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1.
Cell ; 169(3): 523-537.e15, 2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28431250

RESUMO

The distribution of sense and antisense strand DNA mutations on transcribed duplex DNA contributes to the development of immune and neural systems along with the progression of cancer. Because developmentally matured B cells undergo biologically programmed strand-specific DNA mutagenesis at focal DNA/RNA hybrid structures, they make a convenient system to investigate strand-specific mutagenesis mechanisms. We demonstrate that the sense and antisense strand DNA mutagenesis at the immunoglobulin heavy chain locus and some other regions of the B cell genome depends upon localized RNA processing protein complex formation in the nucleus. Both the physical proximity and coupled activities of RNA helicase Mtr4 (and senataxin) with the noncoding RNA processing function of RNA exosome determine the strand-specific distribution of DNA mutations. Our study suggests that strand-specific DNA mutagenesis-associated mechanisms will play major roles in other undiscovered aspects of organismic development.


Assuntos
Linfócitos B/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Núcleo Celular/metabolismo , DNA Helicases/metabolismo , Exorribonucleases/genética , Instabilidade Genômica , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Enzimas Multifuncionais , Proteínas Nucleares/genética , RNA Helicases , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/genética
2.
Cell ; 162(4): 697-8, 2015 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-26276622

RESUMO

Nussenzweig and colleagues evaluate genomic instability and germinal center derived lymphomagenesis in mice infected with Plasmodium to recreate some of the hallmark characteristics of Burkitt lymphoma, a form of cancer more common in parts of Africa where malaria is endemic.


Assuntos
Citidina Desaminase/metabolismo , Linfoma de Células B/enzimologia , Linfoma de Células B/genética , Translocação Genética , Animais , Humanos
3.
Cell ; 161(4): 774-89, 2015 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-25957685

RESUMO

We have ablated the cellular RNA degradation machinery in differentiated B cells and pluripotent embryonic stem cells (ESCs) by conditional mutagenesis of core (Exosc3) and nuclear RNase (Exosc10) components of RNA exosome and identified a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) with emergent functionality. Unexpectedly, eRNA-expressing regions accumulate R-loop structures upon RNA exosome ablation, thus demonstrating the role of RNA exosome in resolving deleterious DNA/RNA hybrids arising from active enhancers. We have uncovered a distal divergent eRNA-expressing element (lncRNA-CSR) engaged in long-range DNA interactions and regulating IgH 3' regulatory region super-enhancer function. CRISPR-Cas9-mediated ablation of lncRNA-CSR transcription decreases its chromosomal looping-mediated association with the IgH 3' regulatory region super-enhancer and leads to decreased class switch recombination efficiency. We propose that the RNA exosome protects divergently transcribed lncRNA expressing enhancers by resolving deleterious transcription-coupled secondary DNA structures, while also regulating long-range super-enhancer chromosomal interactions important for cellular function.


Assuntos
Linfócitos B/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Regulação da Expressão Gênica , RNA Longo não Codificante/metabolismo , Animais , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Instabilidade Genômica , Heterocromatina/metabolismo , Switching de Imunoglobulina , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Sequências Reguladoras de Ácido Nucleico
4.
Mol Cell ; 81(19): 3949-3964.e7, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34450044

RESUMO

Immunoglobulin heavy chain (IgH) locus-associated G-rich long noncoding RNA (SµGLT) is important for physiological and pathological B cell DNA recombination. We demonstrate that the METTL3 enzyme-catalyzed N6-methyladenosine (m6A) RNA modification drives recognition and 3' end processing of SµGLT by the RNA exosome, promoting class switch recombination (CSR) and suppressing chromosomal translocations. The recognition is driven by interaction of the MPP6 adaptor protein with nuclear m6A reader YTHDC1. MPP6 and YTHDC1 promote CSR by recruiting AID and the RNA exosome to actively transcribe SµGLT. Direct suppression of m6A modification of SµGLT or of m6A reader YTHDC1 reduces CSR. Moreover, METTL3, an essential gene for B cell development in the bone marrow and germinal center, suppresses IgH-associated aberrant DNA breaks and prevents genomic instability. Taken together, we propose coordinated and central roles for MPP6, m6A modification, and m6A reader proteins in controlling long noncoding RNA processing, DNA recombination, and development in B cells.


