The integrity of the HMR complex is necessary for centromeric binding and reproductive isolation in Drosophila

Postzygotic isolation by genomic conflict is a major cause for the formation of species. Despite its importance, the molecular mechanisms that result in the lethality of interspecies hybrids are still largely unclear. The genus Drosophila, which contains over 1600 different species, is one of the best characterized model systems to study these questions. We showed in the past that the expression levels of the two hybrid incompatibility factors Hmr and Lhr diverged in the two closely related Drosophila species, D. melanogaster and D. simulans, resulting in an increased level of both proteins in interspecies hybrids. The overexpression of the two proteins also leads to mitotic defects, a misregulation in the expression of transposable elements and decreased fertility in pure species. In this work, we describe a distinct six subunit protein complex containing HMR and LHR and analyse the effect of Hmr mutations on complex integrity and function. Our experiments suggest that HMR needs to bring together components of centromeric and pericentromeric chromatin to fulfil its physiological function and to cause hybrid male lethality

The Integrity of the HMR complex is necessary for centromeric binding and reproductive isolation in Drosophila

Postzygotic isolation by genomic conflict is a major cause for the formation of species. Despite its importance, the molecular mechanisms that result in the lethality of interspecies hybrids are still largely unclear. The genus Drosophila, which contains over 1600 different species, is one of the best characterized model systems to study these questions. We showed in the past that the expression levels of the two hybrid incompatibility factors Hmr and Lhr diverged in the two closely related Drosophila species, D. melanogaster and D. simulans, resulting in an increased level of both proteins in interspecies hybrids. The overexpression of the two proteins also leads to mitotic defects, a misregulation in the expression of transposable elements and decreased fertility in pure species. In this work, we describe a distinct six subunit protein complex containing HMR and LHR and analyse the effect of Hmr mutations on complex integrity and function. Our experiments suggest that HMR needs to bring together components of centromeric and pericentromeric chromatin to fulfil its physiological function and to cause hybrid male lethality. Author summary: A major cause of biological speciation is the sterility and/or lethality of hybrids. This hybrid lethality is thought to be the consequence of two incompatible genomes of the two different species. We used the fruit fly Drosophila melanogaster as a model system to isolate a defined protein complex, which mediates this hybrid lethality. Our data suggest that this complex containing six subunits has evolved in one Drosophila species (Drosophila melanogaster) to bring together components of centromeric and pericentromeric chromatin. We show that the integrity of the complex is necessary for its genomic binding patterns and its ability to maintain fertility in female Drosophila melanogaster flies. Hybrid males between Drosophila melanogaster and the very closely related species Drosophila simulans die because they contain elevated levels of this complex. These high levels result in mitotic defects and a misregulation in the expression of transposable elements in those hybrids. Our results show that mutations that interfere with the complex’s function in Drosophila melanogaster also fail to induce lethality in hybrids suggesting that its evolutionary acquired functions in one species induce lethality in interspecies hybrids

Identification of Drosophila centromere associated proteins by quantitative affinity purification-mass spectrometry

AbstractCentromeres of higher eukaryotes are epigenetically defined by the centromere specific histone H3 variant CENP-ACID. CENP-ACID builds the foundation for the assembly of a large network of proteins. In contrast to mammalian systems, the protein composition of Drosophila centromeres has not been comprehensively investigated. Here we describe the proteome of Drosophila melanogaster centromeres as analyzed by quantitative affinity purification-mass spectrometry (AP-MS). The AP-MS input chromatin material was prepared from D. melanogaster cell lines expressing CENP-ACID or H3.3 fused to EGFP as baits. Centromere chromatin enriched proteins were identified based on their relative abundance in CENP-ACID–GFP compared to H3.3-GFP or mock affinity-purifications. The analysis yielded 86 proteins specifically enriched in centromere chromatin preparations.The data accompanying the manuscript on this approach (Barth et al., 2015, Proteomics 14:2167-78, DOI: 10.1002/pmic.201400052) has been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000758

Developmental regulation of N-terminal H2B methylation in Drosophila melanogaster

Histone post-translational modifications play an important role in regulating chromatin structure and gene expression in vivo. Extensive studies investigated the post-translational modifications of the core histones H3 and H4 or the linker histone H1. Much less is known on the regulation of H2A and H2B modifications. Here, we show that a major modification of H2B in Drosophila melanogaster is the methylation of the N-terminal proline, which increases during fly development. Experiments performed in cultured cells revealed higher levels of H2B methylation when cells are dense, regardless of their cell cycle distribution. We identified dNTMT (CG1675) as the enzyme responsible for H2B methylation. We also found that the level of N-terminal methylation is regulated by dART8, an arginine methyltransferase that physically interacts with dNTMT and asymmetrically methylates H3R2. Our results demonstrate the existence of a complex containing two methyltransferases enzymes, which negatively influence each other’s activity

The Drosophila speciation factor HMR localizes to genomic insulator sites

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 better understand the molecular mechanisms that mediate HMR's function, we measured genome-wide localization of HMR in D. melanogaster tissue culture cells by chromatin immunoprecipitation. Interestingly, we find HMR localizing to genomic insulator sites that can be classified into two groups. One group belongs to gypsy insulators and another one borders HP1a bound regions at active genes. The transcription of the latter group genes is strongly affected in larvae and ovaries of Hmr mutant flies. Our data suggest a novel link between HMR and insulator proteins, a finding that implicates a potential role for genome organization in the formation of species

Als der Ruhm-erfüllte Greis, Der weiland ... Herr Johann Michael Thomä, ... durch Seinen plötzlichen, doch seligen Tod den 5. May, 1747. und durch die den 9. darauf erfolgte Beerdigung ein allgemeines Leidwesen erregte, Sollte die gewohnte Hochachtung annoch gegen den Wohlseligen Herrn Rath, wie auch gegen das vornehme Trauer-Haus seine schmerzlichen Empfindungen mitleidigst zu erkennen geben

ALS DER RUHM-ERFÜLLTE GREIS, DER WEILAND ... HERR JOHANN MICHAEL THOMÄ, ... DURCH SEINEN PLÖTZLICHEN, DOCH SELIGEN TOD DEN 5. MAY, 1747. UND DURCH DIE DEN 9. DARAUF ERFOLGTE BEERDIGUNG EIN ALLGEMEINES LEIDWESEN ERREGTE, SOLLTE DIE GEWOHNTE HOCHACHTUNG ANNOCH GEGEN DEN WOHLSELIGEN HERRN RATH, WIE AUCH GEGEN DAS VORNEHME TRAUER-HAUS SEINE SCHMERZLICHEN EMPFINDUNGEN MITLEIDIGST ZU ERKENNEN GEBEN Als der Ruhm-erfüllte Greis, Der weiland ... Herr Johann Michael Thomä, ... durch Seinen plötzlichen, doch seligen Tod den 5. May, 1747. und durch die den 9. darauf erfolgte Beerdigung ein allgemeines Leidwesen erregte, Sollte die gewohnte Hochachtung annoch gegen den Wohlseligen Herrn Rath, wie auch gegen das vornehme Trauer-Haus seine schmerzlichen Empfindungen mitleidigst zu erkennen geben ([1]