RESUMEN
Cohesin extrusion is thought to play a central role in establishing the architecture of mammalian genomes. However, extrusion has not been visualized in vivo, and thus, its functional impact and energetics are unknown. Using ultra-deep Hi-C, we show that loop domains form by a process that requires cohesin ATPases. Once formed, however, loops and compartments are maintained for hours without energy input. Strikingly, without ATP, we observe the emergence of hundreds of CTCF-independent loops that link regulatory DNA. We also identify architectural "stripes," where a loop anchor interacts with entire domains at high frequency. Stripes often tether super-enhancers to cognate promoters, and in B cells, they facilitate Igh transcription and recombination. Stripe anchors represent major hotspots for topoisomerase-mediated lesions, which promote chromosomal translocations and cancer. In plasmacytomas, stripes can deregulate Igh-translocated oncogenes. We propose that higher organisms have coopted cohesin extrusion to enhance transcription and recombination, with implications for tumor development.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Genoma , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Línea Celular , Proteoglicanos Tipo Condroitín Sulfato/genética , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Cromosomas/metabolismo , Proteínas de Unión al ADN , Humanos , Ratones , Mutagénesis , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , CohesinasRESUMEN
The RAG1 endonuclease, together with its cofactor RAG2, is essential for V(D)J recombination but is a potent threat to genome stability. The sources of RAG1 mis-targeting and the mechanisms that have evolved to suppress it are poorly understood. Here, we report that RAG1 associates with chromatin at thousands of active promoters and enhancers in the genome of developing lymphocytes. The mouse and human genomes appear to have responded by reducing the abundance of "cryptic" recombination signals near RAG1 binding sites. This depletion operates specifically on the RSS heptamer, whereas nonamers are enriched at RAG1 binding sites. Reversing this RAG-driven depletion of cleavage sites by insertion of strong recombination signals creates an ectopic hub of RAG-mediated V(D)J recombination and chromosomal translocations. Our findings delineate rules governing RAG binding in the genome, identify areas at risk of RAG-mediated damage, and highlight the evolutionary struggle to accommodate programmed DNA damage in developing lymphocytes.
Asunto(s)
Inestabilidad Genómica , Proteínas de Homeodominio/metabolismo , Linfocitos/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Secuencia de Bases , Sitios de Unión , Línea Celular , Proteínas de Unión al ADN/metabolismo , Humanos , Linfocitos/citología , Ratones , Datos de Secuencia Molecular , Translocación Genética , Recombinación V(D)JRESUMEN
The antibody gene mutator activation-induced cytidine deaminase (AID) promiscuously damages oncogenes, leading to chromosomal translocations and tumorigenesis. Why nonimmunoglobulin loci are susceptible to AID activity is unknown. Here, we study AID-mediated lesions in the context of nuclear architecture and the B cell regulome. We show that AID targets are not randomly distributed across the genome but are predominantly grouped within super-enhancers and regulatory clusters. Unexpectedly, in these domains, AID deaminates active promoters and eRNA(+) enhancers interconnected in some instances over megabases of linear chromatin. Using genome editing, we demonstrate that 3D-linked targets cooperate to recruit AID-mediated breaks. Furthermore, a comparison of hypermutation in mouse B cells, AID-induced kataegis in human lymphomas, and translocations in MEFs reveals that AID damages different genes in different cell types. Yet, in all cases, the targets are predominantly associated with topological complex, highly transcribed super-enhancers, demonstrating that these compartments are key mediators of AID recruitment.
Asunto(s)
Linfocitos B/metabolismo , Carcinogénesis , Citidina Desaminasa/genética , Elementos de Facilitación Genéticos , Animales , Daño del ADN , Humanos , Linfoma/metabolismo , RatonesRESUMEN
Lymphocyte activation is initiated by a global increase in messenger RNA synthesis. However, the mechanisms driving transcriptome amplification during the immune response are unknown. By monitoring single-stranded DNA genome wide, we show that the genome of naive cells is poised for rapid activation. In G0, â¼90% of promoters from genes to be expressed in cycling lymphocytes are polymerase loaded but unmelted and support only basal transcription. Furthermore, the transition from abortive to productive elongation is kinetically limiting, causing polymerases to accumulate nearer to transcription start sites. Resting lymphocytes also limit the expression of the transcription factor IIH complex, including XPB and XPD helicases involved in promoter melting and open complex extension. To date, two rate-limiting steps have been shown to control global gene expression in eukaryotes: preinitiation complex assembly and polymerase pausing. Our studies identify promoter melting as a third key regulatory step and propose that this mechanism ensures a prompt lymphocyte response to invading pathogens.
