RESUMEN
Idiopathic scoliosis (IS) is a spinal disorder affecting up to 3% of otherwise healthy children. IS has a strong familial genetic component and is believed to be genetically complex due to significant variability in phenotype and heritability. Previous studies identified putative loci and variants possibly contributing to IS susceptibility, including within extracellular matrix, cilia, and actin networks, but the genetic architecture and underlying mechanisms remain unresolved. Here, we used whole-exome sequencing from three affected individuals in a multigenerational family with IS and identified 19 uncommon variants (minor allele frequency < 0.05). Genotyping of additional family members identified a candidate heterozygous variant (H1115Q, G>C, rs142032413) within the ciliary gene KIF7, a regulator within the hedgehog (Hh) signaling pathway. Resequencing of the second cohort of unrelated IS individuals and controls identified several severe mutations in KIF7 in affected individuals only. Subsequently, we generated a mutant zebrafish model of kif7 using CRISPR-Cas9. kif7co63/co63 zebrafish displayed severe scoliosis, presenting in juveniles and progressing through adulthood. We observed no deformities in the brain, Reissner fiber, or central canal cilia in kif7co63/co63 embryos, although alterations were seen in Hh pathway gene expression. This study suggests defects in KIF7-dependent Hh signaling, which may drive pathogenesis in a subset of individuals with IS.
Asunto(s)
Cinesinas , Escoliosis , Pez Cebra , Animales , Cilios/metabolismo , Humanos , Cinesinas/genética , Mutación , Escoliosis/genética , Pez Cebra/genética , Proteínas de Pez CebraRESUMEN
The neurotropic herpesvirus varicella-zoster virus (VZV) establishes a lifelong latent infection in humans following primary infection. The low abundance of VZV nucleic acids in human neurons has hindered an understanding of the mechanisms that regulate viral gene transcription during latency. To overcome this critical barrier, we optimized a targeted capture protocol to enrich VZV DNA and cDNA prior to whole-genome/transcriptome sequence analysis. Since the VZV genome is remarkably stable, it was surprising to detect that VZV32, a VZV laboratory strain with no discernible growth defect in tissue culture, contained a 2,158-bp deletion in open reading frame (ORF) 12. Consequently, ORF 12 and 13 protein expression was abolished and Akt phosphorylation was inhibited. The discovery of the ORF 12 deletion, revealed through targeted genome sequencing analysis, points to the need to authenticate the VZV genome when the virus is propagated in tissue culture.IMPORTANCE Viruses isolated from clinical samples often undergo genetic modifications when cultured in the laboratory. Historically, VZV is among the most genetically stable herpesviruses, a notion supported by more than 60 complete genome sequences from multiple isolates and following multiple in vitro passages. However, application of enrichment protocols to targeted genome sequencing revealed the unexpected deletion of a significant portion of VZV ORF 12 following propagation in cultured human fibroblast cells. While the enrichment protocol did not introduce bias in either the virus genome or transcriptome, the findings indicate the need for authentication of VZV by sequencing when the virus is propagated in tissue culture.
Asunto(s)
ADN Viral/aislamiento & purificación , Genoma Viral , Herpesvirus Humano 3/genética , Sistemas de Lectura Abierta , Eliminación de Secuencia , Línea Celular , ADN Complementario , Herpesvirus Humano 3/crecimiento & desarrollo , Humanos , Análisis de Secuencia de ADN/métodos , Transcriptoma , Proteínas Virales , Virión , Latencia del VirusRESUMEN
Activation-induced deaminase (AID) functions by deaminating cytosines and causing U:G mismatches, a rate-limiting step of Ab gene diversification. However, precise mechanisms regulating AID deamination frequency remain incompletely understood. Moreover, it is not known whether different sequence contexts influence the preferential access of mismatch repair or uracil glycosylase (UNG) to AID-initiated U:G mismatches. In this study, we employed two knock-in models to directly compare the mutability of core Sµ and VDJ exon sequences and their ability to regulate AID deamination and subsequent repair process. We find that the switch (S) region is a much more efficient AID deamination target than the V region. Igh locus AID-initiated lesions are processed by error-free and error-prone repair. S region U:G mismatches are preferentially accessed by UNG, leading to more UNG-dependent deletions, enhanced by mismatch repair deficiency. V region mutation hotspots are largely determined by AID deamination. Recurrent and conserved S region motifs potentially function as spacers between AID deamination hotspots. We conclude that the pattern of mutation hotspots and DNA break generation is influenced by sequence-intrinsic properties, which regulate AID deamination and affect the preferential access of downstream repair. Our studies reveal an evolutionarily conserved role for substrate sequences in regulating Ab gene diversity and AID targeting specificity.