Assuntos
Adenosina/análogos & derivados , Linfócitos B/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Processamento de Terminações 3' de RNA , RNA Longo não Codificante/metabolismo , Recombinação Genética , Adenosina/metabolismo , Animais , Linfócitos B/imunologia , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Feminino , Instabilidade Genômica , Células HEK293 , Humanos , Switching de Imunoglobulina , Cadeias Pesadas de Imunoglobulinas/genética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos Knockout , RNA Longo não Codificante/genética , RNA não Traduzido/genética , RNA não Traduzido/metabolismo
5.
Genome Res ; 31(9): 1663-1679, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34426512

RESUMO

Antibodies offer a powerful means to interrogate specific proteins in a complex milieu. However, antibody availability and reliability can be problematic, whereas epitope tagging can be impractical in many cases. To address these limitations, the Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies (mAbs) against human presumptive chromatin proteins. However, these reagents have not been widely field-tested. We therefore performed a screen to test their ability to enrich genomic regions via chromatin immunoprecipitation (ChIP) and a variety of orthogonal assays. Eight hundred eighty-seven unique antibodies against 681 unique human transcription factors (TFs) were assayed by ultra-high-resolution ChIP-exo/seq, generating approximately 1200 ChIP-exo data sets, primarily in a single pass in one cell type (K562). Subsets of PCRP mAbs were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. About 5% of the tested antibodies displayed high-confidence target (i.e., cognate antigen) enrichment across at least one assay and are strong candidates for additional validation. An additional 34% produced ChIP-exo data that were distinct from background and thus warrant further testing. The remaining 61% were not substantially different from background, and likely require consideration of a much broader survey of cell types and/or assay optimizations. We show and discuss the metrics and challenges to antibody validation in chromatin-based assays.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Fatores de Transcrição , Sítios de Ligação , Imunoprecipitação da Cromatina , Humanos , Indicadores e Reagentes , Reprodutibilidade dos Testes , Fatores de Transcrição/metabolismo
6.
Genes Dev ; 27(1): 1-17, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23307864

RESUMO

The mechanisms by which B cells somatically engineer their genomes to generate the vast diversity of antibodies required to challenge the nearly infinite number of antigens that immune systems encounter are of tremendous clinical and academic interest. The DNA cytidine deaminase activation-induced deaminase (AID) catalyzes two of these mechanisms: class switch recombination (CSR) and somatic hypermutation (SHM). Recent discoveries indicate a significant promiscuous targeting of this B-cell mutator enzyme genome-wide. Here we discuss the various regulatory elements that control AID activity and prevent AID from inducing genomic instability and thereby initiating oncogenesis.


Assuntos
Linfócitos B/enzimologia , Linfócitos B/imunologia , Citidina Desaminase/metabolismo , Genoma , Mutagênese , Imunidade Adaptativa , Animais , Instabilidade Cromossômica , Ativação Enzimática , Humanos , Switching de Imunoglobulina , Hipermutação Somática de Imunoglobulina
7.
Trends Genet ; 33(2): 143-154, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28087167

RESUMO

Intergenic and intragenic enhancers found inside topologically associated regulatory domains (TADs) express noncoding RNAs, known as enhancer RNAs (eRNAs). Recent studies have indicated these eRNAs play a role in gene regulatory networks by controlling promoter and enhancer interactions and topology of higher-order chromatin structure. Misregulation of enhancer and promoter associated noncoding RNAs (ncRNAs) could stabilize deleterious secondary DNA structures, noncoding RNA associated DNA/RNA hybrid formation, and promote collisions of transcription complexes with replisomes. It is revealing that many chromosomal aberrations, some associated with malignancies, are present inside enhancer and/or promoter sequences. Here, we expand on current concepts to discuss enhancer RNAs and enhancer transcription, and how enhancer transcription influences genomic organization and integrity.


