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1.
Front Immunol ; 10: 1572, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31333681

RESUMO

V(D)J recombination generates antigen receptor diversity by mixing and matching individual variable (V), diversity (D), and joining (J) gene segments. An obligate by-product of many of these reactions is the excised signal circle (ESC), generated by excision of the DNA from between the gene segments. Initially, the ESC was believed to be inert and formed to protect the genome from reactive broken DNA ends but more recent work suggests that the ESC poses a substantial threat to genome stability. Crucially, the recombinase re-binds to the ESC, which can result in it being re-integrated back into the genome, to cause potentially oncogenic insertion events. In addition, very recently, the ESC/recombinase complex was found to catalyze breaks at recombination signal sequences (RSSs) throughout the genome, via a "cut-and-run" mechanism. Remarkably, the ESC/recombinase complex triggers these breaks at key leukemia driver genes, implying that this reaction could be a significant cause of lymphocyte genome instability. Here, we explore these alternate pathways and discuss their relative dangers to lymphocyte genome stability.

2.
Mol Cell Oncol ; 6(4): 1610323, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31211241

RESUMO

A newly identified process by which mistargeted V(D)J recombination could cause genome instability in childhood leukemia has been discovered. In this mechanism, called cut-and-run, the excised DNA by-products of V(D)J recombination are re-bound by the recombinase proteins and erroneously trigger double-strand breaks at multiple locations throughout the genome. Many of these breakpoints co-localize with known chromosome alterations in acute lymphoblastic leukemia (ALL).

3.
Front Pediatr ; 7: 122, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31058115

RESUMO

Proteins expressed by recombination activating genes 1 and 2 (RAG1/2) are essential in the process of V(D)J recombination that leads to generation of the T and B cell repertoires. Clinical and immunological phenotypes of patients with RAG deficiencies correlate well to the degree of impaired RAG activity and this has been expanding to variants of combined immunodeficiency (CID) or even milder antibody deficiency syndromes. Pathogenic variants that severely impair recombinase activity of RAG1/2 determine a severe combined immunodeficiency (SCID) phenotype, whereas hypomorphic variants result in leaky (partial) SCID and other immunodeficiencies. We report a patient with novel pathogenic compound heterozygous RAG2 variants that result in a CID phenotype with two distinctive characteristics: late-onset progressive hypogammaglobulinemia and highly elevated B cell count. In addition, the patient had early onset of infections, T cell lymphopenia and expansion of lymphocytes after exposure to herpes family viruses. This case highlights the importance of considering pathogenic RAG variants among patients with preserved B cell count and CID phenotype.

4.
Mol Cell ; 74(3): 584-597.e9, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30905508

RESUMO

V(D)J recombination is essential to generate antigen receptor diversity but is also a potent cause of genome instability. Many chromosome alterations that result from aberrant V(D)J recombination involve breaks at single recombination signal sequences (RSSs). A long-standing question, however, is how such breaks occur. Here, we show that the genomic DNA that is excised during recombination, the excised signal circle (ESC), forms a complex with the recombinase proteins to efficiently catalyze breaks at single RSSs both in vitro and in vivo. Following cutting, the RSS is released while the ESC-recombinase complex remains intact to potentially trigger breaks at further RSSs. Consistent with this, chromosome breaks at RSSs increase markedly in the presence of the ESC. Notably, these breaks co-localize with those found in acute lymphoblastic leukemia patients and occur at key cancer driver genes. We have named this reaction "cut-and-run" and suggest that it could be a significant cause of lymphocyte genome instability.