Asunto(s)
Linfocitos B/metabolismo , Regulación de la Expresión Génica , Activación de Linfocitos , Linfocitos/metabolismo , Regiones Promotoras Genéticas , Animales , Linfocitos B/inmunología , Línea Celular Tumoral , ADN de Cadena Simple/metabolismo , Elementos de Facilitación Genéticos , Estudio de Asociación del Genoma Completo , Humanos , Linfocitos/citología , Linfocitos/inmunología , Ratones , Factor de Transcripción TFIIH/metabolismo , Transcripción GenéticaRESUMEN
A key finding of the ENCODE project is that the enhancer landscape of mammalian cells undergoes marked alterations during ontogeny. However, the nature and extent of these changes are unclear. As part of the NIH Mouse Regulome Project, we here combined DNaseI hypersensitivity, ChIP-seq, and ChIA-PET technologies to map the promoter-enhancer interactomes of pluripotent ES cells and differentiated B lymphocytes. We confirm that enhancer usage varies widely across tissues. Unexpectedly, we find that this feature extends to broadly transcribed genes, including Myc and Pim1 cell-cycle regulators, which associate with an entirely different set of enhancers in ES and B cells. By means of high-resolution CpG methylomes, genome editing, and digital footprinting, we show that these enhancers recruit lineage-determining factors. Furthermore, we demonstrate that the turning on and off of enhancers during development correlates with promoter activity. We propose that organisms rely on a dynamic enhancer landscape to control basic cellular functions in a tissue-specific manner.
Asunto(s)
Linfocitos B/metabolismo , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Regiones Promotoras Genéticas , Regulón , Animales , Linaje de la Célula , Células Cultivadas , Islas de CpG , Metilación de ADN , Técnicas Genéticas , Ratones , Especificidad de Órganos , ARN Largo no Codificante/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The c-Myc HLH-bZIP protein has been implicated in physiological or pathological growth, proliferation, apoptosis, metabolism, and differentiation at the cellular, tissue, or organismal levels via regulation of numerous target genes. No principle yet unifies Myc action due partly to an incomplete inventory and functional accounting of Myc's targets. To observe Myc target expression and function in a system where Myc is temporally and physiologically regulated, the transcriptomes and the genome-wide distributions of Myc, RNA polymerase II, and chromatin modifications were compared during lymphocyte activation and in ES cells as well. A remarkably simple rule emerged from this quantitative analysis: Myc is not an on-off specifier of gene activity, but is a nonlinear amplifier of expression, acting universally at active genes, except for immediate early genes that are strongly induced before Myc. This rule of Myc action explains the vast majority of Myc biology observed in literature.
Asunto(s)
Células Madre Embrionarias/metabolismo , Linfocitos/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Activación Transcripcional , Animales , Linfocitos B/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Genoma , Humanos , Ratones , Regiones Promotoras Genéticas , Bazo/citologíaRESUMEN
Chromosomal rearrangements, including translocations, require formation and joining of DNA double strand breaks (DSBs). These events disrupt the integrity of the genome and are frequently involved in producing leukemias, lymphomas and sarcomas. Despite the importance of these events, current understanding of their genesis is limited. To examine the origins of chromosomal rearrangements we developed Translocation Capture Sequencing (TC-Seq), a method to document chromosomal rearrangements genome-wide, in primary cells. We examined over 180,000 rearrangements obtained from 400 million B lymphocytes, revealing that proximity between DSBs, transcriptional activity and chromosome territories are key determinants of genome rearrangement. Specifically, rearrangements tend to occur in cis and to transcribed genes. Finally, we find that activation-induced cytidine deaminase (AID) induces the rearrangement of many genes found as translocation partners in mature B cell lymphoma.
