RESUMO
Eukaryotic DNA replicates asynchronously, with discrete genomic loci replicating during different stages of S phase. Drosophila larval tissues undergo endoreplication without cell division, and the latest replicating regions occasionally fail to complete endoreplication, resulting in underreplicated domains of polytene chromosomes. Here we show that linker histone H1 is required for the underreplication (UR) phenomenon in Drosophila salivary glands. H1 directly interacts with the Suppressor of UR (SUUR) protein and is required for SUUR binding to chromatin in vivo. These observations implicate H1 as a critical factor in the formation of underreplicated regions and an upstream effector of SUUR. We also demonstrate that the localization of H1 in chromatin changes profoundly during the endocycle. At the onset of endocycle S (endo-S) phase, H1 is heavily and specifically loaded into late replicating genomic regions and is then redistributed during the course of endoreplication. Our data suggest that cell cycle-dependent chromosome occupancy of H1 is governed by several independent processes. In addition to the ubiquitous replication-related disassembly and reassembly of chromatin, H1 is deposited into chromatin through a novel pathway that is replication-independent, rapid, and locus-specific. This cell cycle-directed dynamic localization of H1 in chromatin may play an important role in the regulation of DNA replication timing.
Assuntos
Cromatina/metabolismo , Drosophila/genética , Endorreduplicação , Histonas/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/metabolismo , Heterocromatina/metabolismo , Larva/genética , Larva/metabolismo , Fase S/genética , Glândulas Salivares/metabolismoRESUMO
The Drosophila Nonspecific Lethal (NSL) complex is a major transcriptional regulator of housekeeping genes. It contains at least seven subunits that are conserved in the human KANSL complex: Nsl1/Wah (KANSL1), Dgt1/Nsl2 (KANSL2), Rcd1/Nsl3 (KANSL3), Rcd5 (MCRS1), MBD-R2 (PHF20), Wds (WDR5) and Mof (MOF/KAT8). Previous studies have shown that Dgt1, Rcd1 and Rcd5 are implicated in centrosome maintenance. Here, we analyzed the mitotic phenotypes caused by RNAi-mediated depletion of Rcd1, Rcd5, MBD-R2 or Wds in greater detail. Depletion of any of these proteins in Drosophila S2 cells led to defects in chromosome segregation. Consistent with these findings, Rcd1, Rcd5 and MBD-R2 RNAi cells showed reduced levels of both Cid/CENP-A and the kinetochore component Ndc80. In addition, RNAi against any of the four genes negatively affected centriole duplication. In Wds-depleted cells, the mitotic phenotypes were similar but milder than those observed in Rcd1-, Rcd5- or MBD-R2-deficient cells. RT-qPCR experiments and interrogation of published datasets revealed that transcription of many genes encoding centromere/kinetochore proteins (e.g., cid, Mis12 and Nnf1b), or involved in centriole duplication (e.g., Sas-6, Sas-4 and asl) is substantially reduced in Rcd1, Rcd5 and MBD-R2 RNAi cells, and to a lesser extent in wds RNAi cells. During mitosis, both Rcd1-GFP and Rcd5-GFP accumulate at the centrosomes and the telophase midbody, MBD-R2-GFP is enriched only at the chromosomes, while Wds-GFP accumulates at the centrosomes, the kinetochores, the midbody, and on a specific chromosome region. Collectively, our results suggest that the mitotic phenotypes caused by Rcd1, Rcd5, MBD-R2 or Wds depletion are primarily due to reduced transcription of genes involved in kinetochore assembly and centriole duplication. The differences in the subcellular localizations of the NSL components may reflect direct mitotic functions that are difficult to detect at the phenotypic level, because they are masked by the transcription-dependent deficiency of kinetochore and centriolar proteins.