Assuntos
DNA/genética , Conformação de Ácido Nucleico , RNA não Traduzido/genética , Transcrição Gênica , Cromatina/química , Cromatina/genética , DNA/química , Elementos Facilitadores Genéticos , Redes Reguladoras de Genes/genética , Instabilidade Genômica/genética , Genômica , Humanos , Regiões Promotoras Genéticas , RNA não Traduzido/química
8.
Nature ; 514(7522): 389-93, 2014 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-25119026

RESUMO

The vast majority of the mammalian genome has the potential to express noncoding RNA (ncRNA). The 11-subunit RNA exosome complex is the main source of cellular 3'-5' exoribonucleolytic activity and potentially regulates the mammalian noncoding transcriptome. Here we generated a mouse model in which the essential subunit Exosc3 of the RNA exosome complex can be conditionally deleted. Exosc3-deficient B cells lack the ability to undergo normal levels of class switch recombination and somatic hypermutation, two mutagenic DNA processes used to generate antibody diversity via the B-cell mutator protein activation-induced cytidine deaminase (AID). The transcriptome of Exosc3-deficient B cells has revealed the presence of many novel RNA exosome substrate ncRNAs. RNA exosome substrate RNAs include xTSS-RNAs, transcription start site (TSS)-associated antisense transcripts that can exceed 500 base pairs in length and are transcribed divergently from cognate coding gene transcripts. xTSS-RNAs are most strongly expressed at genes that accumulate AID-mediated somatic mutations and/or are frequent translocation partners of DNA double-strand breaks generated at Igh in B cells. Strikingly, translocations near TSSs or within gene bodies occur over regions of RNA exosome substrate ncRNA expression. These RNA exosome-regulated, antisense-transcribed regions of the B-cell genome recruit AID and accumulate single-strand DNA structures containing RNA-DNA hybrids. We propose that RNA exosome regulation of ncRNA recruits AID to single-strand DNA-forming sites of antisense and divergent transcription in the B-cell genome, thereby creating a link between ncRNA transcription and overall maintenance of B-cell genomic integrity.


Assuntos
Linfócitos B/metabolismo , Citidina Desaminase/metabolismo , RNA não Traduzido/biossíntese , RNA não Traduzido/genética , Transcrição Gênica/genética , Animais , Pareamento de Bases , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/deficiência , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Exossomos/metabolismo , Feminino , Genoma/genética , Instabilidade Genômica/genética , Switching de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas/genética , Masculino , Camundongos , Hibridização de Ácido Nucleico , RNA Antissenso/biossíntese , RNA Antissenso/química , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA não Traduzido/química , RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/genética , Hipermutação Somática de Imunoglobulina/genética , Especificidade por Substrato , Sítio de Iniciação de Transcrição , Translocação Genética/genética
9.
J Cell Sci ; 125(Pt 9): 2172-84, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22328497

RESUMO

Integrin-ß1-null keratinocytes can adhere to fibronectin through integrin αvß6, but form large peripheral focal adhesions and exhibit defective cell spreading. Here we report that, in addition to the reduced avidity of αvß6 integrin binding to fibronectin, the inability of integrin ß6 to efficiently bind and recruit kindlin-2 to focal adhesions directly contributes to these phenotypes. Kindlins regulate integrins through direct interactions with the integrin-ß cytoplasmic tail and keratinocytes express kindlin-1 and kindlin-2. Notably, although both kindlins localize to focal adhesions in wild-type cells, only kindlin-1 localizes to the integrin-ß6-rich adhesions of integrin-ß1-null cells. Rescue of these cells with wild-type and chimeric integrin constructs revealed a correlation between kindlin-2 recruitment and cell spreading. Furthermore, despite the presence of kindlin-1, knockdown of kindlin-2 in wild-type keratinocytes impaired cell spreading. Our data reveal unexpected functional consequences of differences in the association of two homologous kindlin isoforms with two closely related integrins, and suggest that despite their similarities, different kindlins are likely to have unique functions.


