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.
Project description:In this study we described the protein-protein interaction network of the Drosophila Speciation Core Complex by analysing the interactome of its subunit: HMR, LHR, NLP, BOH1 (CG33213), BOH2 (CG4788) and HP1a. For this purpose we performed Affinity Purification coupled with Mass Spectrometry (AP-MS) in D. melanogaster SL2 cells using as bait the two hybrid incompatibility proteins HMR (n = 8) and LHR (n = 4), as well as NLP (n = 3), BOH1(CG33213, n = 4), BOH2 (CG4788, n = 5) and HP1a (n = 4). Each bait was targeted with at least one antibody (rat anti-LHR 12F4, mouse anti-HP1a 2C09, rabbit anti-Nlp, anti-FLAG-M2 for FLAG-CG33213 and FLAG-CG4788), while HMR was targeted with three different antibodies (rat anti-HMR 2C10 and 12F1, anti-FLAG-M2 for FLAG-HMR). Individual replicates and antibodies used are listed in samples_table.
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:Chromatin insulators are DNA-protein complexes that can prevent the spread of repressive chromatin and block communication between enhancers and promoters to regulate gene expression. In Drosophila, the gypsy chromatin insulator complex consists of three core proteins: CP190, Su(Hw), and Mod(mdg4)67.2. These factors concentrate at nuclear foci termed insulator bodies, and their normal localization is correlated with proper insulator function. Here, we identified NURF301/E(bx), a nucleosome remodeling factor, as a novel regulator of gypsy insulator body localization through a high-throughput RNAi imaging screen. NURF301 promotes gypsy-dependent insulator barrier activity and physically interacts with gypsy insulator proteins. Using ChIP-seq, we found that NURF301 co-localizes with insulator proteins genome-wide, and NURF301 promotes chromatin association of Su(Hw) and CP190 at gypsy insulator binding sites. These effects correlate with NURF301-dependent nucleosome repositioning. At the same time, CP190 and Su(Hw) are also required for recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy binding site DNA, and NURF301 is required for proper nuclear positioning of gypsy binding sites. Our data provide new insights into how a nucleosome remodeling factor and insulator proteins cooperatively contribute to nuclear organization.
Project description:Although the majority of genomic binding sites for the insulator protein CTCF are constitutively occupied, a subset show variable occupancy. Such variable sites provide an opportunity to assess context-specific CTCF functions in gene regulation. Here we have identified a variably occupied CTCF site in the Ultrabithorax (Ubx) gene in Drosophila. This site is occupied in tissues where Ubx is active (third thoracic imaginal leg disc) but is not bound in tissues where the Ubx gene is repressed (first thoracic imaginal leg disc).
Project description:Insulators are considered as chromosome organizers. BEAF, one of the insulator proteins, is highly conserved in Drosophila speies but also limited to Drosophila spcies. BEAF associates with TSS of active genes. Comparative study of BEAF binding landscapes in four Drosophila species reveals BEAF association with gene pairs, and the results suggest the role of gain or loss of BEAF binding during the speciation of Drosophila species.
Project description:Insulators are considered as chromosome organizers. BEAF, one of the insulator proteins, is highly conserved in Drosophila speies but also limited to Drosophila spcies. BEAF associates with TSS of active genes. Comparative study of BEAF binding landscapes in four Drosophila species reveals BEAF association with gene pairs, and the results suggest the role of gain or loss of BEAF binding during the speciation of Drosophila species. DNA sample from ChIP for BEAF and input are collected for each of four Drosophila species
Project description:Although the majority of genomic binding sites for the insulator protein CTCF are constitutively occupied, a subset show variable occupancy. Such variable sites provide an opportunity to assess context-specific CTCF functions in gene regulation. Here we have identified a variably occupied CTCF site in the Ultrabithorax (Ubx) gene in Drosophila. This site is occupied in tissues where Ubx is active (third thoracic imaginal leg disc) but is not bound in tissues where the Ubx gene is repressed (first thoracic imaginal leg disc). Comparison of CTCF binding in T1 leg disc vs T3 leg disc in from 3rd instar larva