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1.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(10): 965-969, 2019 Oct 10.
Artigo em Chinês | MEDLINE | ID: mdl-31598937

RESUMO

OBJECTIVE: To explore the genetic basis for a family with non-syndromic autosomal recessive deafness. METHODS: The proband and her parents were subjected to physical and audiological examinations. With genomic DNA extracted from peripheral blood samples, next-generation sequencing was carried out using a panel for deafness genes. Suspected mutation was validated by Sanger sequencing and qPCR analysis of her parents. RESULTS: The proband presented bilateral severe sensorineural hearing loss at three days after birth. Her auditory threshold was 110-120 dBnHL but with absence of vestibular and retinal symptoms. Her brother also had deafness but her parents were normal. No abnormality was found upon physical examination of her family members, while audiological examination showed no middle ear or retrocochlear diseases. Next-generation sequencing identified compound heterozygous mutations of the MYO7A gene, including a previously known c.462C>A (p. Cys154Ter) and a novel EX43_46 Del, which were respectively derived from her mother and father. CONCLUSION: The compound heterozygous mutations of the MYO7A gene probably underlie the disease in this family. Our findings has enriched the mutation spectrum for non-syndromic autosomal recessive deafness 2.

2.
Nan Fang Yi Ke Da Xue Xue Bao ; 39(8): 883-890, 2019 Aug 30.
Artigo em Chinês | MEDLINE | ID: mdl-31511206

RESUMO

OBJECTIVE: To investigate the effect of the chemoprotectant tempol on the anti-tumor activity of cisplatin (DDP). METHODS: The cellular toxicity of tempol in human colon cancer SW480 cells and mouse colon cancer CT26 cells were evaluated using MTT and cell counting kit-8 assays. CalcuSyn software analysis was used to determine the interaction between tempol and DDP in inhibition of the cell viability. A subcutaneous homograft mouse model of colon cancer was established. The mice were randomly divided into control group, tempol group, cisplatin group and tempol + DDP treatment group with intraperitoneal injections of the indicated agents. The tumor size, body weight and lifespan of the mice were measured, and HE staining was used to analyze the cytotoxic effect of the agents on the kidney and liver. Immunohistochemistry and Western blotting were performed to detect the expression of Bax and Bcl2 in the tumor tissue, and TUNEL staining was used to analyze the tumor cell apoptosis. The level of reactive oxygen species (ROS) in the tumor tissue was determined using flow cytometry. RESULTS: Tempol showed inhibitory effects on the viability of SW480 and CT26 cells. CalcuSyn software analysis showed that tempol had a synergistic anti-tumor effect with DDP (CI < 1). In the homograft mouse model, tempol treatment alone did not produce obvious anti-tumor effect. HE staining showed that the combined use of tempol and DDP alleviated DDP-induced fibrogenesis in the kidneys, but tempol also reduced the anti-tumor activity of DDP. Compared with the mice treated with DDP alone, the mice treated with both tempol and DDP had a significantly larger tumor size (P < 0.01) and a shorter lifespan (P < 0.05). Tempol significantly reversed DDP-induced expression of Bax and Bcl2 in the tumor tissue and tumor cell apoptosis (P < 0.001), and obviously reduced the elevation of ROS level in the tumor tissue induced by DDP treatment (P < 0.05). CONCLUSIONS: Tempol can attenuate the anti-tumor effect of DDP while reducing the side effects of DDP. Caution must be taken and the risks and benefits should be carefully weighed when considering the use of tempol as an anti-oxidant to reduce the toxicities of DDP.


