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1.
J Biol Chem ; 295(27): 9052-9060, 2020 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-32414844

RESUMEN

V(D)J recombination is initiated by the recombination-activating gene protein (RAG) recombinase, consisting of RAG-1 and RAG-2 subunits. The susceptibility of gene segments to cleavage by RAG is associated with gene transcription and with epigenetic marks characteristic of active chromatin, including histone H3 trimethylated at lysine 4 (H3K4me3). Binding of H3K4me3 by a plant homeodomain (PHD) in RAG-2 induces conformational changes in RAG-1, allosterically stimulating substrate binding and catalysis. To better understand the path of allostery from the RAG-2 PHD finger to RAG-1, here we employed phylogenetic substitution. We observed that a chimeric RAG-2 protein in which the mouse PHD finger is replaced by the corresponding domain from the shark Chiloscyllium punctatum binds H3K4me3 but fails to transmit an allosteric signal, indicating that binding of H3K4me3 by RAG-2 is insufficient to support recombination. By substituting residues in the C. punctatum PHD with the corresponding residues in the mouse PHD and testing for rescue of allostery, we demonstrate that H3K4me3 binding and transmission of an allosteric signal to RAG-1 are separable functions of the RAG-2 PHD finger.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Regulación Alostérica/genética , Regulación Alostérica/fisiología , Animales , Sitios de Unión , Cromatina/metabolismo , Histonas/fisiología , Lisina/metabolismo , Metilación , Ratones , Filogenia , Unión Proteica , Recombinasas/metabolismo , Tiburones/metabolismo , Especificidad por Sustrato , Recombinación V(D)J/genética , Recombinación V(D)J/fisiología , VDJ Recombinasas/metabolismo
2.
Nat Med ; 26(2): 236-243, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31959990

RESUMEN

Drug-induced hypersensitivity syndrome/drug reaction with eosinophilia and systemic symptoms (DiHS/DRESS) is a potentially fatal multiorgan inflammatory disease associated with herpesvirus reactivation and subsequent onset of autoimmune diseases1-4. Pathophysiology remains elusive and therapeutic options are limited. Cases refractory to corticosteroid therapy pose a clinical challenge1,5 and approximately 30% of patients with DiHS/DRESS develop complications, including infections and inflammatory and autoimmune diseases1,2,5. Progress in single-cell RNA sequencing (scRNA-seq) provides an opportunity to dissect human disease pathophysiology at unprecedented resolutions6, particularly in diseases lacking animal models, such as DiHS/DRESS. We performed scRNA-seq on skin and blood from a patient with refractory DiHS/DRESS, identifying the JAK-STAT signaling pathway as a potential target. We further showed that central memory CD4+ T cells were enriched with DNA from human herpesvirus 6b. Intervention via tofacitinib enabled disease control and tapering of other immunosuppressive agents. Tofacitinib, as well as antiviral agents, suppressed culprit-induced T cell proliferation in vitro, further supporting the roles of the JAK-STAT pathway and herpesviruses in mediating the adverse drug reaction. Thus, scRNA-seq analyses guided successful therapeutic intervention in the patient with refractory DiHS/DRESS. scRNA-seq may improve our understanding of complicated human disease pathophysiology and provide an alternative approach in personalized medicine.


Asunto(s)
Síndrome de Hipersensibilidad a Medicamentos/terapia , Análisis de la Célula Individual , Transcriptoma , Corticoesteroides/uso terapéutico , Adulto , Antivirales/uso terapéutico , Enfermedades Autoinmunes/complicaciones , Linfocitos T CD4-Positivos/citología , Proliferación Celular , Separación Celular , Citometría de Flujo , Herpesvirus Humano 6/inmunología , Humanos , Inmunosupresores/uso terapéutico , Leucocitos Mononucleares/citología , Linfocitos/citología , Masculino , Piperidinas/uso terapéutico , Pirimidinas/uso terapéutico , Pirroles/uso terapéutico , RNA-Seq , Transducción de Señal , Linfocitos T Reguladores/citología , VDJ Recombinasas/metabolismo
3.
Mol Cell Biol ; 38(15)2018 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-29760281

