during which progression through the cycle can be delayed in response to either DNA damage or to the incompletion of prior cell cycle events, such as DNA replication. Grapes protein, a Drosophila homolog of the Chk1 protein of fission yeast, is involved in a developmentally regulated interphase checkpoint during the late syncytial divisions in early embryogenesis. This delay may function to allow both the completion of S phase and the transcription of genes that initiate the switch to zygotic control of embryogenesis (Sibon, 1997 and Fogarty, 1997). grapes mutation causes a metaphase arrest at nuclear cycle 13, early in embryogenesis; grapes mutants undergo cessation of cell cycles upon irradiation of syncytial embryos (Fogarty, 1994). This stage in Drosophila development is termed the midblastula transition, and takes place during blastoderm cellularization. It is marked by the initiation of zygotic transcription. Before describing Grapes in detail, the biochemical pathways for the regulation of G2-M checkpoint control will be described in some detail, since Grapes function in Drosophila is part of an conserved pathway for mitotic control that is present in many species, from yeast to mammals.

Many proteins active in checkpoint pathways have been identified by genetic analysis of yeast, and their mammalian counterparts have also been identified. Mitotic checkpoints require three distinct functions: a detection system to determine the change in DNA structure; a signal pathway to transmit this information; and an effector mechanism to interact with the cell cycle machinery (Carr, 1996). One effector, Chk1, is required in yeast for mitotic arrest after DNA damage. Chk1 is phosphorylated in response to DNA damage and has been shown in various studies to target Wee1 kinase or Cdc2 in the DNA damage checkpoint, Wee 1 kinase (see Drosophila wee) inhibits the cyclin cdc2 (see Drosophila cdc2) by phosphorylation on tyrosine 15, preventing the G2-M transition (O'Connell, 1997).

How is Chk1 phosphorylation regulated? What are the upstream components of DNA damage control? Experimental evidence points to the involvement of two families of proteins. The first family consists of BRCT domain proteins. The conserved BRCT domain, a protein interaction motif, was first described at the carboxyl terminus of the breast cancer protein BRCA1, a p53 binding protein (BP531), and the yeast cell cycle checkpoint protein RAD9. Because BRCT proteins are often quite large, they are almost certainly multifunctional proteins. Many of them could function as scaffolding proteins that assemble into multiple protein complexes at the site of DNA damage and many may not serve as the immediate sensors of DNA damage. Functional domains found in different BRCT domain proteins include DNA binding domains, DNA ligases, PCNA binding domains, RNA binding domains, ankyin repeats and ring fingers (Bork, 1997).

A second family associated with upstream components of checkpoint pathways includes the ATM protein deficient in ataxia-telangiectasia patients rendering them sensitive to ionizing radiation. Sharing structural homology with Atm is the Atr (for ataxia-telangiectaia- and rad3-related) protein and related yeast proteins Rad3/Mec1 and Tel1. In Drosophila, mei-41 encodes an Atr homolog and mei-41 flies have reduced meiotic recombination frequencies and aberrant recombination nodules (Hari, 1995). Atm protein is similar to a family of phosphatidylinositol 3-kinases. Members of this family, which includes the DNA-dependent protein kinase catalytic subunit, appear to act as sensors for defects in various cell-cycle transitions or aberrations in chromosomal mechanisms. Mec1 from S. cerevisiae, Rad3 from S. pombe, and Mei-41 from Drosophila appear to be structural and functional homologs that play important roles in mitotic and meiotic cell-cycle checkpoints and DNA repair. These three proteins play roles in G2 checkpoint control in mitotic cells and also play important roles in meiosis, suggesting important functional links between the two processes (Keegan, 1996 and references).

