RESUMEN
ATRX is an X-linked gene of the SWI/SNF family, mutations in which cause syndromal mental retardation and downregulation of α-globin expression. Here we show that ATRX binds to tandem repeat (TR) sequences in both telomeres and euchromatin. Genes associated with these TRs can be dysregulated when ATRX is mutated, and the change in expression is determined by the size of the TR, producing skewed allelic expression. This reveals the characteristics of the affected genes, explains the variable phenotypes seen with identical ATRX mutations, and illustrates a new mechanism underlying variable penetrance. Many of the TRs are G rich and predicted to form non-B DNA structures (including G-quadruplex) in vivo. We show that ATRX binds G-quadruplex structures in vitro, suggesting a mechanism by which ATRX may play a role in various nuclear processes and how this is perturbed when ATRX is mutated.
Asunto(s)
ADN Helicasas/metabolismo , Proteínas Nucleares/metabolismo , Animales , Células Cultivadas , Inmunoprecipitación de Cromatina , Cromosomas de los Mamíferos/metabolismo , Islas de CpG , ADN Helicasas/genética , ADN Ribosómico/metabolismo , G-Cuádruplex , Expresión Génica , Estudio de Asociación del Genoma Completo , Histonas/metabolismo , Humanos , Ratones , Repeticiones de Minisatélite , Mutación , Proteínas Nucleares/genética , Telómero/metabolismo , Proteína Nuclear Ligada al Cromosoma XRESUMEN
The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state. Using genome editing with zinc-finger nucleases to tag endogenous H3.3, we report genome-wide profiles of H3 variants in mammalian embryonic stem cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acid sequence and change with cellular differentiation at developmentally regulated loci. The H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for localization of H3.3 at telomeres and many transcription factor binding sites. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Atrx is required for Hira-independent localization of H3.3 at telomeres and for the repression of telomeric RNA. Our data demonstrate that multiple and distinct factors are responsible for H3.3 localization at specific genomic locations in mammalian cells.
Asunto(s)
Histonas/análisis , Telómero/química , Animales , Sitios de Unión , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Madre Embrionarias/metabolismo , Genoma , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/genética , Histonas/metabolismo , Ratones , Ratones Endogámicos C57BL , Telómero/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Sitio de Iniciación de la TranscripciónRESUMEN
Telomere maintenance is a hallmark of malignant cells and allows cancers to divide indefinitely. In some cancers, this is achieved through the alternative lengthening of telomeres (ALT) pathway. Whilst loss of ATRX is a near universal feature of ALT-cancers, it is insufficient in isolation. As such, other cellular events must be necessary - but the exact nature of the secondary events has remained elusive. Here, we report that trapping of proteins (such as TOP1, TOP2A and PARP1) on DNA leads to ALT induction in cells lacking ATRX. We demonstrate that protein-trapping chemotherapeutic agents, such as etoposide, camptothecin and talazoparib, induce ALT markers specifically in ATRX-null cells. Further, we show that treatment with G4-stabilising drugs cause an increase in trapped TOP2A levels which leads to ALT induction in ATRX-null cells. This process is MUS81-endonuclease and break-induced replication dependent, suggesting that protein trapping leads to replication fork stalling, with these forks being aberrantly processed in the absence of ATRX. Finally, we show ALT-positive cells harbour a higher load of genome-wide trapped proteins, such as TOP1, and knockdown of TOP1 reduced ALT activity. Taken together, these findings suggest that protein trapping is a fundamental driving force behind ALT-biology in ATRX-deficient malignancies.
A key feature of all cancer cells is their ability to divide indefinitely, and this is dependent on circumvention of telomere shortening through induction of a telomere maintenance mechanism, such as the telomerase-independent, Alternative Lengthening of Telomeres (ALT) pathway. The ALT pathway is characterised by loss of the ATRX chromatin remodeler. The current study provides evidence that, in the absence of ATRX, increased trapping of proteins on DNA leads to replication fork stalling and collapse. At telomeres, this leads to ALT pathway activity. These results help to better understand ALT tumours and might, eventually, be instrumental in developing new therapeutic strategies.