Asunto(s)
Sitios de Unión , Citidina Desaminasa/metabolismo , Reparación del ADN , Motivos de Nucleótidos , Alelos , Animales , Secuencia de Bases , Reparación de la Incompatibilidad de ADN , Técnicas de Sustitución del Gen , Orden Génico , Marcación de Gen , Sitios Genéticos , Ratones , Ratones Noqueados , Proteína 2 Homóloga a MutS/metabolismo , Mutación , Tasa de Mutación , Especificidad por Sustrato , Uracil-ADN Glicosidasa/metabolismo , Exones VDJ/genéticaRESUMEN
Class switch recombination (CSR) generates isotype-switched Abs with distinct effector functions. B cells express phosphatase and tensin homolog (PTEN) and multiple isoforms of class IA PI3K catalytic subunits, including p110α and p110δ, whose roles in CSR remain unknown or controversial. In this article, we demonstrate a direct effect of PTEN on CSR signaling by acute deletion of Pten specifically in mature B cells, thereby excluding the developmental impact of Pten deletion. We show that mature B cell-specific PTEN overexpression enhances CSR. More importantly, we establish a critical role for p110α in CSR. Furthermore, we identify a cooperative role for p110α and p110δ in suppressing CSR. Mechanistically, dysregulation of p110α or PTEN inversely affects activation-induced deaminase expression via modulating AKT activity. Thus, our study reveals that a signaling balance between PTEN and PI3K isoforms is essential to maintain normal CSR.
Asunto(s)
Linfocitos B/inmunología , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Cambio de Clase de Inmunoglobulina/inmunología , Fosfohidrolasa PTEN/metabolismo , Animales , Células Cultivadas , Citidina Desaminasa/biosíntesis , Citidina Desaminasa/metabolismo , Cambio de Clase de Inmunoglobulina/genética , Inmunoglobulina E/genética , Inmunoglobulina E/inmunología , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Ratones , Ratones Noqueados , Fosfohidrolasa PTEN/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
Activation-induced deaminase (AID) initiates U:G mismatches, causing point mutations or DNA double-stranded breaks at Ig loci. How AID-initiated lesions are prevented from inducing genome-wide damage remains elusive. A differential DNA repair mechanism might protect certain non-Ig loci such as c-myc from AID attack. However, determinants regulating such protective mechanisms are largely unknown. To test whether target DNA sequences modulate protective mechanisms via altering the processing manner of AID-initiated lesions, we established a knock-in model by inserting an Sγ2b region, a bona fide AID target, into the first intron of c-myc. Unexpectedly, we found that the inserted S region did not mutate or enhance c-myc genomic instability, due to error-free repair of AID-initiated lesions, in Ag-stimulated germinal center B cells. In contrast, in vitro cytokine-activated B cells display a much higher level of c-myc genomic instability in an AID- and S region-dependent manner. Furthermore, we observe a comparable frequency of AID deamination events between the c-myc intronic sequence and inserted S region in different B cell populations, demonstrating a similar frequency of AID targeting. Thus, our study reveals a clear difference between germinal center and cytokine-activated B cells in their ability to develop genomic instability, attributable to a differential processing of AID-initiated lesions in distinct B cell populations. We propose that locus-specific regulatory mechanisms (e.g., transcription) appear to not only override the effects of S region sequence on AID targeting frequency but also influence the repair manner of AID-initiated lesions.
