RESUMO
The epithelial to mesenchymal transition (EMT) is a biological process in which cells lose cell-cell contacts and become motile. EMT is used during development, for example, in triggering neural crest migration, and in cancer metastasis. Despite progress, the dynamics of JNK signaling, its role in genomewide transcriptional reprogramming, and involved downstream effectors during EMT remain largely unknown. Here, we show that JNK is not required for initiation, but progression of phenotypic changes associated with EMT. Such dependency resulted from JNK-driven transcriptional reprogramming of critical EMT genes and involved changes in their chromatin state. Furthermore, we identified eight novel JNK-induced transcription factors that were required for proper EMT. Three of these factors were also highly expressed in invasive cancer cells where they function in gene regulation to maintain mesenchymal identity. These factors were also induced during neuronal development and function in neuronal migration in vivo. These comprehensive findings uncovered a kinetically distinct role for the JNK pathway in defining the transcriptome that underlies mesenchymal identity and revealed novel transcription factors that mediate these responses during development and disease.
Assuntos
Diferenciação Celular , Redes Reguladoras de Genes , MAP Quinase Quinase 4/metabolismo , Sistema de Sinalização das MAP Quinases , Mesoderma/fisiologia , Ciclo Celular , Linhagem Celular , Perfilação da Expressão Gênica , Humanos , Imagem com Lapso de Tempo , Fatores de Transcrição/metabolismoRESUMO
B cell receptor (BCR) signaling is a key for survival of chronic lymphocytic leukemia (CLL) cells, and BCR signaling inhibitors are clinically active. However, relapse and resistance to treatment require novel treatment options. To detect novel candidate therapeutic targets, we performed a genome-wide DNA methylation screen with custom arrays and identified aberrant promoter DNA methylation in 2,192 genes. The transcription factor NFATC1 that is a downstream effector of BCR signaling was among the top hypomethylated genes and was concomitantly transcriptionally upregulated in CLL. Intriguingly, NFATC1 promoter DNA hypomethylation levels were significantly variant in clinical trial cohorts from different disease progression stages and furthermore correlated with Binet disease staging and thymidine kinase levels, strongly suggesting a central role of NFATC1 in CLL development. Functionally, DNA hypomethylation at NFATC1 promoter inversely correlated with RNA levels of NFATC1 and dysregulation correlated with expression of target genes BCL-2, CCND1 and CCR7. The inhibition of the NFAT regulator calcineurin with tacrolimus and cyclosporin A and the BCR signaling inhibitor ibrutinib significantly reduced NFAT activity in leukemic cell lines, and NFAT inhibition resulted in increased apoptosis of primary CLL cells. In summary, our results indicate that the aberrant activation of NFATC1 by DNA hypomethylation and BCR signaling plays a major role in the pathomechanism of CLL.
Assuntos
Metilação de DNA , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Fatores de Transcrição NFATC/genética , Recidiva Local de Neoplasia/genética , Pirazóis/farmacologia , Pirimidinas/farmacologia , Adenina/análogos & derivados , Idoso , Biomarcadores Tumorais , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Fatores de Transcrição NFATC/antagonistas & inibidores , Fatores de Transcrição NFATC/metabolismo , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Piperidinas , Regiões Promotoras Genéticas , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Gene regulation in mammals involves a complex interplay between promoters and distal regulatory elements that function in concert to drive precise spatiotemporal gene expression programs. However, the dynamics of the distal gene regulatory landscape and its function in the transcriptional reprogramming that underlies neurogenesis and neuronal activity remain largely unknown. Here, we performed a combinatorial analysis of genome-wide data sets for chromatin accessibility (FAIRE-seq) and the enhancer mark H3K27ac, revealing the highly dynamic nature of distal gene regulation during neurogenesis, which gets progressively restricted to distinct genomic regions as neurons acquire a post-mitotic, terminally differentiated state. We further find that the distal accessible and active regions serve as target sites for distinct transcription factors that function in a stage-specific manner to contribute to the transcriptional program underlying neuronal commitment and maturation. Mature neurons respond to a sustained activity of NMDA receptors by epigenetic reprogramming at a large number of distal regulatory regions as well as dramatic reorganization of super-enhancers. Such massive remodeling of the distal regulatory landscape in turn results in a transcriptome that confers a transient loss of neuronal identity and gain of cellular plasticity. Furthermore, NMDA receptor activity also induces many novel prosurvival genes that function in neuroprotective pathways. Taken together, these findings reveal the dynamics of the distal regulatory landscape during neurogenesis and uncover novel regulatory elements that function in concert with epigenetic mechanisms and transcription factors to generate the transcriptome underlying neuronal development and activity.
