RESUMO
Transport of macromolecules through the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs) consisting of nucleoporins (Nups). Elys/Mel-28 is the Nup that binds and connects the decondensing chromatin with the reassembled NPCs at the end of mitosis. Whether Elys links chromatin with the NE during interphase is unknown. Here, using DamID-seq, we identified Elys binding sites in Drosophila late embryos and divided them into those associated with nucleoplasmic or with NPC-linked Elys. These Elys binding sites are located within active or inactive chromatin, respectively. Strikingly, Elys knockdown in S2 cells results in peripheral chromatin displacement from the NE, in decondensation of NE-attached chromatin, and in derepression of genes within. It also leads to slightly more compact active chromatin regions. Our findings indicate that NPC-linked Elys, together with the nuclear lamina, anchors peripheral chromatin to the NE, whereas nucleoplasmic Elys decompacts active chromatin.
Assuntos
Cromatina , Proteínas de Drosophila , Interfase , Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Animais , Sítios de Ligação , Núcleo Celular/metabolismo , Cromatina/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/embriologia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genéticaRESUMO
Heterochromatin and euchromatin form different spatial compartments in the interphase nucleus, with heterochromatin being localized mainly at the nuclear periphery. The mechanisms responsible for peripheral localization of heterochromatin are still not fully understood. The nuclear lamina and nuclear pore complexes were obvious candidates for the role of heterochromatin binders. This review is focused on recent studies showing that heterochromatin interactions with the nuclear lamina and nuclear pore complexes maintain its peripheral localization. Differences in chromatin interactions with the nuclear envelope in cell populations and in individual cells are also discussed.
Assuntos
Lâmina Nuclear , Poro Nuclear , Heterocromatina , Cromatina , Núcleo Celular , Membrana NuclearRESUMO
The chromatin interaction assays, particularly Hi-C, enable detailed studies of genome architecture in multiple organisms and model systems, resulting in a deeper understanding of gene expression regulation mechanisms mediated by epigenetics. However, the analysis and interpretation of Hi-C data remain challenging due to technical biases, limiting direct comparisons of datasets obtained in different experiments and laboratories. As a result, removing biases from Hi-C-generated chromatin contact matrices is a critical data analysis step. Our novel approach, HiConfidence, eliminates biases from the Hi-C data by weighing chromatin contacts according to their consistency between replicates so that low-quality replicates do not substantially influence the result. The algorithm is effective for the analysis of global changes in chromatin structures such as compartments and topologically associating domains. We apply the HiConfidence approach to several Hi-C datasets with significant technical biases, that could not be analyzed effectively using existing methods, and obtain meaningful biological conclusions. In particular, HiConfidence aids in the study of how changes in histone acetylation pattern affect chromatin organization in Drosophila melanogaster S2 cells. The method is freely available at GitHub: https://github.com/victorykobets/HiConfidence.
Assuntos
Drosophila melanogaster , Genoma , Animais , Drosophila melanogaster/genética , Cromatina/genética , Cromossomos , ViésRESUMO
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.
Assuntos
Proteínas de Drosophila , Drosophila , Acetiltransferases/genética , Animais , Mecanismo Genético de Compensação de Dose , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Masculino , Proteínas Nucleares/genética , Cromossomo X/genéticaRESUMO
Eukaryotic chromosomes are spatially segregated into topologically associating domains (TADs). Some TADs are attached to the nuclear lamina (NL) through lamina-associated domains (LADs). Here, we identified LADs and TADs at two stages of Drosophila spermatogenesis - in bamΔ86 mutant testes which is the commonly used model of spermatogonia (SpG) and in larval testes mainly filled with spermatocytes (SpCs). We found that initiation of SpC-specific transcription correlates with promoters' detachment from the NL and with local spatial insulation of adjacent regions. However, this insulation does not result in the partitioning of inactive TADs into sub-TADs. We also revealed an increased contact frequency between SpC-specific genes in SpCs implying their de novo gathering into transcription factories. In addition, we uncovered the specific X chromosome organization in the male germline. In SpG and SpCs, a single X chromosome is stronger associated with the NL than autosomes. Nevertheless, active chromatin regions in the X chromosome interact with each other more frequently than in autosomes. Moreover, despite the absence of dosage compensation complex in the male germline, randomly inserted SpG-specific reporter is expressed higher in the X chromosome than in autosomes, thus evidencing that non-canonical dosage compensation operates in SpG.
