Project description:Hybrid incompatibility between Drosophila melanogaster and D. simulans is caused by a lethal interaction of the proteins encoded by the Hmr and Lhr genes. In D. melanogaster the loss of HMR results in mitotic defects, an increase in transcription of transposable elements and a deregulation of heterochromatic genes. To investigate the molecular mechanisms that mediate HMRs function, we measured genome-wide localization of HMR in D. melanogaster by chromatin immunoprecipitation. Interestingly, we find HMR localizing to genomic insulator sites that can be classified into two groups. One group that belongs to the gypsy class of insulators and another one that separates HP1a binding regions from active promoters. The activity of these promoters is strongly affected in Hmr mutant flies. Our data provide a novel link between HMR and insulator proteins and suggest a key role for genome organization in the formation of species. Overall design: Examination of HMR binding in Drosophila S2 cells. Experiments were verified with replicates, unrelated antibody (IgG control) and HMR RNAi knock-down. CP190 RNAi knock-down experiments in combination with HMR ChIP and H3 ChIP were further used to dissect HMR binding dependencies.
Project description:Hybrid seed lethality as a consequence of interspecies or interploidy hybridizations is a major mechanism of reproductive isolation in plants. This mechanism is manifested in the endosperm, a dosage sensitive tissue supporting embryo growth. Deregulated expression of imprinted genes like ADMETOS (ADM) underpin the interploidy hybridization barrier in Arabidopsis thaliana, however, the mechanisms of their action remained unknown. In this study we show that ADM interacts with the AT-hook domain protein AHL10 and the SET domain-containing SU(VAR)3-9 homolog SUVH9 and ectopically recruits the heterochromatic mark H3K9me2 to AT-rich transposable elements (TEs), causing deregulated expression of neighboring genes. Several hybrid incompatibility genes identified in Drosophila encode for heterochromatin-interacting proteins, which has led to the suggestion that hybrid incompatibilities evolve as consequence of interspecies divergence of selfish DNA elements and their regulation. Our data showing that imbalance of dosage-sensitive chromatin regulators underpins hybrid incompatibility in Arabidopsis strongly support this view, demonstrating that reproductive isolation as a consequence of epigenetic regulation of TEs is a conserved feature in animals and plants. Overall design: 54 genome-wide ChIP sequencing libraries and 6 genome-wide cytosine methylation libraries. All libraries derive from INTACT purified endosperm from seeds at 4 days after pollination from the cross of maternal 2x Col-0 with paternal 2x Col-0 and the mutant backgrounds osd1-1, osd1-1 adm-2, adm-2, 2x suvh2-2 suvh9-1, 4x suvh2-2 suvh9-, 2x ahl10-1, 4x ahl10-1, and osd1-2.
Project description:Hybridization of eggs and sperm from closely related species can give rise to genetic diversity, or can lead to embryo inviability due to incompatibility. Although central to evolution, the cellular and molecular mechanisms underlying postzygotic barriers that drive reproductive isolation and speciation remain largely unknown. Species of the African Clawed frog Xenopus provide an ideal system to study hybridization and genome evolution. Xenopus laevis is an allotetraploid with 36 chromosomes that arose through interspecific hybridization of diploid progenitors, whereas Xenopus tropicalis is a diploid with 20 chromosomes that diverged from a common ancestor ~48 million years ago. Differences in genome size between the two species are accompanied by organism size differences, and size scaling of the egg and subcellular structures such as nuclei and spindles formed in egg extracts. Nevertheless, early development transcriptional programs, gene expression patterns, and protein sequences are generally conserved. Interestingly, whereas the hybrid produced when X. laevis eggs are fertilized by X. tropicalis sperm (le×ts) is viable, the reverse hybrid (te×ls) dies prior to gastrulation. Here, we applied cell biological tools and high-throughput methods to study the mechanisms underlying hybrid inviability. We reveal that two specific X. laevis chromosomes are incompatible with the X. tropicalis cytoplasm and are mis-segregated during mitosis, leading to unbalanced gene expression at the maternal to zygotic transition, followed by cell-autonomous catastrophic embryo death. Overall design: Collect mRNA from whole embryos; three biological replicates were analyzed
Project description:Speciation involves the reproductive isolation of natural populations due to the sterility or lethality of their hybrids. However, the molecular basis of hybrid lethality and the evolutionary driving forces that provoke it, remain largely elusive. The hybrid male rescue (Hmr) and the lethal hybrid rescue (Lhr) genes serve as a model to study speciation in Drosophilids as their interaction causes lethality in male hybrid offspring. Here we show that HMR and LHR form a centromeric complex necessary for proper chromosome segregation. We find that the Hmr expression level is substantially higher in D. melanogaster whereas Lhr expression levels are increased in D. simulans. The resulting elevated amount of HMR/LHR complex in hybrids results in an extensive mislocalisation of the complex, an interference with the regulation of transposable elements and an impairment of cell proliferation. Our findings provide evidence for a major role of centromere divergence in the generation of biodiversity. Raw data were analysed using the MaxQuant 126.96.36.199 software package. Identified proteins were considered as interation partners if their MaxQuant iBAQ values displayed a greater than 16fold enrichment compared to control anti-FLAG purifications from Schneider cell nuclear extracts not expressing any FLAG-tagged protein.
