RESUMO
Predicting regulatory potential from primary DNA sequences or transcription factor binding patterns is not possible. However, the annotation of the genome by chromatin proteins, histone modifications, and differential compaction is largely sufficient to reveal the locations of genes and their differential activity states. The Polycomb Group (PcG) and Trithorax Group (TrxG) proteins are the central players in this cell type-specific chromatin organization. PcG function was originally viewed as being solely repressive and irreversible, as observed at the homeotic loci in flies and mammals. However, it is now clear that modular and reversible PcG function is essential at most developmental genes. Focusing mainly on recent advances, we review evidence for how PcG and TrxG patterns change dynamically during cell type transitions. The ability to implement cell type-specific transcriptional programming with exquisite fidelity is essential for normal development.
Assuntos
Proteínas Cromossômicas não Histona/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Proteínas do Grupo Polycomb/genética , Transcrição Gênica , Animais , Cromatina/química , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Metilação de DNA , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Embrião de Mamíferos , Embrião não Mamífero , Loci Gênicos , Histonas/genética , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas do Grupo Polycomb/classificação , Proteínas do Grupo Polycomb/metabolismo , Elementos de Resposta , Especificidade da Espécie , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Before zygotic genome activation (ZGA), the quiescent genome undergoes reprogramming to transition into the transcriptionally active state. However, the mechanisms underlying euchromatin establishment during early embryogenesis remain poorly understood. Here, we show that histone H4 lysine 16 acetylation (H4K16ac) is maintained from oocytes to fertilized embryos in Drosophila and mammals. H4K16ac forms large domains that control nucleosome accessibility of promoters prior to ZGA in flies. Maternal depletion of MOF acetyltransferase leading to H4K16ac loss causes aberrant RNA Pol II recruitment, compromises the 3D organization of the active genomic compartments during ZGA, and causes downregulation of post-zygotically expressed genes. Germline depletion of histone deacetylases revealed that other acetyl marks cannot compensate for H4K16ac loss in the oocyte. Moreover, zygotic re-expression of MOF was neither able to restore embryonic viability nor onset of X chromosome dosage compensation. Thus, maternal H4K16ac provides an instructive function to the offspring, priming future gene activation.
Assuntos
Histonas/metabolismo , Lisina/metabolismo , Ativação Transcricional/genética , Acetilação , Animais , Sequência de Bases , Segregação de Cromossomos/genética , Sequência Conservada , Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Evolução Molecular , Feminino , Genoma , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Masculino , Mamíferos/genética , Camundongos , Mutação/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Oócitos/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Cromossomo X/metabolismo , Zigoto/metabolismoRESUMO
Male and female brains display anatomical and functional differences. Such differences are observed in species across the animal kingdom, including humans, but have been particularly well-studied in two classic animal model systems, the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans. Here we summarize recent advances in understanding how the worm and fly brain acquire sexually dimorphic features during development. We highlight the advantages of each system, illustrating how the precise anatomical delineation of sexual dimorphisms in worms has enabled recent analysis into how these dimorphisms become specified during development, and how focusing on sexually dimorphic neurons in the fly has enabled an increasingly detailed understanding of sex-specific behaviors.
Assuntos
Drosophila melanogaster , Sistema Nervoso , Animais , Caenorhabditis elegans/genética , Drosophila melanogaster/genética , Feminino , Masculino , Neurônios/fisiologia , Caracteres SexuaisRESUMO
Heterochromatin is characterized by dimethylated or trimethylated histone H3 Lys9 (H3K9me2 or H3K9me3, respectively) and is found at transposable elements, satellite repeats and genes, where it ensures their transcriptional silencing. The histone methyltransferases (HMTs) that methylate H3K9 - in mammals Suppressor of variegation 3-9 homologue 1 (SUV39H1), SUV39H2, SET domain bifurcated 1 (SETDB1), SETDB2, G9A and G9A-like protein (GLP) - and the 'readers' of H3K9me2 or H3K9me3 are highly conserved and show considerable redundancy. Despite their redundancy, genetic ablation or mistargeting of an individual H3K9 methyltransferase can correlate with impaired cell differentiation, loss of tissue identity, premature aging and/or cancer. In this Review, we discuss recent advances in understanding the roles of the known H3K9-specific HMTs in ensuring transcriptional homeostasis during tissue differentiation in mammals. We examine the effects of H3K9-methylation-dependent gene repression in haematopoiesis, muscle differentiation and neurogenesis in mammals, and compare them with mechanistic insights obtained from the study of model organisms, notably Caenorhabditis elegans and Drosophila melanogaster. In all these organisms, H3K9-specific HMTs have both unique and redundant roles that ensure the maintenance of tissue integrity by restricting the binding of transcription factors to lineage-specific promoters and enhancer elements.