Proteins of both Rad3 and BRCT related families associate with meiotic chromosomes. Meiosis segregates a complete haploid set of chromosomes to each gamete, and this process requires the negotiation of a complex series of interdependent events. Homologous chromosomes must associate, recombine, synapse, and segregate to opposite poles at meiosis I. Although events are less clear in multicellular organisms, DNA double-stranded breaks have been linked to the initiation of meiotic chromosomal synapse in yeast. Atr is localized to the nuclei of primary spermatocytes, cells that are undergoing meiosis I. Both Atr and Atm proteins are present at pairing forks in the meiotic chromosomes of mice, however, they do not colocalize; instead they occupy complementary positions. Atr localizes along unsynapsed chromosome axes and Atm interacts with synapsed axes (Keegan, 1996). The presence of Chk1 along meiotic chromosomes is dependent on Atm (Flaggs, 1997). BRCA1, found in discrete, nuclear foci during S phase of the mitotic cycle is detected on unsynapsed elements of human synaptonemal complexes. BRCA1 associates with Rad51, a homolog of bacterial RecA. Rad51 is a member of a protein family known to mediate DNA strand-exchange functions leading to normal recombination. The association of Rad3 and BRCT family proteins with meiotic chromosomes and the fact that mutations in genes coding for these proteins are involved in defects in DNA damage-induced cell cycle responses, radiation hypersensitivity, and defective meiosis, suggests a role for these proteins in one or more signal transduction cascades involved in meiotic and mitotic checkpoint control (Scully, 1997).

grapes, coding for the Drosophila Chk1 homolog, was identified as a member of a set of maternal-effect mutations in which the affected embryos undergo abnormal divisions only after the nuclei have migrated to the cortex. Each of these mutations uniquely and specifically disrupts the cortical cytoskeleton. In grp mutants, during anaphase and telophase of nuclear cycle 12, the spindle deteriorates and the usually pronounced midbody (the microtubular structure separating chromosomes) never forms. Without the midbody, sister telophase nuclei snap-back and fuse. The fused telophase nuclei undergo an abbreviated nuclear cycle 13 interphase, fail to properly condense their chromosomes during prophase, and arrest in metaphase. X-irradiation experiments demonstrate that the grp induced arrest is not a direct consequence of processes specific to nuclear cycle 13. In normal embryos, X-irradiation induces a high error rate but no arrest. In contrast, X-irradiation of grp-derived embryos results in a metaphase arrest even during the pre-migration and initial cortical nuclear cycles, prior to nuclear cycle 13 (Fogarty, 1994).

During the final syncytial divisions before the midblastula transition (divisions 11-13), the duration of interphase increases from 6 to 14 minutes. Interphase 14 is prolonged, lasting over 60 minutes, during which time invaginating membranes surround the majority of nuclei to form a cellular blastoderm. Embryos deprived of grapes do not show a significant increase in interphase length during syncytial divisions 11-13. After mitosis 13, mutant embryos proceed through at least two extra syncytial cycles of spindle assembly and disassembly, and nuclear envelope breakdown and formation; cellularization is never observed. Thus grapes mutation blocks nearly all the changes in cell-cycle dynamics and embryo morphology associated with the midblastula transition (Sibon, 1997).

The changes in cell-cycle length are associated with changes in phosphorylation of the cyclin-dependent kinase cdc2. Termination of the rapid syncytial divisions is accompanied by accumulation of a low electorphoretic-mobility inhibitory tyrosine-phosphorylated form of Cdc2. In string mutants, this tyrosine-phosphorylated form of Cdc2 does not accumulate. It is known that the Cdc2-cyclin complex is activated by the Cdc25 phosphatase known in Drosophila as String, which removes the inhibitory tyrosine phosphates and thus drives the cell into mitosis. In Drosophila, a dramatic decrease in the level of String accompanies the increase in cell-cycle time at the midblastula transition. In grapes mutants, String protein levels do not decrease significantly during the later syncytial mitoses. As Chk1 is known to target wee1 kinase in yeast (O'Connell, 1997), which in turn targets cdc2 in yeast and Drosophila (Campbell, 1995), it is likely that Chk1 targets Wee kinase in Drosophila which in turn maintains cdc2 in an inactive state.

A simple DNA-replication checkpoint model is suggested for cell-cycle control at the midblastula transition. In his model, a free-running cell-cycle oscillator drives the rapid divisions that initiate embryogenesis. During the first 10 divisions, maternally supplied components of the DNA synthesis/initiation machinery are in excess, and S phase is completed before the oscillator triggers mitosis. During divisions 10-13, in contrast, a component of the maternal replication machinery is titrated by the increasing mass of nuclear DNA and becomes rate limiting. DNA synthesis therefore slows, and S phase cannot be completed before the free-running cell-cycle oscillator would normally trigger mitosis. A Grapes kinase-dependent checkpoint pathway is therefore activated and delays M phase until DNA synthesis is complete. This model is consistent with regulation of the midblastula transition by the nucleocytoplasmic ratio, and explains the absence of a G2 phase and the increase in S phase during the later syncytial mitoses, and accounts for the cell-cycle timing defects observed in grp mutant embryos (Sibon, 1997). This model does not take into account the likelihood that GRP mRNA is actively degraded at the midblastula transition, suggesting that additional regulation of transcript level is involved in the disappearance of Grapes during the transition (Fogarty, 1997).

