RESUMO
Regulation of transcription is the main mechanism responsible for precise control of gene expression. Whereas the majority of transcriptional regulation is mediated by DNA-binding transcription factors that bind to regulatory gene regions, an elegant alternative strategy employs small RNA guides, Piwi-interacting RNAs (piRNAs) to identify targets of transcriptional repression. Here, we show that in Drosophila the small ubiquitin-like protein SUMO and the SUMO E3 ligase Su(var)2-10 are required for piRNA-guided deposition of repressive chromatin marks and transcriptional silencing of piRNA targets. Su(var)2-10 links the piRNA-guided target recognition complex to the silencing effector by binding the piRNA/Piwi complex and inducing SUMO-dependent recruitment of the SetDB1/Wde histone methyltransferase effector. We propose that in Drosophila, the nuclear piRNA pathway has co-opted a conserved mechanism of SUMO-dependent recruitment of the SetDB1/Wde chromatin modifier to confer repression of genomic parasites.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , RNA Interferente Pequeno/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Proteínas Argonautas/metabolismo , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Elementos de DNA Transponíveis , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica/genética , Inativação Gênica/fisiologia , Ligação Proteica , Proteínas Inibidoras de STAT Ativados/genética , RNA Interferente Pequeno/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Transcrição Gênica/genéticaRESUMO
Сhromatin is critical for genome compaction and gene expression. On a coarse scale, the genome is divided into euchromatin, which harbors the majority of genes and is enriched in active chromatin marks, and heterochromatin, which is gene-poor but repeat-rich. The conserved molecular hallmark of heterochromatin is the H3K9me3 modification, which is associated with gene silencing. We found that in Drosophila, deposition of most of the H3K9me3 mark depends on SUMO and the SUMO ligase Su(var)2-10, which recruits the histone methyltransferase complex SetDB1/Wde. In addition to repressing repeats, H3K9me3 influences expression of both hetero- and euchromatic host genes. High H3K9me3 levels in heterochromatin are required to suppress spurious transcription and ensure proper gene expression. In euchromatin, a set of conserved genes is repressed by Su(var)2-10/SetDB1-induced H3K9 trimethylation, ensuring tissue-specific gene expression. Several components of heterochromatin are themselves repressed by this pathway, providing a negative feedback mechanism to ensure chromatin homeostasis.
Assuntos
Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Proteínas Cromossômicas não Histona/metabolismo , Metilação de DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Eucromatina/metabolismo , Retroalimentação Fisiológica , Expressão Gênica/genética , Inativação Gênica/fisiologia , Heterocromatina/genética , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Ligases/genética , Metiltransferases/genética , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Repressoras/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genéticaRESUMO
Members of the conserved Argonaute protein family use small RNA guides to locate their mRNA targets and regulate gene expression and suppress mobile genetic elements in eukaryotes1,2. Argonautes are also present in many bacterial and archaeal species3-5. Unlike eukaryotic proteins, several prokaryotic Argonaute proteins use small DNA guides to cleave DNA, a process known as DNA interference6-10. However, the natural functions and targets of DNA interference are poorly understood, and the mechanisms of DNA guide generation and target discrimination remain unknown. Here we analyse the activity of a bacterial Argonaute nuclease from Clostridium butyricum (CbAgo) in vivo. We show that CbAgo targets multicopy genetic elements and suppresses the propagation of plasmids and infection by phages. CbAgo induces DNA interference between homologous sequences and triggers DNA degradation at double-strand breaks in the target DNA. The loading of CbAgo with locus-specific small DNA guides depends on both its intrinsic endonuclease activity and the cellular double-strand break repair machinery. A similar interaction was reported for the acquisition of new spacers during CRISPR adaptation, and prokaryotic genomes that encode Ago nucleases are enriched in CRISPR-Cas systems. These results identify molecular mechanisms that generate guides for DNA interference and suggest that the recognition of foreign nucleic acids by prokaryotic defence systems involves common principles.