Assuntos
Heterocromatina , Histona-Lisina N-Metiltransferase , Animais , Caenorhabditis elegans/genética , Drosophila melanogaster/genética , Heterocromatina/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Mamíferos/genética , MetilaçãoRESUMO
In this issue of Cell, Cassidy et al. (2019) show that, in Drosophila melanogaster, developmental abnormalities resulting from loss of repressors such as microRNAs can be suppressed by slow metabolism. They additionally provide insight into the underlying mechanism that connects metabolic state with developmental outcomes.
Assuntos
Drosophila melanogaster/genética , MicroRNAs , Animais , Regulação da Expressão Gênica , Fatores de TranscriçãoRESUMO
Metabolic conditions affect the developmental tempo of animals. Developmental gene regulatory networks (GRNs) must therefore synchronize their dynamics with a variable timescale. We find that layered repression of genes couples GRN output with variable metabolism. When repressors of transcription or mRNA and protein stability are lost, fewer errors in Drosophila development occur when metabolism is lowered. We demonstrate the universality of this phenomenon by eliminating the entire microRNA family of repressors and find that development to maturity can be largely rescued when metabolism is reduced. Using a mathematical model that replicates GRN dynamics, we find that lowering metabolism suppresses the emergence of developmental errors by curtailing the influence of auxiliary repressors on GRN output. We experimentally show that gene expression dynamics are less affected by loss of repressors when metabolism is reduced. Thus, layered repression provides robustness through error suppression and may provide an evolutionary route to a shorter reproductive cycle.
Assuntos
Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Neurônios/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Olho/citologia , Feminino , Insulina/metabolismo , Mutação com Perda de Função , MicroRNAs/metabolismo , Modelos Teóricos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transcrição GênicaRESUMO
In developing organisms, spatially prescribed cell identities are thought to be determined by the expression levels of multiple genes. Quantitative tests of this idea, however, require a theoretical framework capable of exposing the rules and precision of cell specification over developmental time. We use the gap gene network in the early fly embryo as an example to show how expression levels of the four gap genes can be jointly decoded into an optimal specification of position with 1% accuracy. The decoder correctly predicts, with no free parameters, the dynamics of pair-rule expression patterns at different developmental time points and in various mutant backgrounds. Precise cellular identities are thus available at the earliest stages of development, contrasting the prevailing view of positional information being slowly refined across successive layers of the patterning network. Our results suggest that developmental enhancers closely approximate a mathematically optimal decoding strategy.
Assuntos
Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética , Animais , Padronização Corporal/genética , Diferenciação Celular/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/genética , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Modelos Genéticos , Fatores de Transcrição/metabolismoRESUMO
Prion-like proteins can assume distinct conformational and physical states in the same cell. Sequence analysis suggests that prion-like proteins are prevalent in various species; however, it remains unclear what functional space they occupy in multicellular organisms. Here, we report the identification of a prion-like protein, Herzog (CG5830), through a multimodal screen in Drosophila melanogaster. Herzog functions as a membrane-associated phosphatase and controls embryonic patterning, likely being involved in TGF-ß/BMP and FGF/EGF signaling pathways. Remarkably, monomeric Herzog is enzymatically inactive and becomes active upon amyloid-like assembly. The prion-like domain of Herzog is necessary for both its assembly and membrane targeting. Removal of the prion-like domain impairs activity, while restoring assembly on the membrane using a heterologous prion-like domain and membrane-targeting motif can restore phosphatase activity. This study provides an example of a prion-like domain that allows an enzyme to gain essential functionality via amyloid-like assembly to control animal development.
