RESUMEN
Duplication is a foundation of molecular evolution and a driver of genomic and complex diseases. Here, we develop a genome editing tool named Amplification Editing (AE) that enables programmable DNA duplication with precision at chromosomal scale. AE can duplicate human genomes ranging from 20 bp to 100 Mb, a size comparable to human chromosomes. AE exhibits activity across various cell types, encompassing diploid, haploid, and primary cells. AE exhibited up to 73.0% efficiency for 1 Mb and 3.4% for 100 Mb duplications, respectively. Whole-genome sequencing and deep sequencing of the junctions of edited sequences confirm the precision of duplication. AE can create chromosomal microduplications within disease-relevant regions in embryonic stem cells, indicating its potential for generating cellular and animal models. AE is a precise and efficient tool for chromosomal engineering and DNA duplication, broadening the landscape of precision genome editing from an individual genetic locus to the chromosomal scale.
Asunto(s)
Duplicación de Gen , Edición Génica , Genoma Humano , Humanos , Edición Génica/métodos , Sistemas CRISPR-Cas/genética , ADN/genética , Animales , Células Madre Embrionarias/metabolismo , Cromosomas Humanos/genéticaRESUMEN
Many regions in the human genome vary in length among individuals due to variable numbers of tandem repeats (VNTRs). To assess the phenotypic impact of VNTRs genome-wide, we applied a statistical imputation approach to estimate the lengths of 9,561 autosomal VNTR loci in 418,136 unrelated UK Biobank participants and 838 GTEx participants. Association and statistical fine-mapping analyses identified 58 VNTRs that appeared to influence a complex trait in UK Biobank, 18 of which also appeared to modulate expression or splicing of a nearby gene. Non-coding VNTRs at TMCO1 and EIF3H appeared to generate the largest known contributions of common human genetic variation to risk of glaucoma and colorectal cancer, respectively. Each of these two VNTRs associated with a >2-fold range of risk across individuals. These results reveal a substantial and previously unappreciated role of non-coding VNTRs in human health and gene regulation.
Asunto(s)
Canales de Calcio , Neoplasias Colorrectales , Factor 3 de Iniciación Eucariótica , Glaucoma , Repeticiones de Minisatélite , Humanos , Canales de Calcio/genética , Neoplasias Colorrectales/genética , Genoma Humano , Glaucoma/genética , Polimorfismo Genético , Factor 3 de Iniciación Eucariótica/genéticaRESUMEN
Short tandem repeat (STR) instability causes transcriptional silencing in several repeat expansion disorders. In fragile X syndrome (FXS), mutation-length expansion of a CGG STR represses FMR1 via local DNA methylation. Here, we find megabase-scale H3K9me3 domains on autosomes and encompassing FMR1 on the X chromosome in FXS patient-derived iPSCs, iPSC-derived neural progenitors, EBV-transformed lymphoblasts, and brain tissue with mutation-length CGG expansion. H3K9me3 domains connect via inter-chromosomal interactions and demarcate severe misfolding of TADs and loops. They harbor long synaptic genes replicating at the end of S phase, replication-stress-induced double-strand breaks, and STRs prone to stepwise somatic instability. CRISPR engineering of the mutation-length CGG to premutation length reverses H3K9me3 on the X chromosome and multiple autosomes, refolds TADs, and restores gene expression. H3K9me3 domains can also arise in normal-length iPSCs created with perturbations linked to genome instability, suggesting their relevance beyond FXS. Our results reveal Mb-scale heterochromatinization and trans interactions among loci susceptible to instability.
Asunto(s)
Síndrome del Cromosoma X Frágil , Humanos , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Expansión de Repetición de Trinucleótido , Metilación de ADN , Mutación , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismoRESUMEN
Biochemical processes often require spatial regulation and specific microenvironments. The general lack of organelles in bacteria limits the potential of bioengineering complex intracellular reactions. Here, we demonstrate synthetic membraneless organelles in Escherichia coli termed transcriptionally engineered addressable RNA solvent droplets (TEARS). TEARS are assembled from RNA-binding protein recruiting domains fused to poly-CAG repeats that spontaneously drive liquid-liquid phase separation from the bulk cytoplasm. Targeting TEARS with fluorescent proteins revealed multilayered structures with composition and reaction robustness governed by non-equilibrium dynamics. We show that TEARS provide organelle-like bioprocess isolation for sequestering biochemical pathways, controlling metabolic branch points, buffering mRNA translation rates, and scaffolding protein-protein interactions. We anticipate TEARS to be a simple and versatile tool for spatially controlling E. coli biochemistry. Particularly, the modular design of TEARS enables applications without expression fine-tuning, simplifying the design-build-test cycle of bioengineering.
