Unknown,Transcriptomics,Genomics,Proteomics

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Chromatin de-compaction by the nucleosomal binding protein HMGN5 impairs nuclear sturdiness.

ABSTRACT: In most metazoan nuclei, heterochromatin is located at the nuclear periphery in contact with the nuclear lamina, which provides mechanical stability to the nucleus. We show that in cultured cells, chromatin de-compaction by the nucleosome binding protein HMGN5 decreases the sturdiness, elasticity, and rigidity of the nucleus. Mice overexpressing HMGN5, either globally or only in the heart, are normal at birth but develop hypertrophic heart with large cardiomyoctyes, deformed nuclei and disrupted lamina, and die of cardiac malfunction. Chromatin de-compaction is seen in cardiomyocytes of newborn mice but misshaped nuclei with disrupted lamina are seen only in adult cardiomyocytes, suggesting that loss of heterochromatin diminishes the ability of the nucleus to withstand the mechanical forces of the contracting heart. Thus, heterochromatin enhances the ability of the nuclear lamina to maintain the sturdiness and shape of the eukaryotic nucleus; a structural role for chromatin that is distinct from its genetic functions. The full length mouse Hmgn5 cDNA, containing the entire 5’ UTR and FLAG tag on the 3’ end was amplified by PCR and inserted between BglII and XhoI sites of the pCALL vector (Novak et al., 2000). This vector contains a floxed LacZ cDNA driven by the CAG (a hybrid CMV enhancer and chicken beta–actin) promoter which is highly active in early mouse embryos. The vector was linearized by NotI digestion and injected into fertilized oocytes of C57BL/6 mice. Total embryonic excision of LacZ sequence was done by breeding floxed HMGN5-TG mice with SoxCre mice (Hayashi et al., 2002). Heart-specific excision was performed by breeding floxed HMGN5-TG mice with alphaMHC-cre mice (Oka et al., 2006).Total RNA from the left ventricle of newborn and adult Hmgn5-hTG mice which overexpress HMGN5 only in the heart as well as control mice was collected from biological triplicates (duplicates, in the case of newborn wild-type mice) and analyzed by Affymetrix expression arrays.

ORGANISM(S): Mus musculus

SUBMITTER: Leila Taher

PROVIDER: E-GEOD-63530 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The mammalian cell nucleus displays a remarkable spatial segregation of active euchromatic from inactive heterochromatic genomic regions. In conventional nuclei, euchromatin is localized in the nuclear interior and heterochromatin at the nuclear periphery. In contrast, rod photoreceptors in nocturnal mammals have inverted nuclei, with a dense heterochromatic core and a thin euchromatic outer shell. This inverted architecture likely converts rod nuclei into microlenses to facilitate nocturnal vision, and may relate to the absence of particular proteins that tether heterochromatin to the lamina. However, both the mechanism of inversion and the role of interactions between different types of chromatin and the lamina in nuclear organization remain unknown. To elucidate this mechanism we performed Hi-C and microscopy on cells with inverted nuclei and their conventional counterparts. Strikingly, despite the inversion evident in microscopy, both types of nuclei display similar Hi-C maps. To resolve this paradox we developed a polymer model of chromosomes and found a universal mechanism that reconciles Hi-C and microscopy for both inverted and conventional nuclei. Based solely on attraction between heterochromatic regions, this mechanism is sufficient to drive phase separation of euchromatin and heterochromatin and faithfully reproduces the 3D organization of inverted nuclei. When interactions between heterochromatin and the lamina are added, the same model recreates the conventional nuclear organization. To further test our models, we eliminated lamina interactions in models of conventional nuclei and found that this triggers a spontaneous process of inversion that qualitatively reproduces the pathway of morphological changes during nuclear inversion in vivo. Together, our experiments and modeling suggest that interactions among heterochromatic regions are central to phase separation of the active and inactive genome in inverted and conventional nuclei, while interactions with the lamina are essential for building the conventional architecture from these segregated phases. Ultimately our data suggest that an inverted organization constitutes the default state of nuclear architecture.

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Chromatin de-compaction by the nucleosomal binding protein HMGN5 impairs nuclear sturdiness.

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