RESUMEN
Different regions of RNA molecules can often engage in specific interactions with distinct RNA-binding proteins (RBPs), giving rise to diverse modalities of RNA regulation and function. However, there are currently no methods for unbiased identification of RBPs that interact with specific RNA regions in living cells and under endogenous settings. Here we introduce TREX (targeted RNase H-mediated extraction of crosslinked RBPs)-a highly sensitive approach for identifying proteins that directly bind to specific RNA regions in living cells. We demonstrate that TREX outperforms existing methods in identifying known interactors of U1 snRNA, and reveals endogenous region-specific interactors of NORAD long noncoding RNA. Using TREX, we generated a comprehensive region-by-region interactome for 45S rRNA, uncovering both established and previously unknown interactions that regulate ribosome biogenesis. With its applicability to different cell types, TREX is an RNA-centric tool for unbiased positional mapping of endogenous RNA-protein interactions in living cells.
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Proteínas de Unión al ARN , ARN , ARN/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
The number of known long noncoding RNA (lncRNA) functions is rapidly growing, but how those functions are encoded in their sequence and structure remains poorly understood. NORAD (noncoding RNA activated by DNA damage) is a recently characterized, abundant, and highly conserved lncRNA that is required for proper mitotic divisions in human cells. NORAD acts in the cytoplasm and antagonizes repressors from the Pumilio family that bind at least 17 sites spread through 12 repetitive units in NORAD sequence. Here we study conserved sequences in NORAD repeats, identify additional interacting partners, and characterize the interaction between NORAD and the RNA-binding protein SAM68 (KHDRBS1), which is required for NORAD function in antagonizing Pumilio. These interactions provide a paradigm for how repeated elements in a lncRNA facilitate function.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Unión al ADN/metabolismo , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/metabolismo , Regiones no Traducidas 3' , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/fisiología , Sitios de Unión , Línea Celular Tumoral , Segregación Cromosómica , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Humanos , ARN Largo no Codificante/química , ARN Largo no Codificante/genética , ARN Largo no Codificante/fisiología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/fisiología , Proteínas Represoras/metabolismoRESUMEN
The cell cycle is controlled by guardian proteins that coordinate the process of cell growth and cell division. Alterations in these processes lead to genome instability, which has a causal link to many human diseases. Beyond their well-characterized role of influencing protein-coding genes, an increasing body of evidence has revealed that long noncoding RNAs (lncRNAs) actively participate in regulation of the cell cycle and safeguarding of genome integrity. LncRNAs are versatile molecules that act via a wide array of mechanisms. In this review, we discuss how lncRNAs are implicated in control of the cell cycle and maintenance of genome stability and how changes in lncRNA-regulatory networks lead to proliferative diseases such as cancer.
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Ciclo Celular/genética , Inestabilidad Genómica/genética , ARN Largo no Codificante/fisiología , Animales , Daño del ADN/genética , Reparación del ADN/genética , Humanos , Neoplasias/genéticaRESUMEN
OBJECTIVE: To better comprehend transcriptional phenotypes of cancer cells, we globally characterised RNA-binding proteins (RBPs) to identify altered RNAs, including long non-coding RNAs (lncRNAs). DESIGN: To unravel RBP-lncRNA interactions in cancer, we curated a list of ~2300 highly expressed RBPs in human cells, tested effects of RBPs and lncRNAs on patient survival in multiple cohorts, altered expression levels, integrated various sequencing, molecular and cell-based data. RESULTS: High expression of RBPs negatively affected patient survival in 21 cancer types, especially hepatocellular carcinoma (HCC). After knockdown of the top 10 upregulated RBPs and subsequent transcriptome analysis, we identified 88 differentially expressed lncRNAs, including 34 novel transcripts. CRISPRa-mediated overexpression of four lncRNAs had major effects on the HCC cell phenotype and transcriptome. Further investigation of four RBP-lncRNA pairs revealed involvement in distinct regulatory processes. The most noticeable RBP-lncRNA connection affected lipid metabolism, whereby the non-canonical RBP CCT3 regulated LINC00326 in a chaperonin-independent manner. Perturbation of the CCT3-LINC00326 regulatory network led to decreased lipid accumulation and increased lipid degradation in cellulo as well as diminished tumour growth in vivo. CONCLUSIONS: We revealed that RBP gene expression is perturbed in HCC and identified that RBPs exerted additional functions beyond their tasks under normal physiological conditions, which can be stimulated or intensified via lncRNAs and affected tumour growth.