Asunto(s)
Escherichia coli , Orgánulos , Escherichia coli/genética , Orgánulos/metabolismo , ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Solventes/análisis , Solventes/metabolismoRESUMEN
Fragile X syndrome (FXS), the most common genetic form of intellectual disability in males, is caused by silencing of the FMR1 gene associated with hypermethylation of the CGG expansion mutation in the 5' UTR of FMR1 in FXS patients. Here, we applied recently developed DNA methylation editing tools to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/single guide RNA (sgRNA) switched the heterochromatin status of the upstream FMR1 promoter to an active chromatin state, restoring a persistent expression of FMR1 in FXS iPSCs. Neurons derived from methylation-edited FXS iPSCs rescued the electrophysiological abnormalities and restored a wild-type phenotype upon the mutant neurons. FMR1 expression in edited neurons was maintained in vivo after engrafting into the mouse brain. Finally, demethylation of the CGG repeats in post-mitotic FXS neurons also reactivated FMR1. Our data establish that demethylation of the CGG expansion is sufficient for FMR1 reactivation, suggesting potential therapeutic strategies for FXS.
Asunto(s)
Metilación de ADN/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Edición Génica , Neuronas/patología , Animales , Proteína 9 Asociada a CRISPR/metabolismo , Epigénesis Genética , Células HEK293 , Heterocromatina/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cinética , Masculino , Ratones , Neuronas/metabolismo , Fenotipo , Regiones Promotoras Genéticas , ARN Guía de Kinetoplastida/metabolismo , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
Tandem repeats (TRs) are generated by DNA replication errors and retain a high level of instability, which in principle would make them unsuitable for integration into gene regulatory networks. However, the appearance of DNA sequence motifs recognized by transcription factors may turn TRs into functional cis-regulatory elements, thus favoring their stabilization in genomes. Here, we show that, in human cells, the transcriptional repressor ZEB1, which promotes the maintenance of mesenchymal features largely by suppressing epithelial genes and microRNAs, occupies TRs harboring dozens of copies of its DNA-binding motif within genomic loci relevant for maintenance of epithelial identity. The deletion of one such TR caused quasi-mesenchymal cancer cells to reacquire epithelial features, partially recapitulating the effects of ZEB1 gene deletion. These data demonstrate that the high density of identical motifs in TRs can make them suitable platforms for recruitment of transcriptional repressors, thus promoting their exaptation into pre-existing cis-regulatory networks.
Asunto(s)
Secuencias Repetidas en Tándem/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Adulto , Animales , Secuencia de Bases , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Femenino , Expresión Génica , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Desnudos , MicroARNs/genética , MicroARNs/metabolismo , Persona de Mediana Edad , Mucosa Bucal/metabolismo , Polimorfismo de Nucleótido Simple , Unión Proteica , Factores de Transcripción/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/deficiencia , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genéticaRESUMEN
Long mammalian introns make it challenging for the RNA processing machinery to identify exons accurately. We find that LINE-derived sequences (LINEs) contribute to this selection by recruiting dozens of RNA-binding proteins (RBPs) to introns. This includes MATR3, which promotes binding of PTBP1 to multivalent binding sites within LINEs. Both RBPs repress splicing and 3' end processing within and around LINEs. Notably, repressive RBPs preferentially bind to evolutionarily young LINEs, which are located far from exons. These RBPs insulate the LINEs and the surrounding intronic regions from RNA processing. Upon evolutionary divergence, changes in RNA motifs within LINEs lead to gradual loss of their insulation. Hence, older LINEs are located closer to exons, are a common source of tissue-specific exons, and increasingly bind to RBPs that enhance RNA processing. Thus, LINEs are hubs for the assembly of repressive RBPs and also contribute to the evolution of new, lineage-specific transcripts in mammals. VIDEO ABSTRACT.