Assuntos
Instabilidade Genômica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocação Genética/genética , Recombinação V(D)J/genética , Animais , Sequência de Bases/genética , Células COS , Cromossomos/genética , DNA/genética , Quebras de DNA de Cadeia Dupla , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Células NIH 3T3 , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Recombinases/genética
5.
Blood ; 133(8): 820-829, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30538136

RESUMO

The Recombination Activating Genes, RAG1 and RAG2, are essential for V(D)J recombination and adaptive immunity. Mutations in these genes often cause immunodeficiency, the severity of which reflects the importance of the altered residue or residues during recombination. Here, we describe a novel RAG1 mutation that causes immunodeficiency in an unexpected way: The mutated protein severely disrupts binding of the accessory protein, HMGB1. Although HMGB1 enhances RAG cutting in vitro, its role in vivo was controversial. We show here that reduced HMGB1 binding by the mutant protein dramatically reduces RAG cutting in vitro and almost completely eliminates recombination in vivo. The RAG1 mutation, R401W, places a bulky tryptophan opposite the binding site for HMG Box A at both 12- and 23-spacer recombination signal sequences, disrupting stable binding of HMGB1. Replacement of R401W with leucine and then lysine progressively restores HMGB1 binding, correlating with increased RAG cutting and recombination in vivo. We show further that knockdown of HMGB1 significantly reduces recombination by wild-type RAG1, whereas its re-addition restores recombination with wild-type, but not the mutant, RAG1 protein. Together, these data provide compelling evidence that HMGB1 plays a critical role during V(D)J recombination in vivo.


Assuntos
Proteína HMGB1 , Proteína HMGB2 , Proteínas de Homeodomínio , Mutação de Sentido Incorreto , Recombinação V(D)J/imunologia , Substituição de Aminoácidos , Animais , Células HEK293 , Proteína HMGB1/genética , Proteína HMGB1/imunologia , Proteína HMGB2/genética , Proteína HMGB2/imunologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Camundongos , Células NIH 3T3
7.
FEBS J ; 281(20): 4583-97, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25124066

RESUMO

Transcription activator-like effectors (TALEs) are immensely powerful new tools for genome engineering that can be directed to bind to almost any DNA sequence of choice. They originate from the Xanthomonas species of plant pathogenic bacteria and, in nature, these proteins increase the virulence of Xanthomonas. However, in 2009, the DNA binding code of TALEs was deciphered and, subsequently, TALE proteins have been exploited for many diverse applications. Custom TALEs that target almost any required DNA sequence can be readily constructed in < 1 week. One major application is gene editing: TALEs fused with the Fok I endonuclease catalytic domain can induce double-stranded breaks at a chosen genomic location, similar to zinc finger nucleases. Designer TALE transcription factors have also been developed by linking TALEs to a transcription AD, such as VP64. More recently, TALEs have been developed that can repress transcription, bind methylated DNA or act as fluorescent chromatin probes. In the present review, we describe the assembly of designer TALEs, their expanding range of current and potential future applications, and briefly discuss alternatives, namely, zinc finger nucleases and clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat associated protein 9.


Assuntos
Regulação da Expressão Gênica , Engenharia Genética , Genoma , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Humanos , Fatores de Transcrição/genética
8.
Biochem J ; 461(1): 1-13, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24927119

RESUMO

Cancers arise through the progression of multiple genetic and epigenetic defects that lead to deregulation of numerous signalling networks. However, the last decade has seen the development of the concept of 'oncogene addiction', where tumours appear to depend on a single oncogene for survival. RNAi has provided an invaluable tool in the identification of these oncogenes and oncogene-dependent cancers, and also presents great potential as a novel therapeutic strategy against them. Although RNAi therapeutics have demonstrated effective killing of oncogene-dependent cancers in vitro, their efficacy in vivo is severely limited by effective delivery systems. Several virus-based RNAi delivery strategies have been explored, but problems arose associated with high immunogenicity, random genome integration and non-specific targeting. This has directed efforts towards non-viral formulations, including delivery systems based on virus-like particles, liposomes and cationic polymers, which can circumvent some of these problems by immunomasking and the use of specific tumour-targeting ligands. This review outlines the prevalence of oncogene-dependent cancers, evaluates the potential of RNAi-based therapeutics and assesses the relative strengths and weaknesses of different approaches to targeted RNAi delivery.