Assuntos
Duplicação Cromossômica/genética , Segregação de Cromossomos/genética , Fatores de Transcrição/genética , Animais , Proteínas de Ciclo Celular/genética , Centrômero/metabolismo , Centrossomo/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitose/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transporte Proteico/fisiologia , Interferência de RNA , Proteínas de Ligação a RNA/genética , Elementos Reguladores de Transcrição/genética , Fuso Acromático/genética , Fatores de Transcrição/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMO
Compared to transcription initiation, much less is known about transcription termination. In particular, large-scale mutagenesis studies have, so far, primarily concentrated on promoter and enhancer, but not terminator sequences. Here, we used a massively parallel reporter assay (MPRA) to systematically analyze the influence of short (8 bp) sequence variants (mutations) located downstream of the polyadenylation signal (PAS) on the steady-state mRNA level of the upstream gene, employing an eGFP reporter and human HEK293T cells as a model system. In total, we evaluated 227,755 mutations located at different overlapping positions within +17..+56 bp downstream of the PAS for their ability to regulate the reporter gene expression. We found that the positions +17..+44 bp downstream of the PAS are more essential for gene upregulation than those located more distal to the PAS, and that the mutation sequences ensuring high levels of eGFP mRNA expression are extremely T-rich. Next, we validated the positive effect of a couple of mutations identified in the MPRA screening on the eGFP and luciferase protein expression. The most promising mutation increased the expression of the reporter proteins 13-fold and sevenfold on average in HEK293T and CHO cells, respectively. Overall, these findings might be useful for further improving the efficiency of production of therapeutic products, e.g., recombinant antibodies.
Assuntos
Poliadenilação , Transcrição Gênica , Cricetinae , Animais , Humanos , Poliadenilação/genética , Células HEK293 , Cricetulus , Células CHO , RNA Mensageiro/genética , TransgenesRESUMO
BACKGROUND: The Drosophila central nervous system (CNS) is a convenient model system for the study of the molecular mechanisms of conserved neurobiological processes. The manipulation of gene activity in specific cell types and subtypes of the Drosophila CNS is frequently achieved by employing the binary Gal4/UAS system. However, many Gal4 driver lines available from the Bloomington Drosophila Stock Center (BDSC) and commonly used in Drosophila neurobiology are still not well characterized. Among these are three lines with Gal4 driven by the elav promoter (BDSC #8760, #8765, and #458), one line with Gal4 driven by the repo promoter (BDSC #7415), and the 69B-Gal4 line (BDSC #1774). For most of these lines, the exact insertion sites of the transgenes and the detailed expression patterns of Gal4 are not known. This study is aimed at filling these gaps. RESULTS: We have mapped the genomic location of the Gal4-bearing P-elements carried by the BDSC lines #8760, #8765, #458, #7415, and #1774. In addition, for each of these lines, we have analyzed the Gal4-driven GFP expression pattern in the third instar larval CNS and eye-antennal imaginal discs. Localizations of the endogenous Elav and Repo proteins were used as markers of neuronal and glial cells, respectively. CONCLUSIONS: We provide a mini-atlas of the spatial activity of Gal4 drivers that are widely used for the expression of UAS-target genes in the Drosophila CNS. The data will be helpful for planning experiments with these drivers and for the correct interpretation of the results.
Assuntos
Sistema Nervoso Central/citologia , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Animais , Sistema Nervoso Central/embriologia , Mapeamento Cromossômico , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Larva , Neurobiologia , Organismos Geneticamente Modificados , Regiões Promotoras Genéticas , Fatores de Transcrição/genéticaRESUMO
Border cell (BC) migration during Drosophila oogenesis is an excellent model for the analysis of the migratory and invasive cell behavior. Most studies on BC migration have exploited a slbo-Gal4 driver to regulate gene expression in these cells or to mark them. Here, we report that the slbo-Gal4 transgene present in the line #6458 from the Bloomington Stock Center is inserted within chickadee (chic), a gene encoding the actin-binding protein Profilin, which promotes actin polymerization and is known to be involved in cell migration. The chic6458 mutation caused by the transgene insertion behaves as a null chic allele and is homozygous lethal. To evaluate possible effects of chic6458 on the assessment of BC behavior, we generated new lines bearing the slbo-Gal4 transgene inserted into different second chromosome loci that do not appear to be involved in cell migration. Using these new lines and the slbo-Gal4-chic6458 line, we defined the functional relationships between the twinfilin (twf) and chic in BC migration. Migration of BCs is substantially reduced by mutations in twf, which encodes an actin-binding protein that inhibits actin filament assembly. The defects caused by twf mutations are significantly suppressed when the slbo-Gal4-chic6458, but not the new slbo-Gal4 drivers were used. These findings indicate twf and chic interact and function antagonistically during BC migration in Drosophila oogenesis.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas de Drosophila/genética , Drosophila/genética , Oogênese/genética , Ovário/citologia , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados , Movimento Celular/genética , Cromossomos de Insetos , Drosophila/citologia , Feminino , Heterozigoto , Proteínas dos Microfilamentos/genética , Mutação , Profilinas/genéticaRESUMO
