RESUMO
How Polycomb repressive complex 2 (PRC2) is regulated by RNA remains an unsolved problem. Although PRC2 binds G-tracts with the potential to form RNA G-quadruplexes (rG4s), whether rG4s fold extensively in vivo and whether PRC2 binds folded or unfolded rG4 are unknown. Using the X-inactivation model in mouse embryonic stem cells, here we identify multiple folded rG4s in Xist RNA and demonstrate that PRC2 preferentially binds folded rG4s. High-affinity rG4 binding inhibits PRC2's histone methyltransferase activity, and stabilizing rG4 in vivo antagonizes H3 at lysine 27 (H3K27me3) enrichment on the inactive X chromosome. Surprisingly, mutagenizing the rG4 does not affect PRC2 recruitment but promotes its release and catalytic activation on chromatin. H3K27me3 marks are misplaced, however, and gene silencing is compromised. Xist-PRC2 complexes become entrapped in the S1 chromosome compartment, precluding the required translocation into the S2 compartment. Thus, Xist rG4 folding controls PRC2 activity, H3K27me3 enrichment, and the stepwise regulation of chromosome-wide gene silencing.
Assuntos
Quadruplex G , Histonas , Complexo Repressor Polycomb 2 , RNA Longo não Codificante , Inativação do Cromossomo X , Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Camundongos , Complexo Repressor Polycomb 2/metabolismo , Complexo Repressor Polycomb 2/genética , Histonas/metabolismo , Histonas/genética , Células-Tronco Embrionárias Murinas/metabolismo , Cromatina/metabolismo , Cromatina/genética , Cromossomo X/genética , Cromossomo X/metabolismo , Inativação Gênica , Dobramento de RNA , Ligação ProteicaRESUMO
G9a is a histone methyltransferase responsible for the dimethylation of histone H3 at lysine 9 (H3K9me2). G9a plays key roles in transcriptional silencing of developmentally regulated genes, but its role in X-chromosome inactivation (XCI) has been under debate. Here, we uncover a female-specific function of G9a and demonstrate that deleting G9a has a disproportionate impact on the X chromosome relative to the rest of the genome. G9a deficiency causes a failure of XCI and female-specific hypersensitivity to drug inhibition of H3K9me2. We show that G9a interacts with Tsix and Xist RNAs, and that competitive inhibition of the G9a-RNA interaction recapitulates the XCI defect. During XCI, Xist recruits G9a to silence X-linked genes on the future inactive X. In parallel on the future Xa, Tsix recruits G9a to silence Xist in cis Thus, RNA tethers G9a for allele-specific targeting of the H3K9me2 modification and the G9a-RNA interaction is essential for XCI.
Assuntos
Cromossomos Humanos X , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Metiltransferases , RNA Longo não Codificante , Feminino , Histonas/metabolismo , Humanos , Metiltransferases/genética , RNA Longo não Codificante/genética , Inativação do Cromossomo X/genéticaRESUMO
Monomethylation of lysine 4 on histone H3 (H3K4me1) is a well-established feature of enhancers and promoters, although its function is unknown. Here, we uncover roles for H3K4me1 in diverse cell types. Remarkably, we find that MLL3/4 provokes monomethylation of promoter regions and the conditional repression of muscle and inflammatory response genes in myoblasts. During myogenesis, muscle genes are activated, lose MLL3 occupancy, and become H3K4-trimethylated through an alternative COMPASS complex. Monomethylation-mediated repression was not restricted to skeletal muscle. Together with H3K27me3 and H4K20me1, H3K4me1 was associated with transcriptional silencing in embryonic fibroblasts, macrophages, and human embryonic stem cells (ESCs). On promoters of active genes, we find that H3K4me1 spatially demarcates the recruitment of factors that interact with H3K4me3, including ING1, which, in turn, recruits Sin3A. Our findings point to a unique role for H3K4 monomethylation in establishing boundaries that restrict the recruitment of chromatin-modifying enzymes to defined regions within promoters.
