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

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

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

Dosage Compensation in <i>Drosophila</i>: Its Canonical and Non-Canonical Mechanisms

Dosage compensation equalizes gene expression in a single male X chromosome with that in the pairs of autosomes and female X chromosomes. In the fruit fly Drosophila, canonical dosage compensation is implemented by the male-specific lethal (MSL) complex functioning in all male somatic cells. This complex contains acetyl transferase males absent on the first (MOF), which performs H4K16 hyperacetylation specifically in the male X chromosome, thus facilitating transcription of the X-linked genes. However, accumulating evidence points to an existence of additional, non-canonical dosage compensation mechanisms operating in somatic and germline cells. In this review, we discuss current advances in the understanding of both canonical and non-canonical mechanisms of dosage compensation in Drosophila

Role of Histone Deacetylases in Gene Regulation at Nuclear Lamina

<div><p>Theoretical models suggest that gene silencing at the nuclear periphery may involve “closing” of chromatin by transcriptional repressors, such as histone deacetylases (HDACs). Here we provide experimental evidence confirming these predictions. Histone acetylation, chromatin compactness, and gene repression in lamina-interacting multigenic chromatin domains were analyzed in Drosophila <em>S2</em> cells in which B-type lamin, diverse HDACs, and lamina-associated proteins were downregulated by dsRNA. Lamin depletion resulted in decreased compactness of the repressed multigenic domain associated with its detachment from the lamina and enhanced histone acetylation. Our data reveal the major role for HDAC1 in mediating deacetylation, chromatin compaction, and gene silencing in the multigenic domain, and an auxiliary role for HDAC3 that is required for retention of the domain at the lamina. These findings demonstrate the manifold and central involvement of class I HDACs in regulation of lamina-associated genes, illuminating a mechanism by which these enzymes can orchestrate normal and pathological development.</p> </div

Effect of LEM domain protein depletion on the expression of the <i>60D1</i> gene-cluster.

<p>Bars show increased expression of the cluster genes in <i>S2</i> cells determined with RT-qPCR after treatment with the mixture of <i>dMAN1</i>, <i>Bocksbeutel</i>, and <i>Otefin</i> dsRNAs. The control <i>LacZ</i> dsRNA-treated cells served as the reference. Gene symbols are shown on the X-axis; the <i>60D1</i> cluster is boxed. n = 6; error bars show SEM; *, p≤0.05 for comparison of individual transcript levels between <i>LacZ</i> RNAi and target RNAi; ††, p≤0.01 for comparison between the <i>60D1</i> cluster and control housekeeping genes. Inserts show the knockdown efficiency of the RNAi at the RNA levels.</p

Effect of HDAC depletion on retention of the 60D1 locus at the nuclear periphery.

<p>(<b>A</b>) Position of the locus was determined by FISH (red) and the nuclear envelope visualized with immunostaining for <i>LamDm_o</i> (green). Figure shows representative nuclei of cells treated with control <i>LacZ</i> dsRNA or a mixture of <i>HDAC1</i> and <i>HDAC3</i> dsRNAs. (<b>B</b>) Bars show the proportion of nuclei with FISH signals ≤0.4 µm apart from the nuclear envelope. dsRNAs used for depletion are indicated below the X-axis. <i>LacZ</i>, n = 256, 3 independent experiments; <i>HDAC1</i>, n = 199, 2 independent experiments; <i>HDAC3</i>, n = 201, 2 independent experiments; <i>HDAC1</i>+<i>HDAC3</i>, n = 208, 2 independent experiments; <i>LamDm_o</i>, n = 89, 2 independent experiments. Error bars show SEM. *, p≤0.05 for comparisons to <i>LacZ</i> control.</p

Effects of Class I HDAC depletion on histone acetylation and chromatin compactness.

<p>(<b>A</b>) ChIP assay shows increased acetylation of histones H3 (left panel) and H4 (right panel) in cells treated with <i>HDAC1</i> dsRNA and <i>HDAC3</i> dsRNAs as compared to the <i>LacZ</i> dsRNA-treated control cells. n = 4; error bars represent SEM. (<b>B</b>) Decreased chromatin compactness revealed by the general sensitivity to DNase I assay in <i>HDAC1</i> dsRNA-treated cells as compared to the control <i>LacZ</i> dsRNA treatment. Gene positions are shown below the X-axis with the <i>60D1</i> cluster framed. n = 2 to 4; error bars show SEM. *, p≤0.05; **, p≤0.01; ***, p≤0.001 for comparisons to the control. Inserts show the knockdown efficiency of the RNAi at the RNA levels.</p