RESUMO
Fertilization is a multistep process that culminates in the fusion of sperm and egg, thus marking the beginning of a new organism in sexually reproducing species. Despite its importance for reproduction, the molecular mechanisms that regulate this singular event, particularly sperm-egg fusion, have remained mysterious for many decades. Here, we summarize our current molecular understanding of sperm-egg interaction, focusing mainly on mammalian fertilization. Given the fundamental importance of sperm-egg fusion yet the lack of knowledge of this process in vertebrates, we discuss hallmarks and emerging themes of cell fusion by drawing from well-studied examples such as viral entry, placenta formation, and muscle development. We conclude by identifying open questions and exciting avenues for future studies in gamete fusion.
Assuntos
Fertilização , Interações Espermatozoide-Óvulo , Animais , Masculino , Mamíferos , Reprodução , Interações Espermatozoide-Óvulo/fisiologia , Espermatozoides/fisiologiaRESUMO
Cell fate transitions depend on balanced rewiring of transcription and translation programs to mediate ordered developmental progression. Components of the nonsense-mediated mRNA decay (NMD) pathway have been implicated in regulating embryonic stem cell (ESC) differentiation, but the exact mechanism is unclear. Here we show that NMD controls expression levels of the translation initiation factor Eif4a2 and its premature termination codon-encoding isoform (Eif4a2PTC ). NMD deficiency leads to translation of the truncated eIF4A2PTC protein. eIF4A2PTC elicits increased mTORC1 activity and translation rates and causes differentiation delays. This establishes a previously unknown feedback loop between NMD and translation initiation. Furthermore, our results show a clear hierarchy in the severity of target deregulation and differentiation phenotypes between NMD effector KOs (Smg5 KO > Smg6 KO > Smg7 KO), which highlights heterodimer-independent functions for SMG5 and SMG7. Together, our findings expose an intricate link between mRNA homeostasis and mTORC1 activity that must be maintained for normal dynamics of cell state transitions.
Assuntos
Proteínas de Transporte , Degradação do RNAm Mediada por Códon sem Sentido , Proteínas de Transporte/genética , Expressão Gênica , Células HeLa , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismoRESUMO
Cells control dynamic transitions in transcript levels by regulating transcription, processing, and/or degradation through an integrated regulatory strategy. Here, we combine RNA metabolic labeling, rRNA-depleted RNA-seq, and DRiLL, a novel computational framework, to quantify the level; editing sites; and transcription, processing, and degradation rates of each transcript at a splice junction resolution during the LPS response of mouse dendritic cells. Four key regulatory strategies, dominated by RNA transcription changes, generate most temporal gene expression patterns. Noncanonical strategies that also employ dynamic posttranscriptional regulation control only a minority of genes, but provide unique signal processing features. We validate Tristetraprolin (TTP) as a major regulator of RNA degradation in one noncanonical strategy. Applying DRiLL to the regulation of noncoding RNAs and to zebrafish embryogenesis demonstrates its broad utility. Our study provides a new quantitative approach to discover transcriptional and posttranscriptional events that control dynamic changes in transcript levels using RNA sequencing data.
Assuntos
Simulação por Computador , Células Dendríticas/metabolismo , Análise de Sequência de RNA/métodos , Animais , Perfilação da Expressão Gênica/métodos , Cinética , Lipopolissacarídeos/metabolismo , Camundongos , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA não Traduzido/metabolismo , Transcrição Gênica , Tristetraprolina/metabolismo , Peixe-Zebra/embriologiaRESUMO
Ribosomes are produced in large quantities during oogenesis and are stored in the egg. However, the egg and early embryo are translationally repressed1-4. Here, using mass spectrometry and cryo-electron microscopy analyses of ribosomes isolated from zebrafish (Danio rerio) and Xenopus laevis eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant state to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four conserved factors that form two modules, consisting of Habp4-eEF2 and death associated protein 1b (Dap1b) or Dap in complex with eIF5a. Both modules occupy functionally important sites and act together to stabilize ribosomes and repress translation. Dap1b (also known as Dapl1 in mammals) is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Addition of recombinant zebrafish Dap1b protein is sufficient to block translation and reconstitute the dormant egg ribosome state in a mammalian translation extract in vitro. Thus, a developmentally programmed, conserved ribosome state has a key role in ribosome storage and translational repression in the egg.