Assuntos
Proteínas Amiloidogênicas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Desenvolvimento Embrionário , Fosfoproteínas Fosfatases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/genética , Animais , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Príons/química , Domínios ProteicosRESUMO
X chromosome regulation represents a prime example of an epigenetic phenomenon where coordinated regulation of a whole chromosome is required. In flies, this is achieved by transcriptional upregulation of X chromosomal genes in males to equalize the gene dosage differences in females. Chromatin-bound proteins and long noncoding RNAs (lncRNAs) constituting a ribonucleoprotein complex known as the male-specific lethal (MSL) complex or the dosage compensation complex mediate this process. MSL complex members decorate the male X chromosome, and their absence leads to male lethality. The male X chromosome is also enriched with histone H4 lysine 16 acetylation (H4K16ac), indicating that the chromatin compaction status of the X chromosome also plays an important role in transcriptional activation. How the X chromosome is specifically targeted and how dosage compensation is mechanistically achieved are central questions for the field. Here, we review recent advances, which reveal a complex interplay among lncRNAs, the chromatin landscape, transcription, and chromosome conformation that fine-tune X chromosome gene expression.
Assuntos
Mecanismo Genético de Compensação de Dose , Cromossomo X/genética , Animais , Cromatina/genética , Cromatina/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Epigênese Genética , Feminino , Genes Ligados ao Cromossomo X , Código das Histonas/genética , Humanos , Masculino , Modelos Genéticos , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Cromossomo X/metabolismoRESUMO
More than 80 years ago, the first Polycomb-related phenotype was identified in Drosophila melanogaster. Later, a group of diverse genes collectively called Polycomb group (PcG) genes were identified based on common mutant phenotypes. PcG proteins, which are well-conserved in animals, were originally characterized as negative regulators of gene transcription during development and subsequently shown to function in various biological processes; their deregulation is associated with diverse phenotypes in development and in disease, especially cancer. PcG proteins function on chromatin and can form two distinct complexes with different enzymatic activities: Polycomb repressive complex 1 (PRC1) is a histone ubiquitin ligase and PRC2 is a histone methyltransferase. Recent studies have revealed the existence of various mutually exclusive PRC1 and PRC2 variants. In this Review, we discuss new concepts concerning the biochemical and molecular functions of these new PcG complex variants, and how their epigenetic activities are involved in mammalian development and cancer.
Assuntos
Proteínas de Ciclo Celular/genética , Desenvolvimento Embrionário/genética , Neoplasias/genética , Complexo Repressor Polycomb 2/genética , Animais , Cromatina/genética , Drosophila melanogaster/genética , Embrião de Mamíferos , Histonas/genética , Humanos , Neoplasias/patologiaRESUMO
The diversity of cell types and regulatory states in the brain, and how these change during aging, remains largely unknown. We present a single-cell transcriptome atlas of the entire adult Drosophila melanogaster brain sampled across its lifespan. Cell clustering identified 87 initial cell clusters that are further subclustered and validated by targeted cell-sorting. Our data show high granularity and identify a wide range of cell types. Gene network analyses using SCENIC revealed regulatory heterogeneity linked to energy consumption. During aging, RNA content declines exponentially without affecting neuronal identity in old brains. This single-cell brain atlas covers nearly all cells in the normal brain and provides the tools to study cellular diversity alongside other Drosophila and mammalian single-cell datasets in our unique single-cell analysis platform: SCope (http://scope.aertslab.org). These results, together with SCope, allow comprehensive exploration of all transcriptional states of an entire aging brain.
Assuntos
Envelhecimento , Encéfalo/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Redes Reguladoras de Genes , Análise de Célula Única/métodos , Transcriptoma , Animais , Drosophila melanogaster/fisiologia , Feminino , Perfilação da Expressão Gênica , MasculinoRESUMO
Although animals have evolved multiple mechanisms to suppress transposons, "leaky" mobilizations that cause mutations and diseases still occur. This suggests that transposons employ specific tactics to accomplish robust propagation. By directly tracking mobilization, we show that, during a short and specific time window of oogenesis, retrotransposons achieve massive amplification via a cell-type-specific targeting strategy. Retrotransposons rarely mobilize in undifferentiated germline stem cells. However, as oogenesis proceeds, they utilize supporting nurse cells-which are highly polyploid and eventually undergo apoptosis-as factories to massively manufacture invading products. Moreover, retrotransposons rarely integrate into nurse cells themselves but, instead, via microtubule-mediated transport, they preferentially target the DNA of the interconnected oocytes. Blocking microtubule-dependent intercellular transport from nurse cells significantly alleviates damage to the oocyte genome. Our data reveal that parasitic genomic elements can efficiently hijack a host developmental process to propagate robustly, thereby driving evolutionary change and causing disease.
