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1.
EMBO J ; 35(3): 335-55, 2016 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-26711177

RESUMEN

Intragenic 5-methylcytosine and CTCF mediate opposing effects on pre-mRNA splicing: CTCF promotes inclusion of weak upstream exons through RNA polymerase II pausing, whereas 5-methylcytosine evicts CTCF, leading to exon exclusion. However, the mechanisms governing dynamic DNA methylation at CTCF-binding sites were unclear. Here, we reveal the methylcytosine dioxygenases TET1 and TET2 as active regulators of CTCF-mediated alternative splicing through conversion of 5-methylcytosine to its oxidation derivatives. 5-hydroxymethylcytosine and 5-carboxylcytosine are enriched at an intragenic CTCF-binding sites in the CD45 model gene and are associated with alternative exon inclusion. Reduced TET levels culminate in increased 5-methylcytosine, resulting in CTCF eviction and exon exclusion. In vitro analyses establish the oxidation derivatives are not sufficient to stimulate splicing, but efficiently promote CTCF association. We further show genomewide that reciprocal exchange of 5-hydroxymethylcytosine and 5-methylcytosine at downstream CTCF-binding sites is a general feature of alternative splicing in naïve and activated CD4(+) T cells. These findings significantly expand our current concept of the pre-mRNA "splicing code" to include dynamic intragenic DNA methylation catalyzed by the TET proteins.


Asunto(s)
5-Metilcitosina/metabolismo , Empalme Alternativo , Proteínas de Unión al ADN/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Factor de Unión a CCCTC , Línea Celular , Dioxigenasas , Humanos , Oxigenasas de Función Mixta , Oxidación-Reducción
2.
Retrovirology ; 11: 119, 2014 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-25519886

RESUMEN

BACKGROUND: Reprogramming cellular gene transcription sustains HTLV-1 viral persistence that ultimately leads to the development of adult T-cell leukemia/lymphoma (ATLL). We hypothesized that besides these quantitative transcriptional effects, HTLV-1 qualitatively modifies the pattern of cellular gene expression. RESULTS: Exon expression analysis shows that patients' untransformed and malignant HTLV-1(+) CD4(+) T-cells exhibit multiple alternate exon usage (AEU) events. These affect either transcriptionally modified or unmodified genes, culminate in ATLL, and unveil new functional pathways involved in cancer and cell cycle. Unsupervised hierarchical clustering of array data permitted to isolate exon expression patterns of 3977 exons that discriminate uninfected, infected, and transformed CD4(+) T-cells. Furthermore, untransformed infected CD4+ clones and ATLL samples shared 486 exon modifications distributed in 320 genes, thereby indicating a role of AEUs in HTLV-1 leukemogenesis. Exposing cells to splicing modulators revealed that Sudemycin E reduces cell viability of HTLV-1 transformed cells without affecting primary control CD4+ cells and HTLV-1 negative cell lines, suggesting that the huge excess of AEU might provide news targets for treating ATLL. CONCLUSIONS: Taken together, these data reveal that HTLV-1 significantly modifies the structure of cellular transcripts and unmask new putative leukemogenic pathways and possible therapeutic targets.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Exones , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Virus Linfotrópico T Tipo 1 Humano/fisiología , Leucemia-Linfoma de Células T del Adulto/patología , Virus Linfotrópico T Tipo 1 Humano/crecimiento & desarrollo , Humanos , Transcripción Genética
4.
Cancers (Basel) ; 15(3)2023 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-36765607

RESUMEN

T-cell lymphoblastic lymphoma (T-LBL) is a rare and aggressive lymphatic cancer, often diagnosed at a young age. Patients are treated with intensive chemotherapy, potentially followed by a hematopoietic stem cell transplantation. Although prognosis of T-LBL has improved with intensified treatment protocols, they are associated with side effects and 10-20% of patients still die from relapsed or refractory disease. Given this, the search toward less toxic anti-lymphoma therapies is ongoing. Here, we targeted the recently described DNA hypermethylated profile in T-LBL with the DNA hypomethylating agent decitabine. We evaluated the anti-lymphoma properties and downstream effects of decitabine, using patient derived xenograft (PDX) models. Decitabine treatment resulted in prolonged lymphoma-free survival in all T-LBL PDX models, which was associated with downregulation of the oncogenic MYC pathway. However, some PDX models showed more benefit of decitabine treatment compared to others. In more sensitive models, differentially methylated CpG regions resulted in more differentially expressed genes in open chromatin regions. This resulted in stronger downregulation of cell cycle genes and upregulation of immune response activating transcripts. Finally, we suggest a gene signature for high decitabine sensitivity in T-LBL. Altogether, we here delivered pre-clinical proof of the potential use of decitabine as a new therapeutic agent in T-LBL.