Asunto(s)
Neoplasias , Telómero , Humanos , ADN , Neoplasias/genética , Telomerasa/genética , Telómero/genética , Telómero/metabolismo , Homeostasis del Telómero , Proteína Nuclear Ligada al Cromosoma X/genética , Proteína Nuclear Ligada al Cromosoma X/metabolismoRESUMEN
DNA replication stress can cause chromosomal instability and tumor progression. One key pathway that counteracts replication stress and promotes faithful DNA replication consists of the Fanconi anemia (FA) proteins. However, how these proteins limit replication stress remains largely elusive. Here we show that conflicts between replication and transcription activate the FA pathway. Inhibition of transcription or enzymatic degradation of transcription-associated R-loops (DNA:RNA hybrids) suppresses replication fork arrest and DNA damage occurring in the absence of a functional FA pathway. Furthermore, we show that simple aldehydes, known to cause leukemia in FA-deficient mice, induce DNA:RNA hybrids in FA-depleted cells. Finally, we demonstrate that the molecular mechanism by which the FA pathway limits R-loop accumulation requires FANCM translocase activity. Failure to activate a response to physiologically occurring DNA:RNA hybrids may critically contribute to the heightened cancer predisposition and bone marrow failure of individuals with mutated FA proteins.
Asunto(s)
Daño del ADN , ADN Helicasas/metabolismo , Replicación del ADN , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Inestabilidad Genómica , Ácidos Nucleicos Heterodúplex/metabolismo , Animales , ADN Helicasas/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Células HeLa , Humanos , Leucemia/genética , Leucemia/metabolismo , Leucemia/patología , Ratones , Ratones Noqueados , Mutación , Ácidos Nucleicos Heterodúplex/genéticaRESUMEN
The oxygen transport function of hemoglobin (HB) is thought to have arisen â¼500 million years ago, roughly coinciding with the divergence between jawless (Agnatha) and jawed (Gnathostomata) vertebrates. Intriguingly, extant HBs of jawless and jawed vertebrates were shown to have evolved twice, and independently, from different ancestral globin proteins. This raises the question of whether erythroid-specific expression of HB also evolved twice independently. In all jawed vertebrates studied to date, one of the HB gene clusters is linked to the widely expressed NPRL3 gene. Here we show that the nprl3-linked hb locus of a jawless vertebrate, the river lamprey (Lampetra fluviatilis), shares a range of structural and functional properties with the equivalent jawed vertebrate HB locus. Functional analysis demonstrates that an erythroid-specific enhancer is located in intron 7 of lamprey nprl3, which corresponds to the NPRL3 intron 7 MCS-R1 enhancer of jawed vertebrates. Collectively, our findings signify the presence of an nprl3-linked multiglobin gene locus, which contains a remote enhancer that drives globin expression in erythroid cells, before the divergence of jawless and jawed vertebrates. Different globin genes from this ancestral cluster evolved in the current NPRL3-linked HB genes in jawless and jawed vertebrates. This provides an explanation of the enigma of how, in different species, globin genes linked to the same adjacent gene could undergo convergent evolution.
