Asunto(s)
Biomarcadores de Tumor/genética , Metilación de ADN , Epigénesis Genética , Regulación Leucémica de la Expresión Génica , Leucemia Mielomonocítica Juvenil/diagnóstico , Mutación , Tamizaje Neonatal/métodos , Estudios de Casos y Controles , Femenino , Estudios de Seguimiento , Humanos , Recién Nacido , Leucemia Mielomonocítica Juvenil/genética , Masculino , PronósticoRESUMEN
Eukaryotes have evolved elaborate mechanisms to ensure that chromosomes segregate with high fidelity during mitosis and meiosis, and yet specific aneuploidies can be adaptive during environmental stress. Here, we identify a chromatin-based system required for inducible aneuploidy in a human pathogen. Candida albicans utilizes chromosome missegregation to acquire tolerance to antifungal drugs and for nonmeiotic ploidy reduction after mating. We discovered that the ancestor of C. albicans and 2 related pathogens evolved a variant of histone 2A (H2A) that lacks the conserved phosphorylation site for kinetochore-associated Bub1 kinase, a key regulator of chromosome segregation. Using engineered strains, we show that the relative gene dosage of this variant versus canonical H2A controls the fidelity of chromosome segregation and the rate of acquisition of tolerance to antifungal drugs via aneuploidy. Furthermore, whole-genome chromatin precipitation analysis reveals that Centromere Protein A/ Centromeric Histone H3-like Protein (CENP-A/Cse4), a centromeric histone H3 variant that forms the platform of the eukaryotic kinetochore, is depleted from tetraploid-mating products relative to diploid parents and is virtually eliminated from cells exposed to aneuploidy-promoting cues. We conclude that genetically programmed and environmentally induced changes in chromatin can confer the capacity for enhanced evolvability via chromosome missegregation.
Asunto(s)
Proteína A Centromérica/metabolismo , Segregación Cromosómica/fisiología , Histonas/metabolismo , Aneugénicos/metabolismo , Aneuploidia , Candida albicans/genética , Candida albicans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Proteína A Centromérica/fisiología , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Evolución Molecular , Histonas/fisiología , Cinetocoros/metabolismo , Meiosis , Mitosis , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
In Schizosaccharomyces pombe, transcripts derived from the pericentromeric dg and dh repeats promote heterochromatin formation via RNAi as well as an RNAi-independent mechanism involving the RNA polymerase II (RNAPII)-associated RNA-binding protein Seb1 and RNA processing activities. We show that Seb1 promotes long-lived RNAPII pauses at pericentromeric repeat regions and that their presence correlates with the heterochromatin-triggering activities of the corresponding dg and dh DNA fragments. Globally increasing RNAPII stalling by other means induces the formation of novel large ectopic heterochromatin domains. Such ectopic heterochromatin occurs even in cells lacking RNAi. These results uncover Seb1-mediated polymerase stalling as a signal necessary for heterochromatin nucleation.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Heterocromatina , Proteínas Nucleares/metabolismo , ARN Polimerasa II/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces , Heterocromatina/genética , Heterocromatina/metabolismo , Repeticiones de Microsatélite/genética , Proteínas Nucleares/genética , Interferencia de ARN , ARN Polimerasa II/metabolismo , Proteínas de Unión al ARN/genética , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
Eukaryotic gene expression requires that RNA Polymerase II (RNAP II) gain access to DNA in the context of chromatin. The C-terminal domain (CTD) of RNAP II recruits chromatin modifying enzymes to promoters, allowing for transcription initiation or repression. Specific CTD phosphorylation marks facilitate recruitment of chromatin modifiers, transcriptional regulators, and RNA processing factors during the transcription cycle. However, the readable code for recruiting such factors is still not fully defined and how CTD modifications affect related families of genes or regional gene expression is not well understood. Here, we examine the effects of manipulating the Y1S2P3T4S5P6S7 heptapeptide repeat of the CTD of RNAP II in Schizosaccharomyces pombe by substituting non-phosphorylatable alanines for Ser2 and/or Ser7 and the phosphomimetic glutamic acid for Ser7. Global gene expression analyses were conducted using splicing-sensitive microarrays and validated via RT-qPCR. The CTD mutations did not affect pre-mRNA splicing or snRNA levels. Rather, the data revealed upregulation of subtelomeric genes and alteration of the repressive histone H3 lysine 9 methylation (H3K9me) landscape. The data further indicate that H3K9me and expression status are not fully correlated, suggestive of CTD-dependent subtelomeric repression mechansims that act independently of H3K9me levels.