Assuntos
Cromatina , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Desenvolvimento Muscular/genética , Mioblastos/metabolismo , Animais , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Fibroblastos/citologia , Fibroblastos/metabolismo , Genoma , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Humanos , Proteína 1 Inibidora do Crescimento , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Mioblastos/citologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Complexo Correpressor Histona Desacetilase e Sin3 , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Rett syndrome (RTT) is a severe neurodevelopmental disorder caused by a mutation in the X-linked methyl-CpG-binding protein 2 (MECP2). There is currently no disease-specific treatment, but MECP2 restoration through reactivation of the inactive X (Xi) has been of considerable interest. Progress toward an Xi-reactivation therapy has been hampered by a lack of suitable female mouse models. Because of cellular mosaicism due to random X-chromosome inactivation (XCI), Mecp2+/- heterozygous females develop only mild RTT. Here, we create an improved female mouse model by introducing a mutation in Tsix, the antisense regulator of XCI allelic choice. Tsix-Mecp2 mice show reduced MECP2 mosaicism and closely phenocopy the severely affected Mecp2-null males. Tsix-Mecp2 females demonstrate shortened lifespan, motor weakness, tremors, and gait disturbance. Intriguingly, they also exhibit repetitive behaviors, as is often seen in human RTT, including excessive grooming and biting that result in self-injury. With a Tsix allelic series, we vary MECP2 levels in brain and demonstrate a direct, but nonlinear correlation between MECP2 levels and phenotypic improvement. As little as 5-10% MECP2 restoration improves neuromotor function and extends lifespan five- to eightfold. Our study thus guides future pharmacological strategies and suggests that partial MECP2 restoration could have disproportionate therapeutic benefit.
Assuntos
Modelos Animais de Doenças , Genes Ligados ao Cromossomo X/genética , Longevidade/genética , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , RNA Longo não Codificante/genética , Síndrome de Rett/genética , Alelos , Animais , Comportamento Animal , Encéfalo/patologia , Feminino , Heterozigoto , Humanos , Masculino , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mosaicismo , Atividade Motora/genética , Mutação , Fenótipo , RNA Longo não Codificante/metabolismo , Síndrome de Rett/mortalidade , Síndrome de Rett/patologia , Inativação do Cromossomo XRESUMO
To identify the compendium of distal regulatory elements that govern myogenic differentiation, we generated chromatin state maps based on histone modifications and recruitment of factors that typify enhancers in myoblasts and myotubes. We found a striking concordance between the locations of these newly defined enhancers, MyoD1-binding events, and noncoding RNA transcripts. These enhancers recruit several sequence-specific transcription factors in a spatially constrained manner around MyoD1-binding sites. Remarkably, MyoD1-null myoblasts show a wholesale loss of recruitment of these factors as well as diminished monomethylation of H3K4 (H3K4me1) and acetylation of H3K27 (H3K27ac) and reduced recruitment of Set7, an H3K4 monomethylase. Surprisingly, we found that H3K4me1, but not H3K27ac, could be restored by re-expression of MyoD1 in MyoD1(-/-) myoblasts, although re-expression of this factor in MyoD1-null myotubes restored both histone modifications. Our studies identified a role for MyoD1 in condition-specific enhancer assembly through recruitment of transcription factors and histone-modifying enzymes that shape muscle differentiation.
Assuntos
Elementos Facilitadores Genéticos/genética , Genoma , Músculo Esquelético/metabolismo , Proteína MyoD/genética , Proteína MyoD/metabolismo , Animais , Diferenciação Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Músculo Esquelético/citologiaRESUMO
Although great advances have been made in the treatment of pediatric acute lymphoblastic leukemia, up to one of five patients will relapse, and their prognosis thereafter is dismal. We have previously identified recurrent deletions in TBL1XR1, which encodes for an F-box like protein responsible for regulating the nuclear hormone repressor complex stability. Here we model TBL1XR1 deletions in B-precursor ALL cell lines and show that TBL1XR1 knockdown results in reduced glucocorticoid receptor recruitment to glucocorticoid responsive genes and ultimately decreased glucocorticoid signaling caused by increased levels of nuclear hormone repressor 1 and HDAC3. Reduction in glucocorticoid signaling in TBL1XR1-depleted lines resulted in resistance to glucocorticoid agonists, but not to other chemotherapeutic agents. Importantly, we show that treatment with the HDAC inhibitor SAHA restores sensitivity to prednisolone in TBL1XR1-depleted cells. Altogether, our data indicate that loss of TBL1XR1 is a novel driver of glucocorticoid resistance in ALL and that epigenetic therapy may have future application in restoring drug sensitivity at relapse.
