RESUMEN
The systematic determination of protein function is a key goal of modern biology, but remains challenging with current approaches. Here we present ORFtag, a versatile, cost-effective and highly efficient method for the massively parallel tagging and functional interrogation of proteins at the proteome scale. ORFtag uses retroviral vectors bearing a promoter, peptide tag and splice donor to generate fusions between the tag and endogenous open reading frames (ORFs). We demonstrate the utility of ORFtag through functional screens for transcriptional activators, repressors and posttranscriptional regulators in mouse embryonic stem cells. Each screen recovers known and identifies new regulators, including long ORFs inaccessible by other methods. Among other hits, we find that Zfp574 is a highly selective transcriptional activator and that oncogenic fusions often function as transactivators.
RESUMEN
Cancer initiation and progression are typically associated with the accumulation of driver mutations and genomic instability. However, recent studies demonstrated that cancer can also be driven purely by epigenetic alterations, without driver mutations. Specifically, a 24-h transient downregulation of polyhomeotic (ph-KD), a core component of the Polycomb complex PRC1, is sufficient to induce epigenetically initiated cancers (EICs) in Drosophila, which are proficient in DNA repair and characterized by a stable genome. Whether genomic instability eventually occurs when PRC1 downregulation is performed for extended periods of time remains unclear. Here, we show that prolonged depletion of PH, which mimics cancer initiating events, results in broad dysregulation of DNA replication and repair genes, along with the accumulation of DNA breaks, defective repair, and widespread genomic instability in the cancer tissue. A broad misregulation of H2AK118 ubiquitylation and to a lesser extent of H3K27 trimethylation also occurs and might contribute to these phenotypes. Together, this study supports a model where DNA repair and replication defects accumulate during the tumorigenic transformation epigenetically induced by PRC1 loss, resulting in genomic instability and cancer progression.
Asunto(s)
Reparación del ADN , Epigénesis Genética , Inestabilidad Genómica , Animales , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Complejo Represivo Polycomb 1/metabolismo , Complejo Represivo Polycomb 1/genética , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patología , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Proteínas del Grupo Polycomb/genéticaRESUMEN
Cancer initiation and progression are typically associated with the accumulation of driver mutations and genomic instability. However, recent studies demonstrated that cancers can also be purely initiated by epigenetic alterations, without driver mutations. Specifically, a 24-hours transient down-regulation of polyhomeotic (ph-KD), a core component of the Polycomb complex PRC1, is sufficient to drive epigenetically initiated cancers (EICs) in Drosophila, which are proficient in DNA repair and are characterized by a stable genome. Whether genomic instability eventually occurs when PRC1 down-regulation is performed for extended periods of time remains unclear. Here we show that prolonged depletion of a PRC1 component, which mimics cancer initiating events, results in broad dysregulation of DNA replication and repair genes, along with the accumulation of DNA breaks, defective repair, and widespread genomic instability in the cancer tissue. A broad mis-regulation of H2AK118 ubiquitylation and to a lesser extent of H3K27 trimethylation also occurs, and might contribute to these phenotypes. Together, this study supports a model where DNA repair and replication defects amplify the tumorigenic transformation epigenetically induced by PRC1 loss, resulting in genomic instability and cancer progression.
RESUMEN
Zygotic genome activation (ZGA) is a crucial step of embryonic development. So far, little is known about the role of chromatin factors during this process. Here, we used an in vivo RNA interference reverse genetic screen to identify chromatin factors necessary for embryonic development in Drosophila melanogaster. Our screen reveals that histone acetyltransferases (HATs) and histone deacetylases are crucial ZGA regulators. We demonstrate that Nejire (CBP/EP300 ortholog) is essential for the acetylation of histone H3 lysine-18 and lysine-27, whereas Gcn5 (GCN5/PCAF ortholog) for lysine-9 of H3 at ZGA, with these marks being enriched at all actively transcribed genes. Nonetheless, these HATs activate distinct sets of genes. Unexpectedly, individual catalytic dead mutants of either Nejire or Gcn5 can activate zygotic transcription (ZGA) and transactivate a reporter gene in vitro. Together, our data identify Nejire and Gcn5 as key regulators of ZGA.