Assuntos
Sequência Conservada , Evolução Molecular , Óvulo , Biossíntese de Proteínas , Ribossomos , Proteínas de Xenopus , Proteínas de Peixe-Zebra , Animais , Microscopia Crioeletrônica/métodos , Peptídeos/metabolismo , Ribossomos/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Espectrometria de Massas , Xenopus laevis/embriologia , Óvulo/metabolismo , Estruturas Embrionárias , Desenvolvimento Embrionário , Feminino , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
The pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11-mediated termination is essential for vertebrate development. A range of genomic analyses, including mNET-seq, 3' mRNA-seq, chromatin RNA-seq, and ChIP-seq, reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome-wide. PCF11 binds preferentially between closely spaced genes, where it prevents transcriptional interference and consequent gene downregulation. Notably, PCF11 is sub-stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto-regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development, PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination.
Assuntos
RNA Mensageiro/biossíntese , Terminação da Transcrição Genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Regulação da Expressão Gênica no Desenvolvimento , Células HeLa , Humanos , Mutação , Poliadenilação , Ligação Proteica , Clivagem do RNA , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
Maternal mRNAs are essential for protein synthesis during oogenesis and early embryogenesis. To adapt translation to specific needs during development, maternal mRNAs are translationally repressed by shortening the polyA tails. While mRNA deadenylation is associated with decapping and degradation in somatic cells, maternal mRNAs with short polyA tails are stable. Here we report that the germline-specific eIF4E paralog, eIF4E1b, is essential for zebrafish oogenesis. eIF4E1b localizes to P-bodies in zebrafish embryos and binds to mRNAs with reported short or no polyA tails, including histone mRNAs. Loss of eIF4E1b results in reduced histone mRNA levels in early gonads, consistent with a role in mRNA storage. Using mouse and human eIF4E1Bs (in vitro) and zebrafish eIF4E1b (in vivo), we show that unlike canonical eIF4Es, eIF4E1b does not interact with eIF4G to initiate translation. Instead, eIF4E1b interacts with the translational repressor eIF4ENIF1, which is required for eIF4E1b localization to P-bodies. Our study is consistent with an important role of eIF4E1b in regulating mRNA dormancy and provides new insights into fundamental post-transcriptional regulatory principles governing early vertebrate development.
Assuntos
RNA Mensageiro Estocado , Peixe-Zebra , Animais , Humanos , Camundongos , RNA Mensageiro Estocado/genética , RNA Mensageiro Estocado/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Histonas/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Biossíntese de ProteínasRESUMO
tRNA genes exist in multiple copies in the genome of all organisms across the three domains of life. Besides the sequence differences across tRNA copies, extensive post-transcriptional modification adds a further layer to tRNA diversification. Whilst the crucial role of tRNAs as adapter molecules in protein translation is well established, whether all tRNAs are actually expressed, and whether the differences across isodecoders play any regulatory role is only recently being uncovered. Here we built upon recent developments in the use of NGS-based methods for RNA modification detection and developed tRAM-seq, an experimental protocol and in silico analysis pipeline to investigate tRNA expression and modification. Using tRAM-seq, we analysed the full ensemble of nucleo-cytoplasmic and mitochondrial tRNAs during embryonic development of the model vertebrate zebrafish. We show that the repertoire of tRNAs changes during development, with an apparent major switch in tRNA isodecoder expression and modification profile taking place around the start of gastrulation. Taken together, our findings suggest the existence of a general reprogramming of the expressed tRNA pool, possibly gearing the translational machinery for distinct stages of the delicate and crucial process of embryo development.