Assuntos
Antígenos de Neoplasias/metabolismo , Integrina beta1/metabolismo , Integrinas/metabolismo , Queratinócitos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Sequência de Aminoácidos , Adesão Celular/fisiologia , Fibronectinas/metabolismo , Citometria de Fluxo , Adesões Focais , Técnicas de Inativação de Genes , Humanos , Integrina beta1/genética , Queratinócitos/citologia , Proteínas de Membrana/química , Dados de Sequência Molecular , Proteínas de Neoplasias/química , Alinhamento de Sequência
10.
Signal Transduct Target Ther ; 8(1): 309, 2023 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-37644009

RESUMO

Major depressive disorder (MDD) is a chronic, generally episodic and debilitating disease that affects an estimated 300 million people worldwide, but its pathogenesis is poorly understood. The heritability estimate of MDD is 30-40%, suggesting that genetics alone do not account for most of the risk of major depression. Another factor known to associate with MDD involves environmental stressors such as childhood adversity and recent life stress. Recent studies have emerged to show that the biological impact of environmental factors in MDD and other stress-related disorders is mediated by a variety of epigenetic modifications. These epigenetic modification alterations contribute to abnormal neuroendocrine responses, neuroplasticity impairment, neurotransmission and neuroglia dysfunction, which are involved in the pathophysiology of MDD. Furthermore, epigenetic marks have been associated with the diagnosis and treatment of MDD. The evaluation of epigenetic modifications holds promise for further understanding of the heterogeneous etiology and complex phenotypes of MDD, and may identify new therapeutic targets. Here, we review preclinical and clinical epigenetic findings, including DNA methylation, histone modification, noncoding RNA, RNA modification, and chromatin remodeling factor in MDD. In addition, we elaborate on the contribution of these epigenetic mechanisms to the pathological trait variability in depression and discuss how such mechanisms can be exploited for therapeutic purposes.


Assuntos
Transtorno Depressivo Maior , Humanos , Transtorno Depressivo Maior/genética , Transtorno Depressivo Maior/terapia , Relevância Clínica , Depressão , Epigênese Genética/genética , Metilação de DNA/genética
11.
Nat Genet ; 55(12): 2160-2174, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38049665

RESUMO

Whole-genome sequencing of longitudinal tumor pairs representing transformation of follicular lymphoma to high-grade B cell lymphoma with MYC and BCL2 rearrangements (double-hit lymphoma) identified coding and noncoding genomic alterations acquired during lymphoma progression. Many of these transformation-associated alterations recurrently and focally occur at topologically associating domain resident regulatory DNA elements, including H3K4me3 promoter marks located within H3K27ac super-enhancer clusters in B cell non-Hodgkin lymphoma. One region found to undergo recurrent alteration upon transformation overlaps a super-enhancer affecting the expression of the PAX5/ZCCHC7 gene pair. ZCCHC7 encodes a subunit of the Trf4/5-Air1/2-Mtr4 polyadenylation-like complex and demonstrated copy number gain, chromosomal translocation and enhancer retargeting-mediated transcriptional upregulation upon lymphoma transformation. Consequently, lymphoma cells demonstrate nucleolar dysregulation via altered noncoding 5.8S ribosomal RNA processing. We find that a noncoding mutation acquired during lymphoma progression affects noncoding rRNA processing, thereby rewiring protein synthesis leading to oncogenic changes in the lymphoma proteome.


