RESUMEN
Eukaryotic gene transcription is regulated at many steps, including RNA polymerase II (Pol II) recruitment, transcription initiation, promoter-proximal Pol II pause release, and transcription termination; however, mechanisms regulating transcription during productive elongation remain poorly understood. Enhancers, which activate gene transcription, themselves undergo Pol II-mediated transcription, but our understanding of enhancer transcription and enhancer RNAs (eRNAs) remains incomplete. Here we show that transcription at intragenic enhancers interferes with and attenuates host gene transcription during productive elongation. While the extent of attenuation correlates positively with nascent eRNA expression, the act of intragenic enhancer transcription alone, but not eRNAs, explains the attenuation. Through CRISPR/Cas9-mediated deletions, we demonstrate a physiological role for intragenic enhancer-mediated transcription attenuation in cell fate determination. We propose that intragenic enhancers not only enhance transcription of one or more genes from a distance but also fine-tune transcription of their host gene through transcription interference, facilitating differential utilization of the same regulatory element for disparate functions.
Asunto(s)
Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Células Madre Embrionarias de Ratones/metabolismo , ARN Polimerasa II/genética , Elongación de la Transcripción Genética , Animales , Sistemas CRISPR-Cas , Línea Celular , Cromatina/química , Cromatina/metabolismo , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Edición Génica , Ratones , Células Madre Embrionarias de Ratones/citología , Regiones Promotoras Genéticas , ARN/genética , ARN/metabolismo , ARN Polimerasa II/metabolismoRESUMEN
Bivalent chromatin is characterized by the simultaneous presence of H3K4me3 and H3K27me3, histone modifications generally associated with transcriptionally active and repressed chromatin, respectively. Prevalent in embryonic stem cells (ESCs), bivalency is postulated to poise/prime lineage-controlling developmental genes for rapid activation during embryogenesis while maintaining a transcriptionally repressed state in the absence of activation cues; however, this hypothesis remains to be directly tested. Most gene promoters DNA hypermethylated in adult human cancers are bivalently marked in ESCs, and it was speculated that bivalency predisposes them for aberrant de novo DNA methylation and irreversible silencing in cancer, but evidence supporting this model is largely lacking. Here, we show that bivalent chromatin does not poise genes for rapid activation but protects promoters from de novo DNA methylation. Genome-wide studies in differentiating ESCs reveal that activation of bivalent genes is no more rapid than that of other transcriptionally silent genes, challenging the premise that H3K4me3 is instructive for transcription. H3K4me3 at bivalent promoters-a product of the underlying DNA sequence-persists in nearly all cell types irrespective of gene expression and confers protection from de novo DNA methylation. Bivalent genes in ESCs that are frequent targets of aberrant hypermethylation in cancer are particularly strongly associated with loss of H3K4me3/bivalency in cancer. Altogether, our findings suggest that bivalency protects reversibly repressed genes from irreversible silencing and that loss of H3K4me3 may make them more susceptible to aberrant DNA methylation in diseases such as cancer. Bivalency may thus represent a distinct regulatory mechanism for maintaining epigenetic plasticity.
RESUMEN
Cell type-specific master transcription factors (TFs) play vital roles in defining cell identity and function. However, the roles ubiquitous factors play in the specification of cell identity remain underappreciated. Here we show that the ubiquitous CCAAT-binding NF-Y complex is required for the maintenance of embryonic stem cell (ESC) identity and is an essential component of the core pluripotency network. Genome-wide studies in ESCs and neurons reveal that NF-Y regulates not only genes with housekeeping functions through cell type-invariant promoter-proximal binding, but also genes required for cell identity by binding to cell type-specific enhancers with master TFs. Mechanistically, NF-Y's distinct DNA-binding mode promotes master/pioneer TF binding at enhancers by facilitating a permissive chromatin conformation. Our studies unearth a conceptually unique function for histone-fold domain (HFD) protein NF-Y in promoting chromatin accessibility and suggest that other HFD proteins with analogous structural and DNA-binding properties may function in similar ways.