Assuntos
Óxidos N-Cíclicos/farmacologia , Animais , Antineoplásicos , Antioxidantes , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Cisplatino , Resistencia a Medicamentos Antineoplásicos , Humanos , Camundongos , Marcadores de Spin
3.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(8): 813-816, 2019 Aug 10.
Artigo em Chinês | MEDLINE | ID: mdl-31400135

RESUMO

OBJECTIVE: To carry out prenatal diagnosis for a fetus with ultrasonographic abnormality. METHODS: Chromosomal karyotyping and array comparative genomic hybridization (array-CGH) analysis were applied for the diagnosis. Peripheral blood samples were also taken from the parents for chromosome karyotyping analysis. RESULTS: The fetal karyotype showed additional material of unknown-origin attached to Yq. Array CGH analysis confirmed that the material was derived from 3q22.1q29. The father was found to carry a balanced translocation 46, X, t(Y;3)(q12;q23) (which was diagnosed as 46,XY,Y≥18 elsewhere), whilst the mother was found to be normal. CONCLUSION: 3q partial trisomy may present as malformation of multiple systems. Combination of chromosome karyotyping and array-CGH can provide reliable diagnosis for fetuses with abnormalities by ultrasonography.


Assuntos
Cromossomos Humanos Par 3/genética , Diagnóstico Pré-Natal , Trissomia , Hibridização Genômica Comparativa , Feminino , Feto , Humanos , Cariotipagem , Masculino , Gravidez
4.
Nan Fang Yi Ke Da Xue Xue Bao ; 39(6): 692-698, 2019 Jun 30.
Artigo em Chinês | MEDLINE | ID: mdl-31270048

RESUMO

OBJECTIVE: To optimize DNA library construction in non-crosslinked chromatin immunoprecipitation coupled with next-generation sequencing (Native ChIP-seq) to obtain high-quality Native ChIP-seq data. METHODS: Human nasopharyngeal carcinoma HONE1 cell lysate was digested with MNase for release of the nucleosomes, and the histone-DNA complexes were immunoprecipitated with specific antibodies. The protein component in the precipitate was digested with proteinase K followed by DNA purification; the DNA library was constructed for sequence analysis. RESULTS: Compared with the conventional DNA library construction, Tn5 transposase method allowed direct enrichment of the target DNA after Tn5 fragmentation, which was simple, time-saving and more efficient. The IGV visualized map showed that the information obtained by the two library construction methods was consistent. The sequencing data obtained by the two methods revealed more signal enrichment with Tn5 transposase library construction than with the conventional approach. H3K4me3 ChIP results showed a good reproducibility after Tn5 transposase library construction with a signal-to-noise ratio above 50%. CONCLUSIONS: Tn5 transposase method improves the efficiency of DNA library construction and the results of subsequent sequence analysis, and is especially suitable for detecting histone modification in the DNA to provide a better technical option for epigenetic studies.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Imunoprecipitação da Cromatina , DNA , Biblioteca Gênica , Humanos , Reprodutibilidade dos Testes , Análise de Sequência de DNA
5.
J Ocul Pharmacol Ther ; 35(4): 235-244, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30994400

RESUMO

Purpose: To investigate retinal gene expression of tetramethylpyrazine (TMP) eye drop-treated endotoxin-induced uveitis (EIU) in mice and to explore the mechanisms. Methods: The inflammatory signs of the anterior segment were evaluated, and clinical scores were graded. The retinal transcriptome from the TMP eye drop-treated and the untreated mice was identified by RNA sequencing (RNA-seq) strategy. Differentially expressed genes (DEGs) were validated by real-time PCR. The protein-protein interaction was analyzed using the STRING software. Results: Compared with the TMP-treated group, the inflammatory responses of the untreated control group were much severe and clinical score was remarkably higher (P < 0.001) at 24 h after lipopolysaccharide administration. RNA-seq assay identified 407 DEGs, among which 356 were upregulated and 51 were downregulated. There were 12 upregulated gene ontology terms enriched and 27 upregulated pathways. Seven DEGs, including inflammation-related, complement system-related, and interferon-related genes, were validated using quantitative PCR. Conclusions: TMP exerted anti-inflammatory effect in EIU. Local application of TMP inhibited retinal inflammatory response by regulating the inflammation-related genes, suggesting that TMP may be a potential novel therapeutic drug for ocular inflammation.

6.
Cell Death Dis ; 10(4): 286, 2019 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-30911068

RESUMO

It was brought to the attention of the Editors that there had been an accidental duplication of the western blot images during the preparation of Figs. 1b and c. The authors were notified about the error and have supplied the correct image for Fig. 1c (below). We apologize for any inconvenience this may have caused readers.