Assuntos
Linfoma de Células B , Linfoma , Humanos , Mutação , Linfoma de Células B/genética , Linfoma de Células B/patologia , Translocação Genética/genética , Linfoma/genética , Sequências Reguladoras de Ácido Nucleico
12.
Nature ; 442(7101): 471-4, 2006 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-16810178

RESUMO

In the developing nervous system, Id2 (inhibitor of DNA binding 2, also known as inhibitor of differentiation 2) enhances cell proliferation, promotes tumour progression and inhibits the activity of neurogenic basic helix-loop-helix (bHLH) transcription factors. The anaphase promoting complex/cyclosome and its activator Cdh1 (APC/C(Cdh1)) restrains axonal growth but the targets of APC/C(Cdh1) in neurons are unknown. Id2 and other members of the Id family are very unstable proteins that are eliminated as cells enter the quiescent state, but how they are targeted for degradation has remained elusive. Here we show that Id2 interacts with the core subunits of APC/C and Cdh1 in primary neurons. APC/C(Cdh1) targets Id2 for degradation through a destruction box motif (D box) that is conserved in Id1 and Id4. Depletion of Cdh1 stabilizes Id proteins in neurons, whereas Id2 D-box mutants are impaired for Cdh1 binding and remain stable in cells that exit from the cell cycle and contain active APC/C(Cdh1). Mutants of the Id2 D box enhance axonal growth in cerebellar granule neurons in vitro and in the context of the cerebellar cortex, and overcome the myelin inhibitory signals for growth. Conversely, activation of bHLH transcription factors induces a cluster of genes with potent axonal inhibitory functions including the gene coding for the Nogo receptor, a key transducer of myelin inhibition. Degradation of Id2 in neurons permits the accumulation of the Nogo receptor, thereby linking APC/C(Cdh1) activity with bHLH target genes for the inhibition of axonal growth. These findings indicate that deregulated Id activity might be useful to reprogramme quiescent neurons into the axonal growth mode.


Assuntos
Axônios/fisiologia , Ciclo Celular , Proteína 2 Inibidora de Diferenciação/metabolismo , Processamento de Proteína Pós-Traducional , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Diferenciação Celular , Linhagem Celular Tumoral , Linhagem da Célula , Proliferação de Células , Humanos , Mitose , Especificidade por Substrato
13.
Sci Immunol ; 7(72): eabn2738, 2022 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-35658015

RESUMO

B cell development is linked to successful V(D)J recombination, allowing B cell receptor expression and ultimately antibody secretion for adaptive immunity. Germline noncoding RNAs (ncRNAs) are produced at immunoglobulin (Ig) loci during V(D)J recombination, but their function and posttranscriptional regulation are incompletely understood. Patients with trichohepatoenteric syndrome, characterized by RNA exosome pathway component mutations, exhibit lymphopenia, thus demonstrating the importance of ncRNA surveillance in B cell development in humans. To understand the role of RNA exosome in early B cell development in greater detail, we generated mouse models harboring a B cell-specific cre allele (Mb1cre), coupled to conditional inversion-deletion alleles of one RNA exosome core component (Exosc3) or RNase catalytic subunits (Exosc10 or Dis3). We noticed increased expression of RNA exosome subunits during V(D)J recombination, whereas a B cell developmental blockade at the pro-B cell stage was observed in the different knockout mice, overlapping with a lack of productive rearrangements of VDJ genes at the Ig heavy chain (Igh). This unsuccessful recombination prevented differentiation into pre-B cells, with accumulation of ncRNAs and up-regulation of the p53 pathway. Introduction of a prearranged Igh VDJ allele partly rescued the pre-B cell population in Dis3-deficient cells, although V-J recombination defects were observed at Ig light chain kappa (Igκ), preventing subsequent B cell development. These observations demonstrated that the RNA exosome complex is important for Igh and Igκ recombination and establish the relevance of RNA processing for optimal diversification at these loci during B cell development.