Assuntos
Drosophila melanogaster/genética , Elementos Nucleotídeos Longos e Dispersos , Oogênese , RNA Interferente Pequeno , Retroelementos , Retroviridae/genética , Animais , Proteínas de Drosophila , Feminino , Biblioteca Gênica , Inativação Gênica , Células Germinativas , Proteínas de Fluorescência Verde/metabolismo , Hibridização in Situ Fluorescente , Masculino , Oócitos/metabolismo , Células-Tronco/metabolismoRESUMO
Transcription factors regulate the molecular, morphological, and physiological characteristics of neurons and generate their impressive cell-type diversity. To gain insight into the general principles that govern how transcription factors regulate cell-type diversity, we used large-scale single-cell RNA sequencing to characterize the extensive cellular diversity in the Drosophila optic lobes. We sequenced 55,000 single cells and assigned them to 52 clusters. We validated and annotated many clusters using RNA sequencing of FACS-sorted single-cell types and cluster-specific genes. To identify transcription factors responsible for inducing specific terminal differentiation features, we generated a "random forest" model, and we showed that the transcription factors Apterous and Traffic-jam are required in many but not all cholinergic and glutamatergic neurons, respectively. In fact, the same terminal characters often can be regulated by different transcription factors in different cell types, arguing for extensive phenotypic convergence. Our data provide a deep understanding of the developmental and functional specification of a complex brain structure.
Assuntos
Drosophila melanogaster/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurogênese/fisiologia , Animais , Diferenciação Celular , Neurônios Colinérgicos/fisiologia , Análise por Conglomerados , Simulação por Computador , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Perfilação da Expressão Gênica/métodos , Proteínas de Homeodomínio , Proteínas com Homeodomínio LIM/metabolismo , Fatores de Transcrição Maf Maior/metabolismo , Neuroglia/fisiologia , Neurônios/fisiologia , Neurotransmissores/genética , Neurotransmissores/fisiologia , Lobo Óptico de Animais não Mamíferos/fisiologia , Fenótipo , Proteínas Proto-Oncogênicas/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
The striking correlation between genome topology and transcriptional activity has for decades made researchers revisit the question, "Does form follow function, or does function follow form?" In a new study, Hug et al. address this question by comparing the timing of zygotic genome activation to the emergence of genome structures during Drosophila embryogenesis.
Assuntos
Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Animais , Desenvolvimento Embrionário , Genoma , ZigotoRESUMO
Chromatin architecture is fundamental in regulating gene expression. To investigate when spatial genome organization is first established during development, we examined chromatin conformation during Drosophila embryogenesis and observed the emergence of chromatin architecture within a tight time window that coincides with the onset of transcription activation in the zygote. Prior to zygotic genome activation, the genome is mostly unstructured. Early expressed genes serve as nucleation sites for topologically associating domain (TAD) boundaries. Activation of gene expression coincides with the establishment of TADs throughout the genome and co-localization of housekeeping gene clusters, which remain stable in subsequent stages of development. However, the appearance of TAD boundaries is independent of transcription and requires the transcription factor Zelda for locus-specific TAD boundary insulation. These results offer insight into when spatial organization of the genome emerges and identify a key factor that helps trigger this architecture.
Assuntos
Cromatina/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Genoma de Inseto , Ativação Transcricional , Zigoto/metabolismo , Animais , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/metabolismo , Genes Essenciais , Proteínas Nucleares , RNA Polimerase II/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Myriad experiences produce transient memory, yet, contingent on the internal state of the organism and the saliency of the experience, only some memories persist over time. How experience and internal state influence the duration of memory at the molecular level remains unknown. A self-assembled aggregated state of Drosophila Orb2A protein is required specifically for long-lasting memory. We report that in the adult fly brain the mRNA encoding Orb2A protein exists in an unspliced non-protein-coding form. The convergence of experience and internal drive transiently increases the spliced protein-coding Orb2A mRNA. A screen identified pasilla, the fly ortholog of mammalian Nova-1/2, as a mediator of Orb2A mRNA processing. A single-nucleotide substitution in the intronic region that reduces Pasilla binding and intron removal selectively impairs long-term memory. We posit that pasilla-mediated processing of unspliced Orb2A mRNA integrates experience and internal state to control Orb2A protein abundance and long-term memory formation.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Íntrons , Memória de Longo Prazo , Ribonucleoproteínas/metabolismo , Fatores de Transcrição/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Animais , Sequência de Bases , Comportamento Animal , Encéfalo/metabolismo , Condicionamento Psicológico , Proteínas de Drosophila/química , Drosophila melanogaster/genética , Aprendizagem , Modelos Animais , Motivação , Mutação , Isoformas de Proteínas/metabolismo , Splicing de RNA , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/química , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Whether and how histone post-translational modifications and the proteins that bind them drive 3D genome organization remains unanswered. Here, we evaluate the contribution of H3K9-methylated constitutive heterochromatin to 3D genome organization in Drosophila tissues. We find that the predominant organizational feature of wild-type tissues is the segregation of euchromatic chromosome arms from heterochromatic pericentromeres. Reciprocal perturbation of HP1aâ H3K9me binding, using a point mutation in the HP1a chromodomain or replacement of the replication-dependent histone H3 with H3K9R mutant histones, revealed that HP1a binding to methylated H3K9 in constitutive heterochromatin is required to limit contact frequency between pericentromeres and chromosome arms and regulate the distance between arm and pericentromeric regions. Surprisingly, the self-association of pericentromeric regions is largely preserved despite the loss of H3K9 methylation and HP1a occupancy. Thus, the HP1aâ H3K9 interaction contributes to but does not solely drive the segregation of euchromatin and heterochromatin inside the nucleus.
