Regulated chromatin domain comprising cluster of co-expressed genes in Drosophila melanogaster

Recently, the phenomenon of clustering of co-expressed genes on chromosomes was discovered in eukaryotes. To explore the hypothesis that genes within clusters occupy shared chromatin domains, we performed a detailed analysis of transcription pattern and chromatin structure of a cluster of co-expressed genes. We found that five non-homologous genes (Crtp, Yu, CK2βtes, Pros28.1B and CG13581) are expressed exclusively in Drosophila melanogaster male germ-line and form a non-interrupted cluster in the 15 kb region of chromosome 2. The cluster is surrounded by genes with broader transcription patterns. Analysis of DNase I sensitivity revealed ‘open’ chromatin conformation in the cluster and adjacent regions in the male germ-line cells, where all studied genes are transcribed. In contrast, in somatic tissues where the cluster genes are silent, the domain of repressed chromatin encompassed four out of five cluster genes and an adjacent non-cluster gene CG13589 that is also silent in analyzed somatic tissues. The fifth cluster gene (CG13581) appears to be excluded from the chromatin domain occupied by the other four genes. Our results suggest that extensive clustering of co-expressed genes in eukaryotic genomes does in general reflect the domain organization of chromatin, although domain borders may not exactly correspond to the margins of gene clusters

Akt1-associated actomyosin remodelling is required for nuclear lamina dispersal and nuclear shrinkage in epidermal terminal differentiation

Keratinocyte cornification and epidermal barrier formation are tightly controlled processes, which require complete degradation of intracellular organelles, including removal of keratinocyte nuclei. Keratinocyte nuclear destruction requires Akt1-dependent phosphorylation and degradation of the nuclear lamina protein, Lamin A/C, essential for nuclear integrity. However, the molecular mechanisms that result in complete nuclear removal and their regulation are not well defined. Post-confluent cultures of rat epidermal keratinocytes (REKs) undergo spontaneous and complete differentiation, allowing visualisation and perturbation of the differentiation process in vitro. We demonstrate that there is dispersal of phosphorylated Lamin A/C to structures throughout the cytoplasm in differentiating keratinocytes. We show that the dispersal of phosphorylated Lamin A/C is Akt1-dependent and these structures are specific for the removal of Lamin A/C from the nuclear lamina; nuclear contents and Lamin B were not present in these structures. Immunoprecipitation identified a group of functionally related Akt1 target proteins involved in Lamin A/C dispersal, including actin, which forms cytoskeletal microfilaments, Arp3, required for actin filament nucleation, and Myh9, a component of myosin IIa, a molecular motor that can translocate along actin filaments. Disruption of actin filament polymerisation, nucleation or myosin IIa activity prevented formation and dispersal of cytoplasmic Lamin A/C structures. Live imaging of keratinocytes expressing fluorescently tagged nuclear proteins showed a nuclear volume reduction step taking less than 40 min precedes final nuclear destruction. Preventing Akt1-dependent Lamin A/C phosphorylation and disrupting cytoskeletal Akt1-associated proteins prevented nuclear volume reduction. We propose keratinocyte nuclear destruction and differentiation requires myosin II activity and the actin cytoskeleton for two intermediate processes: Lamin A/C dispersal and rapid nuclear volume reduction

The large fraction of heterochromatin in Drosophila neurons is bound by both B-type lamin and HP1a

CONCLUSIONS: In various differentiated Drosophila cell types, we discovered the existence of peripheral heterochromatin, similar to that observed in mammals. Our findings support the model that peripheral heterochromatin matures enhancing the repression of unwanted genes as cells terminally differentiate.BACKGROUND: In most mammalian cell lines, chromatin located at the nuclear periphery is represented by condensed heterochromatin, as evidenced by microscopy observations and DamID mapping of lamina-associated domains (LADs) enriched in dimethylated Lys9 of histone H3 (H3K9me2). However, in Kc167 cell culture, the only Drosophilla cell type where LADs have previously been mapped, they are neither H3K9me2-enriched nor overlapped with the domains of heterochromatin protein 1a (HP1a).RESULTS: Here, using cell type-specific DamID we mapped genome-wide LADs, HP1a and Polycomb (Pc) domains from the central brain, Repo-positive glia, Elav-positive neurons and the fat body of Drosophila third instar larvae. Strikingly, contrary to Kc167 cells of embryonic origin, in neurons and, to a lesser extent, in glia and the fat body, HP1a domains appear to overlap strongly with LADs in both the chromosome arms and pericentromeric regions. Accordingly, centromeres reside closer to the nuclear lamina in neurons than in Kc167 cells. As expected, active gene promoters are mostly not present in LADs, HP1a and Pc domains. These domains are occupied by silent or weakly expressed genes with genes residing in the HP1a-bound LADs expressed at the lowest level

Long non-coding RNAs: spatial amplifiers that control nuclear structure and gene expression

Over the past decade, it has become clear that mammalian genomes encode thousands of long non-coding RNAs (lncRNAs), many of which are now implicated in diverse biological processes. Recent work studying the molecular mechanisms of several key examples — including Xist, which orchestrates X chromosome inactivation — has provided new insights into how lncRNAs can control cellular functions by acting in the nucleus. Here we discuss emerging mechanistic insights into how lncRNAs can regulate gene expression by coordinating regulatory proteins, localizing to target loci and shaping three-dimensional (3D) nuclear organization. We explore these principles to highlight biological challenges in gene regulation, in which lncRNAs are well-suited to perform roles that cannot be carried out by DNA elements or protein regulators alone, such as acting as spatial amplifiers of regulatory signals in the nucleus

The Role of Nucleoporin Elys in Nuclear Pore Complex Assembly and Regulation of Genome Architecture

For a long time, the nuclear lamina was thought to be the sole scaffold for the attachment of chromosomes to the nuclear envelope (NE) in metazoans. However, accumulating evidence indicates that nuclear pore complexes (NPCs) comprised of nucleoporins (Nups) participate in this process as well. One of the Nups, Elys, initiates NPC reassembly at the end of mitosis. Elys directly binds the decondensing chromatin and interacts with the Nup107–160 subcomplex of NPCs, thus serving as a seeding point for the subsequent recruitment of other NPC subcomplexes and connecting chromatin with the re-forming NE. Recent studies also uncovered the important functions of Elys during interphase where it interacts with chromatin and affects its compactness. Therefore, Elys seems to be one of the key Nups regulating chromatin organization. This review summarizes the current state of our knowledge about the participation of Elys in the post-mitotic NPC reassembly as well as the role that Elys and other Nups play in the maintenance of genome architecture

The Nuclear Lamina as an Organizer of Chromosome Architecture

The nuclear lamina (NL) is a meshwork of lamins and lamin-associated proteins adjoining the inner side of the nuclear envelope. In early embryonic cells, the NL mainly suppresses background transcription, whereas, in differentiated cell types, its disruption affects gene expression more severely. Normally, the NL serves as a backbone for multiple chromatin anchoring sites, thus shaping the spatial organization of chromosomes in the interphase nucleus. However, upon cell senescence, aging, or in some types of terminally differentiated cells and lamin-associated diseases, the loss of NL-chromatin tethering causes drastic alterations in chromosome architecture. Here, we provide an overview of the recent advances in the field of NL-chromatin interactions, focusing on their impact on chromatin positioning, compaction, repression, and spatial organization