RESUMEN

Accessibility of antigen receptor loci to RAG is correlated with the presence of H3K4me3, which binds to a plant homeodomain (PHD) in the RAG-2 subunit and promotes V(D)J recombination. A point mutation in the PHD, W453A, eliminates binding of H3K4me3 and impairs recombination. The debilitating effect of the W453A mutation is ameliorated by second-site mutations that locate an inhibitory domain in the interval from residues 352 through 405 of RAG-2. Disruption of the inhibitory domain stimulates V(D)J recombination within extrachromosomal substrates and at endogenous antigen receptor loci. Association of RAG-1 and RAG-2 with chromatin at the IgH locus in B cell progenitors is dependent on recognition of H3K4me3 by the PHD. Strikingly, disruption of the inhibitory domain permits association of RAG with the IgH locus in the absence of H3K4me3 binding. Thus, the inhibitory domain acts as a gate that prohibits RAG from accessing the IgH locus unless RAG-2 is engaged by H3K4me3.


Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , VDJ Recombinasas/metabolismo , Inmunidad Adaptativa , Regulación Alostérica , Sustitución de Aminoácidos , Animales , Línea Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Reordenamiento Génico de Cadena Pesada de Linfocito B , Genes de las Cadenas Pesadas de las Inmunoglobulinas , Células HEK293 , Código de Histonas , Humanos , Ratones , Modelos Inmunológicos , Células 3T3 NIH , Mutación Puntual , Células Precursoras de Linfocitos B/inmunología , Células Precursoras de Linfocitos B/metabolismo , Dominios Proteicos
4.
Trends Biochem Sci ; 42(1): 72-84, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27825771

RESUMEN

Development of the adaptive immune system is dependent on V(D)J recombination, which forms functional antigen receptor genes through rearrangement of component gene segments. The V(D)J recombinase, comprising recombination-activating proteins RAG1 and RAG2, guides the initial DNA cleavage events to the recombination signal sequence (RSS), which flanks each gene segment. Although the enzymatic steps for RAG-mediated endonucleolytic activity were established over two decades ago, only recently have high-resolution structural studies of the catalytically active core regions of the RAG proteins shed light on conformational requirements for the reaction. While outstanding questions remain, we have a clearer picture of how RAG proteins function in generating the diverse repertoires of antigen receptors, the underlying foundation of the adaptive immune system.


Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , VDJ Recombinasas/química , VDJ Recombinasas/metabolismo , Animales , Roturas del ADN de Doble Cadena , División del ADN , Conformación Proteica
5.
Genes Dev ; 30(8): 873-5, 2016 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-27083993

RESUMEN

Generation of a diverse repertoire of antigen receptor specificities via DNA recombination underpins adaptive immunity. In this issue ofGenes&Development, Carmona and colleagues (pp. 909-917) provide novel insights into the origin and function of recombination-activating gene 1 (RAG1) and RAG2, the lymphocyte-specific components of the recombinase involved in the process.


Asunto(s)
Inmunidad Adaptativa/fisiología , Inmunidad Adaptativa/genética , Inmunidad Adaptativa/inmunología , Animales , Proteínas de Unión al ADN/inmunología , Proteínas de Homeodominio/inmunología , Humanos , VDJ Recombinasas/genética , VDJ Recombinasas/metabolismo
6.
J Biol Chem ; 290(19): 11802-17, 2015 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-25745109