Assuntos
Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona , Proteínas de Drosophila , Drosophila melanogaster , Heterocromatina , Histonas , Heterocromatina/metabolismo , Heterocromatina/genética , Animais , Histonas/metabolismo , Histonas/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Metilação , Eucromatina/metabolismo , Eucromatina/genética , Centrômero/metabolismo , Centrômero/genética , Ligação Proteica , Genoma de Inseto , Segregação de Cromossomos , Processamento de Proteína Pós-TraducionalRESUMO
INTS11 and CPSF73 are metal-dependent endonucleases for Integrator and pre-mRNA 3'-end processing, respectively. Here, we show that the INTS11 binding partner BRAT1/CG7044, a factor important for neuronal fitness, stabilizes INTS11 in the cytoplasm and is required for Integrator function in the nucleus. Loss of BRAT1 in neural organoids leads to transcriptomic disruption and precocious expression of neurogenesis-driving transcription factors. The structures of the human INTS9-INTS11-BRAT1 and Drosophila dIntS11-CG7044 complexes reveal that the conserved C terminus of BRAT1/CG7044 is captured in the active site of INTS11, with a cysteine residue directly coordinating the metal ions. Inspired by these observations, we find that UBE3D is a binding partner for CPSF73, and UBE3D likely also uses a conserved cysteine residue to directly coordinate the active site metal ions. Our studies have revealed binding partners for INTS11 and CPSF73 that behave like cytoplasmic chaperones with a conserved impact on the nuclear functions of these enzymes.
Assuntos
Núcleo Celular , Citoplasma , Proteínas de Drosophila , Ligação Proteica , Humanos , Animais , Núcleo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Citoplasma/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Endonucleases/metabolismo , Endonucleases/genética , Células HEK293 , Neurogênese/genética , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Fator de Especificidade de Clivagem e Poliadenilação/genética , Domínio CatalíticoRESUMO
RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , RNA Polimerase II , Transcrição Gênica , Fatores de Elongação da Transcrição , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Fator B de Elongação Transcricional Positiva/genética , Regiões Promotoras Genéticas , Sistemas CRISPR-Cas , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Cromossomos Politênicos/genética , Cromossomos Politênicos/metabolismo , Regulação da Expressão Gênica , Fosforilação , Ligação Proteica , Resposta ao Choque Térmico/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Nucleossomos/metabolismo , Nucleossomos/genéticaRESUMO
O-linked N-acetylglucosamine transferase (OGT) is found in all metazoans and plays an important role in development but at the single-cell level is only essential in dividing mammalian cells. Postmitotic mammalian cells and cells of invertebrates such as Caenorhabditis elegans and Drosophila can survive without copies of OGT. Why OGT is required in dividing mammalian cells but not in other cells remains unknown. OGT has multiple biochemical activities. Beyond its well-known role in adding ß-O-GlcNAc to serine and threonine residues of nuclear and cytoplasmic proteins, OGT also acts as a protease in the maturation of the cell cycle regulator host cell factor 1 (HCF-1) and serves as an integral member of several protein complexes, many of them linked to gene expression. In this review, we summarize current understanding of the mechanisms underlying OGT's biochemical activities and address whether known functions of OGT could be related to its essential role in dividing mammalian cells.