Assuntos
Proteínas Argonautas/metabolismo , Clostridium butyricum/enzimologia , DNA/metabolismo , Inativação Gênica , Bacteriófagos/genética , Bacteriófagos/fisiologia , Biocatálise , Sistemas CRISPR-Cas , Clostridium butyricum/genética , Clostridium butyricum/virologia , DNA/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Exodesoxirribonuclease V/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
Small non-coding RNAs called piRNAs serve as guides for an adaptable immune system that represses transposable elements in germ cells of Metazoa. In Drosophila the RDC complex, composed of Rhino, Deadlock and Cutoff (Cuff) bind chromatin of dual-strand piRNA clusters, special genomic regions, which encode piRNA precursors. The RDC complex is required for transcription of piRNA precursors, though the mechanism by which it licenses transcription remained unknown. Here, we show that Cuff prevents premature termination of RNA polymerase II. Cuff prevents cleavage of nascent RNA at poly(A) sites by interfering with recruitment of the cleavage and polyadenylation specificity factor (CPSF) complex. Cuff also protects processed transcripts from degradation by the exonuclease Rat1. Our work reveals a conceptually different mechanism of transcriptional enhancement. In contrast to other factors that regulate termination by binding to specific signals on nascent RNA, the RDC complex inhibits termination in a chromatin-dependent and sequence-independent manner.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/biossíntese , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Adenosina/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Biologia Computacional , Bases de Dados Genéticas , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Exorribonucleases/metabolismo , Genes Reporter , Proteínas Associadas aos Microtúbulos/metabolismo , Complexos Multiproteicos , Polímeros/metabolismo , Ligação Proteica , Estabilidade de RNA , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Terminação da Transcrição GenéticaRESUMO
The conserved THO/TREX (transcription/export) complex is critical for pre-mRNA processing and mRNA nuclear export. In metazoa, TREX is loaded on nascent RNA transcribed by RNA polymerase II in a splicing-dependent fashion; however, how TREX functions is poorly understood. Here we show that Thoc5 and other TREX components are essential for the biogenesis of piRNA, a distinct class of small noncoding RNAs that control expression of transposable elements (TEs) in the Drosophila germline. Mutations in TREX lead to defects in piRNA biogenesis, resulting in derepression of multiple TE families, gametogenesis defects, and sterility. TREX components are enriched on piRNA precursors transcribed from dual-strand piRNA clusters and colocalize in distinct nuclear foci that overlap with sites of piRNA transcription. The localization of TREX in nuclear foci and its loading on piRNA precursor transcripts depend on Cutoff, a protein associated with chromatin of piRNA clusters. Finally, we show that TREX is required for accumulation of nascent piRNA precursors. Our study reveals a novel splicing-independent mechanism for TREX loading on nascent RNA and its importance in piRNA biogenesis.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , Animais , Padronização Corporal/genética , Núcleo Celular/metabolismo , Proteínas de Drosophila/biossíntese , Feminino , Fertilidade/genética , Masculino , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transporte Proteico , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Histone 3 lysine 9 trimethylation (H3K9me3) is a conserved histone modification that is best known for its role in constitutive heterochromatin formation and the repression of repetitive DNA elements. More recently, it has become evident that H3K9me3 is also deposited at certain loci in a tissue-specific manner and plays important roles in regulating cell identity. Notably, H3K9me3 can repress genes encoding silencing factors, pointing to a fundamental principle of repressive chromatin auto-regulation. Interestingly, recent studies have shown that H3K9me3 deposition requires protein SUMOylation in different contexts, suggesting that the SUMO pathway functions as an important module in gene silencing and heterochromatin formation. In this Review, we discuss the role of H3K9me3 in gene regulation in various systems and the molecular mechanisms that guide the silencing machinery to target loci.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Lisina/metabolismo , Animais , Retroalimentação Fisiológica , Heterocromatina/metabolismo , Humanos , MetilaçãoRESUMO
Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.
