Unknown,Transcriptomics,Genomics,Proteomics

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A-type lamins bind both hetero- and euchromatin, the latter being regulated by lamina-associated polypeptide 2alpha [ChIP-Seq]

ABSTRACT: Lamins are components of the peripheral nuclear lamina and interact with heterochromatic genomic regions, termed lamina-associated domains (LADs). In contrast to Lamin B11, lamin A/C also localizes throughout the nucleus, where it associates with the chromatin-binding protein lamina-associated polypeptide (LAP) 2alpha. Here we show lamin A/C also interacts with euchromatin, as determined by chromatin immunoprecipitation analyses of eu- and heterochromatin-enriched samples. By way of contrast, Lamin B11 was only found associated with heterochromatin. Euchromatic regions occupied by lamin A/C overlap with those bound by LAP2alpha, the depletion of which shifts binding of lamin A/C towards more heterochromatic regions. These alterations in lamin A/C chromatin interaction affect epigenetic histone marks in euchromatin without significantly affecting gene expression, while loss of lamin A/C in heterochromatic regions increased gene expression. Our data show a novel role of nucleoplasmic lamin A/C and LAP2alpha in regulating euchromatin. Examination of Lamin A/C, Lamin B1 and Lap2a DNA binding in Lap2alpha +/+ and Lap2a -/- cells and according changes in Histone modifications and gene expression

ORGANISM(S): Mus musculus

SUBMITTER: Philipp Rescheneder

PROVIDER: E-GEOD-70147 | 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.

Project description:Abstract: In the yeast S. pombe, multiple chromatin-modifying enzymes are required for heterochromatin formation, yet how their actions alter chromatin structure to block access to DNAby the transcriptional machinery is unknown. We constructed high-resolution nucleosome occupancy maps of heterochromatic regions in wild-type strains and in mutants lacking the H3K9 methyltransferase Clr4 or one of the two activities of the silencing effector complex SHREC. Fourier analysis reveals that these enzymes do not increase the regularity of nucleosome spacing. Rather, their principal effect was to induce the elimination of nucleosome-free regions (NFRs). Both NFRs associated with transcription initiation sites as well as those not associated with promoters are affected. As in S. cerevisiae, repressed genes in euchromatin retain their NFRs. Thus, NFR elimination cannot be explained as a secondary consequence of repression. The maps also show that TFIIIC boundary elements have NFRs resistant to silencing, suggesting a potential role in preventing lateral spread of heterochromatin. NFR elimination offers a mechanism by which heterochromatin restricts access of the transcriptional machinery to DNA. Nucleosome positions in either heterochromatin or euchromatin were mapped in various mutants to determine whether chromatin structure is altered upon heterochromatic silencing. Eight strains were assayed for nucleosome positioning in heterochromatin while two strains were assayed for nucleosome positioning in euchromatin. Two biological replicates were assayed for each strain. No dye swaps were done. RNA transcripts were also mapped using the heterochromatin tiling arrays to determine where RNA transcripts accumulate in heterochromatin. Three strains were assayed for transcripts in heterochromatin by hybridizing cy5-labeled cDNA created from total RNA against cy3-labeled cDNA created from RNA synthesized from sonicated genomic DNA. Two biological replicates were assayed for each strain.

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A-type lamins bind both hetero- and euchromatin, the latter being regulated by lamina-associated polypeptide 2alpha [ChIP-Seq]

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