Assuntos
Linfócitos B , Complexo Multienzimático de Ribonucleases do Exossomo , Animais , Exorribonucleases/genética , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Processamento Pós-Transcricional do RNA , RNA não Traduzido/genética , Recombinação V(D)J/genética
14.
Mol Cell Biol ; 41(4)2021 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-33526453

RESUMO

FACT (facilitates chromatin transcription), an essential and evolutionarily conserved heterodimer from yeast to humans, controls transcription and is found to be upregulated in various cancers. However, the basis for such upregulation is not clearly understood. Our recent results deciphering a new ubiquitin-proteasome system regulation of the FACT subunit SPT16 in orchestrating transcription in yeast hint at the involvement of the proteasome in controlling FACT in humans, with a link to cancer. To test this, we carried out experiments in human embryonic kidney (HEK293) cells, which revealed that human SPT16 undergoes ubiquitylation and that its abundance is increased following inhibition of the proteolytic activity of the proteasome, thus implying proteasomal regulation of human SPT16. Furthermore, we find that the increased abundance/expression of SPT16 in HEK293 cells alters the transcription of genes, including ones associated with cancer, and that the proteasomal degradation of SPT16 is impaired in kidney cancer (Caki-2) cells to upregulate SPT16. Like human SPT16, murine SPT16 in C2C12 cells also undergoes ubiquitylation and proteasomal degradation to regulate transcription. Collectively, our results reveal a proteasomal regulation of mammalian SPT16, with physiological relevance in controlling transcription, and implicate such proteasomal control in the upregulation of SPT16 in cancer.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Fatores de Elongação da Transcrição/metabolismo , Cromatina/metabolismo , Humanos , Proteólise , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/genética
15.
Nat Cancer ; 2(12): 1372-1386, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-35121903

RESUMO

Only a subset of recurrent glioblastoma (rGBM) responds to anti-PD-1 immunotherapy. Previously, we reported enrichment of BRAF/PTPN11 mutations in 30% of rGBM that responded to PD-1 blockade. Given that BRAF and PTPN11 promote MAPK/ERK signaling, we investigated whether activation of this pathway is associated with response to PD-1 inhibitors in rGBM, including patients that do not harbor BRAF/PTPN11 mutations. Here we show that immunohistochemistry for ERK1/2 phosphorylation (p-ERK), a marker of MAPK/ERK pathway activation, is predictive of overall survival following adjuvant PD-1 blockade in two independent rGBM patient cohorts. Single-cell RNA-sequencing and multiplex immunofluorescence analyses revealed that p-ERK was mainly localized in tumor cells and that high-p-ERK GBMs contained tumor-infiltrating myeloid cells and microglia with elevated expression of MHC class II and associated genes. These findings indicate that ERK1/2 activation in rGBM is predictive of response to PD-1 blockade and is associated with a distinct myeloid cell phenotype.


Assuntos
Glioblastoma , Glioblastoma/tratamento farmacológico , Humanos , Imunoterapia , Sistema de Sinalização das MAP Quinases , Recidiva Local de Neoplasia/tratamento farmacológico , Fosforilação
16.
Dev Biol ; 328(2): 273-84, 2009 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-19389359

RESUMO

Activating mutations of FGFRs1-3 cause craniosynostosis (CS), the premature fusion of cranial bones, in man and mouse. The mechanisms by which such mutations lead to CS have been variously ascribed to increased osteoblast proliferation, differentiation, and apoptosis, but it is not always clear how these disturbances relate to the process of suture fusion. We have reassessed coronal suture fusion in an Apert Fgfr2 (S252W) mouse model. We find that the critical event of CS is the early loss of basal sutural mesenchyme as the osteogenic fronts, expressing activated Fgfr2, unite to form a contiguous skeletogenic membrane. A mild increase in osteoprogenitor proliferation precedes but does not accompany this event, and apoptosis is insignificant. On the other hand, the more apical coronal suture initially forms appropriately but then undergoes fusion, albeit at a slower rate, accompanied by a significant decrease in osteoprogenitor proliferation, and increased osteoblast maturation. Apoptosis now accompanies fusion, but is restricted to bone fronts in contact with one another. We correlated these in vivo observations with the intrinsic effects of the activated Fgfr2 S252W mutation in primary osteoblasts in culture, which show an increased capacity for both proliferation and differentiation. Our studies suggest that the major determinant of Fgfr2-induced craniosynostosis is the failure to respond to signals that would halt the recruitment or the advancement of osteoprogenitor cells at the sites where sutures should normally form.