RESUMO
Methylation of cytosines in the CG context (mCG) is the most abundant DNA modification in vertebrates that plays crucial roles in cellular differentiation and identity. After fertilization, DNA methylation patterns inherited from parental gametes are remodelled into a state compatible with embryogenesis. In mammals, this is achieved through the global erasure and re-establishment of DNA methylation patterns. However, in non-mammalian vertebrates like zebrafish, no global erasure has been observed. To investigate the evolutionary conservation and divergence of DNA methylation remodelling in teleosts, we generated base resolution DNA methylome datasets of developing medaka and medaka-zebrafish hybrid embryos. In contrast to previous reports, we show that medaka display comparable DNA methylome dynamics to zebrafish with high gametic mCG levels (sperm: â¼90%; egg: â¼75%), and adoption of a paternal-like methylome during early embryogenesis, with no signs of prior DNA methylation erasure. We also demonstrate that non-canonical DNA methylation (mCH) reprogramming at TGCT tandem repeats is a conserved feature of teleost embryogenesis. Lastly, we find remarkable evolutionary conservation of DNA methylation remodelling patterns in medaka-zebrafish hybrids, indicative of compatible DNA methylation maintenance machinery in far-related teleost species. Overall, these results suggest strong evolutionary conservation of DNA methylation remodelling pathways in teleosts, which is distinct from the global DNA methylome erasure and reestablishment observed in mammals.
RESUMO
Self-organization is a key feature of many biological and developmental processes, including cell migration. Although cell migration has traditionally been viewed as a biological response to extrinsic signals, advances within the past two decades have highlighted the importance of intrinsic self-organizing properties to direct cell migration on multiple scales. In this Review, we will explore self-organizing mechanisms that lay the foundation for both single and collective cell migration. Based on in vitro and in vivo examples, we will discuss theoretical concepts that underlie the persistent migration of single cells in the absence of directional guidance cues, and the formation of an autonomous cell collective that drives coordinated migration. Finally, we highlight the general implications of self-organizing principles guiding cell migration for biological and medical research.
Assuntos
Movimento Celular/fisiologia , Animais , Quimiocinas , Locomoção/fisiologia , Modelos Biológicos , Morfogênese/fisiologiaRESUMO
A large number of mRNAs of maternal origin are produced during oogenesis and deposited in the oocyte. Since transcription stops at the onset of meiosis during oogenesis and does not resume until later in embryogenesis, maternal mRNAs are the only templates for protein synthesis during this period. To ensure that a protein is made in the right place at the right time, the translation of maternal mRNAs must be activated at a specific stage of development. Here we summarize our current understanding of the sophisticated mechanisms that contribute to the temporal repression of maternal mRNAs, termed maternal mRNA dormancy. We discuss mechanisms at the level of the RNA itself, such as the regulation of polyadenine tail length and RNA modifications, as well as at the level of RNA-binding proteins, which often block the assembly of translation initiation complexes at the 5' end of an mRNA or recruit mRNAs to specific subcellular compartments. We also review microRNAs and other mechanisms that contribute to repressing translation, such as ribosome dormancy. Importantly, the mechanisms responsible for mRNA dormancy during the oocyte-to-embryo transition are also relevant to cellular quiescence in other biological contexts.
Assuntos
Oócitos , Oogênese , Animais , Humanos , Oócitos/metabolismo , Oogênese/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro Estocado/metabolismo , RNA Mensageiro Estocado/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Biossíntese de Proteínas , Regulação da Expressão Gênica no Desenvolvimento , Feminino , Desenvolvimento Embrionário/genéticaRESUMO
Post-transcriptional regulation of gene expression is crucial during the oocyte-to-embryo transition, a highly dynamic process characterized by the absence of nuclear transcription. Thus, changes to the RNA content are solely dependent on RNA degradation. Although several mechanisms that promote RNA decay during embryogenesis have been identified, it remains unclear which machineries contribute to remodeling the maternal transcriptome. Here, we focused on the degradation factor Ski7 in zebrafish. Homozygous ski7 mutant fish had higher proportions of both poor quality eggs and eggs that were unable to develop beyond the one-cell stage. Consistent with the idea that Ski7 participates in remodeling the maternal RNA content, transcriptome profiling identified hundreds of misregulated mRNAs in the absence of Ski7. Furthermore, upregulated genes were generally lowly expressed in wild type, suggesting that Ski7 maintains low transcript levels for this subset of genes. Finally, GO enrichment and proteomic analyses of misregulated factors implicated Ski7 in the regulation of redox processes. This was confirmed experimentally by an increased resistance of ski7 mutant embryos to reductive stress. Our results provide first insights into the physiological role of vertebrate Ski7 as a post-transcriptional regulator during the oocyte-to-embryo transition.