RESUMEN

The RAG endonuclease consists of RAG1, which contains the active site for DNA cleavage, and RAG2, an accessory factor whose interaction with RAG1 is critical for catalytic function. How RAG2 activates RAG1 is not understood. Here, we used biolayer interferometry and pulldown assays to identify regions of RAG1 necessary for interaction with RAG2 and to measure the RAG1-RAG2 binding affinity (KD ∼0.4 µM) (where RAG1 and RAG2 are recombination activating genes 1 or 2). Using the Hermes transposase as a guide, we constructed a 36-kDa "mini" RAG1 capable of interacting robustly with RAG2. Mini-RAG1 consists primarily of the catalytic center and the residues N-terminal to it, but it lacks a zinc finger region in RAG1 previously implicated in binding RAG2. The ability of Mini-RAG1 to interact with RAG2 depends on a predicted α-helix (amino acids 997-1008) near the RAG1 C terminus and a region of RAG1 from amino acids 479 to 559. Two adjacent acidic amino acids in this region (Asp-546 and Glu-547) are important for both the RAG1-RAG2 interaction and recombination activity, with Asp-546 of particular importance. Structural modeling of Mini-RAG1 suggests that Asp-546/Glu-547 lie near the predicted 997-1008 α-helix and components of the active site, raising the possibility that RAG2 binding alters the structure of the RAG1 active site. Quantitative Western blotting allowed us to estimate that mouse thymocytes contain on average ∼1,800 monomers of RAG1 and ∼15,000 molecules of RAG2, implying that nuclear concentrations of RAG1 and RAG2 are below the KD value for their interaction, which could help limit off-target RAG activity.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas Nucleares/metabolismo , Recombinación V(D)J , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Genoma Humano , Células HEK293 , Humanos , Interferometría , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutación , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Secundaria de Proteína , Timo/citología , VDJ Recombinasas/metabolismo
7.
Nature ; 518(7540): 507-11, 2015 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-25707801

RESUMEN

V(D)J recombination in the vertebrate immune system generates a highly diverse population of immunoglobulins and T-cell receptors by combinatorial joining of segments of coding DNA. The RAG1-RAG2 protein complex initiates this site-specific recombination by cutting DNA at specific sites flanking the coding segments. Here we report the crystal structure of the mouse RAG1-RAG2 complex at 3.2 Å resolution. The 230-kilodalton RAG1-RAG2 heterotetramer is 'Y-shaped', with the amino-terminal domains of the two RAG1 chains forming an intertwined stalk. Each RAG1-RAG2 heterodimer composes one arm of the 'Y', with the active site in the middle and RAG2 at its tip. The RAG1-RAG2 structure rationalizes more than 60 mutations identified in immunodeficient patients, as well as a large body of genetic and biochemical data. The architectural similarity between RAG1 and the hairpin-forming transposases Hermes and Tn5 suggests the evolutionary conservation of these DNA rearrangements.


Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Homeodominio/química , VDJ Recombinasas/química , Animales , Sitios de Unión , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Modelos Moleculares , Mutación/genética , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Inmunodeficiencia Combinada Grave/genética , Transposasas/química , VDJ Recombinasas/metabolismo , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/genética
8.
DNA Repair (Amst) ; 27: 19-27, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25625798

RESUMEN

V(D)J recombination of lymphocyte antigen receptor genes occurs via the formation of DNA double strand breaks (DSBs) through the activity of RAG1 and RAG2. The co-existence of RAG-independent DNA DSBs generated by genotoxic stressors potentially increases the risk of incorrect repair and chromosomal abnormalities. However, it is not known whether cellular responses to DSBs by genotoxic stressors affect the RAG complex. Using cellular imaging and subcellular fractionation approaches, we show that formation of DSBs by treating cells with DNA damaging agents causes export of nuclear RAG2. Within the cytoplasm, RAG2 exhibited substantial enrichment at the centrosome. Further, RAG2 export was sensitive to inhibition of ATM, and was reversed following DNA repair. The core region of RAG2 was sufficient for export, but not centrosome targeting, and RAG2 export was blocked by mutation of Thr(490). In summary, DNA damage triggers relocalization of RAG2 from the nucleus to centrosomes, suggesting a novel mechanism for modulating cellular responses to DSBs in developing lymphocytes.