grp (chk1) replication-checkpoint mutations and DNA damage trigger a Chk2-dependent block at the Drosophila midblastula transition

The 13 syncytial cleavage divisions that initiate Drosophila embryogenesis are under maternal genetic control. The switch to zygotic regulation of development at the midblastula transition (MBT) follows mitosis 13, when the cleavage divisions terminate, transcription increases and the blastoderm cellularizes. Embryos mutant for grp, which encodes Checkpoint kinase 1 (Chk1), are DNA-replication-checkpoint defective and fail to cellularize, gastrulate or to initiate high-level zygotic transcription at the MBT. The mnk (also known as loki) gene encodes Checkpoint kinase 2 (Chk2), which functions in DNA-damage signal transduction. mnk grp double-mutant embryos are replication-checkpoint defective but cellularize, gastrulate and activate high levels of zygotic gene expression. grp mutant embryos accumulate DNA double-strand breaks and DNA-damaging agents induce a mnk-dependent block to cellularization and zygotic gene expression. It is concluded that the DNA-replication checkpoint maintains genome integrity during the cleavage divisions, and that checkpoint mutations lead to DNA damage that induces a novel Chk2-dependent block at the MBT (Takada, 2007).

Studies in lower eukaryotes initially defined checkpoints as< non-essential pathways that delay cell cycle progression in response to external stress. Subsequently, checkpoint mutations in higher eukaryotes were found to induce developmental defects and embryonic lethality. In Drosophila, the DNA-replication checkpoint is required to delay the cell cycle during the late cleavage stage, and checkpoint mutants subsequently fail to cellularize or activate zygotic gene expression at the MBT. These findings suggested that the replication checkpoint has a direct role in metazoan developmental. Alternatively, the observed developmental defects could be an indirect consequence of checkpoint failure (Takada, 2007).

A null mutation in mnk, which encodes the conserved DNA-damage signaling kinase Chk2, efficiently suppresses the cellularization and zygotic gene-activation defects in grp, but does not restore wild-type cell cycle timing or replication-checkpoint function. It is therefore concluded that progression through the Drosophila MBT does not directly require Chk1 or checkpoint-dependent cell cycle delays. Instead, the data indicate that the essential function for the replication checkpoint is to prevent DNA damage during the syncytial blastoderm divisions, which triggers a Chk2-dependent block to zygotic gene activation and cellularization. Supporting this proposal, DNA-damaging agents trigger a Chk2-dependent block to cellularization and zygotic gene activation, and grp mutations accumulate DNA double-strand breaks. Chk2 is likely to have multiple targets during this developmental response to DNA damage; these targets may include transcription factors that control the expression of genes implicated in cell cycle control and cellularization (Takada, 2007).

Embryos mutant for grp or mei-41 lack a functional replication checkpoint and progress into mitosis prior to S-phase completion, triggering defects in γ-Tubulin localization and microtubule nucleation. These mitotic defects are suppressed by mnk, raising the possibility that mnk suppresses the grp mutant developmental block at the MBT by restoring mitotic function. However, mnk does not suppress the chromosome-segregation defects associated with grp mutants. More significantly, inducing DNA damage following the final syncytial blastoderm division triggers a Chk2-dependent block to cellularization. DNA damage can therefore induce a Chk2-dependent developmental block that is distinct from the damage and Chk2-dependent block to mitosis (Takada, 2007).

The studies outlined here support a simple model in which the developmental arrest associated with grp mutations results from defects in the established function for this kinase in cell cycle control. The early cleavage-stage divisions have a simplified S-phase/M-phase cell cycle, and it is proposed that the crucial function of Chk1 is to delay mitosis until DNA replication is complete. In grp mutants, progression into mitosis before replication is complete leads to DNA damage, which activates a Chk2-dependent block to developmental progression. Intriguingly, disrupting Chk1 function also leads to early embryonic lethality in frogs, mice and worms. Chk1 knockdown in Xenopus and Chk1 (also known as Chek1 - Mouse Genome Informatics) mutations in mouse lead to apoptotic death of the embryo, consistent with a DNA-damage response. It is therefore speculated that Chk1 has a conserved function in maintaining genome integrity during the cleavage stage, and that the early embryonic lethality in checkpoint mutants is a consequence of DNA-damage signaling (Takada, 2007).