Asunto(s)
Drosophila melanogaster , Factores de Transcripción p300-CBP , Animales , Acetilación , Cromatina , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Histona Acetiltransferasas/genética , Lisina/metabolismo , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Recruitment of RNA polymerase II (Pol II) to promoters is essential for transcription. Despite conflicting evidence, the Pol II preinitiation complex (PIC) is often thought to have a uniform composition and to assemble at all promoters via an identical mechanism. Here, using Drosophila melanogaster S2 cells as a model, we demonstrate that different promoter classes function via distinct PICs. Promoter DNA of developmentally regulated genes readily associates with the canonical Pol II PIC, whereas housekeeping promoters do not, and instead recruit other factors such as DREF. Consistently, TBP and DREF are differentially required by distinct promoter types. TBP and its paralog TRF2 also function at different promoter types in a partially redundant manner. In contrast, TFIIA is required at all promoters, and we identify factors that can recruit and/or stabilize TFIIA at housekeeping promoters and activate transcription. Promoter activation by tethering these factors is sufficient to induce the dispersed transcription initiation patterns characteristic of housekeeping promoters. Thus, different promoter classes utilize distinct mechanisms of transcription initiation, which translate into different focused versus dispersed initiation patterns.
Asunto(s)
Proteínas de Drosophila , Factores de Transcripción , Animales , Factores de Transcripción/genética , Factor de Transcripción TFIIA/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Transcripción Genética , Proteínas de Drosophila/genéticaRESUMEN
Gene duplications and transcriptional enhancer emergence/modifications are thought having greatly contributed to phenotypic innovations during animal evolution. Nevertheless, little is known about how enhancers evolve after gene duplication and how regulatory information is rewired between duplicated genes. The Drosophila melanogaster bric-a-brac (bab) complex, comprising the tandem paralogous genes bab1 and bab2, provides a paradigm to address these issues. We previously characterized an intergenic enhancer (named LAE) regulating bab2 expression in the developing legs. We show here that bab2 regulators binding directly the LAE also govern bab1 expression in tarsal cells. LAE excision by CRISPR/Cas9-mediated genome editing reveals that this enhancer appears involved but not strictly required for bab1 and bab2 co-expression in leg tissues. Instead, the LAE enhancer is critical for paralog-specific bab2 expression along the proximo-distal leg axis. Chromatin features and phenotypic rescue experiments indicate that LAE functions partly redundantly with leg-specific regulatory information overlapping the bab1 transcription unit. Phylogenomics analyses indicate that (i) the bab complex originates from duplication of an ancestral singleton gene early on within the Cyclorrhapha dipteran sublineage, and (ii) LAE sequences have been evolutionarily-fixed early on within the Brachycera suborder thus predating the gene duplication event. This work provides new insights on enhancers, particularly about their emergence, maintenance and functional diversification during evolution.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Elementos de Facilitación Genéticos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
To understand the role of the extensive senescence-associated 3D genome reorganization, we generated genome-wide chromatin interaction maps, epigenome, replication-timing, whole-genome bisulfite sequencing, and gene expression profiles from cells entering replicative senescence (RS) or upon oncogene-induced senescence (OIS). We identify senescence-associated heterochromatin domains (SAHDs). Differential intra- versus inter-SAHD interactions lead to the formation of senescence-associated heterochromatin foci (SAHFs) in OIS but not in RS. This OIS-specific configuration brings active genes located in genomic regions adjacent to SAHDs in close spatial proximity and favors their expression. We also identify DNMT1 as a factor that induces SAHFs by promoting HMGA2 expression. Upon DNMT1 depletion, OIS cells transition to a 3D genome conformation akin to that of cells in replicative senescence. These data show how multi-omics and imaging can identify critical features of RS and OIS and discover determinants of acute senescence and SAHF formation.