Assuntos
Apoptose/fisiologia , Craniossinostoses/embriologia , Osteoblastos/patologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Células-Tronco/patologia , Acrocefalossindactilia/embriologia , Acrocefalossindactilia/genética , Acrocefalossindactilia/patologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Craniossinostoses/genética , Craniossinostoses/patologia , Mesoderma/citologia , Mesoderma/embriologia , Camundongos , Camundongos Mutantes , Osteoblastos/fisiologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Células-Tronco/fisiologia
17.
Heliyon ; 6(3): e03442, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32195383

RESUMO

Loss of function of senataxin (SETX), a bona-fide RNA/DNA helicase, is associated with neuronal degeneration leading to Ataxia and Ocular Apraxia (AOA) in human patients. SETX is proposed to promote transcription termination, DNA replication, DNA repair, and to unwind deleterious RNA:DNA hybrids in the genome. In all the above-mentioned mechanisms, SETX unwinds transcription complex-associated nascent RNA which is then degraded by the RNA exosome complex. Here we have used B cells isolated from a SETX mutant mouse model and compared genomic instability and immunoglobulin heavy chain locus (IgH) class switch recombination (CSR) to evaluate aberrant and programmed genomic rearrangements, respectively. Similar to RNA exosome mutant primary B cells, SETX mutant primary B cells display genomic instability but a modest decrease in efficiency of CSR. Furthermore, knockdown of Setx mRNAs from CH12-F3 B-cell lines leads to a defect in IgA CSR and accumulation of aberrant patterns of mutations in IgH switch sequences. Given that SETX mutant mice do not recapitulate the AOA neurodegenerative phenotype, it is possible that some aspects of SETX biology are rescued by redundant helicases in mice. Overall, our study provides new insights into the role of the SETX/RNA exosome axis in suppressing genomic instability so that programmed DNA breaks are properly orchestrated.

18.
Sci Immunol ; 5(44)2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034089

RESUMO

B cells undergo two types of genomic alterations to increase antibody diversity: introduction of point mutations into immunoglobulin heavy- and light-chain (IgH and IgL) variable regions by somatic hypermutation (SHM) and alteration of antibody effector functions by changing the expressed IgH constant region exons through IgH class switch recombination (CSR). SHM and CSR require the B cell-specific activation-induced cytidine deaminase (AID) protein, the transcription of germline noncoding RNAs, and the activity of the 3' regulatory region (3'RR) super-enhancer. Although many transcription regulatory elements (e.g., promoters and enhancers) reside inside the IgH and IgL sequences, the question remains whether clusters of regulatory elements outside IgH control CSR. Using RNA exosome-deficient mouse B cells where long noncoding RNAs (lncRNAs) are easily detected, we identified a cluster of three RNA-expressing elements that includes lncCSRIgA (that expresses lncRNA-CSRIgA). B cells isolated from a mouse model lacking lncRNA-CSRIgA transcription fail to undergo normal levels of CSR to IgA both in B cells of the Peyer's patches and grown in ex vivo culture conditions. lncRNA-CSRIgA is expressed from an enhancer site (lncCSRIgA ) to facilitate the recruitment of regulatory proteins to a nearby CTCF site (CTCFlncCSR) that alters the chromosomal interactions inside the TADlncCSRIgA and long-range interactions with the 3'RR super-enhancer. Humans with IgA deficiency show polymorphisms in the lncCSRIgA locus compared with the normal population. Thus, we provide evidence for an evolutionarily conserved topologically associated domain (TADlncCSRIgA) that coordinates IgA CSR in Peyer's patch B cells through an lncRNA (lncRNA-CSRIgA) transcription-dependent mechanism.