Assuntos
Cromatina/metabolismo , Resistencia a Medicamentos Antineoplásicos , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Glucocorticoides/metabolismo , Proteínas Repressoras/metabolismo , Adolescente , Linhagem Celular Tumoral , Criança , Pré-Escolar , Cromatina/genética , Feminino , Técnicas de Silenciamento de Genes , Glucocorticoides/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Masculino , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Glucocorticoides/genética , Proteínas Repressoras/genéticaRESUMO
The early 1980s revelation of cis-acting genomic elements, known as transcriptional enhancers, is still regarded as one of the fundamental discoveries in the genomic field. However, only with the emergence of genome-wide techniques has the genuine biological scope of enhancers begun to be fully uncovered. Massive scientific efforts of multiple laboratories rapidly advanced the overall perception that enhancers are typified by common epigenetic characteristics that distinguish their activating potential. Broadly, chromatin modifiers and transcriptional regulators lay down the essential foundations necessary for constituting enhancers in their activated form. Basing on genome-wide ChIP-sequencing of enhancer-related marks we identified myogenic enhancers before and after muscle differentiation and discovered that MyoD was bound to nearly a third of condition-specific enhancers. Experimental studies that tested the deposition patterns of enhancer-related epigenetic marks in MyoD-null myoblasts revealed the high dependency that a specific set of muscle enhancers have towards this transcriptional regulator. Re-expression of MyoD restored the deposition of enhancer-related marks at myotube-specific enhancers and partially at myoblasts-specific enhancers. Our proposed mechanistic model suggests that MyoD is involved in recruitment of methyltransferase Set7, acetyltransferase p300 and deposition of H3K4me1 and H3K27ac at myogenic enhancers. In addition, MyoD binding at enhancers is associated with PolII occupancy and with local noncoding transcription. Modulation of muscle enhancers is suggested to be coordinated via transcription factors docking, including c-Jun and Jdp2 that bind to muscle enhancers in a MyoD-dependent manner. We hypothesize that distinct transcription factors may act as placeholders and mediate the assembly of newly formed myogenic enhancers.
Assuntos
Elementos Facilitadores Genéticos , Epigênese Genética , Proteína MyoD/metabolismo , Mioblastos/citologia , Animais , Diferenciação Celular , Epigenômica , Regulação da Expressão Gênica , Humanos , Mioblastos/metabolismoRESUMO
BACKGROUND: Ikaros, the product of IKZF1, is a regulator of lymphoid development and polymorphisms in the gene have been associated with the acute lymphoblastic leukemia (ALL). Additionally, IKZF1 deletions and mutations identify high-risk biological subsets of childhood ALL [Georgopoulos et al. Cell 1995;83(2):289-299; Mullighan et al. N Engl J Md 2009;360(5):470-480]. PROCEDURES: To discover the underlying pathways modulated by Ikaros we performed gene expression and gene ontology analysis in IKZF1 deleted primary B-ALL pediatric patient samples. To validate downstream targets we performed qPCR on individual patient samples. We also created IKZF1 knockdown B-ALL cell lines with over 50% reduction of Ikaros, mimicking haplosufficient Ikaros deletions, and again performed qPCR to investigate the downstream targets. Finally, to understand the association of Ikaros deletion with a poor prognosis we challenged our IKZF1 knockdown cell lines with chemotherapy and compared responses to IKZF1 wild-type controls. RESULTS: We report a specific gene expression signature of 735 up-regulated and 473 down-regulated genes in IKZF1 deleted primary B-ALL pediatric patient samples. Gene ontology studies revealed an up-regulation of genes associated with cell adhesion, cytoskeletal regulation, and motility in IKZF deleted patient samples. Validated up-regulated target genes in IKZF1 deleted patient samples included CTNND1 and PVRL2 (P = 0.0003 and P = 0.001), and RAB3IP and SPIB (P = 0.005 and P = 0.032) were down-regulated. In further studies in IKZF1 knockdown cell lines, apoptosis assays showed no significant chemoresistance. CONCLUSION: IKZF1 knockdown alone does not impart intrinsic chemotherapy resistance suggesting that the association with a poor prognosis may be due to additional lesions, microenvironmental interactions with the bone marrow niche, or other factors.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Fator de Transcrição Ikaros/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Western Blotting , Criança , Proteínas de Fusão bcr-abl/genética , Deleção de Genes , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TranscriptomaRESUMO
We have examined changes in the chromatin landscape during muscle differentiation by mapping the genome-wide location of ten key histone marks and transcription factors in mouse myoblasts and terminally differentiated myotubes, providing an exceptionally rich dataset that has enabled discovery of key epigenetic changes underlying myogenesis. Using this compendium, we focused on a well-known repressive mark, histone H3 lysine 27 trimethylation, and identified novel regulatory elements flanking the myogenin gene that function as a key differentiation-dependent switch during myogenesis. Next, we examined the role of Polycomb-mediated H3K27 methylation in gene repression by systematically ablating components of both PRC1 and PRC2 complexes. Surprisingly, we found mechanistic differences between transient and permanent repression of muscle differentiation and lineage commitment genes and observed that the loss of PRC1 and PRC2 components produced opposing differentiation defects. These phenotypes illustrate striking differences as compared to embryonic stem cell differentiation and suggest that PRC1 and PRC2 do not operate sequentially in muscle cells. Our studies of PRC1 occupancy also suggested a "fail-safe" mechanism, whereby PRC1/Bmi1 concentrates at genes specifying nonmuscle lineages, helping to retain H3K27me3 in the face of declining Ezh2-mediated methyltransferase activity in differentiated cells.
Assuntos
Epigênese Genética , Estudo de Associação Genômica Ampla , Animais , Diferenciação Celular , Cromatina/metabolismo , Células-Tronco Embrionárias/citologia , Histonas/metabolismo , Humanos , Lisina/química , Metilação , Camundongos , Desenvolvimento Muscular , Músculos/fisiologia , Fenótipo , Proteínas do Grupo Polycomb , Proteínas Repressoras/metabolismo , Transcrição GênicaRESUMO
The isolation of protein-RNA complexes in the "denaturing cross-linked RNA immunoprecipitation" (dCLIP) protocol is based on biotin-tagging proteins of interest, UV cross-linking RNA to protein in vivo, RNase protection assay, and isolating RNA-protein complexes under denaturing conditions over a streptavidin column. Insofar as conventional antibody-based CLIP assays have been challenging to apply to Polycomb complexes, dCLIP has been applied successfully and yields small RNA footprints from which de novo motif analysis can be performed to identify RNA binding motifs. For complete details on the use and execution of this protocol, please refer to Rosenberg et al. (2017).
Assuntos
Imunoprecipitação/métodos , Pegadas de Proteínas/métodos , Proteínas de Ligação a RNA , RNA , Animais , Células-Tronco Embrionárias , Células HEK293 , Humanos , Camundongos , RNA/química , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de RNARESUMO
Although polycomb repressive complex 2 (PRC2) is now recognized as an RNA-binding complex, the full range of binding motifs and why PRC2-RNA complexes often associate with active genes have not been elucidated. Here, we identify high-affinity RNA motifs whose mutations weaken PRC2 binding and attenuate its repressive function in mouse embryonic stem cells. Interactions occur at promoter-proximal regions and frequently coincide with pausing of RNA polymerase II (POL-II). Surprisingly, while PRC2-associated nascent transcripts are highly expressed, ablating PRC2 further upregulates expression via loss of pausing and enhanced transcription elongation. Thus, PRC2-nascent RNA complexes operate as rheostats to fine-tune transcription by regulating transitions between pausing and elongation, explaining why PRC2-RNA complexes frequently occur within active genes. Nascent RNA also targets PRC2 in cis and downregulates neighboring genes. We propose a unifying model in which RNA specifically recruits PRC2 to repress genes through POL-II pausing and, more classically, trimethylation of histone H3 at Lys27.