Asunto(s)
Factor de Unión a CCAAT/fisiología , Cromatina/metabolismo , Histonas/metabolismo , Animales , Sitios de Unión , Factor de Unión a CCAAT/metabolismo , Células Cultivadas , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/ultraestructura , Ratones , Modelos Genéticos , Nucleosomas/química , Nucleosomas/metabolismo , Células Madre Pluripotentes , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiologíaRESUMEN
The developmental potential of cells, termed pluripotency, is highly dynamic and progresses through a continuum of naive, formative and primed states. Pluripotency progression of mouse embryonic stem cells (ESCs) from naive to formative and primed state is governed by transcription factors (TFs) and their target genes. Genomic techniques have uncovered a multitude of TF binding sites in ESCs, yet a major challenge lies in identifying target genes from functional binding sites and reconstructing dynamic transcriptional networks underlying pluripotency progression. Here, we integrated time-resolved 'trans-omic' datasets together with TF binding profiles and chromatin conformation data to identify target genes of a panel of TFs. Our analyses revealed that naive TF target genes are more likely to be TFs themselves than those of formative TFs, suggesting denser hierarchies among naive TFs. We also discovered that formative TF target genes are marked by permissive epigenomic signatures in the naive state, indicating that they are poised for expression prior to the initiation of pluripotency transition to the formative state. Finally, our reconstructed transcriptional networks pinpointed the precise timing from naive to formative pluripotency progression and enabled the spatiotemporal mapping of differentiating ESCs to their in vivo counterparts in developing embryos.
Asunto(s)
Desarrollo Embrionario/genética , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Pluripotentes/metabolismo , Factores de Transcripción/genética , Animales , Sitios de Unión/genética , Diferenciación Celular/genética , Cromatina/genética , Regulación del Desarrollo de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Genoma/genética , RatonesRESUMEN
Identification of genes associated with specific biological phenotypes is a fundamental step toward understanding the molecular basis underlying development and pathogenesis. Although RNAi-based high-throughput screens are routinely used for this task, false discovery and sensitivity remain a challenge. Here we describe a computational framework for systematic integration of published gene expression data to identify genes defining a phenotype of interest. We applied our approach to rank-order all genes based on their likelihood of determining ES cell (ESC) identity. RNAi-mediated loss-of-function experiments on top-ranked genes unearthed many novel determinants of ESC identity, thus validating the derived gene ranks to serve as a rich and valuable resource for those working to uncover novel ESC regulators. Underscoring the value of our gene ranks, functional studies of our top-hit Nucleolin (Ncl), abundant in stem and cancer cells, revealed Ncl's essential role in the maintenance of ESC homeostasis by shielding against differentiation-inducing redox imbalance-induced oxidative stress. Notably, we report a conceptually novel mechanism involving a Nucleolin-dependent Nanog-p53 bistable switch regulating the homeostatic balance between self-renewal and differentiation in ESCs. Our findings connect the dots on a previously unknown regulatory circuitry involving genes associated with traits in both ESCs and cancer and might have profound implications for understanding cell fate decisions in cancer stem cells. The proposed computational framework, by helping to prioritize and preselect candidate genes for tests using complex and expensive genetic screens, provides a powerful yet inexpensive means for identification of key cell identity genes.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Homeostasis/genética , Animales , Diferenciación Celular/genética , Proliferación Celular , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Ratones , Proteína Homeótica Nanog , Estrés Oxidativo/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Interferencia de ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reproducibilidad de los Resultados , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismo , NucleolinaRESUMEN
Osteosarcoma (OS) is a primary bone tumor that is most prevalent during adolescence. RUNX2, which stimulates differentiation and suppresses proliferation of osteoblasts, is deregulated in OS. Here, we define pathological roles of RUNX2 in the etiology of OS and mechanisms by which RUNX2 expression is stimulated. RUNX2 is often highly expressed in human OS biopsies and cell lines. Small interference RNA-mediated depletion of RUNX2 inhibits growth of U2OS OS cells. RUNX2 levels are inversely linked to loss of p53 (which predisposes to OS) in distinct OS cell lines and osteoblasts. RUNX2 protein levels decrease upon stabilization of p53 with the MDM2 inhibitor Nutlin-3. Elevated RUNX2 protein expression is post-transcriptionally regulated and directly linked to diminished expression of several validated RUNX2 targeting microRNAs in human OS cells compared with mesenchymal progenitor cells. The p53-dependent miR-34c is the most significantly down-regulated RUNX2 targeting microRNAs in OS. Exogenous supplementation of miR-34c markedly decreases RUNX2 protein levels, whereas 3'-UTR reporter assays establish RUNX2 as a direct target of miR-34c in OS cells. Importantly, Nutlin-3-mediated stabilization of p53 increases expression of miR-34c and decreases RUNX2. Thus, a novel p53-miR-34c-RUNX2 network controls cell growth of osseous cells and is compromised in OS.