Asunto(s)
Linfocitos B/metabolismo , Genoma , Mutagénesis , Translocación Genética , Animales , Células Cultivadas , Citidina Desaminasa/metabolismo , Genes myc , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Ratones , Neoplasias/genética , Análisis de Secuencia de ADN/métodos , Bazo/citologíaRESUMEN
The critical initial step in V(D)J recombination, binding of RAG1 and RAG2 to recombination signal sequences flanking antigen receptor V, D, and J gene segments, has not previously been characterized in vivo. Here, we demonstrate that RAG protein binding occurs in a highly focal manner to a small region of active chromatin encompassing Ig kappa and Tcr alpha J gene segments and Igh and Tcr beta J and J-proximal D gene segments. Formation of these small RAG-bound regions, which we refer to as recombination centers, occurs in a developmental stage- and lineage-specific manner. Each RAG protein is independently capable of specific binding within recombination centers. While RAG1 binding was detected only at regions containing recombination signal sequences, RAG2 binds at thousands of sites in the genome containing histone 3 trimethylated at lysine 4. We propose that recombination centers coordinate V(D)J recombination by providing discrete sites within which gene segments are captured for recombination.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Reordenamiento Génico de Linfocito B , Reordenamiento Génico de Linfocito T , Proteínas de Homeodominio/metabolismo , Animales , Genes de las Cadenas Pesadas de las Inmunoglobulinas , Genes Codificadores de la Cadena alfa de los Receptores de Linfocito T , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Cadenas kappa de Inmunoglobulina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Recombinación GenéticaRESUMEN
Activation-induced cytidine deaminase (AID) initiates antibody gene diversification by creating U:G mismatches. However, AID is not specific for antibody genes; Off-target lesions can activate oncogenes or cause chromosome translocations. Despite its importance in these transactions little is known about how AID finds its targets. We performed an shRNA screen to identify factors required for class switch recombination (CSR) of antibody loci. We found that Spt5, a factor associated with stalled RNA polymerase II (Pol II) and single stranded DNA (ssDNA), is required for CSR. Spt5 interacts with AID, it facilitates association between AID and Pol II, and AID recruitment to its Ig and non-Ig targets. ChIP-seq experiments reveal that Spt5 colocalizes with AID and stalled Pol II. Further, Spt5 accumulation at sites of Pol II stalling is predictive of AID-induced mutation. We propose that AID is targeted to sites of Pol II stalling in part via its association with Spt5.
Asunto(s)
Linfocitos B/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Citidina Desaminasa/metabolismo , Cambio de Clase de Inmunoglobulina , ARN Polimerasa II/metabolismo , Factores de Elongación Transcripcional/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Fibroblastos/metabolismo , Humanos , Inmunoglobulinas/genética , RatonesRESUMEN
50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.
Asunto(s)
Linfocitos B/metabolismo , Ciclo Celular , Núcleo Celular/metabolismo , Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Histonas/metabolismo , Activación de Linfocitos , Proteínas Proto-Oncogénicas c-myc/metabolismo , Acetilcoenzima A/metabolismo , Acetilación , Adenosina Trifosfato/metabolismo , Animales , Linfocitos B/inmunología , Línea Celular , Cromatina/química , Cromatina/genética , Metilación de ADN , Epigénesis Genética , Genotipo , Histonas/química , Inmunidad Humoral , Metilación , Ratones Endogámicos C57BL , Ratones Noqueados , Conformación de Ácido Nucleico , Fenotipo , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Proto-Oncogénicas c-myc/genética , Imagen Individual de Molécula , Relación Estructura-Actividad , Factores de Tiempo , Transcripción GenéticaRESUMEN
The cytidine deaminase AID hypermutates immunoglobulin genes but can also target oncogenes, leading to tumorigenesis. The extent of AID's promiscuity and its predilection for immunoglobulin genes are unknown. We report here that AID interacted broadly with promoter-proximal sequences associated with stalled polymerases and chromatin-activating marks. In contrast, genomic occupancy of replication protein A (RPA), an AID cofactor, was restricted to immunoglobulin genes. The recruitment of RPA to the immunoglobulin loci was facilitated by phosphorylation of AID at Ser38 and Thr140. We propose that stalled polymerases recruit AID, thereby resulting in low frequencies of hypermutation across the B cell genome. Efficient hypermutation and switch recombination required AID phosphorylation and correlated with recruitment of RPA. Our findings provide a rationale for the oncogenic role of AID in B cell malignancy.