Asunto(s)
Senescencia Celular/genética , ADN (Citosina-5-)-Metiltransferasa 1/genética , Genoma Humano , Oncogenes , Células Cultivadas , Ensamble y Desensamble de Cromatina/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Metilación de ADN , Fibroblastos , Heterocromatina/genética , Humanos , Hibridación Fluorescente in SituRESUMEN
Targeted transitions in chromatin states at thousands of genes are essential drivers of eukaryotic development. Therefore, understanding the in vivo dynamics of epigenetic regulators is crucial for deciphering the mechanisms underpinning cell fate decisions. This review illustrates how, in addition to its cell memory function, the Polycomb group of transcriptional regulators orchestrates temporal, cell and tissue-specific expression of master genes during development. These highly sophisticated developmental transitions are dependent on the context- and tissue-specific assembly of the different types of Polycomb Group (PcG) complexes, which regulates their targeting and/or activities on chromatin. Here, an overview is provided of how PcG complexes function at multiple scales to regulate transcription, local chromatin environment, and higher order structures that support normal differentiation and are perturbed in tumorigenesis.
Asunto(s)
Cromatina/genética , Epigénesis Genética , Genoma , Proteínas del Grupo Polycomb/genética , Animales , Carcinogénesis/genética , Diferenciación Celular , Ensamble y Desensamble de Cromatina/genética , Drosophila , Proteínas de Drosophila/genética , Células Madre Embrionarias/fisiología , Silenciador del Gen , Humanos , Ratones , Transcripción GenéticaRESUMEN
Since 2008, juvenile Crassostrea gigas oysters have suffered from massive mortalities in European farming areas. This disease of complex etiology is still incompletely understood. Triggered by an elevated seawater temperature, it has been associated to infections by a herpes virus named OsHV-1 as well as pathogenic vibrios of the Splendidus clade. Ruling out the complexity of the disease, most of our current knowledge has been acquired in controlled experiments. Among the many unsolved questions, it is still ignored what role immunity plays in the capacity oysters have to survive an infectious episode. Here we show that juvenile oysters susceptible to the disease mount an inefficient immune response associated with microbial permissiveness and death. We found that, in contrast to resistant adult oysters having survived an earlier episode of mortality, susceptible juvenile oysters never exposed to infectious episodes died by more than 90% in a field experiment. Susceptible oysters were heavily colonized by OsHV-1 herpes virus as well as bacteria including vibrios potentially pathogenic for oysters, which proliferated in oyster flesh and body fluids during the mortality event. Nonetheless, susceptible oysters were found to sense microbes as indicated by an overexpression of immune receptors and immune signaling pathways. However, they did not express important immune effectors involved in antimicrobial immunity and apoptosis and showed repressed expression of genes involved in ROS and metal homeostasis. This contrasted with resistant oysters, which expressed those important effectors, controlled bacterial and viral colonization and showed 100% survival to the mortality event. Altogether, our results demonstrate that the immune response mounted by susceptible oysters lacks some important immune functions and fails in controlling microbial proliferation. This study opens the way to more holistic studies on the "mass mortality syndrome", which are now required to decipher the sequence of events leading to oyster mortalities and determine the relative weight of pathogens, oyster genetics and oyster-associated microbiota in the disease.
Asunto(s)
Crassostrea/inmunología , Inmunidad Innata , Animales , Crassostrea/microbiología , Crassostrea/virología , Francia , Herpesviridae/fisiología , Agua de Mar , Temperatura , Vibrio/fisiologíaRESUMEN
Chromatin Immunoprecipitation coupled either to qPCR (qChIP) or high-throughput sequencing (ChIP-Seq) has been extensively used in the last decades to identify the DNA binding sites of transcription factors or the localization of various histone marks along the genome. The ChIP experiment generally includes 7 steps: collection of biological samples (A), cross-linking proteins to DNA (B), chromatin isolation and fragmentation by sonication (C), sonication test (D), immunoprecipitation with antibodies against the protein or the histone mark of interest (E), DNA recovery (E), identification of factor-associated DNA sequences by PCR or sequencing (F). The protocol described here can readily be used for ChIP-seq and ChIP-qPCR experiments. The entire procedure, describing experimental setup conditions to optimize assays in intact Drosophila tissues, can be completed within four days.
