Drosophila melanogaster Set8 and L(3)mbt function in gene expression independently of histone H4 lysine 20 methylation.

Monomethylation of lysine 20 of histone H4 (H4K20me1) is catalyzed by Set8 and thought to play important roles in many aspects of genome function that are mediated by H4K20me binding proteins. We interrogated this model in a developing animal by comparing in parallel the transcriptomes of Set8 null , H4 K20R/A , and l(3)mbt mutant Drosophila melanogaster We found that the gene expression profiles of H4 K20A and H4 K20R larvae are markedly different than Set8 null larvae despite similar reductions in H4K20me1. Set8 null mutant cells have a severely disrupted transcriptome and fail to proliferate in vivo, but these phenotypes are not recapitulated by mutation of H4 K20 , indicating that the developmental defects of Set8 null animals are largely due to H4K20me1-independent effects on gene expression. Furthermore, the H4K20me1 binding protein L(3)mbt is recruited to the transcription start sites of most genes independently of H4K20me even though genes bound by L(3)mbt have high levels of H4K20me1. Moreover, both Set8 and L(3)mbt bind to purified H4K20R nucleosomes in vitro. We conclude that gene expression changes in Set8 null and H4 K20 mutants cannot be explained by loss of H4K20me1 or L(3)mbt binding to chromatin and therefore that H4K20me1 does not play a large role in gene expression.

Heterochromatic 3D genome organization is directed by HP1a- and H3K9-dependent and independent mechanisms.

Whether and how histone post-translational modifications and the proteins that bind them drive 3D genome organization remains unanswered. Here, we evaluate the contribution of H3K9-methylated constitutive heterochromatin to 3D genome organization in Drosophila tissues. We find that the predominant organizational feature of wild-type tissues is the segregation of euchromatic chromosome arms from heterochromatic pericentromeres. Reciprocal perturbation of HP1a⋅H3K9me binding, using a point mutation in the HP1a chromodomain or replacement of the replication-dependent histone H3 with H3K9R mutant histones, revealed that HP1a binding to methylated H3K9 in constitutive heterochromatin is required to limit contact frequency between pericentromeres and chromosome arms and regulate the distance between arm and pericentromeric regions. Surprisingly, the self-association of pericentromeric regions is largely preserved despite the loss of H3K9 methylation and HP1a occupancy. Thus, the HP1a⋅H3K9 interaction contributes to but does not solely drive the segregation of euchromatin and heterochromatin inside the nucleus.

Drosophila melanogaster Set8 and L(3)mbt function in gene expression independently of histone H4 lysine 20 methylation.

Mono-methylation of Lysine 20 of histone H4 (H4K20me1) is catalyzed by Set8 and thought to play important roles in many aspects of genome function that are mediated by H4K20me-binding proteins. We interrogated this model in a developing animal by comparing in parallel the transcriptomes of Set8 null , H4 K20R/A , and l(3)mbt mutant Drosophila melanogaster . We found that the gene expression profiles of H4 K20A and H4 K20R larvae are markedly different than Set8 null larvae despite similar reductions in H4K20me1. Set8 null mutant cells have a severely disrupted transcriptome and fail to proliferate in vivo , but these phenotypes are not recapitulated by mutation of H4 K20 indicating that the developmental defects of Set8 null animals are largely due to H4K20me1-independent effects on gene expression. Further, the H4K20me1 binding protein L(3)mbt is recruited to the transcription start sites of most genes independently of H4K20me even though genes bound by L(3)mbt have high levels of H4K20me1. Moreover, both Set8 and L(3)mbt bind to purified H4K20R nucleosomes in vitro. We conclude that gene expression changes in Set8 null and H4 K20 mutants cannot be explained by loss of H4K20me1 or L(3)mbt binding to chromatin, and therefore that H4K20me1 does not play a large role in gene expression.

Drosophila SUMM4 complex couples insulator function and DNA replication control.

Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early versus late in different cell types remain incompletely understood. Intercalary heterochromatin domains replicate very late in both diploid chromosomes of dividing cells and in endoreplicating polytene chromosomes where they are also underreplicated. Drosophila SNF2-related factor SUUR imparts locus-specific underreplication of polytene chromosomes. SUUR negatively regulates DNA replication fork progression; however, its mechanism of action remains obscure. Here, we developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates SUUR ATPase activity and is required for a normal spatiotemporal distribution of SUUR in vivo. SUUR and Mod(Mdg4)-67.2 together mediate the activities of gypsy insulator that prevent certain enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, SuUR or mod(mdg4) mutations reverse underreplication of intercalary heterochromatin. Thus, SUMM4 can impart late replication of intercalary heterochromatin by attenuating the progression of replication forks through euchromatin/heterochromatin boundaries. Our findings implicate a SNF2 family ATP-dependent motor protein SUUR in the insulator function, reveal that DNA replication can be delayed by a chromatin barrier, and uncover a critical role for architectural proteins in replication control. They suggest a mechanism for the establishment of late replication that does not depend on an asynchronous firing of late replication origins.

Inside cells, molecules of DNA provide the instructions needed to make proteins. Cells carefully maintain and repair their DNA, and typically make a complete copy of the genome before they divide to ensure that after division, each daughter cell has a full set. Within human, fly and other eukaryotic nuclei, DNA is packaged into structures known as chromosomes. Cells follow precisely controlled programs to replicate distinct regions of chromosomes at different times. To start copying a particular region, the cell machinery that replicates DNA binds to a sequence known as the origin of replication. It is thought that as-yet unknown cues from the cell may lead the replication machinery to bind to different origins of replication at different times. In some circumstances, cells make extra copies of their DNA without dividing. For example, many cells in the larvae of fruit flies contain hundreds of extra DNA copies to sustain their increased sizes. However, the entire genome is not copied during this process, so cells end up with more copies of some regions of the genome than others. A protein called SUUR is required for hindering the replication of the 'underrepresented' regions, but it is not clear how it works. To address this question, Andreyeva, Emelyanov et al. developed a new approach based on liquid chromatography and quantitative proteomics to identify the native form of SUUR in fruit flies. This revealed that SUUR exists as a stable complex with a protein called Mod(Mdg4), which is needed to recruit SUUR to the chromosomes. Further experiments suggested that SUUR and Mod(Mdg4) work together to bind to regions of DNA known as gypsy insulator elements, creating a physical barrier that hinders the replication machinery from accessing some parts of the genome. The findings of Andreyeva, Emelyanov et al. provide an alternative explanation for how individual cells may stagger the process of copying their DNA without relying on the replication machinery binding to various replication origins at different times. Rather, late replication timing may be instructed by an insulator-born delay of the progression of replication over particular genomic regions. This mechanism adds to the list of nuclear processes (chromosome partitioning, transcriptional regulation, etc.) that are known to be directed by insulators and associated architectural proteins.

Developmental timing of extreme temperature events (heat waves) disrupts host-parasitoid interactions.

When thermal tolerances differ between interacting species, extreme temperature events (heat waves) will alter the ecological outcomes. The parasitoid wasp Cotesia congregata suffers high mortality when reared throughout development at temperatures that are nonstressful for its host, Manduca sexta. However, the effects of short-term heat stress during parasitoid development are unknown in this host-parasitoid system.Here, we investigate how duration of exposure, daily maximum temperature, and the developmental timing of heat waves impact the performance of C. congregata and its host¸ M. sexta. We find that the developmental timing of short-term heat waves strongly determines parasitoid and host outcomes.Heat waves during parasitoid embryonic development resulted in complete wasp mortality and the production of giant, long-lived hosts. Heat waves during the 1st-instar had little effect on wasp success, whereas heat waves during the parasitoid's nutritionally and hormonally critical 2nd instar greatly reduced wasp emergence and eclosion. The temperature and duration of heat waves experienced early in development determined what proportion of hosts had complete parasitoid mortality and abnormal phenotypes.Our results suggest that the timing of extreme temperature events will be crucial to determining the ecological impacts on this host-parasitoid system. Discrepancies in thermal tolerance between interacting species and across development will have important ramifications on ecosystem responses to climate change.