Asunto(s)
Linfocitos B/metabolismo , Transformación Celular Neoplásica , Citidina Desaminasa/metabolismo , Genes de Inmunoglobulinas , Proteína de Replicación A/metabolismo , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/inmunología , Células Cultivadas , Ensamble y Desensamble de Cromatina/genética , Citidina Desaminasa/genética , Genes de Inmunoglobulinas/genética , Genes myc/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Cambio de Clase de Inmunoglobulina , Interleucina-4/inmunología , Interleucina-4/metabolismo , Lipopolisacáridos/inmunología , Lipopolisacáridos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Regiones Promotoras Genéticas/genética , Proteína de Replicación A/genética , Hipermutación Somática de InmunoglobulinaRESUMEN
Deficiencies in factors that regulate the DNA damage response enhance the incidence of malignancy by destabilizing the genome. However, the precise influence of the DNA damage response on regulation of cancer-associated rearrangements is not well defined. Here we examine the genome-wide impact of tumor protein P53-binding protein 1 (53BP1) deficiency in lymphoma and translocation. While both activation-induced cytidine deaminase (AID) and 53BP1 have been associated with cancer in humans, neither AID overexpression nor loss of 53BP1 is sufficient to produce malignancy. However, the combination of 53BP1 deficiency and AID deregulation results in B cell lymphoma. Deep sequencing of the genome of 53BP1(-/-) cancer cells and translocation capture sequencing (TC-Seq) of primary 53BP1(-/-) B cells revealed that their chromosomal rearrangements differ from those found in wild-type cells in that they show increased DNA end resection. Moreover, loss of 53BP1 alters the translocatome by increasing rearrangements to intergenic regions.
Asunto(s)
Transformación Celular Neoplásica/genética , Proteínas Cromosómicas no Histona/fisiología , Citidina Desaminasa/fisiología , Proteínas de Unión al ADN/fisiología , Reordenamiento Génico , Linfoma de Células B/metabolismo , Animales , Células Cultivadas , Proteínas Cromosómicas no Histona/deficiencia , Proteínas Cromosómicas no Histona/genética , Cromosomas de los Mamíferos/genética , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Epigénesis Genética , Genes Supresores de Tumor , Estudio de Asociación del Genoma Completo , Linfoma de Células B/genética , Ratones , Ratones Noqueados , Mutación , Análisis de Secuencia de ADN , Transcripción Genética , Translocación Genética , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Although the cellular concentration of miRNAs is critical to their function, how miRNA expression and abundance are regulated during ontogeny is unclear. We applied miRNA-, mRNA-, and ChIP-Seq to characterize the microRNome during lymphopoiesis within the context of the transcriptome and epigenome. We show that lymphocyte-specific miRNAs are either tightly controlled by polycomb group-mediated H3K27me3 or maintained in a semi-activated epigenetic state prior to full expression. Because of miRNA biogenesis, the cellular concentration of mature miRNAs does not typically reflect transcriptional changes. However, we uncover a subset of miRNAs for which abundance is dictated by miRNA gene expression. We confirm that concentration of 5p and 3p miRNA strands depends largely on free energy properties of miRNA duplexes. Unexpectedly, we also find that miRNA strand accumulation can be developmentally regulated. Our data provide a comprehensive map of immunity's microRNome and reveal the underlying epigenetic and transcriptional forces that shape miRNA homeostasis.
Asunto(s)
Epigénesis Genética , Regulación de la Expresión Génica/genética , Linfocitos , Linfopoyesis/genética , MicroARNs/genética , Animales , Expresión Génica , Humanos , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Recurrent chromosomal translocations underlie both haematopoietic and solid tumours. Their origin has been ascribed to selection of random rearrangements, targeted DNA damage, or frequent nuclear interactions between translocation partners; however, the relative contribution of each of these elements has not been measured directly or on a large scale. Here we examine the role of nuclear architecture and frequency of DNA damage in the genesis of chromosomal translocations by measuring these parameters simultaneously in cultured mouse B lymphocytes. In the absence of recurrent DNA damage, translocations between Igh or Myc and all other genes are directly related to their contact frequency. Conversely, translocations associated with recurrent site-directed DNA damage are proportional to the rate of DNA break formation, as measured by replication protein A accumulation at the site of damage. Thus, non-targeted rearrangements reflect nuclear organization whereas DNA break formation governs the location and frequency of recurrent translocations, including those driving B-cell malignancies.