7.
Protein Cell ; 10(8): 595-605, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30710319

RESUMO

The E3 ligase HERC4 is overexpressed in human breast cancer and its expression levels correlated with the prognosis of breast cancer patients. However, the roles of HERC4 in mammary tumorigenesis remain unclear. Here we demonstrate that the knockdown of HERC4 in human breast cancer cells dramatically suppressed their proliferation, survival, migration, and tumor growth in vivo, while the overexpression of HERC4 promoted their aggressive tumorigenic activities. HERC4 is a new E3 ligase for the tumor suppressor LATS1 and destabilizes LATS1 by promoting the ubiquitination of LATS1. miRNA-136-5p and miRNA-1285-5p, expression of which is decreased in human breast cancers and is inversely correlated with the prognosis of breast cancer patients, are directly involved in suppressing the expression of HERC4. In summary, we discover a miRNA-HERC4-LATS1 pathway that plays important roles in the pathogenesis of breast cancer and represents new therapeutic targets for human breast cancer.

8.
Protein Cell ; 2018 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-30417224

RESUMO

In the original publication the Supplementary Material and Fig. 2 are incorrect. The correct version is provided in this correction article. The text HBG2 appearing in the article should be read as HBG1.

10.
Mol Vis ; 23: 395-406, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28706439

RESUMO

PURPOSE: Endotoxin-induced uveitis (EIU) is a well-established mouse model for studying human acute inflammatory uveitis. The purpose of this study is to investigate the genome-wide retinal transcriptome profile of EIU. METHODS: The anterior segment of the mice was examined with a slit-lamp, and clinical scores were evaluated simultaneously. The histological changes in the posterior segment of the eyes were evaluated with hematoxylin and eosin (H&E) staining. A high throughput RNA sequencing (RNA-seq) strategy using the Illumina Hiseq 2500 platform was applied to characterize the retinal transcriptome profile from lipopolysaccharide (LPS)-treated and untreated mice. The validation of the differentially expressed genes (DEGs) was analyzed with real-time PCR. RESULTS: At the 24th hour after challenge, the clinical score of the LPS group was significantly higher (3.83±0.75, mean ± standard deviation [SD]) than that of the control group (0.08±0.20, mean ± SD; p<0.001). The histological evaluation showed a large number of inflammatory cells infiltrated into the vitreous cavity in the LPS group compared with the control group. A total of 478 DEGs were identified with RNA-seq. Among these genes, 406 were upregulated and 72 were downregulated in the LPS group. Gene Ontology (GO) enrichment showed three significantly enriched upregulated terms. Twenty-one upregulated and seven downregulated pathways were remarkably enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Eleven inflammatory response-, complement system-, fibrinolytic system-, and cell stress-related genes were validated to show similar results as the RNA-seq. CONCLUSIONS: We first reported the retinal transcriptome profile of the EIU mouse with RNA-seq. The results indicate that the abnormal changes in the inflammatory response-, complement system-, fibrinolytic system-, and cell stress-related genes occurred concurrently in EIU. These genes may play an important role in the pathogenesis of EIU. This study will lead to a better understanding of the underlying mechanisms and shed light on discovering novel therapeutic targets for ocular inflammation.


Assuntos
Perfilação da Expressão Gênica , Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Retina/metabolismo , Retina/patologia , Uveíte/genética , Animais , Câmara Anterior/metabolismo , Câmara Anterior/patologia , Análise por Conglomerados , Regulação para Baixo/genética , Endotoxinas , Feminino , Ontologia Genética , Inflamação/patologia , Lipopolissacarídeos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Regulação para Cima/genética , Uveíte/patologia
11.
Oncotarget ; 8(20): 33047-33063, 2017 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-28380434