Asunto(s)
Eritrocitos/metabolismo , Evolución Molecular , Proteínas de Peces , Regulación de la Expresión Génica/fisiología , Hemoglobinas , Lampreas , Animales , Proteínas de Peces/biosíntesis , Proteínas de Peces/genética , Hemoglobinas/biosíntesis , Hemoglobinas/genética , Lampreas/genética , Lampreas/metabolismo , Familia de MultigenesRESUMEN
BACKGROUND: Congenital dyserythropoietic anaemia type I (CDA-I) is a hereditary anaemia caused by biallelic mutations in the widely expressed genes CDAN1 and C15orf41. Little is understood about either protein and it is unclear in which cellular pathways they participate. METHODS: Genetic analysis of a cohort of patients with CDA-I identifies novel pathogenic variants in both known causative genes. We analyse the mutation distribution and the predicted structural positioning of amino acids affected in Codanin-1, the protein encoded by CDAN1. Using western blotting, immunoprecipitation and immunofluorescence, we determine the effect of particular mutations on both proteins and interrogate protein interaction, stability and subcellular localisation. RESULTS: We identify six novel CDAN1 mutations and one novel mutation in C15orf41 and uncover evidence of further genetic heterogeneity in CDA-I. Additionally, population genetics suggests that CDA-I is more common than currently predicted. Mutations are enriched in six clusters in Codanin-1 and tend to affect buried residues. Many missense and in-frame mutations do not destabilise the entire protein. Rather C15orf41 relies on Codanin-1 for stability and both proteins, which are enriched in the nucleolus, interact to form an obligate complex in cells. CONCLUSION: Stability and interaction data suggest that C15orf41 may be the key determinant of CDA-I and offer insight into the mechanism underlying this disease. Both proteins share a common pathway likely to be present in a wide variety of cell types; however, nucleolar enrichment may provide a clue as to the erythroid specific nature of CDA-I. The surprisingly high predicted incidence of CDA-I suggests that better ascertainment would lead to improved patient care.
Asunto(s)
Anemia Diseritropoyética Congénita/genética , Predisposición Genética a la Enfermedad , Glicoproteínas/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Anemia Diseritropoyética Congénita/patología , Femenino , Regulación de la Expresión Génica/genética , Pruebas Genéticas , Genética de Población , Humanos , Masculino , Complejos Multiproteicos/genética , Mutación/genéticaRESUMEN
A substantial amount of organismal complexity is thought to be encoded by enhancers which specify the location, timing, and levels of gene expression. In mammals there are more enhancers than promoters which are distributed both between and within genes. Here we show that activated, intragenic enhancers frequently act as alternative tissue-specific promoters producing a class of abundant, spliced, multiexonic poly(A)(+) RNAs (meRNAs) which reflect the host gene's structure. meRNAs make a substantial and unanticipated contribution to the complexity of the transcriptome, appearing as alternative isoforms of the host gene. The low protein-coding potential of meRNAs suggests that many meRNAs may be byproducts of enhancer activation or underlie as-yet-unidentified RNA-encoded functions. Distinguishing between meRNAs and mRNAs will transform our interpretation of dynamic changes in transcription both at the level of individual genes and of the genome as a whole.
Asunto(s)
Elementos de Facilitación Genéticos/fisiología , Regulación de la Expresión Génica , Regiones Promotoras Genéticas/fisiología , Animales , Células Cultivadas , Células Eritroides , Ratones , Poli A , ARN/química , ARN/fisiología , Isoformas de ARN/química , ARN Mensajero/química , ARN Mensajero/fisiología , TranscriptomaRESUMEN
Primrose syndrome is a rare autosomal dominant condition caused by heterozygous missense variants within ZBTB20. Through an exome sequencing approach (as part of the Deciphering Developmental Disorders [DDD] study) we have identified five unrelated individuals with previously unreported, de novo ZBTB20 pathogenic missense variants. All five missense variants targeted the C2H2 zinc finger domains. This genotype-up approach has allowed further refinement of the Primrose syndrome phenotype. Major characteristics (>90% individuals) include an intellectual disability (most frequently in the moderate range), a recognizable facial appearance and brain MRI abnormalities, particularly abnormalities of the corpus callosum. Other frequent clinical associations (in 50-90% individuals) include sensorineural hearing loss (83%), hypotonia (78%), cryptorchidism in males (75%), macrocephaly (72%), behavioral issues (56%), and dysplastic/hypoplastic nails (57%). Based upon these clinical data we discuss our current management of patients with Primrose syndrome.