Asunto(s)
Linfocitos B/metabolismo , Linfocitos B/patología , Daño del ADN/genética , Translocación Genética/genética , Animales , Linfocitos B/citología , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Posicionamiento de Cromosoma , Cromosomas de los Mamíferos/genética , Cromosomas de los Mamíferos/metabolismo , Citidina Desaminasa/deficiencia , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Roturas del ADN de Doble Cadena , Genes myc/genética , Genoma/genética , Cadenas Pesadas de Inmunoglobulina/genética , Ratones , Proteína de Replicación A/metabolismoRESUMEN
Mutations of STAT3 underlie the autosomal dominant form of hyperimmunoglobulin E syndrome (HIES). STAT3 has critical roles in immune cells and thus, hematopoietic stem cell transplantation (HSCT), might be a reasonable therapeutic strategy in this disease. However, STAT3 also has critical functions in nonhematopoietic cells and dissecting the protean roles of STAT3 is limited by the lethality associated with germline deletion of Stat3. Thus, predicting the efficacy of HSCT for HIES is difficult. To begin to dissect the importance of STAT3 in hematopoietic and nonhematopoietic cells as it relates to HIES, we generated a mouse model of this disease. We found that these transgenic mice recapitulate multiple aspects of HIES, including elevated serum IgE and failure to generate Th17 cells. We found that these mice were susceptible to bacterial infection that was partially corrected by HSCT using wild-type bone marrow, emphasizing the role played by the epithelium in the pathophysiology of HIES.
Asunto(s)
Modelos Animales de Enfermedad , Síndrome de Job/inmunología , Mutación/inmunología , Factor de Transcripción STAT3/inmunología , Animales , Trasplante de Médula Ósea , Células Cultivadas , Citrobacter rodentium/inmunología , Citrobacter rodentium/fisiología , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Infecciones por Enterobacteriaceae/genética , Infecciones por Enterobacteriaceae/inmunología , Infecciones por Enterobacteriaceae/microbiología , Citometría de Flujo , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Síndrome de Job/genética , Síndrome de Job/cirugía , Lipopolisacáridos , Ratones , Ratones Transgénicos , Mutación/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/genética , Choque Séptico/inducido químicamente , Choque Séptico/genética , Choque Séptico/inmunología , Análisis de Supervivencia , Transcriptoma/genética , Transcriptoma/inmunologíaRESUMEN
Human Burkitt lymphomas are divided into two main clinical variants: the endemic form, affecting African children infected with malaria and the Epstein-Barr virus, and the sporadic form, distributed across the rest of the world. However, whereas sporadic translocations decapitate Myc from 5' proximal regulatory elements, most endemic events occur hundreds of kilobases away from Myc. The origin of these rearrangements and how they deregulate oncogenes at such distances remain unclear. We here recapitulate endemic Burkitt lymphoma-like translocations in plasmacytomas from uracil N-glycosylase and activation-induced cytidine deaminase-deficient mice. Mapping of translocation breakpoints using an acetylated histone H3 lysine 9 chromatin immunoprecipitation sequencing approach reveals Igh fusions up to â¼350 kb upstream of Myc or the related oncogene Mycn. A comprehensive analysis of epigenetic marks, PolII recruitment, and transcription in tumor cells demonstrates that the 3' Igh enhancer (Eα) vastly remodels â¼450 kb of chromatin into translocated sequences, leading to significant polymerase occupancy and constitutive oncogene expression. We show that this long-range epigenetic reprogramming is directly proportional to the physical interaction of Eα with translocated sites. Our studies thus uncover the extent of epigenetic remodeling by Ig 3' enhancers and provide a rationale for the long-range deregulation of translocated oncogenes in endemic Burkitt lymphomas. The data also shed light on the origin of endemic-like chromosomal rearrangements.