5.
Nat Commun ; 11(1): 3045, 2020 06 16.
Artículo en Inglés | MEDLINE | ID: mdl-32546717

RESUMEN

Chronic NF-κB activation in inflammation and cancer has long been linked to persistent activation of NF-κB-responsive gene promoters. However, NF-κB factors also massively bind to gene bodies. Here, we demonstrate that recruitment of the NF-κB factor RELA to intragenic regions regulates alternative splicing upon NF-κB activation by the viral oncogene Tax of HTLV-1. Integrative analyses of RNA splicing and chromatin occupancy, combined with chromatin tethering assays, demonstrate that DNA-bound RELA interacts with and recruits the splicing regulator DDX17, in an NF-κB activation-dependent manner. This leads to alternative splicing of target exons due to the RNA helicase activity of DDX17. Similar results were obtained upon Tax-independent NF-κB activation, indicating that Tax likely exacerbates a physiological process where RELA provides splice target specificity. Collectively, our results demonstrate a physical and direct involvement of NF-κB in alternative splicing regulation, which significantly revisits our knowledge of HTLV-1 pathogenesis and other NF-κB-related diseases.


Asunto(s)
Empalme Alternativo/fisiología , Productos del Gen tax/metabolismo , FN-kappa B/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Regulación de la Expresión Génica , Productos del Gen tax/genética , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Humanos , Leucocitos Mononucleares/virología , FN-kappa B/genética , Oncogenes , Factor de Transcripción ReIA/metabolismo
6.
Blood Cancer Discov ; 1(3): 274-289, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-33179015

RESUMEN

Cancer cells display DNA hypermethylation at specific CpG islands in comparison to their normal healthy counterparts, but the mechanism that drives this so-called CpG island methylator phenotype (CIMP) remains poorly understood. Here, we show that CpG island methylation in human T-cell acute lymphoblastic leukemia (T-ALL) mainly occurs at promoters of Polycomb Repressor Complex 2 (PRC2) target genes that are not expressed in normal or malignant T-cells and which display a reciprocal association with H3K27me3 binding. In addition, we revealed that this aberrant methylation profile reflects the epigenetic history of T-ALL and is established already in pre-leukemic, self-renewing thymocytes that precede T-ALL development. Finally, we unexpectedly uncover that this age-related CpG island hypermethylation signature in T-ALL is completely resistant to the FDA-approved hypomethylating agent Decitabine. Altogether, we here provide conceptual evidence for the involvement of a pre-leukemic phase characterized by self-renewing thymocytes in the pathogenesis of human T-ALL.


Asunto(s)
Envejecimiento , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Timocitos , Islas de CpG/genética , Metilación de ADN/genética , Humanos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética
7.
iScience ; 19: 326-339, 2019 Sep 27.
Artículo en Inglés | MEDLINE | ID: mdl-31404833

RESUMEN

The mechanisms supporting dynamic regulation of CTCF-binding sites remain poorly understood. Here we describe the TET-catalyzed 5-methylcytosine derivative, 5-carboxylcytosine (5caC), as a factor driving new CTCF binding within genomic DNA. Through a combination of in vivo and in vitro approaches, we reveal that 5caC generally strengthens CTCF association with DNA and facilitates binding to suboptimal sequences. Dramatically, profiling of CTCF binding in a cellular model that accumulates genomic 5caC identified ~13,000 new CTCF sites. The new sites were enriched for overlapping 5caC and were marked by an overall reduction in CTCF motif strength. As CTCF has multiple roles in gene expression, these findings have wide-reaching implications and point to induced 5caC as a potential mechanism to achieve differential CTCF binding in cells.