RESUMEN
Polycomb group proteins form two main complexes, PRC2 and PRC1, which generally coregulate their target genes. Here we show that PRC1 components act as neoplastic tumor suppressors independently of PRC2 function. By mapping the distribution of PRC1 components and trimethylation of histone H3 at Lys27 (H3K27me3) across the genome, we identify a large set of genes that acquire PRC1 in the absence of H3K27me3 in Drosophila larval tissues. These genes massively outnumber canonical targets and are mainly involved in the regulation of cell proliferation, signaling and polarity. Alterations in PRC1 components specifically deregulate this set of genes, whereas canonical targets are derepressed in both PRC1 and PRC2 mutants. In human embryonic stem cells, PRC1 components colocalize with H3K27me3 as in Drosophila embryos, whereas in differentiated cell types they are selectively recruited to a large set of proliferation and signaling-associated genes that lack H3K27me3, suggesting that the redeployment of PRC1 components during development is evolutionarily conserved.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/crecimiento & desarrollo , Complejo Represivo Polycomb 1/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Genes de Insecto , Histona Demetilasas/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Proteínas Asociadas a Microtúbulos , Proteínas del Grupo Polycomb/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
BACKGROUND: The use of dried blood spots on filter paper is well documented as an affordable and practical alternative to classical venous sampling for various clinical needs. This technique has indeed many advantages in terms of collection, biological safety, storage, and shipment. Amyloid ß (Aß) peptides are useful cerebrospinal fluid (CSF) biomarkers for Alzheimer disease diagnosis. However, Aß determination is hindered by preanalytical difficulties in terms of sample collection and stability in tubes. METHODS: We compared the quantification of Aß peptides (1-40, 1-42, and 1-38) by simplex and multiplex ELISA, following either a standard operator method (liquid direct quantification) or after spotting CSF onto dried matrix paper card. RESULTS: The use of dried matrix spot (DMS) overcame preanalytical problems and allowed the determination of Aß concentrations that were highly commutable (Bland-Altman) with those obtained using CSF in classical tubes. Moreover, we found a positive and significant correlation (r2=0.83, Pearson coefficient p=0.0329) between the two approaches. CONCLUSIONS: This new DMS method for CSF represents an interesting alternative that increases the quality and efficiency in preanalytics. This should enable the better exploitation of Aß analytes for Alzheimer's diagnosis.
Asunto(s)
Péptidos beta-Amiloides/líquido cefalorraquídeo , Ensayo de Inmunoadsorción Enzimática , Fragmentos de Péptidos/líquido cefalorraquídeo , Enfermedad de Alzheimer/diagnóstico , Biomarcadores/líquido cefalorraquídeo , Pruebas con Sangre Seca , HumanosRESUMEN
Mitochondrial dysfunctions are an internal cause of nuclear genome instability. Because mitochondria are key regulators of cellular metabolism, we have investigated a potential link between external growth conditions and nuclear chromosome instability in cells with mitochondrial defects. Using Saccharomyces cerevisiae, we found that cells lacking mitochondrial DNA (rho0 cells) have a unique feature, with nuclear chromosome instability that occurs in nondividing cells and strongly fluctuates depending on the cellular environment. Calorie restriction, lower growth temperatures, growth at alkaline pH, antioxidants (NAC, Tiron), or presence of nearby wild-type cells all efficiently stabilize nuclear genomes of rho0 cells, whereas high glucose and ethanol boost instability. In contrast, other respiratory mutants that still possess mitochondrial DNA (RHO(+)) keep fairly constant instability rates under the same growth conditions, like wild-type or other RHO(+) controls. Our data identify mitochondrial defects as an important driver of nuclear genome instability influenced by environmental factors.