Asunto(s)
Cromatina/genética , Cromatina/metabolismo , Regulación Fúngica de la Expresión Génica , Mutación , Dominios y Motivos de Interacción de Proteínas , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Análisis por Conglomerados , Perfilación de la Expresión Génica , Genes Fúngicos , Histonas , Metilación , Fosforilación , Unión Proteica , ARN Polimerasa II/química , Empalme del ARN , ARN Nuclear Pequeño/metabolismo , Reproducibilidad de los Resultados , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Empalmosomas/metabolismoRESUMEN
Antibody diversification necessitates targeted mutation of regions within the immunoglobulin locus by activation-induced cytidine deaminase (AID). While AID is known to act on single-stranded DNA (ssDNA), the source, structure, and distribution of these substrates in vivo remain unclear. Using the technique of in situ bisulfite treatment, we characterized these substrates-which we found to be unique to actively transcribed genes-as short ssDNA regions, that are equally distributed on both DNA strands. We found that the frequencies of these ssDNA patches act as accurate predictors of AID activity at reporter genes in hypermutating and class switching B cells as well as in Escherichia coli. Importantly, these ssDNA patches rely on transcription, and we report that transcription-induced negative supercoiling enhances both ssDNA tract formation and AID mutagenesis. In addition, RNaseH1 expression does not impact the formation of these ssDNA tracts indicating that these structures are distinct from R-loops. These data emphasize the notion that these transcription-generated ssDNA tracts are one of many in vivo substrates for AID.
Asunto(s)
Linfocitos B/citología , Linfocitos B/metabolismo , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , ADN de Cadena Simple/genética , ADN/genética , Cambio de Clase de Inmunoglobulina/genética , Animales , Núcleo Celular/genética , Citidina/genética , Citidina/metabolismo , ADN/química , ADN de Cadena Simple/metabolismo , Desaminación , Escherichia coli/genética , Humanos , Región Variable de Inmunoglobulina/genética , Ratones , Ribonucleasa H/genética , Ribonucleasa H/metabolismo , Hipermutación Somática de Inmunoglobulina/genética , Especificidad por Sustrato , Sulfitos/química , Transcripción GenéticaRESUMEN
The germinal center (GC) is a transient lymphoid tissue microenvironment that fosters T cell-dependent humoral immunity. Within the GC, the B cell-specific enzyme, activation-induced cytidine deaminase (AID), mutates the immunoglobulin locus, thereby altering binding affinity for antigen. In the absence of AID, larger GC structures are observed in both humans and mice, but the reason for this phenomenon is unclear. Because significant apoptosis occurs within the GC niche to cull cells that have acquired nonproductive mutations, we have examined whether a defect in apoptosis could account for the larger GC structures in the absence of AID. In this report, we reveal significantly reduced death of B cells in AID(-/-) mice as well as in B cells derived from AID(-/-) bone marrow in mixed bone marrow chimeric mice. Furthermore, AID-expressing B cells show decreased proliferation and survival compared with AID(-/-) B cells, indicating an AID-mediated effect on cellular viability. The GC is an etiologic site for B-cell autoimmunity and lymphomagenesis, both of which have been linked to aberrant AID activity. We report a link between AID-induced DNA damage and B-cell apoptosis that has implications for the development of B-cell disorders.