BACKGROUND: Expression of the CNDP2 gene is frequently up- or down-regulated in different types of human cancers. However, how the product of this gene is involved in cell growth and proliferation is poorly understood. Moreover, our knowledge of the functions of the CNDP2 orthologs in well-established model organisms is scarce. In particular, the function of the D. melanogaster ortholog of CNDP2, encoded by the CG17337 gene (hereafter referred to as dCNDP2), is still unknown. RESULTS: This study was aimed at developing a set of genetic and molecular tools to study the roles of dCNDP2. We generated a dCNDP2 null mutation (hereafter ∆dCNDP2) using CRISPR/Cas9-mediated homologous recombination (HR) and found that the ∆dCNDP2 mutants are homozygous viable, morphologically normal and fertile. We also generated transgenic fly lines expressing eGFP-tagged and non-tagged dCNDP2 protein, all under the control of the UAS promoter, as well as polyclonal antibodies specific to dCNDP2. Using these tools, we demonstrate that only one of the two predicted dCNDP2 isoforms is expressed throughout the different tissues tested. dCNDP2 was detected in both the cytoplasm and the nucleus, and was found to be associated with multiple sites in the salivary gland polytene chromosomes. CONCLUSIONS: The dCNDP2 gene is not essential for fly viability under standard laboratory conditions. The subcellular localization pattern of dCNDP2 suggests that this protein might have roles in both the cytoplasm and the nucleus. The genetic and molecular tools developed in this study will allow further functional characterization of the conserved CNDP2 protein using D. melanogaster as a model system.
Assuntos
Drosophila melanogaster/genética , Animais , Animais Geneticamente Modificados , Linhagem Celular , Proliferação de Células , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Isoformas de Proteínas/genéticaRESUMO
BACKGROUND: S2 cells are one of the most widely used Drosophila melanogaster cell lines. A series of studies has shown that they are particularly suitable for RNAi-based screens aimed at the dissection of cellular pathways, including those controlling cell shape and motility, cell metabolism, and host-pathogen interactions. In addition, RNAi in S2 cells has been successfully used to identify many new mitotic genes that are conserved in the higher eukaryotes, and for the analysis of several aspects of the mitotic process. However, no detailed and complete description of S2 cell mitosis at the ultrastructural level has been done. Here, we provide a detailed characterization of all phases of S2 cell mitosis visualized by transmission electron microscopy (TEM). RESULTS: We analyzed by TEM a random sample of 144 cells undergoing mitosis, focusing on intracellular membrane and microtubule (MT) behaviors. This unbiased approach provided a comprehensive ultrastructural view of the dividing cells, and allowed us to discover that S2 cells exhibit a previously uncharacterized behavior of intracellular membranes, involving the formation of a quadruple nuclear membrane in early prometaphase and its disassembly during late prometaphase. After nuclear envelope disassembly, the mitotic apparatus becomes encased by a discontinuous network of endoplasmic reticulum membranes, which associate with mitochondria, presumably to prevent their diffusion into the spindle area. We also observed a peculiar metaphase spindle organization. We found that kinetochores with attached k-fibers are almost invariably associated with lateral MT bundles that can be either interpolar bundles or k-fibers connected to a different kinetochore. This spindle organization is likely to favor chromosome alignment at metaphase and subsequent segregation during anaphase. CONCLUSIONS: We discovered several previously unknown features of membrane and MT organization during S2 cell mitosis. The genetic determinants of these mitotic features can now be investigated, for instance by using an RNAi-based approach, which is particularly easy and efficient in S2 cells.
Assuntos
Linhagem Celular/ultraestrutura , Drosophila melanogaster/citologia , Membranas Intracelulares/ultraestrutura , Cinetocoros/ultraestrutura , Microtúbulos/ultraestrutura , Mitose , Animais , Microscopia Eletrônica de Transmissão/métodosRESUMO
Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.