RESUMO

Aerobic glycolysis is essential for tumor growth and survival. Activation of multiple carcinogenic signals contributes to metabolism reprogramming during malignant transformation of cancer. Recently nitric oxide has been noted to promote glycolysis but the mechanism remains elusive. We report here the dual role of nitric oxide in glycolysis: low/physiological nitric oxide (≤ 100 nM) promotes glycolysis for ATP production, oxidative defense and cell proliferation of ovary cancer cells, whereas excess nitric oxide (≥ 500 nM) inhibits it. Nitric oxide has a positive effect on glycolysis by inducing PKM2 nuclear translocation in an EGFR/ERK2 signaling-dependent manner. Moreover, iNOS induced by mild inflammatory stimulation increased glycolysis and cell proliferation by producing low doses of nitric oxide, while hyper inflammation induced iNOS inhibited it by producing excess nitric oxide. Finally, iNOS expression is abnormally increased in ovarian cancer tissues and is correlated with PKM2 expression. Overexpression of iNOS is associated with aggressive phenotype and poor survival outcome in ovarian cancer patients. Our study indicated that iNOS/NO play a dual role of in tumor glycolysis and progression, and established a bridge between iNOS/NO signaling pathway and EGFR/ERK2/PKM2 signaling pathway, suggesting that interfering glycolysis by targeting the iNOS/NO/PKM2 axis may be a valuable new therapeutic approach of treating ovarian cancer.


Assuntos
Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Proteínas de Membrana/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Neoplasias Ovarianas/patologia , Hormônios Tireóideos/metabolismo , Adulto , Animais , Linhagem Celular Tumoral , Feminino , Glicólise , Humanos , Camundongos , Pessoa de Meia-Idade , Transplante de Neoplasias , Neoplasias Ovarianas/metabolismo , Transporte Proteico
12.
Cell Death Discov ; 3: 17011, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28243469

RESUMO

Autophagy is a cellular survival mechanism that involves the catabolic degradation of damaged proteins and organelles during periods of metabolic stress, and when overly stimulated, commonly contributes to cell death. Nitric oxide (NO), a potent cellular messenger, participates in a complex mechanism which assists in controlling autophagy. However, the mechanism by which endogenous NO formed by distinct isoforms of nitric oxide synthase (NOS) helps to regulate autophagy in cancer cells remains unclear. Here we report that NOS1 reduces excessive levels of autophagy and promotes the survival of nasopharyngeal carcinoma cells. We found that inhibition of NOS1 increased cell death resulting from siRNA or the use of pharmacologic agents; and this effect was reversed by the autophagy inhibitor, chloroquine. The role of NOS1 in the autophagy process depended on the activation of AKT/mTOR signaling by S-nitrosylation of phosphatase and tensin homolog (PTEN) proteins. The mechanism by which NOS1 modifies PTEN protein might involve a direct interaction between these two molecules. Moreover, in an in vivo study, the NOS1 inhibitor N(G)-nitro-L-arginine methyl ester activated AKT/mTOR signaling and promoted autophagy in xenograph tumors. Our studies demonstrated that NOS1 prevents excessive autophagy via S-nitrosylation of PTEN, and activation of the AKT/mTOR signaling pathway. PTEN and the AKT/mTOR signaling pathway are promising targets for improving the chemotherapeutic treatment of cancer.

13.
Cell Rep ; 15(10): 2159-2169, 2016 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-27239026

RESUMO

V(D)J recombination relies on the presence of proximal enhancers that activate the antigen receptor (AgR) loci in a lineage- and stage-specific manner. Unexpectedly, we find that both active and inactive AgR enhancers cooperate to disseminate their effects in a localized and long-range manner. Here, we demonstrate the importance of short-range contacts between active enhancers that constitute an Igk super-enhancer in B cells. Deletion of one element reduces the interaction frequency between other enhancers in the hub, which compromises the transcriptional output of each component. Furthermore, we establish that, in T cells, long-range contact and cooperation between the inactive Igk enhancer MiEκ and the active Tcrb enhancer Eß alters enrichment of CBFß binding in a manner that impacts Tcrb recombination. These findings underline the complexities of enhancer regulation and point to a role for localized and long-range enhancer-sharing between active and inactive elements in lineage- and stage-specific control.


Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Animais , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Camundongos Endogâmicos C57BL , Ligação Proteica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/imunologia
14.
Adv Immunol ; 128: 123-82, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26477367

RESUMO

Given their essential role in adaptive immunity, antigen receptor loci have been the focus of analysis for many years and are among a handful of the most well-studied genes in the genome. Their investigation led initially to a detailed knowledge of linear structure and characterization of regulatory elements that confer control of their rearrangement and expression. However, advances in DNA FISH and imaging combined with new molecular approaches that interrogate chromosome conformation have led to a growing appreciation that linear structure is only one aspect of gene regulation and in more recent years, the focus has switched to analyzing the impact of locus conformation and nuclear organization on control of recombination. Despite decades of work and intense effort from numerous labs, we are still left with an incomplete picture of how the assembly of antigen receptor loci is regulated. This chapter summarizes our advances to date and points to areas that need further investigation.


Assuntos
Elementos Facilitadores Genéticos , Recombinação V(D)J , Animais , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Hibridização in Situ Fluorescente , Regiões Promotoras Genéticas
15.
J Exp Med ; 212(5): 809-24, 2015 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-25847946

RESUMO

Rag1 and Rag2 gene expression in CD4(+)CD8(+) double-positive (DP) thymocytes depends on the activity of a distant anti-silencer element (ASE) that counteracts the activity of an intergenic silencer. However, the mechanistic basis for ASE activity is unknown. Here, we show that the ASE physically interacts with the distant Rag1 and Rag2 gene promoters in DP thymocytes, bringing the two promoters together to form an active chromatin hub. Moreover, we show that the ASE functions as a classical enhancer that can potently activate these promoters in the absence of the silencer or other locus elements. In thymocytes lacking the chromatin organizer SATB1, we identified a partial defect in Tcra gene rearrangement that was associated with reduced expression of Rag1 and Rag2 at the DP stage. SATB1 binds to the ASE and Rag promoters, facilitating inclusion of Rag2 in the chromatin hub and the loading of RNA polymerase II to both the Rag1 and Rag2 promoters. Our results provide a novel framework for understanding ASE function and demonstrate a novel role for SATB1 as a regulator of Rag locus organization and gene expression in DP thymocytes.


Assuntos
Diferenciação Celular/imunologia , Cromatina/imunologia , Proteínas de Ligação a DNA/imunologia , Proteínas de Homeodomínio/imunologia , Proteínas de Ligação à Região de Interação com a Matriz/imunologia , Elementos de Resposta/imunologia , Timócitos/imunologia , Animais , Diferenciação Celular/genética , Cromatina/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/genética , Proteínas de Homeodomínio/genética , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , Camundongos Knockout , Timócitos/citologia
16.
Immunity ; 37(6): 971-85, 2012 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-23159437

RESUMO

Histone 3 lysine 4 trimethylation (H3K4me3) is associated with promoters of active genes and found at hot spots for DNA recombination. Here we have shown that PAXIP1 (also known as PTIP), a protein associated with MLL3 and MLL4 methyltransferase and the DNA damage response, regulates RAG-mediated cleavage and repair during V(D)J recombination in CD4(+) CD8(+) DP thymocytes. Loss of PAXIP1 in developing thymocytes diminished Jα H3K4me3 and germline transcription, suppressed double strand break formation at 3' Jα segments, but resulted in accumulation of unresolved T cell receptor α-chain gene (Tcra) breaks. Moreover, PAXIP1 was essential for release of mature single positive (SP) αß T cells from the thymus through transcriptional activation of sphingosine-1-phosphate receptor S1pr1 as well as for natural killer T cell development. Thus, in addition to maintaining genome integrity during Tcra rearrangements, PAXIP1 controls distinct transcriptional programs during DP differentiation necessary for Tcra locus accessibility, licensing mature thymocytes for trafficking and natural killer T cell development.