Assuntos
Reprogramação Celular/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Animais , Células Cultivadas , Cromatina/metabolismo , Fator 1 de Modelagem da Cromatina/antagonistas & inibidores , Fator 1 de Modelagem da Cromatina/genética , Regulação da Expressão Gênica/genética , Heterocromatina/metabolismo , Camundongos , Nucleossomos/metabolismo , Interferência de RNA , Transdução GenéticaRESUMO
The piRNA pathway is an adaptive mechanism that maintains genome stability by repression of selfish genomic elements. In the male germline of Drosophila melanogaster repression of Stellate genes by piRNAs generated from Supressor of Stellate (Su(Ste)) locus is required for male fertility, but both Su(Ste) piRNAs and their targets are absent in other Drosophila species. We found that D. melanogaster genome contains multiple X-linked non-coding genomic repeats that have sequence similarity to the protein-coding host gene vasa. In the male germline, these vasa-related AT-chX repeats produce abundant piRNAs that are antisense to vasa; however, vasa mRNA escapes silencing due to imperfect complementarity to AT-chX piRNAs. Unexpectedly, we discovered AT-chX piRNAs target vasa of Drosophila mauritiana in the testes of interspecies hybrids. In the majority of hybrid flies, the testes were strongly reduced in size and germline content. A minority of hybrids maintained wild-type array of premeiotic germ cells in the testes, but in them harmful Stellate genes were derepressed due to the absence of Su(Ste) piRNAs, and meiotic failures were observed. Thus, the piRNA pathway contributes to reproductive isolation between D. melanogaster and closely related species, causing hybrid male sterility via misregulation of two different host protein factors.
Assuntos
Quimera/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila/genética , Inativação Gênica , Genoma de Inseto , Proteínas Quinases/genética , RNA Interferente Pequeno/genética , Animais , Sequência de Bases , Quimera/metabolismo , Cruzamentos Genéticos , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Feminino , Fertilidade , Infertilidade , Masculino , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Isolamento Reprodutivo , Alinhamento de Sequência , Espermatozoides/metabolismo , Espermatozoides/patologia , Testículo/anormalidades , Testículo/metabolismoRESUMO
BACKGROUND: The red flour beetle Tribolium castaneum has emerged as an important model organism for the study of gene function in development and physiology, for ecological and evolutionary genomics, for pest control and a plethora of other topics. RNA interference (RNAi), transgenesis and genome editing are well established and the resources for genome-wide RNAi screening have become available in this model. All these techniques depend on a high quality genome assembly and precise gene models. However, the first version of the genome assembly was generated by Sanger sequencing, and with a small set of RNA sequence data limiting annotation quality. RESULTS: Here, we present an improved genome assembly (Tcas5.2) and an enhanced genome annotation resulting in a new official gene set (OGS3) for Tribolium castaneum, which significantly increase the quality of the genomic resources. By adding large-distance jumping library DNA sequencing to join scaffolds and fill small gaps, the gaps in the genome assembly were reduced and the N50 increased to 4753kbp. The precision of the gene models was enhanced by the use of a large body of RNA-Seq reads of different life history stages and tissue types, leading to the discovery of 1452 novel gene sequences. We also added new features such as alternative splicing, well defined UTRs and microRNA target predictions. For quality control, 399 gene models were evaluated by manual inspection. The current gene set was submitted to Genbank and accepted as a RefSeq genome by NCBI. CONCLUSIONS: The new genome assembly (Tcas5.2) and the official gene set (OGS3) provide enhanced genomic resources for genetic work in Tribolium castaneum. The much improved information on transcription start sites supports transgenic and gene editing approaches. Further, novel types of information such as splice variants and microRNA target genes open additional possibilities for analysis.