Assuntos
Cromatina , Drosophila , Animais , Diferenciação Celular/genética , Cromatina/genética , Mecanismo Genético de Compensação de Dose , Drosophila/genética , Células Germinativas , MasculinoRESUMO
Mammalian and Drosophila genomes are partitioned into topologically associating domains (TADs). Although this partitioning has been reported to be functionally relevant, it is unclear whether TADs represent true physical units located at the same genomic positions in each cell nucleus or emerge as an average of numerous alternative chromatin folding patterns in a cell population. Here, we use a single-nucleus Hi-C technique to construct high-resolution Hi-C maps in individual Drosophila genomes. These maps demonstrate chromatin compartmentalization at the megabase scale and partitioning of the genome into non-hierarchical TADs at the scale of 100 kb, which closely resembles the TAD profile in the bulk in situ Hi-C data. Over 40% of TAD boundaries are conserved between individual nuclei and possess a high level of active epigenetic marks. Polymer simulations demonstrate that chromatin folding is best described by the random walk model within TADs and is most suitably approximated by a crumpled globule build of Gaussian blobs at longer distances. We observe prominent cell-to-cell variability in the long-range contacts between either active genome loci or between Polycomb-bound regions, suggesting an important contribution of stochastic processes to the formation of the Drosophila 3D genome.
Assuntos
Drosophila melanogaster/genética , Genoma de Inseto , Conformação de Ácido Nucleico , Animais , Biopolímeros/metabolismo , Cromatina/genética , Bases de Dados Genéticas , Epigênese Genética , Haploidia , Modelos Genéticos , Processos Estocásticos , Cromossomo X/genéticaRESUMO
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.
Assuntos
Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Regulação da Expressão Gênica/fisiologia , Histonas/metabolismo , Humanos , Mitose/genética , Mitose/fisiologia , Corno Dorsal da Medula Espinal/metabolismoRESUMO
Heterochromatin protein 1a (HP1a) is a well-known component of pericentromeric and telomeric heterochromatin in Drosophila. However, its role and the mechanisms of its binding in the chromosome arms (ChAs) remain largely unclear. Here, we identified HP1a-interacting domains in the somatic cells of Drosophila ovaries using a DamID-seq approach and compared them with insertion sites of transposable elements (TEs) revealed by genome sequencing. Although HP1a domains cover only 13% of ChAs, they non-randomly associate with 42% of TE insertions. Furthermore, HP1a on average propagates at 2-kb distances from the TE insertions. These data confirm the role of TEs in formation of HP1a islands in ChAs. However, only 18% of HP1a domains have adjacent TEs, indicating the existence of other mechanisms of HP1a domain formation besides spreading from TEs. In particular, many TE-independent HP1a domains correspond to the regions attached to the nuclear pore complexes (NPCs) or contain active gene promoters. However, HP1a occupancy on the promoters does not significantly influence expression of corresponding genes. At the same time, the steady-state transcript level of many genes located outside of HP1a domains was altered upon HP1a knockdown in the somatic cells of ovaries, thus pointing to the strong indirect effect of HP1a depletion. Collectively, our results support an existence of at least three different mechanisms of HP1a domain emergence in ChAs: spreading from TE insertions, transient interactions with the chromatin located near NPCs, and targeting to the promoters of moderately expressed genes.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Drosophila/fisiologia , Eucromatina/metabolismo , Ovário/fisiologia , Animais , Sítios de Ligação , Sequenciamento de Cromatina por Imunoprecipitação , Homólogo 5 da Proteína Cromobox , Mapeamento Cromossômico , Cromossomos/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Eucromatina/genética , Feminino , Imunofluorescência , Genômica/métodos , Ligação ProteicaRESUMO
In Drosophila ovarian somatic cells, PIWI-interacting small RNAs (piRNAs) against transposable elements are mainly produced from the â¼180-kb flamenco (flam) locus. flam transcripts are gathered into foci, located close to the nuclear envelope, and processed into piRNAs in the cytoplasmic Yb bodies. The mechanism of Yb body formation remains unknown. Using RNA fluorescence in situ hybridization, we found that in the follicle cells of ovaries the 5'-ends of flam transcripts are usually located in close proximity to the nuclear envelope and outside of Yb bodies, whereas their extended downstream regions mostly overlap with Yb bodies. In flamKG mutant ovaries, flam transcripts containing the first and, partially, second exons but lacking downstream regions are gathered into foci at the nuclear envelope, but Yb bodies are not assembled. Strikingly, piRNAs from the protein-coding gene transcripts accumulate at higher levels in flamKG ovaries indicating that piRNA biogenesis may occur without Yb bodies. We propose that normally in follicle cells, flam downstream transcript regions function not only as a substrate for generation of piRNAs but also as a scaffold for Yb body assembly, which competitively decreases piRNA production from the protein-coding gene transcripts. By contrast, in ovarian somatic cap and escort cells Yb body assembly does not require flam transcription.