Asunto(s)
Núcleo Celular/metabolismo , Centrosoma/metabolismo , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas Nucleares/metabolismo , Células Precursoras de Linfocitos B/metabolismo , Transporte Activo de Núcleo Celular , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/fisiología , Células Cultivadas , ADN/efectos de los fármacos , ADN/efectos de la radiación , Reparación del ADN , Proteínas de Unión al ADN/genética , Técnicas de Silenciamiento del Gen , Humanos , Microscopía Fluorescente , Mutación , Proteínas Nucleares/genética , Radiación Ionizante , Fracciones Subcelulares/metabolismo , VDJ Recombinasas/genética , VDJ Recombinasas/metabolismo
9.
J Exp Med ; 212(1): 107-20, 2015 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-25512470

RESUMEN

Gene regulation relies on dynamic changes in three-dimensional chromatin conformation, which are shaped by composite regulatory and architectural elements. However, mechanisms that govern such conformational switches within chromosomal domains remain unknown. We identify a novel mechanism by which cis-elements promote long-range interactions, inducing conformational changes critical for diversification of the TCRß antigen receptor locus (Tcrb). Association between distal Vß gene segments and the highly expressed DßJß clusters, termed the recombination center (RC), is independent of enhancer function and recruitment of V(D)J recombinase. Instead, we find that tissue-specific folding of Tcrb relies on two distinct architectural elements located upstream of the RC. The first, a CTCF-containing element, directly tethers distal portions of the Vß array to the RC. The second element is a chromatin barrier that protects the tether from hyperactive RC chromatin. When the second element is removed, active RC chromatin spreads upstream, forcing the tether to serve as a new barrier. Acquisition of barrier function by the CTCF element disrupts contacts between distal Vß gene segments and significantly alters Tcrb repertoires. Our findings reveal a separation of function for RC-flanking regions, in which anchors for long-range recombination must be cordoned off from hyperactive RC landscapes by chromatin barriers.


Asunto(s)
Cromatina/genética , Células Precursoras de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Timocitos/metabolismo , Animales , Factor de Unión a CCCTC , Linaje de la Célula/genética , Células Cultivadas , Cromatina/metabolismo , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica , Histonas/metabolismo , Hibridación Fluorescente in Situ/métodos , Metilación , Ratones Endogámicos C57BL , Ratones Noqueados , Células Precursoras de Linfocitos B/citología , Regiones Promotoras Genéticas/genética , Unión Proteica , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timocitos/citología , Recombinación V(D)J/genética , VDJ Recombinasas/metabolismo
10.
Cell Rep ; 10(1): 29-38, 2015 Jan 06.
Artículo en Inglés | MEDLINE | ID: mdl-25543141

RESUMEN

V(D)J recombination is initiated by a specialized transposase consisting of the subunits RAG-1 and RAG-2. The susceptibility of gene segments to DNA cleavage by the V(D)J recombinase is correlated with epigenetic modifications characteristic of active chromatin, including trimethylation of histone H3 on lysine 4 (H3K4me3). Engagement of H3K4me3 by a plant homeodomain (PHD) in RAG-2 promotes recombination in vivo and stimulates DNA cleavage by RAG in vitro. We now show that H3K4me3 acts allosterically at the PHD finger to relieve autoinhibition imposed by a separate domain within RAG-2. Disruption of this autoinhibitory domain was associated with constitutive increases in recombination frequency, DNA cleavage activity, substrate binding affinity, and catalytic rate, thus mimicking the stimulatory effects of H3K4me3. Our observations support a model in which allosteric control of RAG is enforced by an autoinhibitory domain whose action is relieved by engagement of active chromatin.


Asunto(s)
Cromatina/genética , Proteínas de Unión al ADN/genética , Histonas/genética , Recombinación V(D)J/genética , VDJ Recombinasas/genética , Animales , Sitios de Unión , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Histonas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Metilación , Ratones , Células 3T3 NIH , Unión Proteica , VDJ Recombinasas/metabolismo
11.
J Exp Med ; 211(9): 1821-32, 2014 Aug 25.
Artículo en Inglés | MEDLINE | ID: mdl-25135298