Asunto(s)
Neoplasias Óseas/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , MicroARNs/metabolismo , Osteosarcoma/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Ciclo Celular/genética , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Proliferación Celular/efectos de la radiación , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Daño del ADN , Regulación hacia Abajo/genética , Regulación hacia Abajo/efectos de la radiación , Rayos gamma , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Ratones , Osteosarcoma/genética , Osteosarcoma/patología , Estabilidad Proteica/efectos de la radiación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína p14ARF Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/deficienciaRESUMEN
As H. pylori infection progresses, intestinal metaplasia (IM), a key event in gastric carcinogenesis, develops in the stomach. The mechanism by which H. pylori infection causes the trans-differentiation of gastric cells to intestinal-type cells remains an important question. In the current study, we found that RUNX3 is deregulated in all human IM specimens examined by either down regulation or mislocalization; Aberrant localization of a gastric tumor suppressor RUNX3 is observed in most human cases of IM with concurrent H. pylori infection, and RUNX3 is down-regulated in most cases of IM without H. pylori-infection. The cytoplasmic mislocalization of a RUNX3 was associated with H. pylori-induced c-Src activation and RUNX tyrosine phosphorylation. Moreover, gastric epithelial cells of Runx3(-/-) mice expressed the intestinal markers Muc2 and Li-Cadherin, which suggests that the deregulation of Runx3 is a key event in the intestinalization of the gastric epithelium. Collectively, the results of the current study suggest that RUNX3 deregulation is associated with H. pylori-induced pathogenesis and the development of IM.
Asunto(s)
Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Citoplasma/metabolismo , Gastritis/metabolismo , Infecciones por Helicobacter/metabolismo , Helicobacter pylori , Animales , Línea Celular Tumoral , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Citoplasma/microbiología , Citoplasma/patología , Femenino , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Gastritis/genética , Gastritis/patología , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/patología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación/genética , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/patología , Neoplasias Gástricas/prevención & controlRESUMEN
RUNX3 is a transcription factor that functions as a tumor suppressor. In some cancers, RUNX3 expression is down-regulated, usually due to promoter hypermethylation. Recently, it was found that RUNX3 can also be inactivated by the mislocalization of the protein in the cytoplasm. The molecular mechanisms controlling this mislocalization are poorly understood. In this study, we found that the overexpression of Src results in the tyrosine phosphorylation and cytoplasmic localization of RUNX3. We also found that the tyrosine residues of endogenous RUNX3 are phosphorylated and that the protein is localized in the cytoplasm in Src-activated cancer cell lines. We further showed that the knockdown of Src by small interfering RNA, or the inhibition of Src kinase activity by a chemical inhibitor, causes the re-localization of RUNX3 to the nucleus. Collectively, our results demonstrate that the tyrosine phosphorylation of RUNX3 by activated Src is associated with the cytoplasmic localization of RUNX3 in gastric and breast cancers.