Assuntos
Drosophila melanogaster/genética , Genoma , Animais , Mapeamento Cromossômico , Cromossomos Artificiais Bacterianos , Biologia Computacional , Mapeamento de Sequências Contíguas , Sequenciamento de Nucleotídeos em Larga Escala , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Cromossomos Politênicos , Mapeamento por RestriçãoRESUMO
Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early versus late in different cell types remain incompletely understood. Intercalary heterochromatin domains replicate very late in both diploid chromosomes of dividing cells and in endoreplicating polytene chromosomes where they are also underreplicated. Drosophila SNF2-related factor SUUR imparts locus-specific underreplication of polytene chromosomes. SUUR negatively regulates DNA replication fork progression; however, its mechanism of action remains obscure. Here, we developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates SUUR ATPase activity and is required for a normal spatiotemporal distribution of SUUR in vivo. SUUR and Mod(Mdg4)-67.2 together mediate the activities of gypsy insulator that prevent certain enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, SuUR or mod(mdg4) mutations reverse underreplication of intercalary heterochromatin. Thus, SUMM4 can impart late replication of intercalary heterochromatin by attenuating the progression of replication forks through euchromatin/heterochromatin boundaries. Our findings implicate a SNF2 family ATP-dependent motor protein SUUR in the insulator function, reveal that DNA replication can be delayed by a chromatin barrier, and uncover a critical role for architectural proteins in replication control. They suggest a mechanism for the establishment of late replication that does not depend on an asynchronous firing of late replication origins.
Inside cells, molecules of DNA provide the instructions needed to make proteins. Cells carefully maintain and repair their DNA, and typically make a complete copy of the genome before they divide to ensure that after division, each daughter cell has a full set. Within human, fly and other eukaryotic nuclei, DNA is packaged into structures known as chromosomes. Cells follow precisely controlled programs to replicate distinct regions of chromosomes at different times. To start copying a particular region, the cell machinery that replicates DNA binds to a sequence known as the origin of replication. It is thought that as-yet unknown cues from the cell may lead the replication machinery to bind to different origins of replication at different times. In some circumstances, cells make extra copies of their DNA without dividing. For example, many cells in the larvae of fruit flies contain hundreds of extra DNA copies to sustain their increased sizes. However, the entire genome is not copied during this process, so cells end up with more copies of some regions of the genome than others. A protein called SUUR is required for hindering the replication of the 'underrepresented' regions, but it is not clear how it works. To address this question, Andreyeva, Emelyanov et al. developed a new approach based on liquid chromatography and quantitative proteomics to identify the native form of SUUR in fruit flies. This revealed that SUUR exists as a stable complex with a protein called Mod(Mdg4), which is needed to recruit SUUR to the chromosomes. Further experiments suggested that SUUR and Mod(Mdg4) work together to bind to regions of DNA known as gypsy insulator elements, creating a physical barrier that hinders the replication machinery from accessing some parts of the genome. The findings of Andreyeva, Emelyanov et al. provide an alternative explanation for how individual cells may stagger the process of copying their DNA without relying on the replication machinery binding to various replication origins at different times. Rather, late replication timing may be instructed by an insulator-born delay of the progression of replication over particular genomic regions. This mechanism adds to the list of nuclear processes (chromosome partitioning, transcriptional regulation, etc.) that are known to be directed by insulators and associated architectural proteins.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/genética , Drosophila/metabolismo , Proteínas de Ligação a DNA/metabolismo , Heterocromatina/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Eucromatina/metabolismo , Cromatina/genética , Cromatina/metabolismo , Replicação do DNARESUMO
Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and Drosophila cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but not KDMTR. We examined KDMTR in normal Drosophila S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: mast/orbit/chb (CLASP1), mei-38 (TPX2), mars (HURP), dgt6 (HAUS6), Eb1 (MAPRE1/EB1), Patronin (CAMSAP2), asp (ASPM), and Klp10A (KIF2A). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6, and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin, and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1, and Patronin positively regulate polymerization, bundling, and stabilization of regrowing MTs until a bipolar spindle is reformed.
Assuntos
Proteínas de Drosophila , Cinetocoros , Animais , Demecolcina/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Cinesinas/genética , Cinetocoros/metabolismo , Mamíferos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Fuso Acromático/metabolismoRESUMO
The ATP-dependent chromatin remodeling factor CHD1 is essential for the assembly of variant histone H3.3 into paternal chromatin during sperm chromatin remodeling in fertilized eggs. It remains unclear, however, if CHD1 has a similar role in normal diploid cells. Using a specifically tailored quantitative mass spectrometry approach, we show that Chd1 disruption results in reduced H3.3 levels in heads of Chd1 mutant flies. Chd1 deletion perturbs brain chromatin structure in a similar way as H3.3 deletion and leads to global de-repression of transcription. The physiological consequences are reduced food intake, metabolic alterations, and shortened lifespan. Notably, brain-specific CHD1 expression rescues these phenotypes. We further demonstrate a strong genetic interaction between Chd1 and H3.3 chaperone Hira. Thus, our findings establish CHD1 as a factor required for the assembly of H3.3-containing chromatin in adult cells and suggest a crucial role for CHD1 in the brain as a regulator of organismal health and longevity.