RESUMEN

V(D)J recombination of TCR loci is regulated by chromatin accessibility to RAG1/2 proteins, rendering RAG1/2 targeting a potentially important regulator of lymphoid differentiation. We show that within the human TCR-α/δ locus, Dδ2-Dδ3 rearrangements occur at a very immature thymic, CD34(+)/CD1a(-)/CD7(+dim) stage, before Dδ2(Dδ3)-Jδ1 rearrangements. These strictly ordered rearrangements are regulated by mechanisms acting beyond chromatin accessibility. Importantly, direct Dδ2-Jδ1 rearrangements are prohibited by a B12/23 restriction and ordered human TCR-δ gene assembly requires RUNX1 protein, which binds to the Dδ2-23RSS, interacts with RAG1, and enhances RAG1 deposition at this site. This RUNX1-mediated V(D)J recombinase targeting imposes the use of two Dδ gene segments in human TCR-δ chains. Absence of this RUNX1 binding site in the homologous mouse Dδ1-23RSS provides a molecular explanation for the lack of ordered TCR-δ gene assembly in mice and may underlie differences in early lymphoid differentiation between these species.


Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Reordenamiento Génico de la Cadena delta de los Receptores de Antígenos de los Linfocitos T , Proteínas de Homeodominio/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Diferenciación Celular , Línea Celular , ADN/genética , ADN/metabolismo , Células HEK293 , Humanos , Cinética , Linfopoyesis , Ratones , Datos de Secuencia Molecular , Especificidad de la Especie , Subgrupos de Linfocitos T/citología , VDJ Recombinasas/metabolismo
12.
Mol Cell Biol ; 34(12): 2162-75, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24687855

RESUMEN

DNA-dependent protein kinase (DNA-PK) orchestrates DNA repair by regulating access to breaks through autophosphorylations within two clusters of sites (ABCDE and PQR). Blocking ABCDE phosphorylation (by alanine mutation) imparts a dominant negative effect, rendering cells hypersensitive to agents that cause DNA double-strand breaks. Here, a mutational approach is used to address the mechanistic basis of this dominant negative effect. Blocking ABCDE phosphorylation hypersensitizes cells to most types of DNA damage (base damage, cross-links, breaks, and damage induced by replication stress), suggesting that DNA-PK binds DNA ends that result from many DNA lesions and that blocking ABCDE phosphorylation sequesters these DNA ends from other repair pathways. This dominant negative effect requires DNA-PK's catalytic activity, as well as phosphorylation of multiple (non-ABCDE) DNA-PK catalytic subunit (DNA-PKcs) sites. PSIPRED analysis indicates that the ABCDE sites are located in the only contiguous extended region of this huge protein that is predicted to be disordered, suggesting a regulatory role(s) and perhaps explaining the large impact ABCDE phosphorylation has on the enzyme's function. Moreover, additional sites in this disordered region contribute to the ABCDE cluster. These data, coupled with recent structural data, suggest a model whereby early phosphorylations promote initiation of nonhomologous end joining (NHEJ), whereas ABCDE phosphorylations, potentially located in a "hinge" region between the two domains, lead to regulated conformational changes that initially promote NHEJ and eventually disengage NHEJ.


Asunto(s)
Proteína Quinasa Activada por ADN/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células CHO , Cisplatino/farmacología , Cricetinae , Cricetulus , Aductos de ADN/efectos de los fármacos , Aductos de ADN/metabolismo , Daño del ADN , Proteína Quinasa Activada por ADN/química , Activación Enzimática/efectos de los fármacos , Genes Dominantes , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fenotipo , Fosforilación/efectos de los fármacos , VDJ Recombinasas/metabolismo
13.
Microbiol Spectr ; 2(6)2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26104458

RESUMEN

V(D)J recombination, the mechanism responsible for generating antigen receptor diversity, has the potential to generate aberrant DNA rearrangements in developing lymphocytes. Indeed, the recombinase has been implicated in several different kinds of errors leading to oncogenic transformation. Here we review the basic aspects of V(D)J recombination, mechanisms underlying aberrant DNA rearrangements, and the types of aberrant events uncovered in recent genomewide analyses of lymphoid neoplasms.