Histone concentration regulates the cell cycle and transcription in early development

The early embryos of many animals including flies, fish, and frogs have unusually rapid cell cycles and delayed onset of transcription. These divisions are dependent on maternally supplied RNAs and proteins including histones. Previous work suggests that the pool size of maternally provided histones can alter the timing of zygotic genome activation (ZGA) in frogs and fish. This study examine the effects of under and overexpression of maternal histones in Drosophila embryogenesis. Decreasing histone concentration advances zygotic transcription, cell cycle elongation, Chk1 activation, and gastrulation. Conversely, increasing histone concentration delays transcription and results in an additional nuclear cycle before gastrulation. Numerous zygotic transcripts are sensitive to histone concentration, and the promoters of histone sensitive genes are associated with specific chromatin features linked to increased histone turnover. These include enrichment of the pioneer transcription factor Zelda and lack of SIN3A and associated histone deacetylases. These findings uncover a critical regulatory role for histone concentrations in ZGA of Drosophila (Wilky, 2019).

To understand the effects of histone concentration on the MBT maternally supplied histones were reduced by downregulating the gene encoding a crucial histone regulator, Stem-Loop Binding Protein (Slbp) via maternally driven RNAi. Under these conditions, histone H2B was reduced by ~50% and H3 by ~60% at the MBT. Approximately 50% of embryos laid by Slbp RNAi mothers (henceforth Slbp embryos) that form a successful blastoderm do not undergo the final division and attempt gastrulation in NC13. Another ~30% exhibit an intermediate phenotype of partial arrest, with only part of the embryo entering NC14. A minority of Slbp embryos begin gastrulation with all nuclei in NC14. NC12 duration was predictive of NC13 arrest, with NC12 being an average of ~5min longer in Slbp embryos that went on to arrest compared with those that did not arrest (Wilky, 2019).

Cellularization was first detected in wild-type (WT) embryos ~20 min into NC14. Partially arrested Slbp embryos also began cellularization ~20 min into NC14, with nuclei that arrested in NC13 waiting until the remainder of the embryo had entered NC14 to cellularize. Fully arrested embryos began cellularization ~20min into NC13, initiating cellularization one cycle early and ~20min earlier in overall developmental time than WT. Despite their reduced cell number, these embryos form mitotic domains and gastrulate without obvious defects, however they die before hatching (Wilky, 2019).

To examine the effects of increased histone concentration on developmental timing cell cycle progression was monitored in embryos from abnormal oocyte (abo) mutant mothers (henceforth abo embryos). abo is a histone locus-specific transcription factor, the knockdown of which increases the production of replication-coupled histones, particularly H2A and H2B (Berloco, 2001). abo increased H2B by ~90%, whereas total (combined replication-coupled and replication-independent) H3 was not affected in NC14 embryos. Approximately 60% of abo embryos displayed fertilization defects or catastrophic early nuclear divisions. Of abo embryos that formed a functioning blastoderm, ~6% underwent a complete extra nuclear division before gastrulating in NC15, whereas ~4% underwent a partial extra nuclear division. Embryos from abo mothers that completed total extra divisions had faster NC14s in which they did not cellularize and spent 40-60 min in NC15 before gastrulating. This suggests an alteration of the normal transcription-dependent developmental program. In some cases, the cell cycle program and transcriptional program may be decoupled, evidenced by the fact that some abo embryos attempted to gastrulate while still in the process of division. abo embryos that underwent extra divisions exhibited a range of gastrulation defects including expanded mitotic domains and ectopic furrow formation (Wilky, 2019).

Since alterations in histone levels can both decrease and increase the number of divisions before cell cycle slowing, it was reasoned that histone levels might affect activation of checkpoint kinase 1 (Chk1, also known as grp), which is required for cell cycle slowing at the MBT. To test this, a fluorescent biosensor of Chk1 activity was crosses into the Slbp background. Even in Slbp embryos that did not undergo early gastrulation, Chk1 activity was higher than in WT, consistent with the lengthened cell cycle. This result indicates that the observed cell cycle phenotypes in the histone-manipulated embryos are likely mediated through changes in Chk1 activity (Wilky, 2019).

