RESUMO
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.
Assuntos
Proteínas de Drosophila/genética , Isolamento Reprodutivo , Animais , Centrômero/metabolismo , Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila simulans/genética , Drosophila simulans/metabolismo , Genes Letais/genética , Especiação Genética , Hibridização Genética/genética , Reprodução/genéticaRESUMO
Most eukaryotic centromeres are located within heterochromatic regions. Paradoxically, heterochromatin can also antagonize de novo centromere formation, and some centromeres lack it altogether. In order to investigate the importance of heterochromatin at centromeres, we used epigenetic engineering of a synthetic alphoidtetO human artificial chromosome (HAC), to which chimeric proteins can be targeted. By tethering the JMJD2D demethylase (also known as KDM4D), we removed heterochromatin mark H3K9me3 (histone 3 lysine 9 trimethylation) specifically from the HAC centromere. This caused no short-term defects, but long-term tethering reduced HAC centromere protein levels and triggered HAC mis-segregation. However, centromeric CENP-A was maintained at a reduced level. Furthermore, HAC centromere function was compatible with an alternative low-H3K9me3, high-H3K27me3 chromatin signature, as long as residual levels of H3K9me3 remained. When JMJD2D was released from the HAC, H3K9me3 levels recovered over several days back to initial levels along with CENP-A and CENP-C centromere levels, and mitotic segregation fidelity. Our results suggest that a minimal level of heterochromatin is required to stabilize mitotic centromere function but not for maintaining centromere epigenetic memory, and that a homeostatic pathway maintains heterochromatin at centromeres.This article has an associated First Person interview with the first authors of the paper.
Assuntos
Cromossomos Artificiais Humanos , Centrômero/genética , Centrômero/metabolismo , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Segregação de Cromossomos/genética , Cromossomos Artificiais Humanos/genética , Cromossomos Artificiais Humanos/metabolismo , Epigênese Genética , Heterocromatina , Histonas/genética , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji , Cinetocoros/metabolismoRESUMO
During interphase centromeres often coalesce into a small number of chromocenters, which can be visualized as distinct, DAPI dense nuclear domains. Intact chromocenters play a major role in maintaining genome stability as they stabilize the transcriptionally silent state of repetitive DNA while ensuring centromere function. Despite its biological importance, relatively little is known about the molecular composition of the chromocenter or the processes that mediate chromocenter formation and maintenance. To provide a deeper molecular insight into the composition of the chromocenter and to demonstrate the usefulness of proximity-based biotinylation as a tool to investigate those questions, we performed super resolution microscopy and proximity-based biotinylation experiments of three distinct proteins associated with the chromocenter in Drosophila. Our work revealed an intricate internal architecture of the chromocenter suggesting a complex multilayered structure of this intranuclear domain.
Assuntos
Proteína Centromérica A/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Interfase/genética , Adenosina Trifosfatases/metabolismo , Animais , Biotinilação , Proteínas de Ciclo Celular/análise , Linhagem Celular , Núcleo Celular/metabolismo , Proteína Centromérica A/genética , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/análise , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Complexos Multiproteicos/metabolismo , Proteômica , Proteínas Recombinantes de Fusão/análise , CoesinasRESUMO
Background: Unicorn™ software on Äkta liquid chromatography instruments outputs chromatography profiles of purified biological macromolecules. While the plots generated by the instrument software are very helpful to inspect basic chromatogram properties, they lack a range of useful annotation, customization and export options. Methods: We use the R Shiny framework to build an interactive app that facilitates the interpretation of chromatograms and the generation of figures for publications. Results: The app allows users to fit a baseline, to highlight selected fractions and elution volumes inside or under the plot (e.g. those used for downstream biochemical/biophysical/structural analysis) and to zoom into the plot. The app is freely available at https://ChromatoShiny.bio.ed.ac.uk. Conclusions: It requires no programming experience, so we anticipate that it will enable chromatography users to create informative, annotated chromatogram plots quickly and simply.
RESUMO
Ki-67 is one of the most famous marker proteins used by histologists to identify proliferating cells. Indeed, over 30 000 articles referring to Ki-67 are listed on PubMed. Here, we review some of the current literature regarding the protein. Despite its clinical importance, our knowledge of the molecular biology and biochemistry of Ki-67 is far from complete, and its exact molecular function(s) remain enigmatic. Furthermore, reports describing Ki-67 function are often contradictory, and it has only recently become clear that this proliferation marker is itself dispensable for cell proliferation. We discuss the unusual organization of the protein and its mRNA and how they relate to various models for its function. In particular, we focus on ways in which the intrinsically disordered structure of Ki-67 might aid in the assembly of the still-mysterious mitotic chromosome periphery compartment by controlling liquid-liquid phase separation of nucleolar proteins and RNAs.