Asunto(s)
Receptores de Antígenos/genética , Recombinación V(D)J , Animales , Variación Genética , Humanos , VDJ Recombinasas/metabolismo
14.
Proc Natl Acad Sci U S A ; 111(1): 397-402, 2014 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-24368847

RESUMEN

The highly diversified repertoire of antigen receptors in the vertebrate immune system is generated via proteins encoded by the recombination activating genes (RAGs) RAG1 and RAG2 by a process known as variable, diversity, and joining [V(D)J] gene recombination. Based on the study of vertebrate RAG proteins, many hypotheses have been proposed regarding the origin and evolution of RAG. This issue remains unresolved, leaving a significant gap in our understanding of the evolution of adaptive immunity. Here, we show that the amphioxus genome contains an ancient RAG1-like DNA fragment (bfRAG1L) that encodes a virus-related protein that is much shorter than vertebrate RAG1 and harbors a region homologous to the central domain of core RAG1 (cRAG1). bfRAG1L also contains an unexpected retroviral type II nuclease active site motif, DXN(D/E)XK, and is capable of degrading both DNA and RNA. Moreover, bfRAG1L shares important functional properties with the central domain of cRAG1, including interaction with RAG2 and localization to the nucleus. Remarkably, the reconstitution of bfRAG1L into a cRAG1-like protein yielded an enzyme capable of recognizing recombination signal sequences and performing V(D)J recombination in the presence of mouse RAG2. Moreover, this reconstituted cRAG1-like protein could mediate the assembly of antigen receptor genes in RAG1-deficient mice. Together, our results demonstrate that amphioxus bfRAG1L encodes a protein that is functionally equivalent to the central domain of cRAG1 and is well prepared for further evolution to mediate V(D)J recombination. Thus, our findings provide unique insights into the evolutionary origin of RAG1.


Asunto(s)
ADN/genética , Genes RAG-1 , Proteínas de Homeodominio/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , ADN/metabolismo , ADN Complementario/metabolismo , Proteínas de Unión al ADN/química , Células HEK293 , Proteínas de Homeodominio/genética , Humanos , Anfioxos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , ARN/metabolismo , Proteínas Recombinantes/química , Recombinación Genética , Homología de Secuencia de Aminoácido , VDJ Recombinasas/metabolismo , Vertebrados/fisiología
15.
J Exp Med ; 210(13): 2823-32, 2013 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-24297995

RESUMEN

Pre-B cell receptor (pre-BCR) signaling and migration from IL-7-rich environments cooperate to drive pre-B cell differentiation via transcriptional programs that remain unclear. We show that the Ikaros transcription factor is required for the differentiation of large pre-B to small pre-B cells. Mice deleted for Ikaros in pro/pre-B cells show a complete block of differentiation at the fraction C' stage, and Ikaros-null pre-B cells cannot differentiate upon withdrawal of IL-7 in vitro. Restoration of Ikaros function rescues pre-B cell differentiation in vitro and in vivo and depends on DNA binding. Ikaros is required for the down-regulation of the pre-BCR, Igκ germline transcription, and Ig L chain recombination. Furthermore, Ikaros antagonizes the IL-7-dependent regulation of >3,000 genes, many of which are up- or down-regulated between fractions C' and D. Affected genes include those important for survival, metabolism, B cell signaling, and function, as well as transcriptional regulators like Ebf1, Pax5, and the Foxo1 family. Our data thus identify Ikaros as a central regulator of IL-7 signaling and pre-B cell development.


Asunto(s)
Linfocitos B/citología , Diferenciación Celular , Factor de Transcripción Ikaros/metabolismo , Interleucina-7/metabolismo , Animales , Separación Celular , Regulación hacia Abajo , Citometría de Flujo , Cadenas kappa de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/genética , Leucemia/genética , Leucemia/metabolismo , Ratones , Ratones Noqueados , Mutación , Fenotipo , Recombinación Genética , Retroviridae/genética , Transducción de Señal , Transcripción Genética , Transcriptoma , VDJ Recombinasas/metabolismo
16.
Artículo en Inglés | MEDLINE | ID: mdl-24584058