Asunto(s)
Apoptosis , Linfocitos B/citología , Citidina Desaminasa/fisiología , Centro Germinal/citología , Animales , Autoinmunidad/genética , Médula Ósea , Proliferación Celular , Supervivencia Celular , Citidina Desaminasa/deficiencia , Citidina Desaminasa/genética , Daño del ADN , Linfoma de Células B/etiología , Linfoma de Células B/genética , Ratones , Ratones NoqueadosRESUMEN
Activation-induced cytidine deaminase (AID) produces DNA breaks in immunoglobulin genes during antibody diversification. Double-stranded breaks (DSB) in the switch region mediate class switch recombination, and contribute to gene conversion and somatic hypermutation in the variable regions. However, the relative extent to which AID induces DSB in these regions or between these and other actively expressed sequences is unknown. Here, we exploited an enhancer-trap plasmid that identifies DSB in actively expressed loci to investigate the frequency and position of AID-induced vector integration events in mouse hybridoma cells. Compared to control cells, wild-type AID stimulates plasmid integration into the genome by as much as 29-fold. Southern and digestion-circularization PCR analysis revealed non-uniformity in the integration sites, with biases of 30- and 116-fold for the immunoglobulin kappa light chain and mu heavy chain genes, respectively. Further, within the immunoglobulin mu gene, 73% of vector integrations map to the mu switch region, an enhancement of five- and 12-fold compared to the adjacent heavy chain variable and mu gene constant regions, respectively. Thus, among potential highly transcribed genes in mouse hybridoma cells, the immunoglobulin heavy and light chain genes are important AID targets, with the immunoglobulin mu switch region being preferred compared to other genomic sites.
Asunto(s)
Citidina Desaminasa/fisiología , Roturas del ADN de Doble Cadena , Reparación del ADN , Región de Cambio de la Inmunoglobulina , Cadenas mu de Inmunoglobulina/genética , Animales , Secuencia de Bases , Citidina Desaminasa/genética , ADN Recombinante/metabolismo , Elementos de Facilitación Genéticos , Hibridomas/enzimología , Cadenas mu de Inmunoglobulina/metabolismo , Ratones , Datos de Secuencia Molecular , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa , Hipermutación Somática de Inmunoglobulina , Especificidad por SustratoRESUMEN
It is unknown how activation-induced cytidine deaminase (AID) targets immunoglobulin (Ig) genes during somatic hypermutation. Results to date are difficult to interpret: while some results argue that Ig genes have special sequences that mobilize AID, other work shows that non-Ig transgenes mutate. In this report, we have examined the effects of the intronic mu enhancer on the somatic hypermutation rates of a retroviral vector. For this analysis, we used centroblast-like Ramos cells to capture as much of the natural process as possible, used AIDhi and AIDlow Ramos variants to ensure that mutations are AID induced, and measured mutation of a GFP-provirus to achieve greater sensitivity. We found that mutation rates of the non-Ig provirus were AID-dependent, were similar at different genomic loci, but were approximately 10-fold lower than the V-region suggesting that AID can mutate non-Ig genes at low rates. However, the intronic mu enhancer did not increase the mutation rates of the provirus. Interestingly, exogenous over-expression of AID revealed that the V-region mutation rate can be saturated by lower levels of AID than the provirus, suggesting that selective mutation of Ig sequences is compromised in cells that over-express AID.
Asunto(s)
Citidina Desaminasa/genética , Genes de Inmunoglobulinas , Región Variable de Inmunoglobulina/genética , Hipermutación Somática de Inmunoglobulina , Animales , Línea Celular Tumoral , Cromosomas Humanos , Citidina Desaminasa/metabolismo , Elementos de Facilitación Genéticos , Vectores Genéticos , Proteínas Fluorescentes Verdes , Humanos , Ratones , Provirus , Retroviridae , TransgenesRESUMEN
Antibody diversification processes play a major role in protecting humans from pathogens. Somatic hypermutation and gene conversion increase the affinity of pathogen-specific antibodies by changing the sequence within antibody variable genes, while the class switch recombination (CSR) process changes the antibody's effector function by replacing the constant region of the antibody gene with a different constant region. Activation-induced cytidine deaminase (AID) initiates each of these three processes by deaminating cytidines within antibody genes, while a host of other DNA transacting factors are involved in either creating new mutations or repairing DNA lesions introduced during these processes. This review will discuss the main features of antibody diversification and their role in lymphomagenesis, highlight outstanding issues and questions that remain in the field, and discuss our contributions to this field.