Assuntos
Marcação de Genes/tendências , Neoplasias/genética , Neoplasias/terapia , Oncogenes/genética , RNA Interferente Pequeno/uso terapêutico , Animais , Terapia Genética/métodos , Terapia Genética/tendências , Humanos , Interferência de RNA/fisiologia , RNA Interferente Pequeno/administração & dosagem
9.
Biochem J ; 458(1): 153-8, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24438328

RESUMO

TALE (transcription activator-like effector) proteins can be tailored to bind to any DNA sequence of choice and thus are of immense utility for genome editing and the specific delivery of transcription activators. However, to perform these functions, they need to occupy their sites in chromatin. In the present study, we have systematically assessed TALE binding to chromatin substrates and find that in vitro TALEs bind to their target site on nucleosomes at the more accessible entry/exit sites, but not at the nucleosome dyad. We show further that in vivo TALEs bind to transcriptionally repressed chromatin and that transcription increases binding by only 2-fold. These data therefore imply that TALEs are likely to bind to their target in vivo even at inactive loci.


Assuntos
Cromatina/metabolismo , Transativadores/metabolismo , Acetilação , Animais , Sítios de Ligação , Camundongos , Células NIH 3T3 , Ligação Proteica
10.
EMBO J ; 32(10): 1381-92, 2013 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-23463099

RESUMO

Initiation of V(D)J recombination critically relies on the formation of an accessible chromatin structure at recombination signal sequences (RSSs) but how this accessibility is generated is poorly understood. Immunoglobulin light-chain loci normally undergo recombination in pre-B cells. We show here that equipping (earlier) pro-B cells with the increased pre-B-cell levels of just one transcription factor, IRF4, triggers the entire cascade of events leading to premature light-chain recombination. We then used this finding to dissect the critical events that generate RSS accessibility and show that the chromatin modifications previously associated with recombination are insufficient. Instead, we establish that non-coding transcription triggers IgL RSS accessibility and find that the accessibility is transient. Transcription transiently evicts H2A/H2B dimers, releasing 35-40 bp of nucleosomal DNA, and we demonstrate that H2A/H2B loss can explain the RSS accessibility observed in vivo. We therefore propose that the transcription-mediated eviction of H2A/H2B dimers is an important mechanism that makes RSSs accessible for the initiation of recombination.


Assuntos
Histonas/metabolismo , Fatores Reguladores de Interferon/metabolismo , Células Precursoras de Linfócitos B/fisiologia , Recombinação V(D)J/fisiologia , Animais , Montagem e Desmontagem da Cromatina , Regulação da Expressão Gênica , Histonas/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Cadeias Leves de Imunoglobulina/genética , Fatores Reguladores de Interferon/genética , Camundongos , Camundongos Transgênicos , Nucleossomos/genética , Nucleossomos/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Transcrição Genética
11.
Biochem Biophys Res Commun ; 441(2): 482-7, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-24383080

RESUMO

Enhancers are essential for long range chromatin opening and the activation of V(D)J recombination at the antigen receptor loci. The murine immunoglobulin lambda light chain locus is a duplicated locus and, using a bacterial artificial chromosome spanning the 3' half of the locus to generate transgenic mice, we have identified a critical enhancer element for lambda locus recombination. Four hypersensitive sites had been previously mapped downstream of the JCλ1 gene segment (HS1-4). Systematic deletion of these individual hypersensitive sites showed that HS1, which forms the major part of the transcription enhancer, Eλ3­1, is essential for Igλ recombination and that it also helps to restrict Igλ stage-specific recombination.