Assuntos
Regulação da Expressão Gênica/genética , Complexo Repressor Polycomb 2/metabolismo , Biossíntese de Proteínas/fisiologia , RNA Polimerase II/metabolismo , RNA/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Histonas/metabolismo , Metilação , Camundongos , Motivos de Nucleotídeos/genética , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas/genética , RNA/genética , Transcrição Gênica/genética , Ativação Transcricional/genéticaRESUMO
Deregulation of Ras pathways results in complex abnormalities of multiple signaling cascades that contribute to human malignancies. Ras is therefore considered an appropriate target for cancer therapy. In light of the complexity of the deregulated Ras pathway, it is important to decipher at the molecular level the response of cancer cells to Ras inhibitors that would reregulate it. In the present study, we used gene expression profiling as a robust method for the global dissection of gene expression alterations that resulted from treatment with the Ras inhibitor S-farnesylthiosalicylic acid (FTS; salirasib). Use of a ranking-based procedure, combined with functional analysis and promoter sequence analysis, enabled us to decipher the common and most prominent patterns of the transcriptional response of five different human cancer cell lines to FTS. Remarkably, the analysis identified a distinctive core transcriptional response to FTS that was common to all cancer cell lines tested. This signature fits well to a recently described deregulated Ras pathway signature that predicted sensitivity to FTS. Taken together, these studies provide strong support for the conclusion that FTS specifically reregulates defective Ras pathways in human tumor cells. Ras pathway reregulation by FTS was manifested by repression of E2F-regulated and NF-Y-regulated genes and of the transcription factor FOS (all of which control cell proliferation), repression of survivin expression (which blocks apoptosis), and induction of activating transcription factor-regulated and Bach2-regulated genes (which participate in translation and stress responses). Our results suggest that cancer patients with deregulated Ras pathway tumors might benefit from FTS treatment.
Assuntos
Antineoplásicos/farmacologia , Farneseno Álcool/análogos & derivados , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Salicilatos/farmacologia , Proteínas ras/antagonistas & inibidores , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Análise por Conglomerados , Regulação para Baixo/efeitos dos fármacos , Farneseno Álcool/farmacologia , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Neoplasias/metabolismo , Regiões Promotoras Genéticas , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Proteínas ras/metabolismoRESUMO
The noncoding RNA Xist recruits silencing factors to the inactive X chromosome (Xi) and facilitates re-organization of Xi structure. Here, we examine the mouse epigenomic landscape of Xi and assess how Xist alters chromatin accessibility. Xist deletion triggers a gain of accessibility of select chromatin regions that is regulated by BRG1, an ATPase subunit of the SWI/SNF chromatin-remodeling complex. In vitro, RNA binding inhibits nucleosome-remodeling and ATPase activities of BRG1, while in cell culture Xist directly interacts with BRG1 and expels BRG1 from the Xi. Xist ablation leads to a selective return of BRG1 in cis, starting from pre-existing BRG1 sites that are free of Xist. BRG1 re-association correlates with cohesin binding and restoration of topologically associated domains (TADs) and results in the formation of de novo Xi 'superloops'. Thus, Xist binding inhibits BRG1's nucleosome-remodeling activity and results in expulsion of the SWI/SNF complex from the Xi.
Assuntos
Cromatina/metabolismo , RNA Longo não Codificante/metabolismo , Cromossomo X/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Linhagem Celular , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Epigênese Genética/genética , Epigênese Genética/fisiologia , Feminino , Camundongos , Nucleossomos/genética , Nucleossomos/metabolismo , RNA Longo não Codificante/genética , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Cromossomo X/genéticaRESUMO
Active Ras and phosphatidylinositol-3-kinase-dependent pathways contribute to the malignant phenotype of glioblastoma multiformes (GBM). Here we show that the Ras inhibitor trans-farnesylthiosalicylic acid (FTS) exhibits profound antioncogenic effects in U87 GBM cells. FTS inhibited active Ras and attenuated Ras signaling to extracellular signal-regulated kinase, phosphatidylinositol-3-kinase, and Akt. Concomitantly, hypoxia-inducible factor 1alpha (HIF-1alpha) disappeared, expression of key glycolysis pathway enzymes and of other HIF-1alpha-regulated genes (including vascular endothelial growth factor and the Glut-1 glucose transporter) was down-regulated, and glycolysis was halted. This led to a dramatic reduction in ATP, resulting in a severe energy crisis. In addition, the expression of E2F-regulated genes was down-regulated in the FTS-treated cells. Consequently, U87 cell growth was arrested and the cells died. These results show that FTS is a potent down-regulator of HIF-1alpha and might therefore block invasiveness, survival, and angiogenesis in GBM.