Asunto(s)
Subgrupos de Linfocitos B/fisiología , Linfocitos B/fisiología , Citidina Desaminasa/metabolismo , Centro Germinal/inmunología , Animales , Células Cultivadas , Citidina Desaminasa/genética , Citocinas/metabolismo , Reparación del ADN/inmunología , Técnicas de Sustitución del Gen , Sitios Genéticos/genética , Inestabilidad Genómica , Humanos , Intrones/genética , Ratones de la Cepa 129 , Mutación/genética , Proteínas Proto-Oncogénicas c-myc/genética , Hipermutación Somática de Inmunoglobulina , Especificidad por SustratoRESUMEN
The specification of mammalian chromosomal replication origins is incompletely understood. To analyze the assembly and activation of prereplicative complexes (pre-RCs), we tested the effects of tethered binding of chromatin acetyltransferases and replication proteins on chromosomal c-myc origin deletion mutants containing a GAL4-binding cassette. GAL4(DBD) (DNA binding domain) fusions with Orc2, Cdt1, E2F1 or HBO1 coordinated the recruitment of the Mcm7 helicase subunit, the DNA unwinding element (DUE)-binding protein DUE-B and the minichromosome maintenance (MCM) helicase activator Cdc45 to the replicator, and restored origin activity. In contrast, replication protein binding and origin activity were not stimulated by fusion protein binding in the absence of flanking c-myc DNA. Substitution of the GAL4-binding site for the c-myc replicator DUE allowed Orc2 and Mcm7 binding, but eliminated origin activity, indicating that the DUE is essential for pre-RC activation. Additionally, tethering of DUE-B was not sufficient to recruit Cdc45 or activate pre-RCs formed in the absence of a DUE. These results show directly in a chromosomal background that chromatin acetylation, Orc2 or Cdt1 suffice to recruit all downstream replication initiation activities to a prospective origin, and that chromosomal origin activity requires singular DNA sequences.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Origen de Réplica , Proteínas de Ciclo Celular/metabolismo , Cromosomas Humanos/química , Cromosomas Humanos/metabolismo , Factor de Transcripción E2F1/metabolismo , Genes myc , Células HeLa , Histona Acetiltransferasas/metabolismo , Humanos , Componente 7 del Complejo de Mantenimiento de Minicromosoma , Proteínas Nucleares/metabolismo , Complejo de Reconocimiento del Origen/metabolismoRESUMEN
DNA sequences prone to forming noncanonical structures (hairpins, triplexes, G-quadruplexes) cause DNA replication fork stalling, activate DNA damage responses, and represent hotspots of genomic instability associated with human disease. The 88-bp asymmetric polypurine-polypyrimidine (Pu-Py) mirror repeat tract from the human polycystic kidney disease (PKD1) intron 21 forms non-B DNA secondary structures in vitro. We show that the PKD1 mirror repeat also causes orientation-dependent fork stalling during replication in vitro and in vivo. When integrated alongside the c-myc replicator at an ectopic chromosomal site in the HeLa genome, the Pu-Py mirror repeat tract elicits a polar replication fork barrier. Increased replication protein A (RPA), Rad9, and ataxia telangiectasia- and Rad3-related (ATR) checkpoint protein binding near the mirror repeat sequence suggests that the DNA damage response is activated upon replication fork stalling. Moreover, the proximal c-myc origin of replication was not required to cause orientation-dependent checkpoint activation. Cells expressing the replication fork barrier display constitutive Chk1 phosphorylation and continued growth, i.e. checkpoint adaptation. Excision of the Pu-Py mirror repeat tract abrogates the DNA damage response. Adaptation to Chk1 phosphorylation in cells expressing the replication fork barrier may allow the accumulation of mutations that would otherwise be remediated by the DNA damage response.
Asunto(s)
Canales Catiónicos TRPP/metabolismo , Inmunoprecipitación de Cromatina , Citosol/metabolismo , ADN/química , ADN/metabolismo , Daño del ADN , Cartilla de ADN/genética , Replicación del ADN , Inestabilidad Genómica , Células HeLa , Humanos , Intrones , Conformación de Ácido Nucleico , Fosforilación , Proteínas Proto-Oncogénicas c-myc/metabolismo , Purinas/química , Pirimidinas/química , Canales Catiónicos TRPP/genéticaRESUMEN
Instability of (CTG) x (CAG) microsatellite trinucleotide repeat (TNR) sequences is responsible for more than a dozen neurological or neuromuscular diseases. TNR instability during DNA synthesis is thought to involve slipped-strand or hairpin structures in template or nascent DNA strands, although direct evidence for hairpin formation in human cells is lacking. We have used targeted recombination to create a series of isogenic HeLa cell lines in which (CTG) x (CAG) repeats are replicated from an ectopic copy of the Myc (also known as c-myc) replication origin. In this system, the tendency of chromosomal (CTG) x (CAG) tracts to expand or contract was affected by origin location and the leading or lagging strand replication orientation of the repeats, and instability was enhanced by prolonged cell culture, increased TNR length and replication inhibition. Hairpin cleavage by synthetic zinc finger nucleases in these cells has provided the first direct evidence for the formation of hairpin structures during replication in vivo.
