TADs pair homologous chromosomes to promote interchromosomal gene regulation, bioRxiv, 2018-10-17

AbstractHomologous chromosomes colocalize to regulate gene expression in processes including genomic imprinting and X-inactivation, but the mechanisms driving these interactions are poorly understood. In Drosophila, homologous chromosomes pair throughout development, promoting an interchromosomal gene regulatory mechanism called transvection. Despite over a century of study, the molecular features that facilitate chromosome-wide pairing are unknown. The “button” model of pairing proposes that specific regions along chromosomes pair with a higher affinity than their surrounding regions, but only a handful of DNA elements that drive homologous pairing between chromosomes have been described. Here, we identify button loci interspersed across the fly genome that have the ability to pair with their homologous sequences. Buttons are characterized by topologically associated domains (TADs), which drive pairing with their endogenous loci from multiple locations in the genome. Fragments of TADs do not pair, suggesting a model in which combinations of elements interspersed along the full length of a TAD are required for pairing. Though DNA-binding insulator proteins are not associated with pairing, buttons are enriched for insulator cofactors, suggesting that these proteins may mediate higher order interactions between homologous TADs. Using a TAD spanning the spinelessd gene as a paradigm, we find that pairing is necessary but not sufficient for transvection. spineless pairing and transvection are cell-type-specific, suggesting that local buttoning and unbuttoning regulates transvection efficiency between cell types. Together, our data support a model in which specialized TADs button homologous chromosomes together to facilitate cell-type-specific interchromosomal gene regulation.

biorxiv molecular-biology 0-100-users 2018

Altered chromatin localization of hybrid lethality proteins in Drosophila, bioRxiv, 2018-10-09

AbstractUnderstanding hybrid incompatibilities is a fundamental pursuit in evolutionary genetics. In crosses between Drosophila melanogaster females and Drosophila simulans males, the interaction of at least three genes is necessary for hybrid male lethality Hmr mel, Lhr sim, and gfzf sim. All three hybrid incompatibility genes are chromatin associated factors. While HMR and LHR physically bind each other and function together in a single complex, the connection between either of these proteins and gfzf remains mysterious. Here, we investigate the allele specific chromatin binding patterns of gfzf. First, our cytological analyses show that there is little difference in protein localization of GFZF between the two species except at telomeric sequences. In particular, GFZF binds the telomeric retrotransposon repeat arrays, and the differential binding of GFZF at telomeres reflects the rapid changes in sequence composition at telomeres between D. melanogaster and D. simulans. Second, we investigate the patterns of GFZF and HMR co-localization and find that the two proteins do not normally co-localize in D. melanogaster. However, in inter-species hybrids, HMR shows extensive mis-localization to GFZF sites, and this altered localization requires the presence of gfzf sim. Third, we find by ChIP-Seq that over-expression of HMR and LHR within species is sufficient to cause HMR to mis-localize to GFZF binding sites, indicating that HMR has a natural low affinity for GFZF sites. Together, these studies provide the first insights into the different properties of gfzf between D. melanogaster and D. simulans as well as a molecular interaction between gfzf and Hmr in the form of altered protein localization.

biorxiv molecular-biology 0-100-users 2018

MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo, bioRxiv, 2018-09-24

ABSTRACTMitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell-types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially-localized 3XHA epitope-tag (“MITO-Tag”) for the fast isolation of mitochondria from cultured cells to now generate “MITO-Tag Mice.” Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology and our strategy should be generally applicable for studying other mammalian organelles in specific cell-types in vivo.

biorxiv molecular-biology 100-200-users 2018

 

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