RESUMEN

Lymphocytes recognize a vast variety of pathogens by expressing a diverse repertoire of antigen receptor genes that are assembled by V(D)J recombination in immature B cells (Igh, Igk) and T cells (Tcrb, Tcra/d). V(D)J recombination takes place in the 3' proximal domain containing the D, J, and C gene segments, whereas 31 (Tcrb) to 200 (Igh) V genes are spread over a large region of 0.67 (Tcrb) to 3 (Igk) Mb pairs. All antigen receptor loci undergo reversible contraction at the developmental stage, where they engage in V-(D)J recombination. This long-range looping promotes the participation of all V genes in V-(D)J recombination by juxtaposing distant V genes next to (D)J segments in the proximal recombination center. The B-cell-specific Pax5, ubiquitous YY1, and architectural CTCF/cohesin proteins promote Igh locus contraction in pro-B cells by binding to multiple sites in the VH gene cluster. These regulators also control the pro-B-cell-specific activity of the distally located PAIR elements, which are likely involved in the regulation of VH-DJH recombination by mediating locus contraction. Notably, the large VH gene cluster of the Igh locus undergoes flexible long-range looping that ensures similar participation of all VH genes in VH-DJH recombination to generate a diverse antibody repertoire.


Asunto(s)
Receptores de Antígenos/genética , Recombinación V(D)J , VDJ Recombinasas/metabolismo , Alelos , Animales , Anticuerpos/inmunología , Linfocitos B/inmunología , Cromatina/metabolismo , Elementos de Facilitación Genéticos , Humanos , Linfocitos/inmunología , Ratones , Modelos Genéticos , Familia de Multigenes , Mutagénesis , Unión Proteica , Receptores de Antígenos/inmunología , Factores de Transcripción/metabolismo
17.
J Biol Chem ; 287(43): 36488-98, 2012 Oct 19.
Artículo en Inglés | MEDLINE | ID: mdl-22942284

RESUMEN

The first step in V(D)J recombination is the formation of specific DNA double-strand breaks (DSBs) by the RAG1 and RAG2 proteins, which form the RAG recombinase. DSBs activate a complex network of proteins termed the DNA damage response (DDR). A key early event in the DDR is the phosphorylation of histone H2AX around DSBs, which forms a binding site for the tandem BRCA1 C-terminal (tBRCT) domain of MDC1. This event is required for subsequent signal amplification and recruitment of additional DDR proteins to the break site. RAG1 bears a histone H2AX-like motif at its C terminus (R1Ct), making it a putative MDC1-binding protein. In this work we show that the tBRCT domain of MDC1 binds the R1Ct motif of RAG1. Surprisingly, we also observed a second binding interface between the two proteins that involves the Proline-Serine-Threonine rich (PST) repeats of MDC1 and the N-terminal non-core region of RAG1 (R1Nt). The repeats-R1Nt interaction is constitutive, whereas the tBRCT-R1Ct interaction likely requires phosphorylation of the R1Ct motif of RAG1. As the C terminus of RAG1 has been implicated in inhibition of RAG activity, we propose a model in which phosphorylation of the R1Ct motif of RAG1 functions as a self-initiated regulatory signal.


Asunto(s)
Proteínas de Homeodominio/metabolismo , Modelos Biológicos , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , VDJ Recombinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencias de Aminoácidos , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodominio/genética , Humanos , Proteínas Nucleares/genética , Mapeo Peptídico/métodos , Fosforilación , Estructura Terciaria de Proteína , Secuencias Repetitivas de Aminoácido , Transactivadores/genética , VDJ Recombinasas/genética
18.
Nat Struct Mol Biol ; 19(8): 834-6, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22773102

RESUMEN

The formation of diverse immunoglobulin genes results in part from Rag protein-mediated DNA double-strand breaks at the edges of immunoglobulin gene segments, followed by combinatorial reassembly of these segments. We report that a Transib transposase from the insect Helicoverpa zea is active in vitro and that its breakage and joining activities mimic those of Rag, providing strong evidence that Rag and Transib transposases were derived from a common progenitor.