Project description:In higher eukaryotes centromeres often coalesce into a large intranuclear domain called the chromocenter. Chromocenters are important for the organization of pericentric heterochromatin and a disturbance of their formation results in an upregulation of repetitive elements and causes defects in chromosome segregation. Mutations in the gene encoding for the centromere associated Drosophila speciation factor HMR show very similar phenotypes suggesting a role of HMR in chromocenter architecture and function. We performed confocal and super resolution microscopy as well as proximity based biotinylation experiments of HMR, centromeric protein dCenpA and heterochromatic protein HP1a to generate a molecular map of HMR, dCenpA and HP1a bound chromatin. Our work reveals an intricate internal structure of the centromeric chromatin region, which suggests a role of HMR in separating heterochromatin from centromeric chromatin.
Project description:Interspecific hybridization often induces epigenetic remodeling that leads to transposon activation, gene expression changes, and loss of imprinting. These genomic changes can be deleterious and lead to postzygotic hybrid incompatibility. In Arabidopsis, loss of genomic imprinting of PHERES1 and presumed failure of Polycomb Repressive Complex is partially responsible for seed inviability observed in A. thaliana X A. arenosa interspecific hybrids. We used this species pair to further analyze the relationship between parent-specific gene expression and postzygotic hybrid incompatibility using two A. thaliana ecotypes, Col-0 and C24, with differential seed survival. We found that maternal imprinting was perturbed for PHERES1, HDG3, and six other genes in both A. thaliana hybrids and paternal imprinting was lost for MEDEA as observed previously. Three classes of retroelements; Sadhu, Athila, and Copia, maintained proper repression patterns suggesting some regulatory mechanisms are not disrupted early in development. We propose that early genome remodeling and loss of imprinting of seed development genes induces lethality in both compatible and incompatible hybrids. Overall design: We examined gene expression in A. thaliana intraspecific hybrid crosses to determine normal patterns of maternal and paternal expression early in seed development.
Project description:Interspecific hybridization often induces epigenetic remodeling that leads to transposon activation, gene expression changes, and loss of imprinting. These genomic changes can be deleterious and lead to postzygotic hybrid incompatibility. In Arabidopsis, loss of genomic imprinting of PHERES1 and presumed failure of Polycomb Repressive Complex is partially responsible for seed inviability observed in A. thaliana X A. arenosa interspecific hybrids. We used this species pair to further analyze the relationship between parent-specific gene expression and postzygotic hybrid incompatibility using two A. thaliana ecotypes, Col-0 and C24, with differential seed survival. We found that maternal imprinting was perturbed for PHERES1, HDG3, and six other genes in both A. thaliana hybrids and paternal imprinting was lost for MEDEA as observed previously. Three classes of retroelements; Sadhu, Athila, and Copia, maintained proper repression patterns suggesting some regulatory mechanisms are not disrupted early in development. We propose that early genome remodeling and loss of imprinting of seed development genes induces lethality in both compatible and incompatible hybrids. We examined gene expression in A. thaliana intraspecific hybrid crosses to determine normal patterns of maternal and paternal expression early in seed development.