Assuntos
Encéfalo/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/metabolismo , Histonas/metabolismo , Metaboloma/fisiologia , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Comportamento Alimentar , Feminino , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/análise , Longevidade , Masculino , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismoRESUMO
The disruption of the protective intestinal barrier-the 'leaky gut'-is a common complication of the inflammatory bowel disease. There is limited data on the mechanisms of the intestinal barrier disruption upon low-grade inflammation characteristic of patients with inflammatory bowel disease in clinical remission. Thus, animal models that recapitulate the complexity of chronic intestinal inflammation in vivo are of particular interest. In this study, we used Mucin-2 (Muc2) knockout mice predisposed to colitis to study intestinal barrier upon chronic inflammation. We used 4-kDa FITC-Dextran assay and transmission electron microscopy to demonstrate the increased intestinal permeability and morphological defects in intercellular junctions in Muc2 knockout mice. Confocal microscopy revealed the disruption of the apical F-actin cytoskeleton and delocalization of tight junction protein Claudin-3 from the membrane. We further demonstrate mitochondrial damage, impaired oxygen consumption and the reduction of the intestinal ATP content in Muc2 knockout mice. Finally, we show that chemically induced mitochondrial uncoupling in the wild type mice mimics the intestinal barrier disruption in vivo and causes partial loss of F-actin and membrane localization of Claudin-3. We propose that mitochondrial damage and metabolic shifts during chronic inflammation contribute to the leaky gut syndrome in Muc2 knockout animal model of colitis.
Assuntos
Trifosfato de Adenosina/metabolismo , Colite/genética , Mucosa Intestinal/metabolismo , Mitocôndrias/patologia , Mucina-2/fisiologia , Junções Íntimas/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Mucina-2/genéticaRESUMO
Moonlighting proteins can perform one or more additional functions besides their primary role. It has been posited that a protein can acquire a moonlighting function through a gradual evolutionary process, which is favored when the primary and secondary functions are exerted in different cellular compartments. Transcription factors (TFs) and splicing factors (SFs) control processes that occur in interphase nuclei and are strongly reduced during cell division, and are therefore in a favorable situation to evolve moonlighting mitotic functions. However, recently published moonlighting protein databases, which comprise almost 400 proteins, do not include TFs and SFs with secondary mitotic functions. We searched the literature and found several TFs and SFs with bona fide moonlighting mitotic functions, namely they localize to specific mitotic structure(s), interact with proteins enriched in the same structure(s), and are required for proper morphology and functioning of the structure(s). In addition, we describe TFs and SFs that localize to mitotic structures but cannot be classified as moonlighting proteins due to insufficient data on their biochemical interactions and mitotic roles. Nevertheless, we hypothesize that most TFs and SFs with specific mitotic localizations have either minor or redundant moonlighting functions, or are evolving towards the acquisition of these functions.
Assuntos
Mitose/fisiologia , Fatores de Processamento de RNA/metabolismo , Fatores de Transcrição/metabolismo , HumanosRESUMO
During production of the original article [1], there was a technical error that resulted in author corrections not being rendered in the PDF version of the article.