Asunto(s)
Anomalías Múltiples/diagnóstico , Anomalías Múltiples/genética , Calcinosis/diagnóstico , Calcinosis/genética , Enfermedades del Oído/diagnóstico , Enfermedades del Oído/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Variación Genética , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Atrofia Muscular/diagnóstico , Atrofia Muscular/genética , Proteínas del Tejido Nervioso/genética , Fenotipo , Factores de Transcripción/genética , Niño , Preescolar , Facies , Femenino , Sitios Genéticos , Genotipo , Humanos , Imagen por Resonancia Magnética , Masculino , MutaciónRESUMEN
ATRX is a chromatin remodelling factor found at a wide range of tandemly repeated sequences including telomeres (TTAGGG)n ATRX mutations are found in nearly all tumours that maintain their telomeres via the alternative lengthening of telomere (ALT) pathway, and ATRX is known to suppress this pathway. Here, we show that recruitment of ATRX to telomeric repeats depends on repeat number, orientation and, critically, on repeat transcription. Importantly, the transcribed telomeric repeats form RNA-DNA hybrids (R-loops) whose abundance correlates with the recruitment of ATRX Here, we show loss of ATRX is also associated with increased R-loop formation. Our data suggest that the presence of ATRX at telomeres may have a central role in suppressing deleterious DNA secondary structures that form at transcribed telomeric repeats, and this may account for the increased DNA damage, stalling of replication and homology-directed repair previously observed upon loss of ATRX function.
Asunto(s)
Ensamble y Desensamble de Cromatina , ADN/genética , ARN/genética , Telómero/genética , Telómero/metabolismo , Proteína Nuclear Ligada al Cromosoma X/metabolismo , Cromatina , ADN/química , Daño del ADN , Replicación del ADN , G-Cuádruplex , Humanos , Homeostasis del Telómero/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Proteína Nuclear Ligada al Cromosoma X/deficiencia , Proteína Nuclear Ligada al Cromosoma X/genéticaRESUMEN
Since the publication of the human reference genome, the identities of specific genes associated with human diseases are being discovered at a rapid rate. A central problem is that the biological activity of these genes is often unclear. Detailed investigations in model vertebrate organisms, typically mice, have been essential for understanding the activities of many orthologues of these disease-associated genes. Although gene-targeting approaches and phenotype analysis have led to a detailed understanding of nearly 6,000 protein-coding genes, this number falls considerably short of the more than 22,000 mouse protein-coding genes. Similarly, in zebrafish genetics, one-by-one gene studies using positional cloning, insertional mutagenesis, antisense morpholino oligonucleotides, targeted re-sequencing, and zinc finger and TAL endonucleases have made substantial contributions to our understanding of the biological activity of vertebrate genes, but again the number of genes studied falls well short of the more than 26,000 zebrafish protein-coding genes. Importantly, for both mice and zebrafish, none of these strategies are particularly suited to the rapid generation of knockouts in thousands of genes and the assessment of their biological activity. Here we describe an active project that aims to identify and phenotype the disruptive mutations in every zebrafish protein-coding gene, using a well-annotated zebrafish reference genome sequence, high-throughput sequencing and efficient chemical mutagenesis. So far we have identified potentially disruptive mutations in more than 38% of all known zebrafish protein-coding genes. We have developed a multi-allelic phenotyping scheme to efficiently assess the effects of each allele during embryogenesis and have analysed the phenotypic consequences of over 1,000 alleles. All mutant alleles and data are available to the community and our phenotyping scheme is adaptable to phenotypic analysis beyond embryogenesis.