Assuntos
Proteínas de Transporte/genética , Diferenciação Celular , Movimento Celular , Dano ao DNA , Regulação da Expressão Gênica , Proteínas Nucleares/genética , Timócitos/citologia , Timócitos/imunologia , Animais , Proteínas de Transporte/metabolismo , Linhagem da Célula/genética , Movimento Celular/genética , Histonas/metabolismo , Camundongos , Células T Matadoras Naturais/citologia , Células T Matadoras Naturais/metabolismo , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Lisoesfingolipídeo/genética , Recombinação Genética , Linfócitos T/citologia , Linfócitos T/metabolismo , Timócitos/metabolismo , Transcrição Genética
17.
Nature ; 476(7361): 467-71, 2011 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-21832993

RESUMO

Cohesin enables post-replicative DNA repair and chromosome segregation by holding sister chromatids together from the time of DNA replication in S phase until mitosis. There is growing evidence that cohesin also forms long-range chromosomal cis-interactions and may regulate gene expression in association with CTCF, mediator or tissue-specific transcription factors. Human cohesinopathies such as Cornelia de Lange syndrome are thought to result from impaired non-canonical cohesin functions, but a clear distinction between the cell-division-related and cell-division-independent functions of cohesion--as exemplified in Drosophila--has not been demonstrated in vertebrate systems. To address this, here we deleted the cohesin locus Rad21 in mouse thymocytes at a time in development when these cells stop cycling and rearrange their T-cell receptor (TCR) α locus (Tcra). Rad21-deficient thymocytes had a normal lifespan and retained the ability to differentiate, albeit with reduced efficiency. Loss of Rad21 led to defective chromatin architecture at the Tcra locus, where cohesion-binding sites flank the TEA promoter and the Eα enhancer, and demarcate Tcra from interspersed Tcrd elements and neighbouring housekeeping genes. Cohesin was required for long-range promoter-enhancer interactions, Tcra transcription, H3K4me3 histone modifications that recruit the recombination machinery and Tcra rearrangement. Provision of pre-rearranged TCR transgenes largely rescued thymocyte differentiation, demonstrating that among thousands of potential target genes across the genome, defective Tcra rearrangement was limiting for the differentiation of cohesin-deficient thymocytes. These findings firmly establish a cell-division-independent role for cohesin in Tcra locus rearrangement and provide a comprehensive account of the mechanisms by which cohesin enables cellular differentiation in a well-characterized mammalian system.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Proteínas Cromossômicas não Histona/metabolismo , Rearranjo Gênico do Linfócito T , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Timo/citologia , Animais , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Regulação da Expressão Gênica , Rearranjo Gênico do Linfócito T/genética , Genes RAG-1/genética , Camundongos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Recombinases/metabolismo , Timo/metabolismo , Transcrição Genética
18.
J Immunol ; 187(5): 2484-91, 2011 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-21784972

RESUMO

Murine Tcra and Tcrd gene segments are organized into a single genetic locus (Tcra/Tcrd locus) that undergoes V(D)J recombination in CD4(-)CD8(-) double-negative (DN) thymocytes to assemble Tcrd genes and in CD4(+)CD8(+) double-positive thymocytes to assemble Tcra genes. Recombination events are regulated by two developmental stage-specific enhancers, E(δ) and E(α). Effects of E(α) on Trca/Tcrd locus chromatin have been well documented, but effects of E(δ) have not. In this regard, E(α) acts over long distances to activate many V(α) and J(α) segments for recombination in double-positive thymocytes. However, in DN thymocytes, it is unclear whether E(δ) functions over long distances to regulate V(δ) gene segments or functions only locally to regulate D(δ) and J(δ) gene segments. In this study, we analyzed germline transcription, histone modifications, and recombination on wild-type and E(δ)-deficient alleles in adult and fetal thymocytes. We found that E(δ) functions as a local enhancer whose influence is limited to no more than ∼10 kb in either direction (including D(δ), J(δ), and TRDV5 gene segments) in adult DN thymocytes. However, we identified a unique long-distance role for E(δ) promoting accessibility and recombination of fetal V(δ) gene segment TRDV4, over a distance of 55 kb, in fetal thymocytes. TRDV4 recombination is specifically repressed in adult thymocytes. We found that this repression is enforced by a developmentally regulated loss of histone acetylation. Constitutively high levels of a suppressive modification, histone H3 lysine 9 dimethylation, may contribute to repression as well.