Assuntos
Farneseno Álcool/análogos & derivados , Farneseno Álcool/farmacologia , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Salicilatos/farmacologia , Fatores de Transcrição/fisiologia , Proteínas ras/antagonistas & inibidores , Morte Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/patologia , Glicólise/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Fosfatidilinositol 3-Quinases/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
The Ras inhibitor farnesylthiosalicylic acid (FTS) has been shown to induce apoptosis in glioblastoma multiforme, but its mechanism of action was unknown. We show that FTS or dominant-negative Ras, by deregulating extracellular signal-regulated kinase and Akt signaling, decreases survivin gene transcripts in U87 glioblastoma multiforme, leading to disappearance of survivin protein and cell death. FTS affected both Ras-controlled regulators of survivin transcription and Ras-regulated survival signals. Thus, Ras inhibition by FTS resulted in release of the survivin "brake" on apoptosis and in activation of the mitochondrial apoptotic pathway: dephosphorylation of Bad, activation of Bax, release of cytochrome c, and caspase activation. FTS-induced apoptosis of U87 cells was strongly attenuated by forced expression of survivin or by caspase inhibitors. These results show that resistance to apoptosis in glioblastoma multiforme can be abolished by a single Ras inhibitor, which targets both survivin, a critical inhibitor of apoptosis, and the intrinsic mitochondrial apoptotic machinery.
Assuntos
Apoptose/efeitos dos fármacos , Farneseno Álcool/análogos & derivados , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Salicilatos/farmacologia , Proteínas ras/antagonistas & inibidores , Caspases/metabolismo , Linhagem Celular Tumoral , Farneseno Álcool/farmacologia , Expressão Gênica , Glioblastoma/genética , Humanos , Proteínas Inibidoras de Apoptose , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Survivina , Transfecção , Proteínas ras/genéticaRESUMO
Chen et al (Reports, 28 October 2016, p. 468) proposed that an interaction between Xist RNA and Lamin B receptor (LBR) is necessary and sufficient for Xist spreading during X-chromosome inactivation. We reanalyzed their data and found that reported genotypes of mutants are not supported by the sequencing data. These inconsistencies preclude assessment of the role of LBR in Xist spreading.
Assuntos
Inativação Gênica , Lâmina Nuclear , RNA Longo não Codificante/genética , RNA não Traduzido/genética , Cromossomo X , Inativação do Cromossomo XRESUMO
Interaction networks between chromatin complexes and long noncoding RNAs have become a recurrent theme in epigenetic regulation. However, technical limitations have precluded identification of RNA binding motifs for chromatin-associated proteins. Here, we add a denaturation step to UV-crosslink RNA immunoprecipitation (dCLIP) and apply dCLIP to mouse and human chromobox homolog 7 (CBX7), an RNA binding subunit of Polycomb repressive complex 1 (PRC1). In both species, CBX7 predominantly binds 3' UTRs of messenger RNAs. CBX7 binds with a median RNA "footprint" of 171-183 nucleotides, the small size of which facilitates motif identification by bioinformatics. We find four families of consensus RNA motifs in mouse, and independent analysis of human CBX7 dCLIP data identifies similar motifs. Their mutation abolishes CBX7 binding in vitro. Pharmacological intervention with antisense oligonucleotides paradoxically increases CBX7 binding and enhances gene expression. These data support the utility of dCLIP and reveal an unexpected functional interaction between CBX7 and the 3' UTRs of mRNA.