As cellularization and gastrulation require zygotic transcription, it was suspected that embryos with altered development likely have altered gene expression. Single-embryo RNA-seq was performed on staged Slbp embryos that remained in NC13 for more than 30 min. These were compared with either NC-matched (NC13) or time-matched (NC14) WT embryos. To control for maternal effects of Slbp RNAi, pre-blastoderm stage WT and Slbp embryos were compared. The Slbp embryos underwent ZGA one NC earlier than WT. ~5000 genes were identified that were differentially expressed between Slbp and WT NC13, with ~60% being upregulated. The upregulated genes have largely previously been identified as new zygotic transcripts, including cell cycle regulators such as fruhstart (frs, also known as Z600) and signaling molecules such as four-jointed (fj), whereas the downregulated genes are enriched for maternally degraded transcripts. This is thought to represent a coherent change in ZGA timing instead of global transcription dysregulation, as 98% of the genes that are overexpressed in Slbp are expressed before the end of NC14 in the control or previously published datasets. Indeed, the transcriptomes of histone-depleted embryos that stopped in NC13 are more similar to WT NC14 than WT NC13, which suggests a role for cell cycle elongation in ZGA. Nonetheless, ~1500 genes are differentially expressed between Slbp NC13 and WT NC14 without accounting for differences in ploidy. Of these, the majority of the ~1000 overexpressed genes are again associated with zygotic transcription, and downregulated genes associated with maternal products. Thus, ZGA is even further accelerated in the histone knockdown than can be explained by purely time alone (Wilky, 2019).

As ZGA is accelerated by histone depletion, it was asked whether ZGA would be delayed in the histone overexpression mutant. RNA-seq was performed on pools of abo and WT embryos collected 15-30 min into NC14. >1000 genes were identified that were differentially expressed between abo and WT, with approximately equal numbers of genes up- and down-regulated. As expected, the downregulated genes in abo were enriched for previously identified zygotically expressed transcripts, and upregulated transcripts were enriched for maternally deposited genes. Thus, histone overexpression delays the onset of ZGA (Wilky, 2019).

Zygotic genes, the transcription of which is upregulated by histone depletion and downregulated by histone overexpression, contain many important developmental and cell cycle regulators including: frs, hairy (h), fushi tarazu (ftz) and odd-skipped (odd). Conversely, the maternally degraded transcripts that are destabilized by histone depletion and stabilized by histone overexpression include several cell cycle regulators such as Cyclin B (CycB), string (stg, also known as Cdc25string) and Myt1. Therefore, histone concentration can modulate the expression and stability of specific cell cycle regulators, which may contribute to the onset of MBT (Wilky, 2019).

Since histone concentration has previously been implicated in sensing the nuclear-cytoplasmic (N/C) ratio (Amodeo, 2015), this study compared the genes that are changed in both the histone under- and overexpression embryos with those that had previously been found to be dependent on either the N/C ratio or developmental time (Lu, 2009). Both previously identified N/C ratio-dependent and time-dependent genes (Lu, 2009) followed the same general trends as the total zygotic gene sets, indicating that histone availability cannot explain these previous classifications (Wilky, 2019).

Next, attempts were made to disentangle the effects of cell cycle length from transcription in the histone overexpression mutant. Single-embryo time-course RNA-seq was performed on abo and WT embryos collected 3 min before mitosis of NC10-NC13 and 3 min into NC14. In addition, unfertilized embryos (henceforth NC0) of both genotypes were collected to control for differences in maternal contribution. Even with a stringent selection process that accounted for cell cycle time and embryo health, a small set of robustly upregulated (179) and downregulated (260) genes was detected across NC10-NC14. Of the newly transcribed genes, 111 genes were detected with delayed transcription, including frs and only 37 that are upregulated. These results were confirmed using qPCR. When compared with previous datasets, zygotic genes tend to be underexpressed, as was the case for the pooled abo dataset; however, the majority of these enrichments are not statistically significant. Nonetheless the majority of these underexpressed genes are expressed during NC14 in WT. This geneset, in combination with the time-matched Slbp comparison, enables further examination of the chromatin features that underlie histone sensitivity for transcription independent of cell cycle changes (Wilky, 2019).