Assuntos
Genes de Insetos , Genoma de Inseto , Genômica , Tribolium/genética , Animais , Sítios de Ligação , Biologia Computacional/métodos , Genômica/métodos , MicroRNAs/genética , Anotação de Sequência Molecular , Filogenia , Interferência de RNA , Reprodutibilidade dos TestesRESUMO
Members of the conserved Argonaute (Ago) protein family provide defence against invading nucleic acids in eukaryotes in the process of RNA interference. Many prokaryotes also contain Ago proteins that are predicted to be active nucleases; however, their functional activities in host cells remain poorly understood. Here, we characterize the in vitro and in vivo properties of the SeAgo protein from the mesophilic cyanobacterium Synechococcus elongatus. We show that SeAgo is a DNA-guided nuclease preferentially acting on single-stranded DNA targets, with non-specific guide-independent activity observed for double-stranded substrates. The SeAgo gene is steadily expressed in S. elongatus; however, its deletion or overexpression does not change the kinetics of cell growth. When purified from its host cells or from heterologous E. coli, SeAgo is loaded with small guide DNAs whose formation depends on the endonuclease activity of the argonaute protein. SeAgo co-purifies with SSB proteins suggesting that they may also be involved in DNA processing. The SeAgo-associated small DNAs are derived from diverse genomic locations, with certain enrichment for the proposed sites of chromosomal replication initiation and termination, but show no preference for an endogenous plasmid. Therefore, promiscuous genome sampling by SeAgo does not have great effects on cell physiology and plasmid maintenance.
Assuntos
Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Genoma Bacteriano , Genômica , Synechococcus/genética , Synechococcus/metabolismo , Proteínas Argonautas/química , Sequência de Bases , Sítios de Ligação , DNA/química , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Genômica/métodos , Modelos Biológicos , Modelos Moleculares , Conformação Molecular , Proteínas Recombinantes , Relação Estrutura-AtividadeRESUMO
MicroRNAs are well-established players in the development of multicellular animals. Most of our understanding of microRNA function in arthropod development comes from studies in Drosophila. Despite their advantages as model systems, the long germband embryogenesis of fruit flies is an evolutionary derived state restricted to several holometabolous insect lineages. MicroRNA evolution and expression across development in animals exhibiting the ancestral and more widespread short germband mode of embryogenesis has not been characterized. We sequenced small RNA libraries of oocytes and successive intervals covering the embryonic development of the short germband model organism, Tribolium castaneum. We analyzed the evolution and temporal expression of the microRNA complement and sequenced libraries of total RNA to investigate the relationships with microRNA target expression. We show microRNA maternal loading and sequence-specific 3' end nontemplate oligoadenylation of maternally deposited microRNAs that is conserved between Tribolium and Drosophila. We further uncover large clusters encoding multiple paralogs from several Tribolium-specific microRNA families expressed during a narrow interval of time immediately after the activation of zygotic transcription. These novel microRNAs, together with several early expressed conserved microRNAs, target a significant number of maternally deposited transcripts. Comparison with Drosophila shows that microRNA-mediated maternal transcript targeting is a conserved process in insects, but the number and sequences of microRNAs involved have diverged. The expression of fast-evolving and species-specific microRNAs in the early blastoderm of T. castaneum is consistent with previous findings in Drosophila and shows that the unique permissiveness for microRNA innovation at this stage is a conserved phenomenon.