Responses of Manduca sexta larvae to heat waves.

Climate change is increasing the frequency of heat waves and other extreme weather events experienced by organisms. How does the number and developmental timing of heat waves affect survival, growth and development of insects? Do heat waves early in development alter performance later in development? We addressed these questions using experimental heat waves with larvae of the tobacco hornworm, Manduca sexta. The experiments used diurnally fluctuating temperature treatments differing in the number (0-3) and developmental timing (early, middle and/or late in larval development) of heat waves, in which a single heat wave involved three consecutive days with a daily maximum temperature of 42°C. Survival to pupation declined with increasing number of heat waves. Multiple (but not single) heat waves significantly reduced development time and pupal mass; the best models for the data indicated that both the number and developmental timing of heat waves affected performance. In addition, heat waves earlier in development significantly reduced growth and development rates later in larval development. Our results illustrate how the frequency and developmental timing of sublethal heat waves can have important consequences for life history traits in insects.

Responses of Manduca sexta larvae to heat waves.

Climate change is increasing the frequency of heat waves and other extreme weather events experienced by organisms. How does the number and developmental timing of heat waves affect survival, growth and development of insects? Do heat waves early in development alter performance later in development? We addressed these questions using experimental heat waves with larvae of the tobacco hornworm, Manduca sexta. The experiments used diurnally fluctuating temperature treatments differing in the number (0-3) and developmental timing (early, middle and/or late in larval development) of heat waves, in which a single heat wave involved three consecutive days with a daily maximum temperature of 42°C. Survival to pupation declined with increasing number of heat waves. Multiple (but not single) heat waves significantly reduced development time and pupal mass; the best models for the data indicated that both the number and developmental timing of heat waves affected performance. In addition, heat waves earlier in development significantly reduced growth and development rates later in larval development. Our results illustrate how the frequency and developmental timing of sublethal heat waves can have important consequences for life history traits in insects.

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

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

Growth, stress, and acclimation responses to fluctuating temperatures in field and domesticated populations of Manduca sexta.

Diurnal fluctuations in temperature are ubiquitous in terrestrial environments, and insects and other ectotherms have evolved to tolerate or acclimate to such fluctuations. Few studies have examined whether ectotherms acclimate to diurnal temperature fluctuations, or how natural and domesticated populations differ in their responses to diurnal fluctuations. We examine how diurnally fluctuating temperatures during development affect growth, acclimation, and stress responses for two populations of Manduca sexta: a field population that typically experiences wide variation in mean and fluctuations in temperature, and a laboratory population that has been domesticated in nearly constant temperatures for more than 300 generations. Laboratory experiments showed that diurnal fluctuations throughout larval development reduced pupal mass for the laboratory but not the field population. The differing effects of diurnal fluctuations were greatest at higher mean temperature (30°C): Here diurnal fluctuations reduced pupal mass and increased pupal development time for the laboratory population, but had little effect for the field population. We also evaluated how mean and fluctuations in temperature during early larval development affected growth rate during the final larval instar as a function of test temperature. At an intermediate (25°C) mean temperature, both the laboratory and field population showed a positive acclimation response to diurnal fluctuations, in which subsequent growth rate was significantly higher at most test temperatures. In contrast at higher mean temperature (30°C), diurnal fluctuations significantly reduced subsequent growth rate at most test temperatures for the laboratory population, but not for the field population. These results suggest that during domestication in constant temperatures, the laboratory population has lost the capacity to tolerate or acclimate to high and fluctuating temperatures. Population differences in acclimation capacity in response to temperature fluctuations have not been previously demonstrated, but they may be important for understanding the evolution of reaction norms and performance curves.