Asunto(s)
Proteínas de Insectos/metabolismo , Transposasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Elementos Transponibles de ADN/genética , Genes de Insecto , Humanos , Proteínas de Insectos/genética , Datos de Secuencia Molecular , Mariposas Nocturnas/enzimología , Mariposas Nocturnas/crecimiento & desarrollo , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Transposasas/genética , VDJ Recombinasas/genética , VDJ Recombinasas/metabolismo
19.
Nucleic Acids Res ; 40(13): 6082-96, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22434887

RESUMEN

During V(D)J recombination, the RAG1/2 recombinase is thought to play an active role in transferring newly excised recombination ends from the RAG post-cleavage complex (PCC) to the non-homologous end joining (NHEJ) machinery to promote appropriate antigen receptor gene assembly. However, this transfer mechanism is poorly understood, partly because of the technical difficulty in revealing weak association of coding ends (CEs) with one of the PCCs, coding end complex (CEC). Using fluorescence resonance energy transfer (FRET) and anisotropy measurement, we present here real-time monitoring of the RAG1/2-catalyzed cleavage reaction, and provide unequivocal evidence that CEs are retained within the CEC in the presence of Mg(2+). By examining the dynamic fluorescence changes during the cleavage reaction, we compared the stability of CEC assembled with core RAG1 paired with full-length RAG2, core RAG2 or a frameshift RAG2 mutant that was speculated to destabilize the PCC, leading to increased aberrant joining. While the latter two CECs exhibit similar stability, the full-length RAG2 renders a less stable CEC unless H3K4me3 peptides are added. Interestingly, the RAG2 mutant appears to modulate the structure of the RAG-12RSS pre-cleavage complex. Thus, the fluorescence-based detection offers a sensitive, quantitative and continuous assessment of pre-cleavage complex assembly and CEC stability.


Asunto(s)
División del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/metabolismo , VDJ Recombinasas/metabolismo , Biocatálisis , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/genética , Polarización de Fluorescencia , Transferencia Resonante de Energía de Fluorescencia , Proteínas de Homeodominio/genética , Magnesio/química , Mutación
20.
Proc Natl Acad Sci U S A ; 109(10): 3903-8, 2012 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-22355127

RESUMEN

Nonhomologous end joining (NHEJ), a major pathway of DNA double-strand break (DSB) repair, is required during lymphocyte development to resolve the programmed DSBs generated during Variable, Diverse, and Joining [V(D)J] recombination. XRCC4-like factor (XLF) (also called Cernunnos or NHEJ1) is a unique component of the NHEJ pathway. Although germ-line mutations of other NHEJ factors abrogate lymphocyte development and lead to severe combined immunodeficiency (SCID), XLF mutations cause a progressive lymphocytopenia that is generally less severe than SCID. Accordingly, XLF-deficient murine lymphocytes show no measurable defects in V(D)J recombination. We reported earlier that ATM kinase and its substrate histone H2AX are both essential for V(D)J recombination in XLF-deficient lymphocytes, despite moderate role in V(D)J recombination in WT cells. p53-binding protein 1 (53BP1) is another substrate of ATM. 53BP1 deficiency led to small reduction of peripheral lymphocyte number by compromising both synapse and end-joining at modest level during V(D)J recombination. Here, we report that 53BP1/XLF double deficiency blocks lymphocyte development at early progenitor stages, owing to severe defects in end joining during chromosomal V(D)J recombination. The unrepaired DNA ends are rapidly degraded in 53BP1(-/-)XLF(-/-) cells, as reported for H2AX(-/-)XLF(-/-) cells, revealing an end protection role for 53BP1 reminiscent of H2AX. In contrast to the early embryonic lethality of H2AX(-/-)XLF(-/-) mice, 53BP1(-/-)XLF(-/-) mice are born alive and develop thymic lymphomas with translocations involving the T-cell receptor loci. Together, our findings identify a unique function for 53BP1 in end-joining and tumor suppression.


Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Linfocitos/citología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Supresoras de Tumor/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Daño del ADN , Ratones , Ratones SCID , Ratones Transgénicos , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Recombinación Genética , Proteína 1 de Unión al Supresor Tumoral P53 , VDJ Recombinasas/metabolismo
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