Assuntos
Estruturas Citoplasmáticas/metabolismo , Elementos de DNA Transponíveis/genética , Drosophila melanogaster/genética , RNA Interferente Pequeno/genética , Animais , Éxons/genética , Feminino , Mutação/genética , Membrana Nuclear/metabolismo , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
How the nuclear lamina (NL) impacts on global chromatin architecture is poorly understood. Here, we show that NL disruption in Drosophila S2 cells leads to chromatin compaction and repositioning from the nuclear envelope. This increases the chromatin density in a fraction of topologically-associating domains (TADs) enriched in active chromatin and enhances interactions between active and inactive chromatin. Importantly, upon NL disruption the NL-associated TADs become more acetylated at histone H3 and less compact, while background transcription is derepressed. Two-colour FISH confirms that a TAD becomes less compact following its release from the NL. Finally, polymer simulations show that chromatin binding to the NL can per se compact attached TADs. Collectively, our findings demonstrate a dual function of the NL in shaping the 3D genome. Attachment of TADs to the NL makes them more condensed but decreases the overall chromatin density in the nucleus by stretching interphase chromosomes.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Cromatina/metabolismo , Histonas/metabolismo , Lâmina Nuclear/metabolismo , Animais , Linhagem Celular , Cromossomos de Insetos/metabolismo , Regulação para Baixo , Drosophila melanogaster , Perfilação da Expressão Gênica , Genes de Insetos/genética , Hibridização in Situ Fluorescente , Modelos Animais , Regulação para CimaRESUMO
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.
Assuntos
Cromossomos/metabolismo , Lâmina Nuclear/metabolismo , Animais , Diferenciação Celular , Heterocromatina/metabolismo , Humanos , Modelos BiológicosRESUMO
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. 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.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Heterocromatina/genética , Lamina Tipo B/metabolismo , Neurônios/metabolismo , Animais , Linhagem Celular , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Drosophila , Proteínas de Drosophila/genética , Histonas/metabolismo , Lamina Tipo B/genética , Ligação ProteicaRESUMO
Piwi in a complex with Piwi-interacting RNAs (piRNAs) triggers transcriptional silencing of transposable elements (TEs) in Drosophila ovaries, thus ensuring genome stability. To do this, Piwi must scan the nascent transcripts of genes and TEs for complementarity to piRNAs. The mechanism of this scanning is currently unknown. Here we report the DamID-seq mapping of multiple Piwi-interacting chromosomal domains in somatic cells of Drosophila ovaries. These domains significantly overlap with genomic regions tethered to Nuclear Pore Complexes (NPCs). Accordingly, Piwi was coimmunoprecipitated with the component of NPCs Elys and with the Xmas-2 subunit of RNA transcription and export complex, known to interact with NPCs. However, only a small Piwi fraction has transient access to DNA at nuclear pores. Importantly, although 36% of the protein-coding genes overlap with Piwi-interacting domains and RNA-immunoprecipitation results demonstrate promiscuous Piwi binding to numerous genic and TE nuclear transcripts, according to available data Piwi does not silence these genes, likely due to the absence of perfect base-pairing between piRNAs and their transcripts.