Asunto(s)
Ribonucleoproteínas Nucleares Heterogéneas/química , Elementos de Nucleótido Esparcido Largo , Proteínas Asociadas a Matriz Nuclear/química , Poliadenilación , Proteína de Unión al Tracto de Polipirimidina/química , Proteínas de Unión al ARN/química , ARN/química , Empalme Alternativo , Animales , Sitios de Unión , Exones , Células HeLa , Humanos , Intrones , Ratones , Mutación , Motivos de Nucleótidos , Filogenia , Unión Proteica , Mapeo de Interacción de Proteínas , Empalme del ARNRESUMEN
More than 25 inherited human disorders are caused by the unstable expansion of repetitive DNA sequences termed short tandem repeats (STRs). A fundamental unresolved question is why some STRs are susceptible to pathologic expansion, whereas thousands of repeat tracts across the human genome are relatively stable. Here, we discover that nearly all disease-associated STRs (daSTRs) are located at boundaries demarcating 3D chromatin domains. We identify a subset of boundaries with markedly higher CpG island density compared to the rest of the genome. daSTRs specifically localize to ultra-high-density CpG island boundaries, suggesting they might be hotspots for epigenetic misregulation or topological disruption linked to STR expansion. Fragile X syndrome patients exhibit severe boundary disruption in a manner that correlates with local loss of CTCF occupancy and the degree of FMR1 silencing. Our data uncover higher-order chromatin architecture as a new dimension in understanding repeat expansion disorders.
Asunto(s)
Cromatina/genética , Repeticiones de Microsatélite/fisiología , Expansión de Repetición de Trinucleótido/fisiología , Adulto , Encéfalo/citología , Encéfalo/patología , Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/fisiología , Línea Celular , Cromatina/fisiología , Ensamble y Desensamble de Cromatina/genética , Ensamble y Desensamble de Cromatina/fisiología , Islas de CpG/genética , Islas de CpG/fisiología , ADN/genética , Enfermedad/etiología , Enfermedad/genética , Femenino , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/fisiología , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Genoma Humano/genética , Humanos , Masculino , Repeticiones de Microsatélite/genética , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
Nuclear pore complexes (NPCs) are â¼100 MDa transport channels assembled from multiple copies of â¼30 nucleoporins (Nups). One-third of these Nups contain phenylalanine-glycine (FG)-rich repeats, forming a diffusion barrier, which is selectively permeable for nuclear transport receptors that interact with these repeats. Here, we identify an additional function of FG repeats in the structure and biogenesis of the yeast NPC. We demonstrate that GLFG-containing FG repeats directly bind to multiple scaffold Nups in vitro and act as NPC-targeting determinants in vivo. Furthermore, we show that the GLFG repeats of Nup116 function in a redundant manner with Nup188, a nonessential scaffold Nup, to stabilize critical interactions within the NPC scaffold needed for late steps of NPC assembly. Our results reveal a previously unanticipated structural role for natively unfolded GLFG repeats as Velcro to link NPC subcomplexes and thus add a new layer of connections to current models of the NPC architecture.
Asunto(s)
Poro Nuclear/química , Saccharomyces cerevisiae/citología , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Biogénesis de Organelos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Alterations in transcriptional regulators can orchestrate oncogenic gene expression programs in cancer. Here, we show that the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, which is mutated in over 20% of human tumors, interacts with EWSR1, a member of a family of proteins with prion-like domains (PrLD) that are frequent partners in oncogenic fusions with transcription factors. In Ewing sarcoma, we find that the BAF complex is recruited by the EWS-FLI1 fusion protein to tumor-specific enhancers and contributes to target gene activation. This process is a neomorphic property of EWS-FLI1 compared to wild-type FLI1 and depends on tyrosine residues that are necessary for phase transitions of the EWSR1 prion-like domain. Furthermore, fusion of short fragments of EWSR1 to FLI1 is sufficient to recapitulate BAF complex retargeting and EWS-FLI1 activities. Our studies thus demonstrate that the physical properties of prion-like domains can retarget critical chromatin regulatory complexes to establish and maintain oncogenic gene expression programs.