To identify chromatin features associated with histone sensitivity, the presence was compared of 143 modENCODE chromatin signals near the transcriptional start site (TSS±500 bp) of genes whose expression was altered by changes in histone concentration independent of cell cycle time. A clear pattern was found of unique chromatin features for the histone-sensitive genes, compared with all newly transcribed genes, that was highly similar between the histone over- and underexpression experiments. The pioneer transcription factor Zld, known to be important for nucleosome eviction during ZGA, was enriched in the promoters of histone-sensitive genes. Insulator proteins such as BEAF-32 and CP190 were depleted in histone-sensitive genes. Promoters of histone-sensitive genes also show a strong reduction for SIN3A, a transcriptional repressor associated with cell cycle regulation. SIN3A is known to recruit HDACs to TSSs, and almost all HDACs also show significant de-enrichment at the TSSs of histone-sensitive genes. Taken together, these marks make up a unique chromatin signature that may sensitize a locus to changes in histone concentration, as is likely for pioneer factors such as Zld. Other aspects of this signature may indicate that these genes are subsequently subject to later developmental regulation, as indicated by H3K4me3 and H3K27me3 (Wilky, 2019).

This study has demonstrated that histone concentration regulates the timing of the MBT in Drosophila, resulting in both early gastrulation and extra pre-MBT divisions from histone reduction and increase, respectively. Histone concentration also regulates ZGA. Thousands of genes are prematurely transcribed in histone-depleted embryos and hundreds of genes are delayed in histone-overexpressing embryos. The majority of these genes appear to be downstream of changes in cell cycle duration, suggesting a model in which histones directly regulate cell cycle progression. In other cell types, histone loss halts the cell cycle via accumulation of DNA damage and stalled replication forks. In the early embryo, changes in histone availability may similarly create replication stress to directly or indirectly activate Chk1 as this study has shown. In turn, Chk1 would inhibit Stg and/or Twine to slow the cell cycle. This mechanism is supported by previous observations that loss of zygotic histones causes the downregulation of Stg in the first post-MBT cell cycle. In this case, the observed transcriptional changes would be independent or downstream of the altered cell cycle (Wilky, 2019).

Alternatively, direct changes in transcription downstream of histone availability may feed into the cell cycle. In bulk, histone-sensitive transcripts might underlie the replication stress that has been previously proposed to slow the cell cycle at the MBT. Consistent with this, the cell cycle lengthening and partial arrest phenotypes observed in mutant RNA Pol II embryos occur at a similar frequency to those observed as the result of histone depletion. Another possibility is that specific histone-sensitive transcripts are responsible for cell cycle elongation. One promising candidate for a histone-sensitive cell cycle regulator is the N/C ratio-sensitive CDK inhibitor frs, as zygotic transcription of frs plays a crucial role in stopping the cell cycle at the MBT. In contrast, tribbles, an N/C ratio-dependent inhibitor of Twine that has also been implicated in cell cycle slowing, does not show a consistent response between histone perturbations. In this previously proposed model, maternal histone stores may compete with pioneer transcription factors to set the timing of transcription initiation. Indeed, the central Drosophila pioneer transcription factor Zld is enriched at the promoters of histone-sensitive genes. Moreover, this study has identified a broader set of chromatin features that may sensitize individual loci to changes in histone concentrations. These include less obvious candidates for global early transcriptional regulators, such as SIN3A, HDACs and class I insulator proteins that may protect transcripts from changes in histone concentrations. This work highlights the importance of histone concentration in regulating the timing of MBT and provides evidence that promoters of histone-sensitive genes possess a unique chromatin signature. However, future studies will be required to isolate the specific downstream effectors that respond to changes in histone concentrations in the early embryo (Wilky, 2019).


GENE STRUCTURE

Sequencing of additional grp cDNAs reveals differences in the 5' untranslated regions. These differences are presumably due to alternative splicing or alternative promoter usage at the grp locus, but do not affect the translation product (Fogarty, 1997).

cDNA clone length - 2567 bases

Bases in 5' UTR - 737

Bases in 3' UTR - 290


PROTEIN STRUCTURE

Amino Acids - 513

Structural Domains

Grapes has extensive structural similarity to the catalytic domain of the serine/threonine family of protein kinases. One member of this family, the Chk1/Rad27 DNA damage checkpoint kinase of S. pombe has significant homology to the predicted Grp protein, extending beyond the kinase domain. Over a contiguous 97 amino acid region within the kinase domain, the proteins exhibit 59% identity and 82% conservation. Over the entire protein sequence, the two proteins exhibit 26% amino acid identity. Outside the kinase domain, five regions, ranging in length from 9 to 24 amino acids, exhibit a greater than 30% amino acid sequence identity (Fogarty, 1997).


grapes: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 3 January 2020

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.