8.
Nat Commun ; 9(1): 4281, 2018 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-30323192

RESUMEN

T-acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with complicated heterogeneity. Although expression profiling reveals common elevated genes in distinct T-ALL subtypes, little is known about their functional role(s) and regulatory mechanism(s). We here show that SHQ1, an H/ACA snoRNP assembly factor involved in snRNA pseudouridylation, is highly expressed in T-ALL. Mechanistically, oncogenic NOTCH1 directly binds to the SHQ1 promoter and activates its transcription. SHQ1 depletion induces T-ALL cell death in vitro and prolongs animal survival in murine T-ALL models. RNA-Seq reveals that SHQ1 depletion impairs widespread RNA splicing, and MYC is one of the most prominently downregulated genes due to inefficient splicing. MYC overexpression significantly rescues T-ALL cell death resulted from SHQ1 inactivation. We herein report a mechanism of NOTCH1-SHQ1-MYC axis in T-cell leukemogenesis. These findings not only shed light on the role of SHQ1 in RNA splicing and tumorigenesis, but also provide additional insight into MYC regulation.


Asunto(s)
Proteínas Portadoras/metabolismo , Regulación Leucémica de la Expresión Génica , Leucemia de Células T/genética , Leucemia de Células T/patología , Empalme del ARN/genética , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , Proteínas Portadoras/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Técnicas de Inactivación de Genes , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones Endogámicos C57BL , Modelos Biológicos , Unión Proteica , Precursores del ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Notch1/metabolismo
9.
Oncotarget ; 7(3): 2889-909, 2016 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-26284582

RESUMEN

In addition to spliceosome gene mutations, oncogene expression and drug resistance in AML might influence exon expression. We performed exon-array analysis and exon-specific PCR (ESPCR) to identify specific landscapes of exon expression that are associated with DEK and WT1 oncogene expression and the resistance of AML cells to AraC, doxorubicin or azacitidine. Data were obtained for these five conditions through exon-array analysis of 17 cell lines and 24 patient samples and were extended through qESPCR of samples from 152 additional AML cases. More than 70% of AEUs identified by exon-array were technically validated through ESPCR. In vitro, 1,130 to 5,868 exon events distinguished the 5 conditions from their respective controls while in vivo 6,560 and 9,378 events distinguished chemosensitive and chemoresistant AML, respectively, from normal bone marrow. Whatever the cause of this effect, 30 to 80% of mis-spliced mRNAs involved genes unmodified at the whole transcriptional level. These AEUs unmasked new functional pathways that are distinct from those generated by transcriptional deregulation. These results also identified new putative pathways that could help increase the understanding of the effects mediated by DEK or WT1, which may allow the targeting of these pathways to prevent resistance of AML cells to chemotherapeutic agents.


Asunto(s)
Empalme Alternativo/genética , Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Anciano , Antraciclinas/farmacología , Azacitidina/farmacología , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/metabolismo , Citarabina/farmacología , Doxorrubicina/farmacología , Exones/genética , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Masculino , Persona de Mediana Edad , Proteínas Oncogénicas/metabolismo , Oncogenes/genética , Proteínas de Unión a Poli-ADP-Ribosa , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas WT1/metabolismo
10.
Oncotarget ; 5(20): 9534-45, 2014 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-25375204

RESUMEN

Approximately one-third of expressed genes are misspliced in AML, opening the possibility that additional factors than splicing factor mutations might cause RNA missplicing in these diseases. AML cells harbor a constellation of epigenetic modifications and regularly express large amounts of WT1 transcripts. Histone acetylation/methylation and DNA CpG methylation favor either exon skipping or inclusion, mainly through interfering with RNA Pol II-mediated elongation. This can result either from the binding of various factors on Pol II or alternatively from the recruitment of DNA binding factors that create roadblocks to Pol II-induced elongation. WT1 exhibits pleiotropic effects on mRNA splicing, which mainly result from the binding properties of WT1 via its zinc fingers domains to DNA, RNA, and proteins. Through the repression of the kinase SRPK1, WT1 modifies the splicing of VEGF, which plays important roles in hematopoiesis and angiogenesis. At the protein level, WT1 interacts with the splicing factors U2AF2, WTAP, and RPM4. Therefore, AML cells appear to have acquired numerous properties known to interfere with mRNA splicing. The challenge is now to elucidate these links in order to trigger mRNA splicing at the therapeutic level.