Asunto(s)
Proteínas de Unión a Calmodulina/química , Proteínas de Unión a Calmodulina/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Sarcoma de Ewing/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , Repeticiones de Microsatélite , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Priónicas/metabolismo , Dominios Proteicos , Sarcoma de Ewing/patologíaRESUMEN
Inverted Alu repeats (IRAlus) are abundantly found in the transcriptome, especially in introns and 3' untranslated regions (UTRs). Yet, the biological significance of IRAlus embedded in 3' UTRs remains largely unknown. Here, we find that 3' UTR IRAlus silences genes involved in essential signaling pathways. We utilize J2 antibody to directly capture and map the double-stranded RNA structure of 3' UTR IRAlus in the transcriptome. Bioinformatic analysis reveals alternative polyadenylation as a major axis of IRAlus-mediated gene regulation. Notably, the expression of mouse double minute 2 (MDM2), an inhibitor of p53, is upregulated by the exclusion of IRAlus during UTR shortening, which is exploited to silence p53 during tumorigenesis. Moreover, the transcriptome-wide UTR lengthening in neural progenitor cells results in the global downregulation of genes associated with neurodegenerative diseases, including amyotrophic lateral sclerosis, via IRAlus inclusion. Our study establishes the functional landscape of 3' UTR IRAlus and its role in human pathophysiology.
Asunto(s)
Poliadenilación , Proteína p53 Supresora de Tumor , Humanos , Ratones , Animales , Proteína p53 Supresora de Tumor/genética , Regiones no Traducidas 3'/genética , Regulación de la Expresión Génica , IntronesRESUMEN
The multi-pass transmembrane protein ACCELERATED CELL DEATH 6 (ACD6) is an immune regulator in Arabidopsis thaliana with an unclear biochemical mode of action. We have identified two loci, MODULATOR OF HYPERACTIVE ACD6 1 (MHA1) and its paralog MHA1-LIKE (MHA1L), that code for â¼7 kDa proteins, which differentially interact with specific ACD6 variants. MHA1L enhances the accumulation of an ACD6 complex, thereby increasing the activity of the ACD6 standard allele for regulating plant growth and defenses. The intracellular ankyrin repeats of ACD6 are structurally similar to those found in mammalian ion channels. Several lines of evidence link increased ACD6 activity to enhanced calcium influx, with MHA1L as a direct regulator of ACD6, indicating that peptide-regulated ion channels are not restricted to animals.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Ancirinas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Muerte Celular , Canales Iónicos/genética , Canales Iónicos/metabolismo , Inmunidad de la Planta/genéticaRESUMEN
Nuclear pore complexes (NPCs) direct the nucleocytoplasmic transport of macromolecules. Here, we provide a composite multiscale structure of the yeast NPC, based on improved 3D density maps from cryogenic electron microscopy and AlphaFold2 models. Key features of the inner and outer rings were integrated into a comprehensive model. We resolved flexible connectors that tie together the core scaffold, along with equatorial transmembrane complexes and a lumenal ring that anchor this channel within the pore membrane. The organization of the nuclear double outer ring reveals an architecture that may be shared with ancestral NPCs. Additional connections between the core scaffold and the central transporter suggest that under certain conditions, a degree of local organization is present at the periphery of the transport machinery. These connectors may couple conformational changes in the scaffold to the central transporter to modulate transport. Collectively, this analysis provides insights into assembly, transport, and NPC evolution.
Asunto(s)
Poro Nuclear , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Transporte de MembranaRESUMEN
DNA mismatch repair is a conserved antimutagenic pathway that maintains genomic stability through rectification of DNA replication errors and attenuation of chromosomal rearrangements. Paradoxically, mutagenic action of mismatch repair has been implicated as a cause of triplet repeat expansions that cause neurological diseases such as Huntington disease and myotonic dystrophy. This mutagenic process requires the mismatch recognition factor MutSß and the MutLα (and/or possibly MutLγ) endonuclease, and is thought to be triggered by the transient formation of unusual DNA structures within the expanded triplet repeat element. This review summarizes the current knowledge of DNA mismatch repair involvement in triplet repeat expansion, which encompasses in vitro biochemical findings, cellular studies, and various in vivo transgenic animal model experiments. We present current mechanistic hypotheses regarding mismatch repair protein function in mediating triplet repeat expansions and discuss potential therapeutic approaches targeting the mismatch repair pathway.