RESUMEN
Loss-of-function (LOF) methods such as RNA interference (RNAi), antisense oligonucleotides or CRISPR-based genome editing provide unparalleled power for studying the biological function of genes of interest. However, a major concern is non-specific targeting, which involves depletion of transcripts other than those intended. Little work has been performed to characterize the off-target effects of these common LOF methods at the whole-transcriptome level. Here, we experimentally compared the non-specific activity of RNAi, antisense oligonucleotides and CRISPR interference (CRISPRi). All three methods yielded non-negligible off-target effects in gene expression, with CRISPRi also exhibiting strong clonal effects. As an illustrative example, we evaluated the performance of each method for determining the role of an uncharacterized long noncoding RNA (lncRNA). Several LOF methods successfully depleted the candidate lncRNA but yielded different sets of differentially expressed genes as well as a different cellular phenotype upon depletion. Similar discrepancies between methods were observed with a protein-coding gene (Ch-TOG/CKAP5) and another lncRNA (MALAT1). We suggest that the differences between methods arise due to method-specific off-target effects and provide guidelines for mitigating such effects in functional studies. Our recommendations provide a framework with which off-target effects can be managed to improve functional characterization of genes of interest.
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Sistemas CRISPR-Cas , Técnicas de Silenciamiento del Gen , Oligonucleótidos Antisentido , Oligonucleótidos , Interferencia de ARN , Transcripción Genética , Perfilación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Oligonucleótidos Antisentido/química , Proteínas/genética , ARN Largo no Codificante/metabolismoRESUMEN
Imprinted gene clusters are regulated by long noncoding RNAs (lncRNAs), CCCTC binding factor (CTCF)-mediated boundaries, and DNA methylation. DIRAS3 (also known as ARH1 or NOEY1) is an imprinted gene encoding a protein belonging to the RAS superfamily of GTPases and is located within an intron of a lncRNA called GNG12-AS1. In this study, we investigated whether GNG12-AS1 is imprinted and coregulated with DIRAS3. We report that GNG12-AS1 is coexpressed with DIRAS3 in several tissues and coordinately downregulated with DIRAS3 in breast cancers. GNG12-AS1 has several splice variants, all of which initiate from a single transcription start site. In placenta tissue and normal cell lines, GNG12-AS1 is biallelically expressed but some isoforms are allele-specifically spliced. Cohesin plays a role in allele-specific splicing of GNG12-AS1. In breast cancer cell lines with loss of DIRAS3 imprinting, DIRAS3 and GNG12-AS1 are silenced in cis and the remaining GNG12-AS1 transcripts are predominantly monoallelic. The GNG12-AS1 locus, which includes DIRAS3, provides an example of imprinted cotranscriptional splicing and a potential model system for studying the long-range effects of CTCF-cohesin binding on splicing and transcriptional interference.
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Neoplasias de la Mama/genética , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Impresión Genómica , ARN Largo no Codificante/genética , Proteínas de Unión al GTP rho/genética , Alelos , Empalme Alternativo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/metabolismo , Metilación de ADN , Femenino , Regulación de la Expresión Génica , Humanos , Intrones , Placenta/citología , Placenta/metabolismo , Embarazo , ARN Largo no Codificante/metabolismo , Sitio de Iniciación de la Transcripción , Transcripción Genética , Proteínas de Unión al GTP rho/metabolismo , CohesinasRESUMEN
Iroquois transcription factor gene IRX3 is highly expressed in 20-30% of acute myeloid leukemia (AML) and contributes to the pathognomonic differentiation block. Intron 8 FTO sequences â¼220kB downstream of IRX3 exhibit histone acetylation, DNA methylation, and contacts with the IRX3 promoter, which correlate with IRX3 expression. Deletion of these intronic elements confirms a role in positively regulating IRX3. RNAseq revealed long non-coding (lnc) transcripts arising from this locus. FTO-lncAML knockdown (KD) induced differentiation of AML cells, loss of clonogenic activity, and reduced FTO intron 8:IRX3 promoter contacts. While both FTO-lncAML KD and IRX3 KD induced differentiation, FTO-lncAML but not IRX3 KD led to HOXA downregulation suggesting transcript activity in trans. FTO-lncAMLhigh AML samples expressed higher levels of HOXA and lower levels of differentiation genes. Thus, a regulatory module in FTO intron 8 consisting of clustered enhancer elements and a long non-coding RNA is active in human AML, impeding myeloid differentiation.