Asunto(s)
Leucemia Mieloide Aguda/genética , Empalme del ARN , ARN Mensajero/genética , Animales , Epigenómica , Expresión Génica , Humanos
11.
Cancer Res ; 74(21): 6082-93, 2014 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-25205102

RESUMEN

Viruses disrupt the host cell microRNA (miRNA) network to facilitate their replication. Human T-cell leukemia virus type I (HTLV-1) replication relies on the clonal expansion of its host CD4(+) and CD8(+) T cells, yet this virus causes adult T-cell leukemia/lymphoma (ATLL) that typically has a CD4(+) phenotype. The viral oncoprotein Tax, which is rarely expressed in ATLL cells, has long been recognized for its involvement in tumor initiation by promoting cell proliferation, genetic instability, and miRNA dysregulation. Meanwhile, HBZ is expressed in both untransformed infected cells and ATLL cells and is involved in sustaining cell proliferation and silencing virus expression. Here, we show that an HBZ-miRNA axis promotes cell proliferation and genetic instability, as indicated by comet assays that showed increased numbers of DNA-strand breaks. Expression profiling of miRNA revealed that infected CD4(+) cells, but not CD8(+) T cells, overexpressed oncogenic miRNAs, including miR17 and miR21. HBZ activated these miRNAs via a posttranscriptional mechanism. These effects were alleviated by knocking down miR21 or miR17 and by ectopic expression of OBFC2A, a DNA-damage factor that is downregulated by miR17 and miR21 in HTLV-1-infected CD4(+) T cells. These findings extend the oncogenic potential of HBZ and suggest that viral expression might be involved in the remarkable genetic instability of ATLL cells.


Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Proliferación Celular/genética , Inestabilidad Genómica , Leucemia-Linfoma de Células T del Adulto/genética , Proteínas Virales/genética , Adulto , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Regulación Viral de la Expresión Génica , Genes pX/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Leucemia-Linfoma de Células T del Adulto/inmunología , Leucemia-Linfoma de Células T del Adulto/patología , Proteínas de los Retroviridae , Proteínas Virales/metabolismo
12.
Neoplasia ; 16(1): 21-30, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24563617

RESUMEN

Although numerous factors have been found to modulate hTERT transcription, the mechanism of its repression in certain leukemias remains unknown. We show here that DEK represses hTERT transcription through its enrichment on the hTERT promoter in cells from chronic and acute myeloid leukemias, chronic lymphocytic leukemia, but not acute lymphocytic leukemias where hTERT is overexpressed. We isolated DEK from the hTERT promoter incubated with nuclear extracts derived from fresh acute myelogenous leukemia (AML) cells and from cells expressing Tax, an hTERT repressor encoded by the human T cell leukemia virus type 1. In addition to the recruitment of DEK, the displacement of two potent known hTERT transactivators from the hTERT promoter characterized both AML cells and Tax-expressing cells. Reporter and chromatin immunoprecipitation assays permitted to map the region that supports the repressive effect of DEK on hTERT transcription, which was proportionate to the level of DEK-promoter association but not with the level of DEK expression. Besides hTERT repression, this context of chromatin redistribution of DEK was found to govern about 40% of overall transcriptional modifications, including those of cancer-prone genes. In conclusion, DEK emerges as an hTERT repressor shared by various leukemia subtypes and seems involved in the deregulation of numerous genes associated with leukemogenesis.


Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/genética , Proteínas Oncogénicas/metabolismo , Telomerasa/genética , Telomerasa/metabolismo , Células de la Médula Ósea/citología , Núcleo Celular/metabolismo , Perfilación de la Expresión Génica , Productos del Gen tax/metabolismo , Células HeLa , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Células Jurkat , Leucemia Mieloide Aguda/metabolismo , Hibridación de Ácido Nucleico , Proteínas Oncogénicas/genética , Proteínas de Unión a Poli-ADP-Ribosa , Regiones Promotoras Genéticas
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