Asunto(s)
Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Regulación Neoplásica de la Expresión Génica , Tirosina/química , Familia-src Quinasas/metabolismo , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Células HeLa , Humanos , Fosforilación , Transporte de Proteínas , ARN Interferente Pequeño/metabolismo , Neoplasias Gástricas/metabolismo , Tirosina/genética , Tirosina/metabolismoRESUMEN
The high incidence and fatality rate of uterine cervical cancer warrant effective diagnostic and therapeutic target identification for this disease. Here, we have found a novel oncoprotein FTS (Fused Toes Homolog), which is involved in cervical cancer pathogenesis. Immunohistochemical analysis of human cervical biopsy samples revealed that the expression of FTS is absent in normal cervical epithelium but progressively overexpressed in human cervical intraneoplastic lesions (CIN-I to CIN-III), this characteristic phenomenon put this protein, a potential diagnostic marker for the screening of early neoplastic changes of cervix. Using FTS-specific small hairpin RNA (shRNA) in cervical cancer cells, we determined a specific role for FTS protein in, cervical neoplasia. Targeted stable knock down of FTS in HeLa cells led to the growth inhibition, cell-cycle arrest, and apoptosis with concurrent increase in p21 protein. FTS effectively represses the p21 mRNA expression in dual luciferase assay which indicates that p21 is transcriptionally regulated by this oncoprotein which in turn affect the regular cell-cycle process and its components. Consistent with this we found a reciprocal association between these proteins in early cervical neoplastic tissues. These data unraveled the involvement of new oncoprotein FTS in cervical cancer which plays a central role in carcinogenesis. Targeted inhibition of FTS lead to the shutdown of key elemental characteristics of cervical cancer and could lead to an effective therapeutic strategy for cervical cancer.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Biomarcadores de Tumor/metabolismo , Lesiones Precancerosas/diagnóstico , Lesiones Precancerosas/patología , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/patología , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Apoptosis , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Fase G1 , Técnicas de Silenciamiento del Gen , Humanos , Lesiones Precancerosas/metabolismo , Fase de Descanso del Ciclo Celular , Transcripción Genética , Neoplasias del Cuello Uterino/metabolismoRESUMEN
Radiotherapy is the major treatment modality for uterine cervical cancer, but in some cases, the disease is radioresistant. Defining the molecular events that contribute to radioresistance and progression of cancer are of critical importance. Here we evaluated the role of Fused Toes Homolog (FTS) in radiation resistance of cervical carcinoma. Immunostaning of cervical cancer cells and tissues revealed that FTS localization and expression was changed after radiation. Targeted stable knockdown of FTS in HeLa cells led to the growth inhibition after radiation. Radiation induced AKT mediated cytoprotective effect was countered by FTS knockdown which leads to PARP cleavage and caspase-3 activation leading to cell death. FTS knockdown promotes radiation induced cell cycle arrest at G0/G1 and apoptosis of HeLa cells with concurrent alterations in the display of cell cycle regulatory proteins. This study revealed FTS is involved in radioresistance of cervical cancer. Targeted inhibition of FTS led to the shutdown of key elemental characteristics of cervical cancer and could lead to an effective therapeutic strategy.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Tolerancia a Radiación , Radiación , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Anexina A5/metabolismo , Caspasa 3/metabolismo , Proteínas de Ciclo Celular/metabolismo , Muerte Celular , Línea Celular Tumoral , Supervivencia Celular , Células Clonales , Regulación hacia Abajo , Femenino , Citometría de Flujo , Fase G1 , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias del Cuello Uterino/enzimologíaRESUMEN
Survivin, an antiapoptotic protein, is elevated in most malignancies and attributes to radiation resistance in tumors including glioblastoma multiforme. The downregulation of survivin could sensitize glioblastoma cells to radiation therapy. In this study, we investigated the effect of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), in attenuating survivin and enhancing the therapeutic efficacy for glioblastoma cells, and elucidated the underlying mechanisms. Here we tested various concentrations of rapamycin (1-8 nM) in combination with radiation dose 4 Gy. Rapamycin effectively modulated the protein kinase B (Akt)/mTOR pathway by inhibiting the phosphorylation of Akt and mTOR proteins, and this inhibition was further enhanced by radiation. The expression level of survivin was decreased in rapamycin pre-treatment glioblastoma cells followed by radiation; meanwhile, the phosphorylation of H2A histone family member X (H2AX) at serine-139 (γ-H2AX) was increased. p21 protein was also induced on radiation with rapamycin pre-treatment, which enhanced G1 arrest and the accumulation of cells at G0/subG1 phase. Furthermore, the clonogenic cell survival assay revealed a significant dose-dependent decrease in the surviving fraction for all three cell lines pre-treated with rapamycin. Our studies demonstrated that targeting survivin may be an effective approach for radiosensitization of malignant glioblastoma.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/metabolismo , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Western Blotting , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Citometría de Flujo , Fase G1/efectos de los fármacos , Fase G1/efectos de la radiación , Glioblastoma/metabolismo , Glioblastoma/patología , Histonas/metabolismo , Humanos , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación , Survivin , Serina-Treonina Quinasas TOR/metabolismo , Factores de TiempoRESUMEN
The development of the heart follows a synergic action of several signaling pathways during gestational, pre- & postnatal stages. The current study aimed to investigate whether the myocardium experiences transcriptional changes during the transition from post-natal to adult hood stages. Herein, we used C57/B16/J mice at 4 (28- days; post-natal/PN) and 20 weeks (adulthood/AH) of ages and employed the next generation RNAseq (NGS) to profile the transcriptome and echocardiography analysis to monitor the structural/functional changes in the heart. NGS-based RNA-seq revealed that 1215 genes were significantly upregulated and 2549 were down regulated in the AH versus PN hearts, indicating a significant transcriptional change during this transition. A synchronized cardiac transcriptional regulation through cell cycle, growth hormones, redox homeostasis and metabolic pathways was noticed in both PN and AH hearts. Echocardiography reveals significant structural and functional (i.e. systolic/diastolic) changes during the transition of PN to adult stage. Particularly, a progressive decline in ejection fraction and cardiac output was observed in AH hearts. These structural adaptations are in line with critical signaling pathways that drive the maturation of heart during AH. Overall, we have presented a comprehensive transcriptomic analysis along with structural-functional relationship during the myocardial development in adult mice.