Asunto(s)
Replicación del ADN/fisiología , ADN/biosíntesis , ADN/genética , Inestabilidad de Microsatélites , Conformación de Ácido Nucleico , Repeticiones de Trinucleótidos/genética , Células Cultivadas , ADN/química , Endonucleasas/genética , Endonucleasas/metabolismo , Células HeLa , Humanos , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Origen de Réplica/genética , Origen de Réplica/fisiología , Dedos de Zinc/genética , Dedos de Zinc/fisiologíaRESUMEN
Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine; however, their potential clinical application is hampered by the low efficiency of somatic cell reprogramming. Here, we show that the synergistic activity of synthetic modified mRNAs encoding reprogramming factors and miRNA-367/302s delivered as mature miRNA mimics greatly enhances the reprogramming of human primary fibroblasts into iPSCs. This synergistic activity is dependent upon an optimal RNA transfection regimen and culturing conditions tailored specifically to human primary fibroblasts. As a result, we can now generate up to 4,019 iPSC colonies from only 500 starting human primary neonatal fibroblasts and reprogram up to 90.7% of individually plated cells, producing multiple sister colonies. This methodology consistently generates clinically relevant, integration-free iPSCs from a variety of human patient's fibroblasts under feeder-free conditions and can be applicable for the clinical translation of iPSCs and studying the biology of reprogramming.
Asunto(s)
Técnicas de Reprogramación Celular , Línea Celular , Fibroblastos , Humanos , Células Madre Pluripotentes Inducidas , ARNRESUMEN
(CTG)(n) · (CAG)(n) trinucleotide repeat (TNR) expansion in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene causes myotonic dystrophy type 1. However, a direct link between TNR instability, the formation of noncanonical (CTG)(n) · (CAG)(n) structures, and replication stress has not been demonstrated. In a human cell model, we found that (CTG)(45) · (CAG)(45) causes local replication fork stalling, DNA hairpin formation, and TNR instability. Oligodeoxynucleotides (ODNs) complementary to the (CTG)(45) · (CAG)(45) lagging-strand template eliminated DNA hairpin formation on leading- and lagging-strand templates and relieved fork stalling. Prolonged cell culture, emetine inhibition of lagging-strand synthesis, or slowing of DNA synthesis by low-dose aphidicolin induced (CTG)(45) · (CAG)(45) expansions and contractions. ODNs targeting the lagging-strand template blocked the time-dependent or emetine-induced instability but did not eliminate aphidicolin-induced instability. These results show directly that TNR replication stalling, replication stress, hairpin formation, and instability are mechanistically linked in vivo.
Asunto(s)
Replicación del ADN , ADN/genética , Inestabilidad Genómica , Oligodesoxirribonucleótidos/metabolismo , Expansión de Repetición de Trinucleótido , ADN/química , ADN/metabolismo , Células HeLa , Humanos , Distrofia Miotónica/genética , Proteína Quinasa de Distrofia Miotónica , Conformación de Ácido Nucleico , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Myotonic dystrophy type 1 (DM1) is associated with expansion of (CTG)(n) · (CAG)(n) trinucleotide repeats (TNRs) in the 3' untranslated region (UTR) of the DMPK gene. Replication origins are cis-acting elements that potentiate TNR instability; therefore, we mapped replication initiation sites and prereplication complex protein binding within the ~10-kb DMPK/SIX5 locus in non-DM1 and DM1 cells. Two origins, IS(DMPK) and IS(SIX5), flanked the (CTG)(n) · (CAG)(n) TNRs in control cells and in DM1 cells. Orc2 and Mcm4 bound near each of the replication initiation sites, but a dramatic change in (CTG)(n) · (CAG)(n) replication polarity was not correlated with TNR expansion. To test whether (CTG)(n) · (CAG)(n) TNRs are cis-acting elements of instability in human cells, model cell lines were created by integration of cassettes containing the c-myc replication origin and (CTG)(n) · (CAG)(n) TNRs in HeLa cells. Replication forks were slowed by (CTG)(n) · (CAG)(n) TNRs in a length-dependent manner independent of replication polarity, implying that expanded (CTG)(n) · (CAG)(n) TNRs lead to replication stress. Consistent with this prediction, TNR instability increased in the HeLa model cells and DM1 cells upon small interfering RNA (siRNA) knockdown of the fork stabilization protein Claspin, Timeless, or Tipin. These results suggest that aberrant DNA replication and TNR instability are linked in DM1 cells.