Assuntos
Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Tribolium/embriologia , Tribolium/genética , Animais , Regulação para Baixo , Drosophila/genética , Desenvolvimento Embrionário/genética , Biblioteca Gênica , MicroRNAs/metabolismo , Anotação de Sequência Molecular , Análise de Sequência de RNARESUMO
MicroRNAs are short non-protein-coding RNAs that regulate gene expression at the post-transcriptional level and are essential for the embryonic development of multicellular animals. Comparative genome-scale analyses have revealed that metazoan evolution is accompanied by the continuous acquisition of novel microRNA genes. This suggests that novel microRNAs may promote innovation and diversity in development. We determined the evolutionary origins of extant Drosophila microRNAs and estimated the sequence divergence between the 130 orthologous microRNAs in Drosophila melanogaster and Drosophila virilis, separated by 63 million years of evolution. We then generated small RNA sequencing data sets covering D. virilis development and explored the relationship between microRNA conservation and expression in a developmental context. We find that late embryonic, larval, and adult stages are dominated by conserved microRNAs. This pattern, however, does not hold for the early embryo, where rapidly evolving microRNAs are uniquely present at high levels in both species. The group of fast-evolving microRNAs that are highly expressed in the early embryo belong to two Drosophilid lineage-specific clusters: mir-310 â¼ 313 and mir-309 â¼ 6. These clusters have particularly complex evolutionary histories of duplication, gain, and loss. Our analyses suggest that the early embryo is a more permissive environment for microRNA changes and innovations. Fast-evolving microRNAs, therefore, have the opportunity to become preferentially integrated in early developmental processes, and may impact the evolution of development. The relationship between microRNA conservation and expression throughout the development of Drosophila differs from that previously observed for protein-coding genes.
Assuntos
Drosophila/genética , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Animais , Sequência de Bases , Sequência Conservada , Drosophila/classificação , Drosophila/crescimento & desenvolvimento , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Feminino , Hibridização In Situ , Dados de Sequência Molecular , Filogenia , Homologia de Sequência do Ácido NucleicoRESUMO
Genetic linkage may result in the expression of multiple products from a polycistronic transcript, under the control of a single promoter. In animals, protein-coding polycistronic transcripts are rare. However, microRNAs are frequently clustered in the genomes of animals, and these clusters are often transcribed as a single unit. The evolution of microRNA clusters has been the subject of much speculation, and a selective advantage of clusters of functionally related microRNAs is often proposed. However, the origin of microRNA clusters has not been so far explored. Here, we study the evolution of microRNA clusters in Drosophila melanogaster. We observed that the majority of microRNA clusters arose by the de novo formation of new microRNA-like hairpins in existing microRNA transcripts. Some clusters also emerged by tandem duplication of a single microRNA. Comparative genomics show that these clusters are unlikely to split or undergo rearrangements. We did not find any instances of clusters appearing by rearrangement of pre-existing microRNA genes. We propose a model for microRNA cluster evolution in which selection over one of the microRNAs in the cluster interferes with the evolution of the other linked microRNAs. Our analysis suggests that the study of microRNAs and small RNAs must consider linkage associations.
Assuntos
Evolução Molecular , MicroRNAs/genética , Animais , Drosophila melanogaster/genética , Genoma , MicroRNAs/químicaRESUMO
A single transcript sometimes codes for more than one product. In bacteria, and in a few exceptional animal lineages, many genes are organized into operons: clusters of open reading frames that are transcribed together in a single polycistronic transcript. However, polycistronic transcripts are rare in eukaryotes. One notable exception is that of miRNAs (microRNAs), small RNAs that regulate gene expression at the post-transcriptional level. The primary transcripts of miRNAs commonly produce more than one functional product, by at least three different mechanisms. miRNAs are often produced from polycistronic transcripts together with other miRNA precursors. Also, miRNAs frequently derive from protein-coding gene introns. Finally, each miRNA precursor can produce two mature miRNA products. We argue, in the present review, that miRNAs are frequently hosted in transcripts coding for multiple products because new miRNA precursor sequences that arise by chance in transcribed regions are more likely to become functional miRNAs during evolution.