Assuntos
Regiões 3' não Traduzidas/genética , Cromatina/genética , Complexo Repressor Polycomb 1/genética , RNA Mensageiro/genética , Animais , Epigênese Genética/genética , Células HEK293 , Humanos , Camundongos , Proteínas do Grupo Polycomb/genética , Ligação Proteica/genética , RNA Longo não Codificante/genéticaRESUMO
Prostate cancer is one of the most frequently diagnosed cancers in human males. Progression of these tumors is facilitated by autocrine/paracrine growth factors which activate critical signaling cascades that promote prostate cancer cell growth, survival and migration. Among these, Ras pathways have a major role. Here we examined the effect of the Ras inhibitor S-trans, trans-farnesylthiosalicylic acid (FTS), on growth and viability of androgen-dependent and androgen-independent prostate cancer cells. FTS downregulated Ras, inhibited signaling to Akt and reduced the levels of cell-cycle regulatory proteins including cyclin D1, p-RB, E2F-1 and cdc42 in LNCaP and PC3 cells. Consequently the anchorage-dependent and anchorage-independent growth of LNCaP and PC3 cells were inhibited. FTS also induced apoptotic cell death which was inhibited by the broad-spectrum caspases inhibitor, Boc-asp-FMK. Our study demonstrated that androgen-dependent and androgen-independent prostate cancer cells require active Ras for growth and survival. Ras inhibition by FTS results in growth arrest and cell death. FTS may be qualified as a potential agent for the treatment of prostate cancer.
Assuntos
Androgênios/fisiologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Farneseno Álcool/análogos & derivados , Salicilatos/farmacologia , Proteínas ras/antagonistas & inibidores , Clorometilcetonas de Aminoácidos/farmacologia , Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ciclina D1/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Fator de Transcrição E2F1/metabolismo , Farneseno Álcool/farmacologia , Humanos , Masculino , Miotonina Proteína Quinase , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Inibidores de Serina Proteinase/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/metabolismo , Proteínas ras/fisiologiaRESUMO
The ability to regulate gene expression in response to environmental alterations is vital for the endurance of all cells. However, unlike bacteria and unicellular organisms, cells of multicellular eukaryotes have developed this competency in a highly sophisticated manner, which ultimately allows for multiple lineages of differentiated cells. To maintain stability and generate progeny, differentiated cells must remain lineage-committed through numerous cell generations, and therefore their transcriptional modus operandi ought to be memorized and transmittable. To preserve the specialized characteristics of differentiated cells, it is crucial that transcriptional alterations that are triggered by specific external or intrinsic stimuli can last also after stimuli fading and propagate onto daughter cells. The unique composition of DNA and histones, and their ability to acquire a variety of epigenetic modifications, enables eukaryotic chromatin to assimilate cellular plasticity and molecular memory. The most well-studied types of epigenetic modifiers are covalently modifying DNA or histones, mostly in a reversible manner. Additional epigenetic mechanisms include histone variant replacement, energy-utilizing remodeling factors, and noncoding transcripts assembled with modifying complexes. Working with multifunctional complexes including transcription factors, epigenetic modifiers have the potential to dictate a variety of transcriptional programs underlying all cellular lineages, while utilizing in each the same source DNA as their substrates.
Assuntos
DNA/metabolismo , Enzimas/metabolismo , Epigênese Genética , Eucariotos/genética , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Eucariotos/metabolismo , Humanos , Metilação , RNA Longo não Codificante/metabolismoRESUMO
MyoD1 is a key regulator that orchestrates skeletal muscle differentiation through the regulation of gene expression. Although many studies have focused on its role in transcriptional control at gene promoters, less is known regarding the role of MyoD1 in the assembly of active enhancers. Here, we discuss novel data that point to the ability of MyoD1 to mediate the assembly of active enhancers that augment the transcription of genes essential for muscle development and lineage specification. Based on genome-wide studies of epigenetic marks that typify active enhancers, we recently identified the compendium of distal regulatory elements that dictate transcriptional programs during myogenesis. Superimposition of MyoD1 binding sites upon the locations of muscle enhancers revealed its unequivocal binding to a core region of nearly a third of condition-specific muscle enhancers. Further studies exploring deposition of enhancer-related epigenetic marks in myoblasts lacking MyoD1 demonstrate the dependence of muscle enhancer assembly on the presence of MyoD1. We propose a model wherein MyoD1 mediates recruitment of Set7, H3K4me1, H3K27ac, p300, and RNAP II to MyoD1-bound enhancers to establish condition-specific activation of muscle genes. Moreover, muscle enhancers are modulated through coordinated binding of transcription factors, including c-Jun, Jdp2, Meis, and Runx1, which are recruited to muscle enhancers in a MyoD1-dependent manner. Thus, MyoD1 and enhancer-associated transcription factors function coordinately to assemble and regulate enhancers, thereby augmenting expression of muscle-related genes.