Asunto(s)
Genoma/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Alelos , Animales , Exoma/genética , Femenino , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Genómica , Masculino , Anotación de Secuencia Molecular , Mutagénesis , Mutación/genética , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Pez Cebra/fisiología , Proteínas de Pez Cebra/metabolismoRESUMEN
Methyl-CpG binding protein 2 (MeCP2) is a multi-function factor involved in locus-specific transcriptional modulation and the regulation of genome architecture, e.g., pericentric heterochromatin (PCH) organization. MECP2 mutations are responsible for Rett syndrome (RTT), a devastating postnatal neurodevelopmental disorder, the pathogenetic mechanisms of which are still unknown. MeCP2, together with Alpha-thalassemia/mental retardation syndrome X-linked protein (ATRX), accumulates at chromocenters, which are repressive PCH domains. As with MECP2, mutations in ATRX cause ATR-X syndrome which is associated with severe intellectual disability. We exploited two murine embryonic stem cell lines, in which the expression of MeCP2 or ATRX is abolished. Through immunostaining, chromatin immunoprecipitation and western blot, we show that MeCP2 and ATRX are reciprocally dependent both for their expression and targeting to chromocenters. Moreover, ATRX plays a role in the accumulation of members of the heterochromatin protein 1 (HP1) family at PCH and, as MeCP2, modulates their expression. Furthermore, ATRX and HP1 targeting to chromocenters depends on an RNA component. 3D-DNA fluorescence in situ hybridization (FISH) highlighted, for the first time, a contribution of ATRX in MeCP2-mediated chromocenter clustering during neural differentiation. Overall, we provide a detailed dissection of the functional interplay between MeCP2 and ATRX in higher-order PCH organization in neurons. Our findings suggest molecular defects common to RTT and ATR-X syndrome, including an alteration in PCH.
Asunto(s)
Diferenciación Celular/fisiología , Heterocromatina/metabolismo , Proteína 2 de Unión a Metil-CpG/metabolismo , Neuronas/metabolismo , Proteína Nuclear Ligada al Cromosoma X/metabolismo , Animales , Diferenciación Celular/genética , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Modelos Animales de Enfermedad , Células Madre Embrionarias , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Heterocromatina/química , Heterocromatina/genética , Hibridación Fluorescente in Situ , Discapacidad Intelectual/genética , Discapacidad Intelectual Ligada al Cromosoma X/genética , Proteína 2 de Unión a Metil-CpG/genética , Ratones , Mutación , Síndrome de Rett/genética , Proteína Nuclear Ligada al Cromosoma X/química , Proteína Nuclear Ligada al Cromosoma X/genética , Talasemia alfa/genéticaRESUMEN
The regulation of chromatin structure is of paramount importance for a variety of fundamental nuclear processes, including gene expression, DNA repair, replication, and recombination. The ATP-dependent chromatin-remodelling factor ATRX (α thalassaemia/mental retardation X-linked) has emerged as a key player in each of these processes. Exciting recent developments suggest that ATRX plays a variety of key roles at tandem repeat sequences within the genome, including the deposition of a histone variant, prevention of replication fork stalling, and the suppression of a homologous recombination-based pathway of telomere maintenance. Here, we provide a mechanistic overview of the role of ATRX in each of these processes, and propose how they may be connected to give rise to seemingly disparate human diseases.
Asunto(s)
Cromatina/metabolismo , ADN Helicasas/metabolismo , Proteínas Nucleares/metabolismo , Ensamble y Desensamble de Cromatina , ADN Helicasas/genética , Replicación del ADN/genética , Replicación del ADN/fisiología , Histonas/metabolismo , Humanos , Proteínas Nucleares/genética , Telómero/metabolismo , Proteína Nuclear Ligada al Cromosoma XRESUMEN
The thalassemias, together with sickle cell anemia and its variants, are the world's most common form of inherited anemia, and in economically undeveloped countries, they still account for tens of thousands of premature deaths every year. In developed countries, treatment of thalassemia is also still far from ideal, requiring lifelong transfusion or allogeneic bone marrow transplantation. Clinical and molecular genetic studies over the course of the last 50 years have demonstrated how coinheritance of modifier genes, which alter the balance of α-like and ß-like globin gene expression, may transform severe, transfusion-dependent thalassemia into relatively mild forms of anemia. Most attention has been paid to pathways that increase γ-globin expression, and hence the production of fetal hemoglobin. Here we review the evidence that reduction of α-globin expression may provide an equally plausible approach to ameliorating clinically severe forms of ß-thalassemia, and in particular, the very common subgroup of patients with hemoglobin E ß-thalassemia that makes up approximately half of all patients born each year with severe ß-thalassemia.