Assuntos
MicroRNAs/genética , RNA Mensageiro/genética , Animais , Humanos , ÍntronsRESUMO
The conserved family of Transcription Intermediary Factors (TIF1) proteins consists of key transcriptional regulators that control transcription of target genes by modulating chromatin state. Unlike mammals that have four TIF1 members, Drosophila only encodes one member of the family, Bonus. Bonus has been implicated in embryonic development and organogenesis and shown to regulate several signaling pathways, however, its targets and mechanism of action remained poorly understood. We found that knockdown of Bonus in early oogenesis results in severe defects in ovarian development and in ectopic expression of genes that are normally repressed in the germline, demonstrating its essential function in the ovary. Recruitment of Bonus to chromatin leads to silencing associated with accumulation of the repressive H3K9me3 mark. We show that Bonus associates with the histone methyltransferase SetDB1 and the chromatin remodeler NuRD and depletion of either component releases Bonus-induced repression. We further established that Bonus is SUMOylated at a single site at its N-terminus that is conserved among insects and this modification is indispensable for Bonus's repressive activity. SUMOylation influences Bonus's subnuclear localization, its association with chromatin and interaction with SetDB1. Finally, we showed that Bonus SUMOylation is mediated by the SUMO E3-ligase Su(var)2-10, revealing that although SUMOylation of TIF1 proteins is conserved between insects and mammals, both the mechanism and specific site of modification is different in the two taxa. Together, our work identified Bonus as a regulator of tissue-specific gene expression and revealed the importance of SUMOylation as a regulator of complex formation in the context of transcriptional repression.
Assuntos
Cromatina , Proteínas de Drosophila , Animais , Feminino , Cromatina/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Células Germinativas/metabolismo , Mamíferos/genética , Análise de Mediação , SumoilaçãoRESUMO
Genome regulation involves complex protein interactions that are often mediated through post-translational modifications (PTMs). SUMOylation-modification by the small ubiquitin-like modifier (SUMO)-has been implicated in numerous essential processes in eukaryotes. In Drosophila, SUMO is required for viability and fertility, with its depletion from ovaries leading to heterochromatin loss and ectopic transposon and gene activation. Here, we developed a proteomics-based strategy to uncover the Drosophila ovarian "SUMOylome," which revealed that SUMOylation is widespread among proteins involved in heterochromatin regulation and different aspects of the Piwi-interacting small RNA (piRNA) pathway that represses transposons. Furthermore, we show that SUMOylation of several piRNA pathway proteins occurs in a Piwi-dependent manner. Together, these data highlight broad implications of protein SUMOylation in epigenetic regulation and indicate novel roles of this modification in the cellular defense against genomic parasites. Finally, this work provides a resource for the study of SUMOylation in other biological contexts in the Drosophila model.
RESUMO
The conserved family of Transcription Intermediary Factors (TIF1) proteins consists of key transcriptional regulators that control transcription of target genes by modulating chromatin state. Unlike mammals that have four TIF1 members, Drosophila only encodes one member of the family, Bonus. Bonus has been implicated in embryonic development and organogenesis and shown to regulate several signaling pathways, however, its targets and mechanism of action remained poorly understood. We found that knockdown of Bonus in early oogenesis results in severe defects in ovarian development and in ectopic expression of genes that are normally repressed in the germline, demonstrating its essential function in the ovary. Recruitment of Bonus to chromatin leads to silencing associated with accumulation of the repressive H3K9me3 mark. We show that Bonus associates with the histone methyltransferase SetDB1 and the chromatin remodeler NuRD and depletion of either component releases Bonus-induced repression. We further established that Bonus is SUMOylated at a single site at its N-terminus that is conserved among insects and this modification is indispensable for Bonus's repressive activity. SUMOylation influences Bonus's subnuclear localization, its association with chromatin and interaction with SetDB1. Finally, we showed that Bonus SUMOylation is mediated by the SUMO E3-ligase Su(var)2-10, revealing that although SUMOylation of TIF1 proteins is conserved between insects and mammals, both the mechanism and specific site of modification is different in the two taxa. Together, our work identified Bonus as a regulator of tissue-specific gene expression and revealed the importance of SUMOylation as a regulator of complex formation in the context of transcriptional repression.