Assuntos
Regulação da Expressão Gênica , Neurogênese/genética , Plasticidade Neuronal/genética , Elementos Reguladores de Transcrição , Sequências Reguladoras de Ácido Nucleico , Animais , Reprogramação Celular/genética , Cromatina/genética , Cromatina/metabolismo , Análise por Conglomerados , Epigênese Genética , Perfilação da Expressão Gênica , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Histonas/metabolismo , Camundongos , Neurônios/metabolismo , Especificidade de Órgãos , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais , Fatores de Transcrição , Transcrição Gênica , TranscriptomaRESUMO
Epigenetic mechanisms determine the access of regulatory factors to DNA during events such as transcription and the DNA damage response. However, the global response of histone modifications and chromatin accessibility to UV exposure remains poorly understood. Here, we report that UV exposure results in a genome-wide reduction in chromatin accessibility, while the distribution of the active regulatory mark H3K27ac undergoes massive reorganization. Genomic loci subjected to epigenetic reprogramming upon UV exposure represent target sites for sequence-specific transcription factors. Most of these are distal regulatory regions, highlighting their importance in the cellular response to UV exposure. Furthermore, UV exposure results in an extensive reorganization of super-enhancers, accompanied by expression changes of associated genes, which may in part contribute to the stress response. Taken together, our study provides the first comprehensive resource for genome-wide chromatin changes upon UV irradiation in relation to gene expression and elucidates new aspects of this relationship.
Assuntos
Montagem e Desmontagem da Cromatina/efeitos da radiação , Cromatina/metabolismo , Dano ao DNA , Epigênese Genética/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Animais , Cromatina/genética , Cromatina/patologia , Camundongos , Células NIH 3T3RESUMO
Non-coding RNAs are much more common than previously thought. However, for the vast majority of non-coding RNAs, the cellular function remains enigmatic. The two long non-coding RNA (lncRNA) genes DLEU1 and DLEU2 map to a critical region at chromosomal band 13q14.3 that is recurrently deleted in solid tumors and hematopoietic malignancies like chronic lymphocytic leukemia (CLL). While no point mutations have been found in the protein coding candidate genes at 13q14.3, they are deregulated in malignant cells, suggesting an epigenetic tumor suppressor mechanism. We therefore characterized the epigenetic makeup of 13q14.3 in CLL cells and found histone modifications by chromatin-immunoprecipitation (ChIP) that are associated with activated transcription and significant DNA-demethylation at the transcriptional start sites of DLEU1 and DLEU2 using 5 different semi-quantitative and quantitative methods (aPRIMES, BioCOBRA, MCIp, MassARRAY, and bisulfite sequencing). These epigenetic aberrations were correlated with transcriptional deregulation of the neighboring candidate tumor suppressor genes, suggesting a coregulation in cis of this gene cluster. We found that the 13q14.3 genes in addition to their previously known functions regulate NF-kB activity, which we could show after overexpression, siRNA-mediated knockdown, and dominant-negative mutant genes by using Western blots with previously undescribed antibodies, by a customized ELISA as well as by reporter assays. In addition, we performed an unbiased screen of 810 human miRNAs and identified the miR-15/16 family of genes at 13q14.3 as the strongest inducers of NF-kB activity. In summary, the tumor suppressor mechanism at 13q14.3 is a cluster of genes controlled by two lncRNA genes that are regulated by DNA-methylation and histone modifications and whose members all regulate NF-kB. Therefore, the tumor suppressor mechanism in 13q14.3 underlines the role both of epigenetic aberrations and of lncRNA genes in human tumorigenesis and is an example of colocalization of a functionally related gene cluster.