Project description:Background: The Dobzhansky-Muller (D-M) model of speciation by genic incompatibility is widely accepted as the primary cause of interspecific postzygotic isolation. Since the introduction of this model, there have been theoretical and experimental data supporting the existence of such incompatibilities. However, speciation genes have been largely elusive, with only a handful of candidate genes identified in a few organisms. The Saccharomyces sensu stricto yeasts have small genomes, can be easily cultured, and can mate interspecifically to produce sterile hybrids, are thus an ideal model for studying postzygotic isolation. Among them, only a single D-M pair has been found, between S. bayanus and S. cerevisiae, comprising the mitochondrially targeted product of a nuclear gene, AEP2, and a mitochondrially encoded locus, OLI1, the 5' region of whose transcript is bound by Aep2. Thus far, no D-M pair of nuclear genes has been identified between any sensu stricto yeasts. Methods: We report here the first detailed genome-wide analysis of rare F2 progeny from an otherwise sterile hybrid, and show that no classic D-M pairs of speciation genes exist between the nuclear genomes of the closely related yeasts S. cerevisiae and S. paradoxus. Instead, our analyses suggest that more complex interactions may be responsible for their post-zygotic separation. These interactions most likely involve multiple loci having weak effects, as there were multiple significant pairwise combinations of loci, with no single combination being completely excluded from the viable F2s. Conclusions: The lack of a nuclear encoded classic D-M pair between these two yeasts, yet the existence of multiple loci that may each exert a small effect through complex interactions, suggests that initial speciation events might not always be mediated by D-M pairs. An alternative explanation may be that "death by a thousand cuts" leads to speciation, whereby an accumulation of polymorphisms can lead to an incompatibility between the species "transcriptional and metabolic networks, with no single pair at least initially being responsible for the incompatibility. After such a speciation event, it is possible that one or more D-M pairs might subsequently arise following isolation. Genotypes for hybrids between S. cerevisiae and S. paradoxus. A genotyping experiment design type classifies an individual or group of individuals on the basis of alleles, haplotypes, SNP's. Overall design: Genotyping design
Project description:DNA methylation is a conserved epigenetic mark in plants and many animals. How parental alleles interact in progeny to influence the epigenome is poorly understood. We analyzed the DNA methylomes of Arabidopsis Col and C24 ecotypes, and their hybrid progeny. Hy- brids displayed nonadditive DNA methylation levels, termed meth- ylation interactions, throughout the genome. Approximately 2,500 methylation interactions occurred at regions where parental DNA methylation levels are similar, whereas almost 1,000 were at differ- entially methylated regions in parents. Methylation interactions were characterized by an abundance of 24-nt small interfering RNAs. Furthermore, dysfunction of the RNA-directed DNA methylation pathway abolished methylation interactions but did not affect the increased biomass observed in hybrid progeny. Methylation interac- tions correlated with altered genetic variation within the genome, suggesting that they may play a role in genome evolution. Whole genome bisulfite sequencing and small RNA sequencing of the wild type and nrpd1nrpe1 double mutant background of parent Col ,C24, the hybrid ColXC24 and C24XCol to explore the role of the RdDM pathway in DNA methylation interactions.
Project description:The hemera (hmr) mutant was identified as the first photomorphogenetic mutant with the combination of long hypocotyl and albino phenotypes in the light. Phytochrome-Interacting bHLH transcription Factors (PIFs), which are repressors of photomorphogenesis accumulate in darkness and are degraded in the light in a phytochrome-dependent manner. Two PIFs, PIF1 and PIF3 accumulated in the light in hmr mutants. In order to determine the gene expression of PIF-dependent genes in hmr mutants in the light, we have performed whole-genome expression analysis on two hmr mutants: a null allele, hmr-5; and a weak allele, hmr-22. Wild-type (Col-0) and hmr mutant seeds were surface-sterilized and plated on half-strength Murashige and Skoog (MS) growth medium without sucrose. The seeds were stratified in the dark at 4ºC for 4d. Seedlings were grown in constant red light (Rc, 10μmol/m2/s) at 21°C for 4d.