Asunto(s)
Reparación de la Incompatibilidad de ADN , Expansión de Repetición de Trinucleótido , Animales , Cromatina/metabolismo , Escherichia coli , Inestabilidad Genómica , Histonas/metabolismo , Humanos , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/metabolismo , Conformación de Ácido NucleicoRESUMEN
The vaccine strain against smallpox, vaccinia virus (VACV), is highly immunogenic yet causes relatively benign disease. These attributes are believed to be caused by gene loss in VACV. Using a targeted small interfering RNA (siRNA) screen, we identified a viral inhibitor found in cowpox virus (CPXV) and other orthopoxviruses that bound to the host SKP1-Cullin1-F-box (SCF) machinery and the essential necroptosis kinase receptor interacting protein kinase 3 (RIPK3). This "viral inducer of RIPK3 degradation" (vIRD) triggered ubiquitination and proteasome-mediated degradation of RIPK3 and inhibited necroptosis. In contrast to orthopoxviruses, the distantly related leporipoxvirus myxoma virus (MYXV), which infects RIPK3-deficient hosts, lacks a functional vIRD. Introduction of vIRD into VACV, which encodes a truncated and defective vIRD, enhanced viral replication in mice. Deletion of vIRD reduced CPXV-induced inflammation, viral replication, and mortality, which were reversed in RIPK3- and MLKL-deficient mice. Hence, vIRD-RIPK3 drives pathogen-host evolution and regulates virus-induced inflammation and pathogenesis.
Asunto(s)
Virus de la Viruela Vacuna/fisiología , Viruela Vacuna/inmunología , ARN Interferente Pequeño/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Virus Vaccinia/metabolismo , Proteínas Virales/metabolismo , Animales , Evolución Molecular , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Inflamación , Ratones , Ratones Noqueados , Necroptosis/genética , Orthopoxvirus , Filogenia , Proteínas Quinasas/genética , Proteolisis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Análisis de Secuencia de ARN , Proteínas Virales/genética , Replicación ViralRESUMEN
Discovering potent human monoclonal antibodies (mAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) on sporozoites (SPZ) and elucidating their mechanisms of neutralization will facilitate translation for passive prophylaxis and aid next-generation vaccine development. Here, we isolated a neutralizing human mAb, L9 that preferentially bound NVDP minor repeats of PfCSP with high affinity while cross-reacting with NANP major repeats. L9 was more potent than six published neutralizing human PfCSP mAbs at mediating protection against mosquito bite challenge in mice. Isothermal titration calorimetry and multiphoton microscopy showed that L9 and the other most protective mAbs bound PfCSP with two binding events and mediated protection by killing SPZ in the liver and by preventing their egress from sinusoids and traversal of hepatocytes. This study defines the subdominant PfCSP minor repeats as neutralizing epitopes, identifies an in vitro biophysical correlate of SPZ neutralization, and demonstrates that the liver is an important site for antibodies to prevent malaria.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antiprotozoarios/inmunología , Antimaláricos/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Esporozoítos/inmunología , Adolescente , Adulto , Animales , Línea Celular , Línea Celular Tumoral , Epítopos/inmunología , Femenino , Células HEK293 , Hepatocitos/inmunología , Hepatocitos/parasitología , Humanos , Hígado/inmunología , Hígado/parasitología , Malaria/inmunología , Malaria/parasitología , Vacunas contra la Malaria/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Adulto JovenRESUMEN
Expansion mutations in polyalanine stretches are associated with a growing number of diseases sharing a high degree of genotypic and phenotypic commonality. These similarities prompted us to query the normal function of physiological polyalanine stretches and to investigate whether a common molecular mechanism is involved in these diseases. Here, we show that UBA6, an E1 ubiquitin-activating enzyme, recognizes a polyalanine stretch within its cognate E2 ubiquitin-conjugating enzyme USE1. Aberrations in this polyalanine stretch reduce ubiquitin transfer to USE1 and, subsequently, polyubiquitination and degradation of its target, the ubiquitin ligase E6AP. Furthermore, we identify competition for the UBA6-USE1 interaction by various proteins with polyalanine expansion mutations in the disease state. The deleterious interactions of expanded polyalanine tract proteins with UBA6 in mouse primary neurons alter the levels and ubiquitination-dependent degradation of E6AP, which in turn affects the levels of the synaptic protein Arc. These effects are also observed in induced pluripotent stem cell-derived autonomic neurons from patients with polyalanine expansion mutations, where UBA6 overexpression increases neuronal resilience to cell death. Our results suggest a shared mechanism for such mutations that may contribute to the congenital malformations seen in polyalanine tract diseases.