Assuntos
Elementos Facilitadores Genéticos , Cadeias lambda de Imunoglobulina/genética , Recombinação V(D)J , Animais , Mapeamento Cromossômico , Cromossomos Artificiais Bacterianos/genética , Loci Gênicos , Camundongos , Camundongos Transgênicos , Deleção de Sequência
12.
Mol Cell Biol ; 30(15): 3805-15, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20498275

RESUMO

During development, gene activation is stringently regulated to restrict expression only to the correct cell type and correct developmental stage. Here, we present mechanistic evidence that suggests DNA methylation contributes to this regulation by suppressing premature gene activation. Using the mouse Myogenin promoter as an example of the weak CpG island class of promoters, we find that it is initially methylated but becomes demethylated as development proceeds. Full hypersensitive site formation of the Myogenin promoter requires both the MEF2 and SIX binding sites, but binding to only one site can trigger the partial chromatin opening of the nonmethylated promoter. DNA methylation markedly decreases hypersensitive site formation that now occurs at a detectable level only when binding to both MEF2 and SIX binding sites is possible. This suggests that the probability of activating the methylated promoter is low until two of the factors are coexpressed within the same cell. Consistent with this, the single-cell analysis of developing somites shows that the coexpression of MEF2A and SIX1, which bind the MEF2 and SIX sites, correlates with the fraction of cells that demethylate the Myogenin promoter. Taken together, these studies imply that DNA methylation helps to prevent inappropriate gene activation until sufficient activating factors are coexpressed.


Assuntos
Metilação de DNA , Miogenina/genética , Animais , Sítios de Ligação/genética , Cromatina , Ilhas de CpG , Embrião de Mamíferos , Genes , Proteínas de Homeodomínio , Fatores de Transcrição MEF2 , Metilação , Camundongos , Camundongos Transgênicos , Fatores de Regulação Miogênica , Miogenina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional
13.
Blood ; 116(1): 85-96, 2010 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-20410505

RESUMO

Heat shock protein 27 (HSP27) is a chaperone whose cellular expression increases in response to various stresses and protects the cell either by inhibiting apoptotic cell death or by promoting the ubiquitination and proteasomal degradation of specific proteins. Here, we show that globin transcription factor 1 (GATA-1) is a client protein of HSP27. In 2 models of erythroid differentiation; that is, in the human erythroleukemia cell line, K562 induced to differentiate into erythroid cells on hemin exposure and CD34(+) human cells ex vivo driven to erythroid differentiation in liquid culture, depletion of HSP27 provokes an accumulation of GATA-1 and impairs terminal maturation. More specifically, we demonstrate that, in the late stages of the erythroid differentiation program, HSP27 is phosphorylated in a p38-dependent manner, enters the nucleus, binds to GATA-1, and induces its ubiquitination and proteasomal degradation, provided that the transcription factor is acetylated. We conclude that HSP27 plays a role in the fine-tuning of terminal erythroid differentiation through regulation of GATA-1 content and activity.


Assuntos
Diferenciação Celular , Células Eritroides/metabolismo , Fator de Transcrição GATA1/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Animais , Antígenos CD34/sangue , Células COS , Núcleo Celular/metabolismo , Células Cultivadas , Células Eritroides/citologia , Células Eritroides/efeitos dos fármacos , Fator de Transcrição GATA1/genética , Proteínas de Choque Térmico HSP27/genética , Células HeLa , Humanos , Imidazóis/farmacologia , Immunoblotting , Interleucina-6/farmacologia , Células K562 , Leupeptinas/farmacologia , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Ligação Proteica , Piridinas/farmacologia , Interferência de RNA , Fator de Crescimento Transformador beta/farmacologia , Ubiquitinação/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
14.
Nucleic Acids Res ; 35(18): 6311-21, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17881376

RESUMO

Targeted chromatin remodelling is essential for many nuclear processes, including the regulation of V(D)J recombination. ATP-dependent nucleosome remodelling complexes are important players in this process whose activity must be tightly regulated. We show here that histone acetylation regulates nucleosome remodelling complex activity to boost RAG cutting during the initiation of V(D)J recombination. RAG cutting requires nucleosome mobilization from recombination signal sequences. Histone acetylation does not stimulate nucleosome mobilization per se by CHRAC, ACF or their catalytic subunit, ISWI. Instead, we find the more open structure of acetylated chromatin regulates the ability of nucleosome remodelling complexes to access their nucleosome templates. We also find that bromodomain/acetylated histone tail interactions can contribute to this targeting at limited concentrations of remodelling complex. We therefore propose that the changes in higher order chromatin structure associated with histone acetylation contribute to the correct targeting of nucleosome remodelling complexes and this is a novel way in which histone acetylation can modulate remodelling complex activity.