RESUMO
BACKGROUND: The calmodulin-regulated spectrin-associated proteins (CAMSAPs) belong to a conserved protein family, which includes members that bind the polymerizing mcrotubule (MT) minus ends and remain associated with the MT lattice formed by minus end polymerization. Only one of the three mammalian CAMSAPs, CAMSAP1, localizes to the mitotic spindle but its function is unclear. In Drosophila, there is only one CAMSAP, named Patronin. Previous work has shown that Patronin stabilizes the minus ends of non-mitotic MTs and is required for proper spindle elongation. However, the precise role of Patronin in mitotic spindle assembly is poorly understood. RESULTS: Here we have explored the role of Patronin in Drosophila mitosis using S2 tissue culture cells as a model system. We show that Patronin associates with different types of MT bundles within the Drosophila mitotic spindle, and that it is required for their stability. Imaging of living cells expressing Patronin-GFP showed that Patronin displays a dynamic behavior. In prometaphase cells, Patronin accumulates on short segments of MT bundles located near the chromosomes. These Patronin "seeds" extend towards the cell poles and stop growing just before reaching the poles. Our data also suggest that Patronin localization is largely independent of proteins acting at the MT minus ends such as Asp and Klp10A. CONCLUSION: Our results suggest a working hypothesis about the mitotic role of Patronin. We propose that Patronin binds the minus ends within MT bundles, including those generated from the walls of preexisting MTs via the augmin-mediated pathway. This would help maintaining MT association within the mitotic bundles, thereby stabilizing the spindle structure. Our data also raise the intriguing possibility that the minus ends of bundled MTs can undergo a limited polymerization.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/fisiologia , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Centrossomo/metabolismo , Segregação de Cromossomos , Cinesinas/metabolismo , Microtúbulos/metabolismo , Polimerização , Ligação Proteica , Fuso Acromático/metabolismoRESUMO
Proper control of DNA replication is essential to ensure faithful transmission of genetic material and prevent chromosomal aberrations that can drive cancer progression and developmental disorders. DNA replication is regulated primarily at the level of initiation and is under strict cell-cycle regulation. Importantly, DNA replication is highly influenced by developmental cues. In Drosophila, specific regions of the genome are repressed for DNA replication during differentiation by the SNF2 domain-containing protein SUUR through an unknown mechanism. We demonstrate that SUUR is recruited to active replication forks and mediates the repression of DNA replication by directly inhibiting replication fork progression instead of functioning as a replication fork barrier. Mass spectrometry identification of SUUR-associated proteins identified the replicative helicase member CDC45 as a SUUR-associated protein, supporting a role for SUUR directly at replication forks. Our results reveal that control of eukaryotic DNA copy number can occur through the inhibition of replication fork progression.
Assuntos
Variações do Número de Cópias de DNA/genética , Replicação do DNA , Drosophila melanogaster/metabolismo , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Dosagem de Genes , Espectrometria de Massas , Ligação Proteica , Estrutura Terciária de Proteína , Transporte ProteicoRESUMO
SUUR (Suppressor of Under-Replication) protein is responsible for late replication and, as a consequence, for DNA underreplication of intercalary and pericentric heterochromatin in Drosophila melanogaster polytene chromosomes. However, the mechanism by which SUUR slows down the replication process is not clear. To identify possible partners for SUUR we performed a yeast two-hybrid screen using full-length SUUR as bait. This identified HP1, the well-studied heterochromatin protein, as a strong SUUR interactor. Furthermore, we have determined that the central region of SUUR is necessary and sufficient for interaction with the C-terminal part of HP1, which contains the hinge and chromoshadow domains. In addition, recruitment of SUUR to ectopic HP1 sites on chromosomes provides evidence for their association in vivo. Indeed, we found that the distributions of SUUR and HP1 on polytene chromosomes are interdependent: both absence and overexpression of HP1 prevent SUUR from chromosomal binding, whereas SUUR overexpression causes redistribution of HP1 to numerous sites occupied by SUUR. Finally, HP1 binds to intercalary heterochromatin when histone methyltransferase activity of SU(VAR)3-9 is increased. We propose that interaction with HP1 is crucial for the association of SUUR with chromatin.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Heterocromatina/metabolismo , Animais , Cromossomos/ultraestrutura , Drosophila melanogaster/genética , Heterocromatina/ultraestrutura , Complexo Repressor Polycomb 1 , Técnicas do Sistema de Duplo-HíbridoRESUMO
The morphological characteristics of intercalary heterochromatin (IH) are compared with those of other types of silenced chromatin in the Drosophila melanogaster genome: pericentric heterochromatin (PH) and regions subject to position effect variegation (PEV). We conclude that IH regions in polytene chromosomes are binding sites of silencing complexes such as PcG complexes and of SuUR protein. Binding of these proteins results in the appearance of condensed chromatin and late replication of DNA, which in turn may result in DNA underreplication. IH and PH as well as regions subject to PEV have in common the condensed chromatin appearance, the localization of specific proteins, late replication, underreplication in polytene chromosomes, and ectopic pairing.