Assuntos
Proteínas Argonautas/metabolismo , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Poro Nuclear/metabolismo , Ovário/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas Argonautas/química , Proteínas Argonautas/genética , Cromatina/genética , Elementos de DNA Transponíveis , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Inativação Gênica , Genoma de Inseto , Instabilidade Genômica , Modelos Biológicos , Poro Nuclear/genética , Ovário/citologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Recent data indicate that eukaryotic chromosomes are organized into Topologically Associating Domains (TADs); however, the mechanisms underlying TAD formation remain obscure. Based on the results of Hi-C analysis performed on 4 Drosophila melanogaster cell lines, we have proposed that specific properties of nucleosomes in active and repressed chromatin play a key role in the formation of TADs. Our computer simulations showed that the ability of "inactive" nucleosomes to stick to each other and the lack of such ability in "active" nucleosomes is sufficient for spatial segregation of these types of chromatin, which is revealed in the Hi-C analysis as TAD/inter-TAD partitioning. However, some Drosophila and mammalian TADs contain both active and inactive chromatin, a fact that does not fit this model. Herein, we present additional arguments for the model by postulating that transcriptionally active chromatin is extruded on the surface of a TAD, and discuss the possible impact of this organization on the enhancer-promoter communication and on the segregation of TADs.
Assuntos
Cromatina/química , Cromatina/metabolismo , Animais , Cromossomos de Insetos/genética , Drosophila melanogaster/genética , HumanosRESUMO
Recent advances enabled by the Hi-C technique have unraveled many principles of chromosomal folding that were subsequently linked to disease and gene regulation. In particular, Hi-C revealed that chromosomes of animals are organized into topologically associating domains (TADs), evolutionary conserved compact chromatin domains that influence gene expression. Mechanisms that underlie partitioning of the genome into TADs remain poorly understood. To explore principles of TAD folding in Drosophila melanogaster, we performed Hi-C and poly(A)(+) RNA-seq in four cell lines of various origins (S2, Kc167, DmBG3-c2, and OSC). Contrary to previous studies, we find that regions between TADs (i.e., the inter-TADs and TAD boundaries) in Drosophila are only weakly enriched with the insulator protein dCTCF, while another insulator protein Su(Hw) is preferentially present within TADs. However, Drosophila inter-TADs harbor active chromatin and constitutively transcribed (housekeeping) genes. Accordingly, we find that binding of insulator proteins dCTCF and Su(Hw) predicts TAD boundaries much worse than active chromatin marks do. Interestingly, inter-TADs correspond to decompacted inter-bands of polytene chromosomes, whereas TADs mostly correspond to densely packed bands. Collectively, our results suggest that TADs are condensed chromatin domains depleted in active chromatin marks, separated by regions of active chromatin. We propose the mechanism of TAD self-assembly based on the ability of nucleosomes from inactive chromatin to aggregate, and lack of this ability in acetylated nucleosomal arrays. Finally, we test this hypothesis by polymer simulations and find that TAD partitioning may be explained by different modes of inter-nucleosomal interactions for active and inactive chromatin.
Assuntos
Cromatina/genética , Drosophila melanogaster/genética , Genoma de Inseto , Transcrição Gênica , Animais , Linhagem Celular , Montagem e Desmontagem da Cromatina , Mapeamento Cromossômico , Simulação por Computador , Modelos Moleculares , Nucleossomos/genética , Nucleossomos/metabolismo , Cromossomos Politênicos/genética , Análise de Sequência de RNARESUMO
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 S2 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.