Assuntos
Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Rearranjo Gênico da Cadeia delta dos Receptores de Antígenos dos Linfócitos T/genética , Genes Codificadores da Cadeia delta de Receptores de Linfócitos T/genética , Linfopoese/genética , Linfócitos T/imunologia , Animais , Southern Blotting , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Feto , Expressão Gênica , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T/genética , Histonas/genética , Histonas/metabolismo , Hibridização In Situ , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
J Immunol ; 186(6): 3556-62, 2011 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-21317385

RESUMO

In CD4(-)CD8(-) double-negative thymocytes, the murine Tcrb locus is composed of alternating blocks of active and inactive chromatin containing Tcrb gene segments and trypsinogen genes, respectively. Although chromatin structure is appreciated to be critical for regulated recombination and expression of Tcrb gene segments, the molecular mechanisms that maintain the integrity of these differentially regulated Tcrb locus chromatin domains are not understood. We localized a boundary between active and inactive chromatin by mapping chromatin modifications across the interval extending from Prss2 (the most 3' trypsinogen gene) to D(ß)1. This boundary, located 6 kb upstream of D(ß)1, is characterized by a transition from repressive (histone H3 lysine 9 dimethylation [H3K9me2]) to active (histone H3 acetylation [H3ac]) chromatin and is marked by a peak of histone H3 lysine 4 dimethylation (H3K4me2) that colocalizes with a retroviral long terminal repeat (LTR). Histone H3 lysine 4 dimethylation is retained and histone H3 lysine 9 dimethylation fails to spread past the LTR even on alleles lacking the Tcrb enhancer (E(ß)) suggesting that these features may be determined by the local DNA sequence. Notably, we found that LTR-containing DNA functions as a barrier-type insulator that can protect a transgene from negative chromosomal position effects. We propose that, in vivo, the LTR blocks the spread of heterochromatin, and thereby helps to maintain the integrity of the E(ß)-regulated chromatin domain. We also identified low-abundance, E(ß)-dependent transcripts that initiate at the border of the LTR and an adjacent long interspersed element. We speculate that this transcription, which extends across D(ß), J(ß) and C(ß) gene segments, may play an additional role promoting initial opening of the E(ß)-regulated chromatin domain.


Assuntos
Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/imunologia , Heterocromatina/metabolismo , Elementos Isolantes/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Ativação Transcricional/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Metilação de DNA/genética , Metilação de DNA/imunologia , Elementos Facilitadores Genéticos/imunologia , Heterocromatina/genética , Histonas/genética , Histonas/metabolismo , Humanos , Elementos Isolantes/genética , Células Jurkat , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fatores do Domínio POU/deficiência , Fatores do Domínio POU/genética , Fatores do Domínio POU/metabolismo , Estrutura Terciária de Proteína/genética , Subpopulações de Linfócitos T/citologia , Tripsinogênio/antagonistas & inibidores , Tripsinogênio/genética
20.
Immunol Res ; 49(1-3): 192-201, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21128009

RESUMO

V(D)J recombination is regulated through changes in chromatin structure that allow recombinase proteins access to recombination signal sequences and through changes in three-dimensional chromatin organization that bring pairs of distant recombination signal sequences into proximity. The Tcra/Tcrd locus is complex and undergoes distinct recombination programs in double negative and double positive thymocytes that lead to the assembly of Tcrd and Tcra genes, respectively. Our studies provide insights into how locus chromatin structure is regulated and how changes in locus chromatin structure can target and then retarget the recombinase to create developmental progressions of recombination events. Our studies also reveal distinct locus conformations in double negative and double positive thymocytes and suggest how these conformations may support the distinct recombination programs in the two compartments.


Assuntos
Cromatina , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Cromatina/química , Cromatina/metabolismo , Rearranjo Gênico , Genes RAG-1/genética , Humanos , Receptores de Antígenos de Linfócitos T gama-delta/genética , Recombinação Genética , Transdução de Sinais/genética , Hipermutação Somática de Imunoglobulina , Linfócitos T/citologia , VDJ Recombinases
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