RESUMO
Cell fate commitment is driven by dynamic changes in chromatin architecture and activity of lineage-specific transcription factors (TFs). The chromatin assembly factor-1 (CAF-1) is a histone chaperone that regulates chromatin architecture by facilitating nucleosome assembly during DNA replication. Accumulating evidence supports a substantial role of CAF-1 in cell fate maintenance, but the mechanisms by which CAF-1 restricts lineage choice remain poorly understood. Here, we investigate how CAF-1 influences chromatin dynamics and TF activity during lineage differentiation. We show that CAF-1 suppression triggers rapid differentiation of myeloid stem and progenitor cells into a mixed lineage state. We find that CAF-1 sustains lineage fidelity by controlling chromatin accessibility at specific loci, and limiting the binding of ELF1 TF at newly-accessible diverging regulatory elements. Together, our findings decipher key traits of chromatin accessibility that sustain lineage integrity and point to a powerful strategy for dissecting transcriptional circuits central to cell fate commitment.
Assuntos
Cromatina , Chaperonas de Histonas , Fator 1 de Modelagem da Cromatina/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Cromossomos/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismoRESUMO
Ribosomal RNAs (rRNAs) are essential components of the ribosome and are among the most abundant macromolecules in the cell. To ensure high rRNA level, eukaryotic genomes contain dozens to hundreds of rDNA genes, however, only a fraction of the rRNA genes seems to be active, while others are transcriptionally silent. We found that individual rDNA genes have high level of cell-to-cell heterogeneity in their expression in Drosophila melanogaster. Insertion of heterologous sequences into rDNA leads to repression associated with reduced expression in individual cells and decreased number of cells expressing rDNA with insertions. We found that SUMO (Small Ubiquitin-like Modifier) and SUMO ligase Ubc9 are required for efficient repression of interrupted rDNA units and variable expression of intact rDNA. Disruption of the SUMO pathway abolishes discrimination of interrupted and intact rDNAs and removes cell-to-cell heterogeneity leading to uniformly high expression of individual rDNA in single cells. Our results suggest that the SUMO pathway is responsible for both repression of interrupted units and control of intact rDNA expression.
Assuntos
DNA Ribossômico/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Genes de RNAr , Proteínas Repressoras/metabolismo , Animais , Elementos de DNA Transponíveis , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Heterocromatina/metabolismo , Redes e Vias Metabólicas , Modelos Genéticos , Proteínas Nucleares/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina , Transgenes , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
BACKGROUND: Piwi-interacting RNAs (piRNAs) are a class of short (~26-31-nucleotide) non-protein-coding RNAs expressed in the metazoan germline. The piRNA pathway in arthropods is best understood in the ovary of Drosophila melanogaster, where it acts to silence active transposable elements (TEs). Maternal loading of piRNAs in oocytes is further required for the inheritance of piRNA-mediated transposon defence. However, our understanding of the diversity, evolution and function of the piRNA complement beyond drosophilids is limited. The red flour beetle, Tribolium castaneum, is an emerging model organism separated from Drosophila by ~ 350 million years of evolution that displays a number of features ancestral to arthropods, including short germ embryogenesis. Here, we characterize the maternally deposited and zygotically expressed small RNA and mRNA complements throughout T. castaneum embryogenesis. RESULTS: We find that beetle oocytes and embryos of all stages are abundant in heterogeneous ~ 28-nucleotide RNAs. These small RNAs originate from discrete genomic loci enriched in TE sequences and display the molecular signatures of transposon-derived piRNAs. In addition to the maternally loaded primary piRNAs, Tribolium embryos produce secondary piRNAs by the cleavage of zygotically activated TE transcripts via the ping-pong mechanism. The two Tribolium piRNA pathway effector proteins, Tc-Piwi/Aub and Tc-Ago3, are also expressed throughout the soma of early embryos. CONCLUSIONS: Our results show that the piRNA pathway in Tribolium is not restricted to the germline, but also operates in the embryo and may act to antagonize zygotically activated transposons. Taken together, these data highlight a functional divergence of the piRNA pathway between insects.