Assuntos
Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Oócitos/metabolismo , RNA/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário/genética , Exossomos/genética , Exossomos/metabolismo , Perfilação da Expressão Gênica/métodos , Mutação , Oócitos/citologia , Ligação Proteica , RNA/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
Fertilization is the fundamental process that initiates the development of a new individual in all sexually reproducing species. Despite its importance, our understanding of the molecular players that govern mammalian sperm-egg interaction is incomplete, partly because many of the essential factors found in nonmammalian species do not have obvious mammalian homologs. We have recently identified the lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) protein Bouncer as an essential fertilization factor in zebrafish [S. Herberg, K. R. Gert, A. Schleiffer, A. Pauli, Science 361, 1029-1033 (2018)]. Here, we show that Bouncer's homolog in mammals, Sperm Acrosome Associated 4 (SPACA4), is also required for efficient fertilization in mice. In contrast to fish, in which Bouncer is expressed specifically in the egg, SPACA4 is expressed exclusively in the sperm. Male knockout mice are severely subfertile, and sperm lacking SPACA4 fail to fertilize wild-type eggs in vitro. Interestingly, removal of the zona pellucida rescues the fertilization defect of Spaca4-deficient sperm in vitro, indicating that SPACA4 is not required for the interaction of sperm and the oolemma but rather of sperm and the zona pellucida. Our work identifies SPACA4 as an important sperm protein necessary for zona pellucida penetration during mammalian fertilization.
Assuntos
Antígenos Ly/metabolismo , Fertilização , Infertilidade Masculina/patologia , Glicoproteínas de Membrana/fisiologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Interações Espermatozoide-Óvulo , Acrossomo/metabolismo , Acrossomo/patologia , Animais , Antígenos Ly/genética , Feminino , Infertilidade Masculina/etiologia , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Zona Pelúcida/metabolismo , Zona Pelúcida/patologiaRESUMO
The organization of nucleosomes influences transcriptional activity by controlling accessibility of DNA binding proteins to the genome. Genome-wide nucleosome binding profiles have identified a canonical nucleosome organization at gene promoters, where arrays of well-positioned nucleosomes emanate from nucleosome-depleted regions. The mechanisms of formation and the function of canonical promoter nucleosome organization remain unclear. Here we analyze the genome-wide location of nucleosomes during zebrafish embryogenesis and show that well-positioned nucleosome arrays appear on thousands of promoters during the activation of the zygotic genome. The formation of canonical promoter nucleosome organization is independent of DNA sequence preference, transcriptional elongation, and robust RNA polymerase II (Pol II) binding. Instead, canonical promoter nucleosome organization correlates with the presence of histone H3 lysine 4 trimethylation (H3K4me3) and affects future transcriptional activation. These findings reveal that genome activation is central to the organization of nucleosome arrays during early embryogenesis.
Assuntos
Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário/genética , Nucleossomos/genética , Ativação Transcricional/genética , Animais , Genoma , Histona-Lisina N-Metiltransferase/genética , Regiões Promotoras Genéticas , RNA Polimerase II/genética , Análise de Sequência de DNA , Transcrição Gênica , Peixe-ZebraRESUMO
Non-coding RNAs (ncRNAs) are emerging as key regulators of embryogenesis. They control embryonic gene expression by several means, ranging from microRNA-induced degradation of mRNAs to long ncRNA-mediated modification of chromatin. Many aspects of embryogenesis seem to be controlled by ncRNAs, including the maternal-zygotic transition, the maintenance of pluripotency, the patterning of the body axes, the specification and differentiation of cell types and the morphogenesis of organs. Drawing from several animal model systems, we describe two emerging themes for ncRNA function: promoting developmental transitions and maintaining developmental states. These examples also highlight the roles of ncRNAs in ensuring a robust commitment to one of two possible cell fates.