RESUMEN
Genomic imprinting is an epigenetic marking of genes in the parental germline that ensures the stable transmission of monoallelic gene expression patterns in a parent-of-origin-specific manner. Epigenetic marking systems are thus able to regulate gene activity independently of the underlying DNA sequence. Several imprinted gene products regulate cell proliferation and fetal growth; loss of their imprinted state, which effectively alters their dosage, might promote or suppress tumourigenic processes. Conversely, global epigenetic changes that underlie tumourigenesis might affect imprinted gene expression. Here, we review imprinted genes with regard to their roles in epigenetic predisposition to cancer, and discuss acquired epigenetic changes (DNA methylation, histone modifications and chromatin conformation) either as a result of cancer or as an early event in neoplasia. We also address recent work showing the potential role of noncoding RNA in modifying chromatin and affecting imprinted gene expression, and summarise the effects of loss of imprinting in cancer with regard to the roles that imprinted genes play in regulating growth signalling cascades. Finally, we speculate on the clinical applications of epigenetic drugs in cancer.
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Impresión Genómica , Neoplasias/genética , Cromatina/química , Cromatina/genética , Metilación de ADN , Epigenómica , Expresión Génica , Histonas/genética , Histonas/metabolismo , Conformación Proteica , ARN no Traducido/genética , ARN no Traducido/metabolismo , Factores de Riesgo , Transducción de SeñalRESUMEN
Long noncoding RNAs (lncRNAs) have emerged as important regulators of gene expression networks. Over 50,000 lncRNA loci have been annotated in the human genome, but only a subset has been involved in regulation of key cellular processes, organismal development, and diseases. Hence, the functional role for the majority of the lncRNA genes remains unknown. With the recent developments of different CRISPR/Cas9 technologies, the function of lncRNAs can now be examined. CRISPR interference (CRISPRi) is one of these methods that can be used to inhibit the expression of any genomic locus including lncRNAs. This system utilizes catalytically inactive (d)Cas9 fused to KRAB repression domain and single guide RNA against targeted genomic locus. Since CRISPRi has negligible off-target effects and does not involve changes in the underlying genomic DNA sequence, it represents a valuable addition to the existing armamentarium used to investigate lncRNA biology.
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Sistemas CRISPR-Cas , Edición Génica/métodos , Interferencia de ARN , ARN Largo no Codificante/genética , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Células HeLa , Humanos , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , ARN Largo no Codificante/metabolismoRESUMEN
Genome stability relies on proper coordination of mitosis and cytokinesis, where dynamic microtubules capture and faithfully segregate chromosomes into daughter cells. With a high-content RNAi imaging screen targeting more than 2,000 human lncRNAs, we identify numerous lncRNAs involved in key steps of cell division such as chromosome segregation, mitotic duration and cytokinesis. Here, we provide evidence that the chromatin-associated lncRNA, linc00899, leads to robust mitotic delay upon its depletion in multiple cell types. We perform transcriptome analysis of linc00899-depleted cells and identify the neuronal microtubule-binding protein, TPPP/p25, as a target of linc00899. We further show that linc00899 binds TPPP/p25 and suppresses its transcription. In cells depleted of linc00899, upregulation of TPPP/p25 alters microtubule dynamics and delays mitosis. Overall, our comprehensive screen uncovers several lncRNAs involved in genome stability and reveals a lncRNA that controls microtubule behaviour with functional implications beyond cell division.
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División Celular/genética , División Celular/fisiología , ARN Largo no Codificante/genética , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Mitosis/genética , Mitosis/fisiología , Proteínas/genética , Interferencia de ARN/fisiologíaRESUMEN
A recent outbreak of Zika virus in Brazil has led to a simultaneous increase in reports of neonatal microcephaly. Zika targets cerebral neural precursors, a cell population essential for cortical development, but the cause of this neurotropism remains obscure. Here we report that the neural RNA-binding protein Musashi-1 (MSI1) interacts with the Zika genome and enables viral replication. Zika infection disrupts the binding of MSI1 to its endogenous targets, thereby deregulating expression of factors implicated in neural stem cell function. We further show that MSI1 is highly expressed in neural progenitors of the human embryonic brain and is mutated in individuals with autosomal recessive primary microcephaly. Selective MSI1 expression in neural precursors could therefore explain the exceptional vulnerability of these cells to Zika infection.