Assuntos
Cromossomos de Mamíferos/genética , Switching de Imunoglobulina/genética , Imunoglobulinas/genética , RNA não Traduzido/genética , Animais , Linfócitos B/imunologia , Linhagem Celular , Cromossomos de Mamíferos/imunologia , Humanos , Switching de Imunoglobulina/imunologia , Imunoglobulinas/imunologia , Camundongos , Camundongos Knockout , RNA não Traduzido/imunologia , Ativação Transcricional/genética , Ativação Transcricional/imunologia
19.
Clin Cancer Res ; 26(16): 4390-4401, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32430477

RESUMO

PURPOSE: Cancer immunoediting shapes tumor progression by the selection of tumor cell variants that can evade immune recognition. Given the immune evasion and intratumor heterogeneity characteristic of gliomas, we hypothesized that CD8+ T cells mediate immunoediting in these tumors. EXPERIMENTAL DESIGN: We developed retrovirus-induced PDGF+ Pten -/- murine gliomas and evaluated glioma progression and tumor immunogenicity in the absence of CD8+ T cells by depleting this immune cell population. Furthermore, we characterized the genomic alterations present in gliomas that developed in the presence and absence of CD8+ T cells. RESULTS: Upon transplantation, gliomas that developed in the absence of CD8+ T cells engrafted poorly in recipients with intact immunity but engrafted well in those with CD8+ T-cell depletion. In contrast, gliomas that developed under pressure from CD8+ T cells were able to fully engraft in both CD8+ T-cell-depleted mice and immunocompetent mice. Remarkably, gliomas developed in the absence of CD8+ T cells exhibited increased aneuploidy, MAPK pathway signaling, gene fusions, and macrophage/microglial infiltration, and showed a proinflammatory phenotype. MAPK activation correlated with macrophage/microglia recruitment in this model and in the human disease. CONCLUSIONS: Our studies indicate that, in these tumor models, CD8+ T cells influence glioma oncogenic pathways, tumor genotype, and immunogenicity. This suggests immunoediting of immunogenic tumor clones through their negative selection by CD8+ T cells during glioma formation.


Assuntos
Neoplasias Encefálicas/imunologia , Glioma/imunologia , Evasão da Resposta Imune/imunologia , Linfócitos T/imunologia , Animais , Antígenos de Neoplasias/imunologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Glioma/genética , Glioma/patologia , Humanos , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Microglia/imunologia , Microglia/patologia , Linfócitos T/patologia
20.
Mol Cell Biol ; 26(11): 4351-61, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16705184

RESUMO

A precise balance between proliferation and differentiation must be maintained during neural development to obtain the correct proportion of differentiated cell types in the adult nervous system. The basic helix-loop-helix (bHLH) transcription factors known as E proteins and their natural inhibitors, the Id proteins, control the timing of differentiation and terminal exit from the cell cycle. Here we show that progression into S phase of human neuroblastoma cells is prevented by E proteins and promoted by Id2. Cyclin-dependent kinase inhibitors (CKI) have been identified as key effectors of cell cycle arrest in differentiating cells. However, p57Kip2 is the only CKI that is absolutely required for normal development. Through the use of global gene expression analysis in neuroblastoma cells engineered to acutely express the E protein E47 and Id2, we find that p57Kip2 is a target of E47. Consistent with the role of Id proteins, Id2 prevents activation of p57Kip2 expression, and the retinoblastoma tumor suppressor protein, a known Id2 inhibitor, counters this activity. The strong E47-mediated inhibition of entry into S phase is entirely reversed in cells in which expression of p57Kip2 is silenced by RNA interference. During brain development, expression of p57Kip2 is opposite that of Id2. Our findings identify p57Kip2 as a functionally relevant target recruited by bHLH transcription factors to induce cell cycle arrest in developing neuroblasts and suggest that deregulated expression of Id proteins may be an epigenetic mechanism to silence expression of this CKI in neural tumors.


Assuntos
Ciclo Celular , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteína 2 Inibidora de Diferenciação/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fatores de Transcrição TCF/metabolismo , Adenoviridae/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proliferação de Células , Cromossomos Humanos Par 11/genética , Humanos , Análise em Microsséries , Neuroblastoma/patologia , Proteína do Retinoblastoma/metabolismo , Proteína 1 Semelhante ao Fator 7 de Transcrição , Transcrição Gênica , Células Tumorais Cultivadas
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