Asunto(s)
Péptidos , Enzimas Activadoras de Ubiquitina , Ubiquitina , Humanos , Animales , Ratones , Ubiquitinación , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/metabolismo , MutaciónRESUMEN
Many bacterial pathogens regulate their virulence genes via phase variation, whereby length-variable simple sequence repeats control the transcription or coding potential of those genes. Here, we have exploited this relationship between DNA structure and physiological function to discover a globally acting small RNA (sRNA) regulator of virulence in the gastric pathogen Helicobacter pylori. Our study reports the first sRNA whose expression is affected by a variable thymine (T) stretch in its promoter. We show the sRNA post-transcriptionally represses multiple major pathogenicity factors of H. pylori, including CagA and VacA, by base pairing to their mRNAs. We further demonstrate transcription of the sRNA is regulated by the nickel-responsive transcriptional regulator NikR (thus named NikS for nickel-regulated sRNA), thereby linking virulence factor regulation to nickel concentrations. Using in-vitro infection experiments, we demonstrate NikS affects host cell internalization and epithelial barrier disruption. Together, our results show NikS is a phase-variable, post-transcriptional global regulator of virulence properties in H. pylori.
Asunto(s)
Helicobacter pylori/genética , Helicobacter pylori/patogenicidad , ARN Bacteriano/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Factores de Virulencia/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Recuento de Colonia Microbiana , Endocitosis/efectos de los fármacos , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Helicobacter pylori/efectos de los fármacos , Interacciones Huésped-Patógeno/efectos de los fármacos , Níquel/farmacología , Fenotipo , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
The failure to repress transcription of repetitive genomic elements can lead to catastrophic genome instability and is associated with various human diseases. As such, multiple parallel mechanisms cooperate to ensure repression and heterochromatinization of these elements, especially during germline development and early embryogenesis. A vital question in the field is how specificity in establishing heterochromatin at repetitive elements is achieved. Apart from trans-acting protein factors, recent evidence points to a role of different RNA species in targeting repressive histone marks and DNA methylation to these sites in mammals. Here, we review recent discoveries on this topic and predominantly focus on the role of RNA methylation, piRNAs, and other localized satellite RNAs.
Asunto(s)
Silenciador del Gen , Heterocromatina , Mamíferos , Metilación , ARN , Secuencias Repetitivas de Ácidos Nucleicos , Heterocromatina/genética , Heterocromatina/metabolismo , ARN/genética , ARN/metabolismo , Mamíferos/genética , Metilación de ADN , Histonas/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos/genética , Elementos Transponibles de ADN/genética , Humanos , AnimalesRESUMEN
Autosomal-dominant ataxia with sensory and autonomic neuropathy is a highly specific combined phenotype that we described in two Swedish kindreds in 2014; its genetic cause had remained unknown. Here, we report the discovery of exonic GGC trinucleotide repeat expansions, encoding poly-glycine, in zinc finger homeobox 3 (ZFHX3) in these families. The expansions were identified in whole-genome datasets within genomic segments that all affected family members shared. Non-expanded alleles carried one or more interruptions within the repeat. We also found ZFHX3 repeat expansions in three additional families, all from the region of Skåne in southern Sweden. Individuals with expanded repeats developed balance and gait disturbances at 15 to 60 years of age and had sensory neuropathy and slow saccades. Anticipation was observed in all families and correlated with different repeat lengths determined through long-read sequencing in two family members. The most severely affected individuals had marked autonomic dysfunction, with severe orthostatism as the most disabling clinical feature. Neuropathology revealed p62-positive intracytoplasmic and intranuclear inclusions in neurons of the central and enteric nervous system, as well as alpha-synuclein positivity. ZFHX3 is located within the 16q22 locus, to which spinocerebellar ataxia type 4 (SCA4) repeatedly had been mapped; the clinical phenotype in our families corresponded well with the unique phenotype described in SCA4, and the original SCA4 kindred originated from Sweden. ZFHX3 has known functions in neuronal development and differentiation n both the central and peripheral nervous system. Our findings demonstrate that SCA4 is caused by repeat expansions in ZFHX3.