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Genoma Viral , Microcefalia/metabolismo , Microcefalia/virología , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ARN/metabolismo , Replicación Viral , Infección por el Virus Zika/virología , Virus Zika/fisiología , Animales , Encéfalo/anomalías , Encéfalo/metabolismo , Encéfalo/virología , Niño , Chlorocebus aethiops , Femenino , Células HEK293 , Humanos , Masculino , Microcefalia/genética , Mutación , Células-Madre Neurales/metabolismo , Células-Madre Neurales/fisiología , Células-Madre Neurales/virología , Células Vero , Virus Zika/genéticaRESUMEN
Aging is characterized by progressive loss of physiological and cellular functions, but the molecular basis of this decline remains unclear. We explored how aging affects transcriptional dynamics using single-cell RNA sequencing of unstimulated and stimulated naïve and effector memory CD4+ T cells from young and old mice from two divergent species. In young animals, immunological activation drives a conserved transcriptomic switch, resulting in tightly controlled gene expression characterized by a strong up-regulation of a core activation program, coupled with a decrease in cell-to-cell variability. Aging perturbed the activation of this core program and increased expression heterogeneity across populations of cells in both species. These discoveries suggest that increased cell-to-cell transcriptional variability will be a hallmark feature of aging across most, if not all, mammalian tissues.
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Envejecimiento/genética , Envejecimiento/inmunología , Linfocitos T CD4-Positivos/inmunología , Memoria Inmunológica/genética , Transcriptoma , Animales , Senescencia Celular/genética , Senescencia Celular/inmunología , Variación Genética , Activación de Linfocitos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T/metabolismo , Análisis de Secuencia de ARN , Análisis de la Célula IndividualRESUMEN
DNA mismatch repair (MMR) plays a key role in the cytotoxic response of human cells to methylating agents, however, the cascade of events leading to cell cycle arrest and cell death has yet to be characterized. We studied the role of MMR in the transcriptional response to DNA methylation damage in two human cellular models: (a). the lymphoblastoid cell line TK6 and its derivative MT1, which is mutated in the MMR gene hMSH6; and (b). the epithelial cell line 293T Lalpha in which the expression of the MMR gene hMLH1 can be tightly regulated and p53 is inactivated. Upon N-methyl-N'-nitro-N-nitrosoguanidine treatment, only cells with functional MMR were killed, but the type of cytotoxic response differed. In TK6 cells, S-phase arrest and apoptosis were accompanied by a dramatic change in gene expression, notably, an up-regulation of several genes encoding growth inhibitors and proapoptotic factors both p53 dependent and independent. In contrast, the MMR-dependent transcriptional response in 293T Lalpha cells was substantially less pronounced than in TK6 cells, despite an efficient induction of a G(2)-M checkpoint and nonapoptotic cell death. Thus, we demonstrate that in human cells of different origin, MMR-mediated killing by methylating agents occurs through different pathways and regardless of the p53 status. Moreover, once DNA methylation damage has been processed by the MMR system, tumor cells might be committed to die, although one or more of their signaling pathways are impaired.
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Disparidad de Par Base/fisiología , Metilación de ADN/efectos de los fármacos , Reparación del ADN/fisiología , Metilnitronitrosoguanidina/toxicidad , Proteínas Adaptadoras Transductoras de Señales , Linfocitos B/efectos de los fármacos , Linfocitos B/fisiología , Proteínas Portadoras , Línea Celular , Reparación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Humanos , Homólogo 1 de la Proteína MutL , Mutación Missense , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/deficiencia , Proteínas Nucleares , Transcripción Genética/efectos de los fármacos , Proteína p53 Supresora de Tumor/fisiología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Most human aneuploidies originate maternally, due in part to the presence of highly stringent checkpoints during male meiosis. Indeed, male sterility is common among aneuploid mice used to study chromosomal abnormalities, and male germline transmission of exogenous DNA has been rarely reported. Here we show that, despite aberrant testis architecture, males of the aneuploid Tc1 mouse strain produce viable sperm and transmit human chromosome 21 to create aneuploid offspring. In these offspring, we mapped transcription, transcriptional initiation, enhancer activity, non-methylated DNA, and transcription factor binding in adult tissues. Remarkably, when compared with mice derived from female passage of human chromosome 21, the chromatin condensation during spermatogenesis and the extensive epigenetic reprogramming specific to male germline transmission resulted in almost indistinguishable patterns of transcriptional deployment. Our results reveal an unexpected tolerance of aneuploidy during mammalian spermatogenesis, and the surprisingly robust ability of mouse developmental machinery to accurately deploy an exogenous chromosome, regardless of germline transmission.