Assuntos
Desenvolvimento Embrionário , Regulação da Expressão Gênica , RNA não Traduzido/genética , Animais , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Dosagem de Genes , Humanos , RNA Mensageiro/genéticaRESUMO
Over the past decade, high-throughput studies have identified many novel transcripts. While their existence is undisputed, their coding potential and functionality have remained controversial. Recent computational approaches guided by ribosome profiling have indicated that translation is far more pervasive than anticipated and takes place on many transcripts previously assumed to be non-coding. Some of these newly discovered translated transcripts encode short, functional proteins that had been missed in prior screens. Other transcripts are translated, but it might be the process of translation rather than the resulting peptides that serves a function. Here, we review annotation studies in zebrafish to discuss the challenges of placing RNAs onto the continuum that ranges from functional protein-encoding mRNAs to potentially non-functional peptide-producing RNAs to non-coding RNAs. As highlighted by the discovery of the novel signaling peptide Apela/ELABELA/Toddler, accurate annotations can give rise to exciting opportunities to identify the functions of previously uncharacterized transcripts.
Assuntos
Peptídeos/metabolismo , RNA não Traduzido/genética , Animais , Humanos , Anotação de Sequência Molecular , Fases de Leitura Aberta/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-Zebra/genéticaRESUMO
Large-scale genomics and computational approaches have identified thousands of putative long non-coding RNAs (lncRNAs). It has been controversial, however, as to what fraction of these RNAs is truly non-coding. Here, we combine ribosome profiling with a machine-learning approach to validate lncRNAs during zebrafish development in a high throughput manner. We find that dozens of proposed lncRNAs are protein-coding contaminants and that many lncRNAs have ribosome profiles that resemble the 5' leaders of coding RNAs. Analysis of ribosome profiling data from embryonic stem cells reveals similar properties for mammalian lncRNAs. These results clarify the annotation of developmental lncRNAs and suggest a potential role for translation in lncRNA regulation. In addition, our computational pipeline and ribosome profiling data provide a powerful resource for the identification of translated open reading frames during zebrafish development.
Assuntos
RNA Longo não Codificante/genética , RNA/genética , Ribossomos/genética , Animais , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Translation is best known as the fundamental mechanism by which the ribosome converts a sequence of nucleotides into a string of amino acids. Extensive research over many years has elucidated the key principles of translation, and the majority of translated regions were thought to be known. The recent discovery of wide-spread translation outside of annotated protein-coding open reading frames (ORFs) came therefore as a surprise, raising the intriguing possibility that these newly discovered translated regions might have unrecognized protein-coding or gene-regulatory functions. Here, we highlight recent findings that provide evidence that some of these newly discovered translated short ORFs (sORFs) encode functional, previously missed small proteins, while others have regulatory roles. Based on known examples we will also speculate about putative additional roles and the potentially much wider impact that these translated regions might have on cellular homeostasis and gene regulation.