Asunto(s)
Globinas alfa/genética , Talasemia beta/genética , Talasemia beta/terapia , Animales , Regulación hacia Abajo/efectos de los fármacos , Terapia Genética/métodos , Humanos , Terapia Molecular Dirigida , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/uso terapéutico , Globinas alfa/metabolismo , Talasemia beta/metabolismo , Talasemia beta/patologíaRESUMEN
Imprinted genes are an exceptional cluster of genes which are expressed in a parent-of-origin dependent fashion. This allele-specific expression is dependent on differential DNA methylation which is established in the parental germlines in a sex-specific manner. The DNA methylation imprint is accompanied by heterochromatin modifications which must be continuously maintained through development. This review summarises the factors which are important for protecting the epigenetic modifications at imprinted differentially methylated regions (DMRs), including PGC7, ZFP57 and the ATRX/Daxx/H3.3 complex. We discuss how these factors maintain heterochromatin silencing, not only at imprinted DMRs, but also other heterochromatic regions in the genome.
Asunto(s)
Silenciador del Gen , Impresión Genómica , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Cromosómicas no Histona , Proteínas Co-Represoras , ADN Helicasas/genética , ADN Helicasas/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Chaperonas Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas Represoras , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína Nuclear Ligada al Cromosoma XRESUMEN
Cancers result from the accumulation of genetic lesions, but the cellular consequences of driver mutations remain unclear, especially during the earliest stages of malignancy. The V617F mutation in the JAK2 non-receptor tyrosine kinase (JAK2V617F) is present as an early somatic event in most patients with myeloproliferative neoplasms (MPNs), and the study of these chronic myeloid malignancies provides an experimentally tractable approach to understanding early tumorigenesis. Introduction of exogenous JAK2V617F impairs replication fork progression and is associated with activation of the intra-S checkpoint, with both effects mediated by phosphatidylinositide 3-kinase (PI3K) signaling. Analysis of clonally derived JAK2V617F-positive erythroblasts from MPN patients also demonstrated impaired replication fork progression accompanied by increased levels of replication protein A (RPA)-containing foci. However, the associated intra-S checkpoint response was impaired in erythroblasts from polycythemia vera (PV) patients, but not in those from essential thrombocythemia (ET) patients. Moreover, inhibition of p53 in PV erythroblasts resulted in more gamma-H2Ax (γ-H2Ax)-marked double-stranded breaks compared with in like-treated ET erythroblasts, suggesting the defective intra-S checkpoint function seen in PV increases DNA damage in the context of attenuated p53 signaling. These results demonstrate oncogene-induced impairment of replication fork progression in primary cells from MPN patients, reveal unexpected disease-restricted differences in activation of the intra-S checkpoint, and have potential implications for the clonal evolution of malignancies.
Asunto(s)
Puntos de Control del Ciclo Celular , Replicación del ADN , Janus Quinasa 2/fisiología , Fase S , Apoptosis , División Celular , Cromosomas/metabolismo , Cromosomas/ultraestructura , Daño del ADN , Reparación del ADN , Diploidia , Fibroblastos/metabolismo , Genotipo , Enfermedades Hematológicas/genética , Humanos , Janus Quinasa 2/genética , Leucemia/metabolismo , Leucemia/patología , Microscopía Fluorescente , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , Fosforilación , ARN Interferente Pequeño/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The role of DNA sequence in determining chromatin state is incompletely understood. We have previously demonstrated that large chromosomal segments from human cells recapitulate their native chromatin state in mouse cells, but the relative contribution of local sequences versus their genomic context remains unknown. In this study, we compare orthologous chromosomal regions for which the human locus establishes prominent sites of Polycomb complex recruitment in pluripotent stem cells, whereas the corresponding mouse locus does not. Using recombination-mediated cassette exchange at the mouse locus, we establish the primacy of local sequences in the encoding of chromatin state. We show that the signal for chromatin bivalency is redundantly encoded across a bivalent domain and that this reflects competition between Polycomb complex recruitment and transcriptional activation. Furthermore, our results suggest that a high density of unmethylated CpG dinucleotides is sufficient for vertebrate Polycomb recruitment. This model is supported by analysis of DNA methyltransferase-deficient embryonic stem cells.