Assuntos
Metilação de DNA/genética , Leucemia , RNA Longo não Codificante , Proteínas Supressoras de Tumor , Adulto , Idoso , Idoso de 80 Anos ou mais , Transformação Celular Neoplásica , Cromatina/genética , Cromossomos Humanos Par 13/genética , Regulação para Baixo , Epigênese Genética/genética , Feminino , Células HEK293 , Humanos , Leucemia/sangue , Leucemia/genética , Leucemia/fisiopatologia , Masculino , Pessoa de Meia-Idade , Mutação , NF-kappa B/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Sítio de Iniciação de Transcrição , Transferases , Proteínas Supressoras de Tumor/sangue , Proteínas Supressoras de Tumor/genética , Regulação para CimaRESUMO
Chronic lymphocytic leukemia is characterized by the accumulation of B cells that are resistant to apoptosis. This resistance is induced by pro-survival stimuli from the microenvironment. TCL1 and ATM are central to the pathogenesis of the disease and associated with more aggressive disease. Their protein products have recently been shown to physically interact in leukemic cells and to impact on NF-κB signaling, which is a key regulator of apoptosis. In the present study we show that TCL1 and ATM are significantly co-expressed and up-regulated in malignant cells compared to non-malignant B cells, and that expression of TCL1 is partially deregulated by aberrant DNA-methylation. In addition, complex external stimuli induce essentially similar TCL1 and ATM time-course kinetics. In line with a coordinative regulation of NF-κB signaling by TCL1, its knockdown induced apoptosis in primary leukemia cells. These findings suggest that both genes functionally cooperate to modulate similar apoptosis-related cellular pathways.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Proteínas Proto-Oncogênicas/genética , Adulto , Idoso , Linhagem Celular Tumoral , Deleção Cromossômica , Cromossomos Humanos Par 11 , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-IdadeAssuntos
DNA de Neoplasias , Fatores de Transcrição Kruppel-Like , Leucemia Linfocítica Crônica de Células B , Proteínas de Neoplasias , Regiões Promotoras Genéticas , Receptores Notch , Transdução de Sinais , Metilação de DNA , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Feminino , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismoRESUMO
Ectopic expression of defined transcription factors can force direct cell-fate conversion from one lineage to another in the absence of cell division. Several transcription factor cocktails have enabled successful reprogramming of various somatic cell types into induced neurons (iNs) of distinct neurotransmitter phenotype. However, the nature of the intermediate states that drive the reprogramming trajectory toward distinct iN types is largely unknown. Here we show that successful direct reprogramming of adult human brain pericytes into functional iNs by Ascl1 and Sox2 encompasses transient activation of a neural stem cell-like gene expression program that precedes bifurcation into distinct neuronal lineages. During this transient state, key signaling components relevant for neural induction and neural stem cell maintenance are regulated by and functionally contribute to iN reprogramming and maturation. Thus, Ascl1- and Sox2-mediated reprogramming into a broad spectrum of iN types involves the unfolding of a developmental program via neural stem cell-like intermediates.
Assuntos
Linhagem da Célula/fisiologia , Reprogramação Celular/fisiologia , Células-Tronco Neurais/fisiologia , Neurônios/fisiologia , Pericitos/fisiologia , Adulto , Idoso , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Células-Tronco Neurais/citologia , Neurônios/citologia , Pericitos/citologia , Fatores de Transcrição SOXB1/genética , Adulto JovemRESUMO
The series of events that allow the conversion from adherent epithelial cells into migratory cells is collectively known as epithelial-mesenchymal transition (EMT). EMT is employed during embryonic development such as for gastrulation and neural crest migration and is misused in diseases, such as cancer metastasis. ERK signalling is known to be essential for EMT, however its influence on the epigenetic and transcriptional programme underlying EMT is poorly understood. Here, using a comprehensive genome-wide analysis of H3K27ac mark and gene expression in mammary epithelial cells undergoing EMT, we found that ERK signalling is essential for the epigenetic reprogramming underlying hallmark gene expression and phenotypic changes of EMT. We show that the chemical inhibition of Erk signalling during EMT prevents the loss and gain of the H3K27ac mark at regulatory regions of epithelial and mesenchymal genes, respectively, and results in a transcriptome and epigenome closer to those of epithelial cells. Further computational analyses identified a distinct set of transcription factor motifs enriched at distal regulatory regions that are epigenetically remodelled by ERK signalling. Altogether, our findings reveal an ERK-dependent epigenetic remodelling of regulatory elements that results in a gene expression programme essential for driving EMT.
Assuntos
Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/genética , Regulação da Expressão Gênica/genética , Sistema de Sinalização das MAP Quinases/genética , Fatores de Transcrição/metabolismo , Animais , Epigenômica , Humanos , Camundongos , Transdução de SinaisRESUMO
Pax6 is a highly conserved transcription factor among vertebrates and is important in various aspects of the central nervous system development. However, the gene regulatory circuitry of Pax6 underlying these functions remains elusive. We find that Pax6 targets a large number of promoters in neural progenitors cells. Intriguingly, many of these sites are also bound by another progenitor factor, Sox2, which cooperates with Pax6 in gene regulation. A combinatorial analysis of Pax6-binding data set with transcriptome changes in Pax6-deficient neural progenitors reveals a dual role for Pax6, in which it activates the neuronal (ectodermal) genes while concurrently represses the mesodermal and endodermal genes, thereby ensuring the unidirectionality of lineage commitment towards neuronal differentiation. Furthermore, Pax6 is critical for inducing activity of transcription factors that elicit neurogenesis and repress others that promote non-neuronal lineages. In addition to many established downstream effectors, Pax6 directly binds and activates a number of genes that are specifically expressed in neural progenitors but have not been previously implicated in neurogenesis. The in utero knockdown of one such gene, Ift74, during brain development impairs polarity and migration of newborn neurons. These findings demonstrate new aspects of the gene regulatory circuitry of Pax6, revealing how it functions to control neuronal development at multiple levels to ensure unidirectionality and proper execution of the neurogenic program.