Assuntos
Fases de Leitura Aberta , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Biologia Computacional/métodos , Regulação da Expressão Gênica , Homeostase , Ribossomos/metabolismoRESUMO
Long noncoding RNAs (lncRNAs) comprise a diverse class of transcripts that structurally resemble mRNAs but do not encode proteins. Recent genome-wide studies in humans and the mouse have annotated lncRNAs expressed in cell lines and adult tissues, but a systematic analysis of lncRNAs expressed during vertebrate embryogenesis has been elusive. To identify lncRNAs with potential functions in vertebrate embryogenesis, we performed a time-series of RNA-seq experiments at eight stages during early zebrafish development. We reconstructed 56,535 high-confidence transcripts in 28,912 loci, recovering the vast majority of expressed RefSeq transcripts while identifying thousands of novel isoforms and expressed loci. We defined a stringent set of 1133 noncoding multi-exonic transcripts expressed during embryogenesis. These include long intergenic ncRNAs (lincRNAs), intronic overlapping lncRNAs, exonic antisense overlapping lncRNAs, and precursors for small RNAs (sRNAs). Zebrafish lncRNAs share many of the characteristics of their mammalian counterparts: relatively short length, low exon number, low expression, and conservation levels comparable to that of introns. Subsets of lncRNAs carry chromatin signatures characteristic of genes with developmental functions. The temporal expression profile of lncRNAs revealed two novel properties: lncRNAs are expressed in narrower time windows than are protein-coding genes and are specifically enriched in early-stage embryos. In addition, several lncRNAs show tissue-specific expression and distinct subcellular localization patterns. Integrative computational analyses associated individual lncRNAs with specific pathways and functions, ranging from cell cycle regulation to morphogenesis. Our study provides the first systematic identification of lncRNAs in a vertebrate embryo and forms the foundation for future genetic, genomic, and evolutionary studies.
Assuntos
Desenvolvimento Embrionário/genética , RNA não Traduzido/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Cromatina , Análise por Conglomerados , Biologia Computacional/métodos , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genômica , Camundongos , Fases de Leitura Aberta , Especificidade de Órgãos/genética , Transcrição GênicaRESUMO
The maintenance of specific gene expression patterns during cellular proliferation is crucial for the identity of every cell type and the development of tissues in multicellular organisms. Such a cellular memory function is conveyed by the complex interplay of the Polycomb and Trithorax groups of proteins (PcG/TrxG). These proteins exert their function at the level of chromatin by establishing and maintaining repressed (PcG) and active (TrxG) chromatin domains. Past studies indicated that a core PcG protein complex is potentially associated with cell type or even cell stage-specific sets of accessory proteins. In order to better understand the dynamic aspects underlying PcG composition and function we have established an inducible version of the biotinylation tagging approach to purify Polycomb and associated factors from Drosophila embryos. This system enabled fast and efficient isolation of Polycomb containing complexes under near physiological conditions, thereby preserving substoichiometric interactions. Novel interacting proteins were identified by highly sensitive mass spectrometric analysis. We found many TrxG related proteins, suggesting a previously unrecognized extent of molecular interaction of the two counteracting chromatin regulatory protein groups. Furthermore, our analysis revealed an association of PcG protein complexes with the cohesin complex and showed that Polycomb-dependent silencing of a transgenic reporter depends on cohesin function.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/isolamento & purificação , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Regulação da Expressão Gênica/fisiologia , Animais , Biotinilação , Cromatina/metabolismo , Cromatografia em Gel , Drosophila/genética , Drosophila/fisiologia , Ligantes , Espectrometria de Massas , Oligonucleotídeos/genética , Complexo Repressor Polycomb 1 , Espectrofotometria , CoesinasRESUMO
Cohesin is a highly conserved multisubunit complex that holds sister chromatids together in mitotic cells. At the metaphase to anaphase transition, proteolytic cleavage of the alpha kleisin subunit (Rad21) by separase causes cohesin's dissociation from chromosomes and triggers sister-chromatid disjunction. To investigate cohesin's function in postmitotic cells, where it is widely expressed, we have created fruit flies whose Rad21 can be cleaved by TEV protease. Cleavage causes precocious separation of sister chromatids and massive chromosome missegregation in proliferating cells, but not disaggregation of polytene chromosomes in salivary glands. Crucially, cleavage in postmitotic neurons is lethal. In mushroom-body neurons, it causes defects in axon pruning, whereas in cholinergic neurons it causes highly abnormal larval locomotion. These data demonstrate essential roles for cohesin in nondividing cells and also introduce a powerful tool by which to investigate protein function in metazoa.