Asunto(s)
Islas de CpG/fisiología , Regulación de la Expresión Génica/genética , Proteínas Represoras/metabolismo , Globinas alfa/genética , Animales , Células Cultivadas/metabolismo , Cromatina/genética , Mapeo Cromosómico , Cromosomas Humanos Par 16 , Metilación de ADN , ADN Recombinante/genética , Células Madre Embrionarias/metabolismo , Humanos , Ratones , Ratones Transgénicos , Células Madre Pluripotentes/metabolismo , Proteínas del Grupo Polycomb , Recombinación Genética , Secuencias Reguladoras de Ácidos Nucleicos , Especificidad de la Especie , Transcripción GenéticaRESUMEN
In this study, we report on 8 compound heterozygotes for mutations in the key erythroid transcription factor Krüppel-like factor 1 in patients who presented with severe, transfusion-dependent hemolytic anemia. In most cases, the red cells were hypochromic and microcytic, consistent with abnormalities in hemoglobin synthesis. In addition, in many cases, the red cells resembled those seen in patients with membrane defects or enzymopathies, known as chronic nonspherocytic hemolytic anemia (CNSHA). Analysis of RNA and protein in primary erythroid cells from these individuals provided evidence of abnormal globin synthesis, with persistent expression of fetal hemoglobin and, most remarkably, expression of large quantities of embryonic globins in postnatal life. The red cell membranes were abnormal, most notably expressing reduced amounts of CD44 and, consequently, manifesting the rare In(Lu) blood group. Finally, all tested patients showed abnormally low levels of the red cell enzyme pyruvate kinase, a known cause of CNSHA. These patients define a new type of severe, transfusion-dependent CNSHA caused by mutations in a trans-acting factor (Krüppel-like factor 1) and reveal an important pathway regulating embryonic globin gene expression in adult humans.
Asunto(s)
Anemia Hemolítica/etiología , Hemoglobina Fetal/genética , Regulación de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/genética , Mutación , Reacción a la Transfusión , Adolescente , Adulto , Secuencia de Aminoácidos , Anemia Hemolítica/sangre , Anemia Hemolítica/genética , Niño , Preescolar , Secuencia Conservada , Índices de Eritrocitos , Eritrocitos/metabolismo , Femenino , Hemoglobina Fetal/química , Orden Génico , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Alineación de Secuencia , Adulto Joven , Globinas alfa/metabolismo , Globinas beta/metabolismoRESUMEN
A 0.8 kb intronic duplication in MAGT1 and a single base pair deletion in the last exon of ATRX were identified using a chromosome X-specific microarray and exome sequencing in a family with five males demonstrating intellectual disability (ID) and unusual skin findings (e.g., generalized pruritus). MAGT1 is an Mg²âº transporter previously associated with primary immunodeficiency and ID, whereas mutations in ATRX cause ATRX-ID syndrome. In patient cells, the function of ATRX was demonstrated to be abnormal based on altered RNA/protein expression, hypomethylation of rDNA, and abnormal cytokinesis. Dysfunction of MAGT1 was reflected in reduced RNA/protein expression and Mg²âº influx. The mutation in ATRX most likely explains the ID, whereas MAGT1 disruption could be linked to abnormal skin findings, as normal magnesium homeostasis is necessary for skin health. This work supports observations that multiple mutations collectively contribute to the phenotypic variability of syndromic ID, and emphasizes the importance of correlating clinical phenotype with genomic and cell function analyses.