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Cromosomas Humanos/metabolismo , Análisis Citogenético , Células Germinativas/fisiología , Meiosis , Transcripción Genética , Animales , Humanos , Masculino , RatonesRESUMEN
Long noncoding RNAs (lncRNAs) regulate gene expression via their RNA product or through transcriptional interference, yet a strategy to differentiate these two processes is lacking. To address this, we used multiple small interfering RNAs (siRNAs) to silence GNG12-AS1, a nuclear lncRNA transcribed in an antisense orientation to the tumour-suppressor DIRAS3. Here we show that while most siRNAs silence GNG12-AS1 post-transcriptionally, siRNA complementary to exon 1 of GNG12-AS1 suppresses its transcription by recruiting Argonaute 2 and inhibiting RNA polymerase II binding. Transcriptional, but not post-transcriptional, silencing of GNG12-AS1 causes concomitant upregulation of DIRAS3, indicating a function in transcriptional interference. This change in DIRAS3 expression is sufficient to impair cell cycle progression. In addition, the reduction in GNG12-AS1 transcripts alters MET signalling and cell migration, but these are independent of DIRAS3. Thus, differential siRNA targeting of a lncRNA allows dissection of the functions related to the process and products of its transcription.
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Subunidades gamma de la Proteína de Unión al GTP/metabolismo , ARN Largo no Codificante/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Ciclo Celular , Subunidades gamma de la Proteína de Unión al GTP/genética , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Isoformas de Proteínas , Interferencia de ARN , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , ARN Largo no Codificante/genética , Proteínas de Unión al GTP rho/genéticaRESUMEN
Postreplicative mismatch repair (MMR) increases the fidelity of DNA replication by up to three orders of magnitude, through correcting DNA polymerase errors that escaped proofreading. MMR also controls homologous recombination (HR) by aborting strand exchange between divergent DNA sequences. In recent years, MMR has also been implicated in the response of mammalian cells to DNA damaging agents. Thus, MMR-deficient cells were shown to be around 100-fold more resistant to killing by methylating agents of the S(N)1type than cells with functional MMR. In the case of cisplatin, the sensitivity difference was lower, typically two- to three-fold, but was observed in all matched MMR-proficient and -deficient cell pairs. More controversial is the role of MMR in cellular response to other DNA damaging agents, such as ionizing radiation (IR), topoisomerase poisons, antimetabolites, UV radiation and DNA intercalators. The MMR-dependent DNA damage signalling pathways activated by the above agents are also ill-defined. To date, signalling cascades involving the Ataxia telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), as well as the stress-activated kinases JNK/SAPK and p38alpha have been linked with methylating agent and 6-thioguanine (TG) treatments, while cisplatin damage was reported to activate the c-Abl and JNK/SAPK kinases in MMR-dependent manner. MMR defects are found in several different cancer types, both familiar and sporadic, and it is possible that the involvement of the MMR system in DNA damage signalling play an important role in transformation. The scope of this article is to provide a brief overview of the recent literature on this subject and to raise questions that could be addressed in future studies.
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Disparidad de Par Base , Daño del ADN , Reparación del ADN , Animales , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/metabolismo , Cisplatino/farmacología , ADN/ultraestructura , Metilación de ADN , Proteínas de Unión al ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Modelos Biológicos , Modelos Genéticos , Proteínas Serina-Treonina Quinasas/metabolismo , Radiación Ionizante , Recombinación Genética , Transducción de Señal , Tioguanina/química , Proteínas Supresoras de Tumor , Rayos Ultravioleta , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: The discovery of cytosine hydroxymethylation (5hmC) as a mechanism that potentially controls DNA methylation changes typical of neoplasia prompted us to investigate its behaviour in colon cancer. 5hmC is globally reduced in proliferating cells such as colon tumours and the gut crypt progenitors, from which tumours can arise. RESULTS: Here, we show that colorectal tumours and cancer cells express Ten-Eleven-Translocation (TET) transcripts at levels similar to normal tissues. Genome-wide analyses show that promoters marked by 5hmC in normal tissue, and those identified as TET2 targets in colorectal cancer cells, are resistant to methylation gain in cancer. In vitro studies of TET2 in cancer cells confirm that these promoters are resistant to methylation gain independently of sustained TET2 expression. We also find that a considerable number of the methylation gain-resistant promoters marked by 5hmC in normal colon overlap with those that are marked with poised bivalent histone modifications in embryonic stem cells. CONCLUSIONS: Together our results indicate that promoters that acquire 5hmC upon normal colon differentiation are innately resistant to neoplastic hypermethylation by mechanisms that do not require high levels of 5hmC in tumours. Our study highlights the potential of cytosine modifications as biomarkers of cancerous cell proliferation.