RESUMO
Rapid nerve conduction in the CNS is facilitated by insulation of axons with myelin, a specialized oligodendroglial compartment distant from the cell body. Myelin is turned over and adapted throughout life; however, the molecular and cellular basis of myelin dynamics remains elusive. Here we performed a comprehensive transcriptome analysis (RNA-seq) of myelin biochemically purified from mouse brains at various ages and find a surprisingly large pool of transcripts enriched in myelin. Further computational analysis showed that the myelin transcriptome is closely related to the myelin proteome but clearly distinct from the transcriptomes of oligodendrocytes and brain tissues, suggesting a highly selective incorporation of mRNAs into the myelin compartment. The mRNA-pool in myelin displays maturation-dependent dynamic changes of composition, abundance, and functional associations; however ageing-dependent changes after 6 months were minor. We suggest that this transcript pool enables myelin turnover and the local adaptation of individual pre-existing myelin sheaths.
Assuntos
Sistema Nervoso Central/metabolismo , Bainha de Mielina/metabolismo , Transcriptoma/genética , Animais , Biomarcadores/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos Endogâmicos C57BL , Análise de Componente Principal , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Topoisomerases resolve torsional stress, while their function in gene regulation, especially during cellular differentiation, remains unknown. Here we find that the expression of topo II isoforms, topoisomerase IIα and topoisomerase IIß, is the characteristic of dividing and postmitotic tissues, respectively. In embryonic stem cells, topoisomerase IIα preferentially occupies active gene promoters. Topoisomerase IIα inhibition compromises genomic integrity, which results in epigenetic changes, altered kinetics of RNA Pol II at target promoters and misregulated gene expression. Common targets of topoisomerase IIα and topoisomerase IIß are housekeeping genes, while unique targets are involved in proliferation/pluripotency and neurogenesis, respectively. Topoisomerase IIα targets exhibiting bivalent chromatin resolve upon differentiation, concomitant with their activation and occupancy by topoisomerase IIß, features further observed for long genes. These long silent genes display accessible chromatin in embryonic stem cells that relies on topoisomerase IIα activity. These findings suggest that topoisomerase IIα not only contributes to stem-cell transcriptome regulation but also primes developmental genes for subsequent activation upon differentiation.
Assuntos
Antígenos de Neoplasias/genética , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Animais , Antígenos de Neoplasias/metabolismo , Diferenciação Celular , Proliferação de Células , Cromatina/química , Cromatina/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Genes Essenciais , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Ligação Proteica , RNA Polimerase II/genética , RNA Polimerase II/metabolismoRESUMO
Aberrant expression of the homeodomain transcription factor CDX2 occurs in most cases of acute myeloid leukemia (AML) and promotes leukemogenesis, making CDX2, in principle, an attractive therapeutic target. Conversely, CDX2 acts as a tumor suppressor in colonic epithelium. The effectors mediating the leukemogenic activity of CDX2 and the mechanism underlying its context-dependent properties are poorly characterized, and strategies for interfering with CDX2 function in AML remain elusive. We report data implicating repression of the transcription factor KLF4 as important for the oncogenic activity of CDX2, and demonstrate that CDX2 differentially regulates KLF4 in AML versus colon cancer cells through a mechanism that involves tissue-specific patterns of promoter binding and epigenetic modifications. Furthermore, we identified deregulation of the PPARγ signaling pathway as a feature of CDX2-associated AML and observed that PPARγ agonists derepressed KLF4 and were preferentially toxic to CDX2+ leukemic cells. These data delineate transcriptional programs associated with CDX2 expression in hematopoietic cells, provide insight into the antagonistic duality of CDX2 function in AML versus colon cancer, and suggest reactivation of KLF4 expression, through modulation of PPARγ signaling, as a therapeutic modality in a large proportion of AML patients.