Assuntos
Montagem e Desmontagem da Cromatina , Rearranjo Gênico do Linfócito B , Rearranjo Gênico do Linfócito T , Histonas/metabolismo , Nucleossomos/metabolismo , Acetilação , Animais , Linhagem Celular , Drosophila/metabolismo , Histonas/química , Proteínas de Homeodomínio/metabolismo , Região de Junção de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Nucleossomos/química , Recombinação Genética
15.
Biochem Biophys Res Commun ; 363(1): 223-8, 2007 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-17868643

RESUMO

Enhancers play an important role in chromatin opening but the temporal relationship between enhancer activation and the generation of an accessible chromatin structure is poorly defined. Recombination enhancers are essential for chromatin opening and subsequent V(D)J recombination at immunoglobulin loci. In mice, the kappa light chain locus displays an open chromatin structure before the lambda locus yet the same proteins, PU.1/PIP, trigger full enhancer activation of both loci. Using primary B cells isolated from distinct developmental stages and an improved method to quantitatively determine hypersensitive site formation, we find the kappa and lambda recombination enhancers become fully hypersensitive soon after transition to large and small pre-B-II cells, respectively. This correlates strictly with the stages at which these loci are activated. Since these cells are short-lived, these data imply that there is a close temporal relationship between full enhancer hypersensitive site formation and locus chromatin opening.


Assuntos
Cromatina/genética , Elementos Facilitadores Genéticos/genética , Genes de Imunoglobulinas , Cadeias kappa de Imunoglobulina/genética , Recombinação Genética/genética , Ativação Transcricional/genética , Animais , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Células Cultivadas , Camundongos
16.
EMBO J ; 25(14): 3264-74, 2006 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-16858405

RESUMO

Regulation of transcription requires mechanisms to both activate and terminate transcription factor activity. GATA-1 is a key haemopoietic transcription factor whose activity is increased by acetylation. We show here that acetylated GATA-1 is targeted for degradation via the ubiquitin/proteasome pathway. Acetylation positively signals ubiquitination, suggesting that activation by acetylation simultaneously marks GATA-1 for degradation. Promoter-specific MAPK phosphorylation then cooperates with acetylation to execute protein loss. The requirement for both modifications is novel and suggests a way by which degradation of the active protein can be specifically regulated in response to external phosphorylation-mediated signalling. As many transcription factors are activated by acetylation, we suggest that this might be a general mechanism to control transcription factor activity.


Assuntos
Fator de Transcrição GATA1/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Acetilação , Animais , Células COS , Fator de Transcrição GATA1/biossíntese , Fator de Transcrição GATA1/genética , Hematopoese/fisiologia , Fosforilação , Ubiquitina/metabolismo
17.
EMBO J ; 22(19): 5197-207, 2003 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-14517257

RESUMO

A key component in the regulation of V(D)J recombination is control of the accessibility of RAG proteins to recombination signal sequences (RSS). Nucleosomes are known to inhibit this accessibility. We show here that the signal sequence itself represses accessibility by causing nucleosome positioning over the RSS. This positioning is mediated, in vitro and in vivo, by the conserved nonamer of the RSS. Consistent with this strong positioning, nucleosomes at RSSs are resistant to remodelling by nucleosome sliding. In vivo we find that consensus RSSs are preferentially protected, whereas those that lack a consensus nonamer, including some cryptic RSSs, fail to position nucleosomes. Decreased protection of these non-consensus RSSs correlates with their increased use in recombination assays. We therefore suggest that nucleosome positioning by RSSs provides a previously unanticipated level of protection and regulation of V(D)J recombination.


Assuntos
Rearranjo Gênico/fisiologia , Região Variável de Imunoglobulina/metabolismo , Nucleossomos/metabolismo , Recombinação Genética/fisiologia , Células 3T3 , Animais , Regulação da Expressão Gênica , Camundongos
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