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Neoplasias del Colon/genética , Citosina/análogos & derivados , Metilación de ADN/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , 5-Metilcitosina/análogos & derivados , Proliferación Celular/genética , Neoplasias del Colon/patología , Citosina/metabolismo , Proteínas de Unión al ADN/genética , Dioxigenasas , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Proteínas Proto-Oncogénicas/genéticaRESUMEN
CONTEXT: Enhancer of zeste homolog 2 (EZH2) is a histone lysine methyltransferase belonging to the polycomb group protein family. Overexpression of EZH2 has been found in several human malignancies including hematological and solid tumors. OBJECTIVES: In this study we investigated the expression levels of EZH2 and its polycomb group protein partners in thyroid carcinoma tissues with different degrees of malignancy to identify potential new therapeutic targets for anaplastic thyroid carcinoma (ATC). RESULTS: We show that high EZH2 expression levels are characteristic of undifferentiated ATC, whereas no significant changes were observed in well-differentiated papillary and follicular thyroid carcinomas as compared with normal thyroid. Knockdown of EZH2 in ATC cell lines results in cell growth inhibition, loss of anchorage-independent growth, migration, and invasion properties. Moreover, we demonstrate that EZH2 directly controls differentiation of ATC cells by silencing the thyroid specific transcription factor paired-box gene 8 (PAX8). CONCLUSIONS: EZH2 is specifically overexpressed in ATC, and it directly contributes to transcriptional silencing of PAX8 gene and ATC differentiation.
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Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción/genética , Animales , Adhesión Celular/genética , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Proteínas de Unión al ADN/metabolismo , Progresión de la Enfermedad , Proteína Potenciadora del Homólogo Zeste 2 , Silenciador del Gen/fisiología , Células HeLa , Humanos , Ratones , Ratones Transgénicos , Factor de Transcripción PAX8 , Factores de Transcripción Paired Box/genética , Complejo Represivo Polycomb 2 , Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Factores de Transcripción/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
BACKGROUND: Polycomb group (PcG) genes code for chromatin multiprotein complexes that are responsible for maintaining gene silencing of transcriptional programs during differentiation and in adult tissues. Despite the large amount of information on PcG function during development and cell identity homeostasis, little is known regarding the dynamics of PcG complexes and their role during terminal differentiation. RESULTS: We show that two distinct polycomb repressive complex (PRC)2 complexes contribute to skeletal muscle cell differentiation: the PRC2-Ezh2 complex, which is bound to the myogenin (MyoG) promoter and muscle creatine kinase (mCK) enhancer in proliferating myoblasts, and the PRC2-Ezh1 complex, which replaces PRC2-Ezh2 on MyoG promoter in post-mitotic myotubes. Interestingly, the opposing dynamics of PRC2-Ezh2 and PRC2-Ezh1 at these muscle regulatory regions is differentially regulated at the chromatin level by Msk1 dependent methyl/phospho switch mechanism involving phosphorylation of serine 28 of the H3 histone (H3S28ph). While Msk1/H3S28ph is critical for the displacement of the PRC2-Ezh2 complex, this pathway does not influence the binding of PRC2-Ezh1 on the chromatin. Importantly, depletion of Ezh1 impairs muscle differentiation and the chromatin recruitment of MyoD to the MyoG promoter in differentiating myotubes. We propose that PRC2-Ezh1 is necessary for controlling the proper timing of MyoG transcriptional activation and thus, in contrast to PRC2-Ezh2, is required for myogenic differentiation. CONCLUSIONS: Our data reveal another important layer of epigenetic control orchestrating skeletal muscle cell terminal differentiation, and introduce a novel function of the PRC2-Ezh1 complex in promoter setting.