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In vitro reconstitution of chromatin architecture reveals principles of Drosophila 3D genome organization [MNase-Seq]

ABSTRACT: Metazoan genomes are organized by folding of the nucleosome fiber into loops and domains that support long-range regulatory interactions. Although cohesin-mediated loop extrusion and architectural DNA-binding proteins are central to current models of genome organization, how these mechanisms integrate to generate higher-order structure remains incompletely understood. Early Drosophila melanogaster embryogenesis provides a unique window into the emergence of chromatin architecture, as rapid syncytial nuclear divisions occur largely in the absence of transcription. However, probing the mechanisms underlying this primordial folding in vivo is technically challenging. Here, we establish an in vitro system that reconstitutes complex chromatin using extracts from syncytial embryos. Nucleosome mapping and Micro-C analyses reveal that long-range interactions, including loops and topologically associating domains (TADs), emerge spontaneously from soluble extract components. While some structures resemble those observed in early embryos, others represent latent interaction potentials that are constrained in vivo. Focusing on the eve locus, we find that TAD formation is incompatible with a simple loop extrusion model and instead requires direct pairing of boundary elements mediated by the insulator protein ‘Suppressor-of-hairy-wing’ Su(Hw). Together, our work demonstrates that key features of 3D genome organization can arise in a cell-free system and provides a tractable platform for mechanistic dissection of chromatin folding in Drosophila.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE317897 | GEO | 2026/09/14

REPOSITORIES: GEO

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In vitro reconstitution of chromatin architecture reveals principles of Drosophila 3D genome organization [MicroC]

Project description:Metazoan genomes are organized by folding of the nucleosome fiber into loops and domains that support long-range regulatory interactions. Although cohesin-mediated loop extrusion and architectural DNA-binding proteins are central to current models of genome organization, how these mechanisms integrate to generate higher-order structure remains incompletely understood. Early Drosophila melanogaster embryogenesis provides a unique window into the emergence of chromatin architecture, as rapid syncytial nuclear divisions occur largely in the absence of transcription. However, probing the mechanisms underlying this primordial folding in vivo is technically challenging. Here, we establish an in vitro system that reconstitutes complex chromatin using extracts from syncytial embryos. Nucleosome mapping and Micro-C analyses reveal that long-range interactions, including loops and topologically associating domains (TADs), emerge spontaneously from soluble extract components. While some structures resemble those observed in early embryos, others represent latent interaction potentials that are constrained in vivo. Focusing on the eve locus, we find that TAD formation is incompatible with a simple loop extrusion model and instead requires direct pairing of boundary elements mediated by the insulator protein ‘Suppressor-of-hairy-wing’ Su(Hw). Together, our work demonstrates that key features of 3D genome organization can arise in a cell-free system and provides a tractable platform for mechanistic dissection of chromatin folding in Drosophila.

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2015-10-23 | E-GEOD-74072 | biostudies-arrayexpress

Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes

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