Asunto(s)
Linfoma de Burkitt/genética , Regulación Neoplásica de la Expresión Génica/genética , Genes de las Cadenas Pesadas de las Inmunoglobulinas/genética , Genes myc/genética , Cambio de Clase de Inmunoglobulina/genética , Translocación Genética/genética , Animales , Linfocitos B/citología , Linfocitos B/fisiología , Linfoma de Burkitt/epidemiología , Células Cultivadas , Citidina/genética , Modelos Animales de Enfermedad , Enfermedades Endémicas , Elementos de Facilitación Genéticos/genética , Epigénesis Genética/genética , Reordenamiento Génico de Linfocito B/genética , Humanos , Ratones , Proteínas de Fusión Oncogénica/genética , Células Plasmáticas/citología , Células Plasmáticas/fisiología , Sitio de Iniciación de la Transcripción/fisiología , Uracil-ADN Glicosidasa/genéticaRESUMEN
Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder characterized by cutaneous fibrofolliculomas, pulmonary cysts, and kidney malignancies. Affected individuals carry germ line mutations in folliculin (FLCN), a tumor suppressor gene that becomes biallelically inactivated in kidney tumors by second-hit mutations. Similar to other factors implicated in kidney cancer, FLCN has been shown to modulate activation of mammalian target of rapamycin (mTOR). However, its precise in vivo function is largely unknown because germ line deletion of Flcn results in early embryonic lethality in animal models. Here, we describe mice deficient in the newly characterized folliculin-interacting protein 1 (Fnip1). In contrast to Flcn, Fnip1(-/-) mice develop normally, are not susceptible to kidney neoplasia, but display a striking pro-B cell block that is entirely independent of mTOR activity. We show that this developmental arrest results from rapid caspase-induced pre-B cell death, and that a Bcl2 transgene reconstitutes mature B-cell populations, respectively. We also demonstrate that conditional deletion of Flcn recapitulates the pro-B cell arrest of Fnip1(-/-) mice. Our studies thus demonstrate that the FLCN-FNIP complex deregulated in BHD syndrome is absolutely required for B-cell differentiation, and that it functions through both mTOR-dependent and independent pathways.
Asunto(s)
Linfocitos B/fisiología , Síndrome de Birt-Hogg-Dubé/genética , Proteínas Portadoras/genética , Diferenciación Celular/genética , Eliminación de Gen , Proteínas Proto-Oncogénicas/genética , Proteínas Supresoras de Tumor/genética , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Diferenciación Celular/inmunología , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Especificidad de la Especie , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
Viruses with large, double-stranded DNA genomes captured the majority of their genes from their hosts at different stages of evolution. The origins of many virus genes are readily detected through significant sequence similarity with cellular homologs. In particular, this is the case for virus enzymes, such as DNA and RNA polymerases or nucleotide kinases, that retain their catalytic activity after capture by an ancestral virus. However, a large fraction of virus genes have no readily detectable cellular homologs, meaning that their origins remain enigmatic. We explored the potential origins of such proteins that are encoded in the genomes of orthopoxviruses, a thoroughly studied virus genus that includes major human pathogens. To this end, we used AlphaFold2 to predict the structures of all 214 proteins that are encoded by orthopoxviruses. Among the proteins of unknown provenance, structure prediction yielded clear indications of origin for 14 of them and validated several inferences that were previously made via sequence analysis. A notable emerging trend is the exaptation of enzymes from cellular organisms for nonenzymatic, structural roles in virus reproduction that is accompanied by the disruption of catalytic sites and by an overall drastic divergence that precludes homology detection at the sequence level. Among the 16 orthopoxvirus proteins that were found to be inactivated enzyme derivatives are the poxvirus replication processivity factor A20, which is an inactivated NAD-dependent DNA ligase; the major core protein A3, which is an inactivated deubiquitinase; F11, which is an inactivated prolyl hydroxylase; and more similar cases. For nearly one-third of the orthopoxvirus virion proteins, no significantly similar structures were identified, suggesting exaptation with subsequent major structural rearrangement that yielded unique protein folds. IMPORTANCE Protein structures are more strongly conserved in evolution than are amino acid sequences. Comparative structural analysis is particularly important for inferring the origins of viral proteins that typically evolve at high rates. We used a powerful protein structure modeling method, namely, AlphaFold2, to model the structures of all orthopoxvirus proteins and compared them to all available protein structures. Multiple cases of recruitment of host enzymes for structural roles in viruses, accompanied by the disruption of catalytic sites, were discovered. However, many viral proteins appear to have evolved unique structural folds.