Assuntos
Cromatina/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Histona Desacetilase 1/genética , Histona Desacetilases/genética , Histonas/genética , Lâmina Nuclear/genética , Acetilação , Animais , Western Blotting , Linhagem Celular , Cromatina/enzimologia , Imunoprecipitação da Cromatina , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/enzimologia , Inativação Gênica , Histona Desacetilase 1/metabolismo , Histona Desacetilases/metabolismo , Histonas/metabolismo , Lamina Tipo B/antagonistas & inibidores , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Família Multigênica , Lâmina Nuclear/enzimologia , RNA de Cadeia Dupla/genética , Transcrição GênicaRESUMO
There is accumulating evidence that the nuclear periphery is a transcriptionally repressive compartment. A surprisingly large fraction of the genome is either in transient or permanent contact with nuclear envelope, where the majority of genes are maintained in a silent state, waiting to be awakened during cell differentiation. The integrity of the nuclear lamina and the histone deacetylase activity appear to be essential for gene repression at the nuclear periphery. However, the molecular mechanisms of silencing, as well as the events that lead to the activation of lamina-tethered genes, require further elucidation. This review summarizes recent advances in understanding of the mechanisms that link nuclear architecture, local chromatin structure, and gene regulation.
Assuntos
Inativação Gênica , Lâmina Nuclear/genética , Animais , Posicionamento Cromossômico/genética , Cromossomos/metabolismo , HumanosRESUMO
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, CK2betates, 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.
Assuntos
Cromatina/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Animais , Proteínas de Drosophila/biossíntese , Drosophila melanogaster/metabolismo , Genes de Insetos , Masculino , Dados de Sequência Molecular , Mutação , Especificidade de Órgãos , Complexo de Endopeptidases do Proteassoma/biossíntese , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Espermatogênese , Testículo/metabolismoRESUMO
Clustering of co-expressed, non-homologous genes on chromosomes implies their co-regulation. In lower eukaryotes, co-expressed genes are often found in pairs. Clustering of genes that share aspects of transcriptional regulation has also been reported in higher eukaryotes. To advance our understanding of the mode of coordinated gene regulation in multicellular organisms, we performed a genome-wide analysis of the chromosomal distribution of co-expressed genes in Drosophila. We identified a total of 1,661 testes-specific genes, one-third of which are clustered on chromosomes. The number of clusters of three or more genes is much higher than expected by chance. We observed a similar trend for genes upregulated in the embryo and in the adult head, although the expression pattern of individual genes cannot be predicted on the basis of chromosomal position alone. Our data suggest that the prevalent mechanism of transcriptional co-regulation in higher eukaryotes operates with extensive chromatin domains that comprise multiple genes.
Assuntos
Drosophila melanogaster/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genes de Insetos/genética , Genoma , Família Multigênica/genética , Animais , Cromatina/genética , Drosophila melanogaster/embriologia , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica no Desenvolvimento , Cabeça , Masculino , Especificidade de Órgãos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Caracteres Sexuais , Processos Estocásticos , Testículo/metabolismo , Cromossomo X/genéticaRESUMO
An earlier described CK2(beta)tes gene of Drosophila melanogaster is shown to encode a male germline specific isoform of regulatory beta subunit of casein kinase 2. Western-analysis using anti-CK2(beta)tes Ig revealed CK2(beta)tes protein in Drosophila testes extract. Expression of a CK2(beta)tes-beta-galactosidase fusion protein driven by the CK2(beta)tes promoter was found in transgenic flies at postmitotic stages of spermatogenesis. Examination of biochemical characteristics of a recombinant CK2(beta)tes protein expressed in Escherichia coli revealed properties similar to those of CK2beta: (a) CK2(beta)tes protein stimulates CK2alpha catalytic activity toward synthetic peptide; (b) it inhibits phosphorylation of calmodulin and mediates stimulation of CK2alpha by polylysine; (c) it is able to form (CK2(beta)tes)2 dimers, as well as (CK2alpha)2(CK2(beta)tes)2 tetramers. Using the yeast two-hybrid system and coimmunoprecipitation analysis of protein extract from Drosophila testes, we demonstrated an association between CK2(beta)tes and CK2alpha. Northern-analysis has shown that another regulatory (beta') subunit found recently in D. melanogaster genome is also testis-specific. Thus, we describe the first example of two tissue-specific regulatory subunits of CK2 which might serve to provide CK2 substrate recognition during spermatogenesis.