Asunto(s)
Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Discapacidad Intelectual Ligada al Cromosoma X/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Prurito/genética , Cromosomas Humanos X , Citocinesis , Metilación de ADN , ADN Ribosómico/metabolismo , Exoma , Femenino , Genes Duplicados , Humanos , Intrones , Magnesio/metabolismo , Masculino , Discapacidad Intelectual Ligada al Cromosoma X/metabolismo , Discapacidad Intelectual Ligada al Cromosoma X/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Linaje , Fenotipo , Mutación Puntual , Prurito/patología , Análisis de Secuencia de ADN , Síndrome , Proteína Nuclear Ligada al Cromosoma XRESUMEN
The identification of structural variants (SVs) in genomic data represents an ongoing challenge because of difficulties in reliable SV calling leading to reduced sensitivity and specificity. We prepared high-quality DNA from 9 parent-child trios, who had previously undergone short-read whole-genome sequencing (Illumina platform) as part of the Genomics England 100,000 Genomes Project. We reanalysed the genomes using both Bionano optical genome mapping (OGM; 8 probands and one trio) and Nanopore long-read sequencing (Oxford Nanopore Technologies [ONT] platform; all samples). To establish a "truth" dataset, we asked whether rare proband SV calls (n = 234) made by the Bionano Access (version 1.6.1)/Solve software (version 3.6.1_11162020) could be verified by individual visualisation using the Integrative Genomics Viewer with either or both of the Illumina and ONT raw sequence. Of these, 222 calls were verified, indicating that Bionano OGM calls have high precision (positive predictive value 95%). We then asked what proportion of the 222 true Bionano SVs had been identified by SV callers in the other two datasets. In the Illumina dataset, sensitivity varied according to variant type, being high for deletions (115/134; 86%) but poor for insertions (13/58; 22%). In the ONT dataset, sensitivity was generally poor using the original Sniffles variant caller (48% overall) but improved substantially with use of Sniffles2 (36/40; 90% and 17/23; 74% for deletions and insertions, respectively). In summary, we show that the precision of OGM is very high. In addition, when applying the Sniffles2 caller, the sensitivity of SV calling using ONT long-read sequence data outperforms Illumina sequencing for most SV types.
Asunto(s)
Benchmarking , Secuenciación de Nanoporos , Secuenciación Completa del Genoma , Humanos , Secuenciación Completa del Genoma/métodos , Secuenciación Completa del Genoma/normas , Secuenciación de Nanoporos/métodos , Benchmarking/métodos , Variación Estructural del Genoma/genética , Mapeo Cromosómico/métodos , Genoma Humano/genética , Genómica/métodos , Programas Informáticos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Femenino , Nanoporos , Masculino , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ADN/normasRESUMEN
Although mutations causing monogenic disorders most frequently lie within the affected gene, sequence variation in complex disorders is more commonly found in noncoding regions. Furthermore, recent genome- wide studies have shown that common DNA sequence variants in noncoding regions are associated with "normal" variation in gene expression resulting in cell-specific and/or allele-specific differences. The mechanism by which such sequence variation causes changes in gene expression is largely unknown. We have addressed this by studying natural variation in the binding of key transcription factors (TFs) in the well-defined, purified cell system of erythropoiesis. We have shown that common polymorphisms frequently directly perturb the binding sites of key TFs, and detailed analysis shows how this causes considerable (~10-fold) changes in expression from a single allele in a tissue-specific manner. We also show how a SNP, located at some distance from the recognized TF binding site, may affect the recruitment of a large multiprotein complex and alter the associated chromatin modification of the variant regulatory element. This study illustrates the principles by which common sequence variation may cause changes in tissue-specific gene expression, and suggests that such variation may underlie an individual's propensity to develop complex human genetic diseases.