RESUMEN
Runt-related (RUNX) transcription factors play pivotal roles in neoplastic development and have tissue-specific developmental roles in hematopoiesis (RUNX1), osteogenesis (RUNX2), as well as neurogenesis and thymopoiesis (RUNX3). RUNX3 is a tumor suppressor in gastric carcinoma, and its expression is frequently inactivated by DNA methylation or its protein mislocalized in many cancer types, including gastric and breast cancer. Jun-activation domain-binding protein 1 (Jab1/CSN5), a component of the COP9 signalosome (CSN), is critical for nuclear export and the degradation of several tumor suppressor proteins, including p53, p27(Kip1), and Smad4. Here, we find that Jab1 facilitates nuclear export of RUNX3 that is controlled by CSN-associated kinases. RUNX3 sequestered in the cytoplasm is rapidly degraded through a proteasome-mediated pathway. Our results identify a novel mechanism of regulating nuclear export and protein stability of RUNX3 by the CSN complex.
Asunto(s)
Subunidad alfa 3 del Factor de Unión al Sitio Principal/análisis , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Citoplasma/enzimología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptido Hidrolasas/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Complejo del Señalosoma COP9 , Núcleo Celular/metabolismo , Células Cultivadas , Células HeLa , Humanos , Transcripción Genética , TransfecciónRESUMEN
Pluripotency is highly dynamic and progresses through a continuum of pluripotent stem cell states. The two states that bookend the pluripotency continuum, naive and primed, are well characterized, but our understanding of the intermediate states and transitions between them remains incomplete. Here, we dissect the dynamics of pluripotent state transitions underlying pre- to post-implantation epiblast differentiation. Through comprehensive mapping of the proteome, phosphoproteome, transcriptome, and epigenome of embryonic stem cells transitioning from naive to primed pluripotency, we find that rapid, acute, and widespread changes to the phosphoproteome precede ordered changes to the epigenome, transcriptome, and proteome. Reconstruction of the kinase-substrate networks reveals signaling cascades, dynamics, and crosstalk. Distinct waves of global proteomic changes mark discrete phases of pluripotency, with cell-state-specific surface markers tracking pluripotent state transitions. Our data provide new insights into multi-layered control of the phased progression of pluripotency and a foundation for modeling mechanisms regulating pluripotent state transitions (www.stemcellatlas.org).
Asunto(s)
Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/fisiología , Animales , Diferenciación Celular/fisiología , Linaje de la Célula , Células Madre Embrionarias/citología , Epigenoma/genética , Regulación del Desarrollo de la Expresión Génica , Estratos Germinativos/citología , Estratos Germinativos/metabolismo , Humanos , Proteoma/metabolismo , Transducción de Señal , Transcriptoma/genéticaRESUMEN
Faithful transcription initiation is critical for accurate gene expression, yet the mechanisms underlying specific transcription start site (TSS) selection in mammals remain unclear. Here, we show that the histone-fold domain protein NF-Y, a ubiquitously expressed transcription factor, controls the fidelity of transcription initiation at gene promoters in mouse embryonic stem cells. We report that NF-Y maintains the region upstream of TSSs in a nucleosome-depleted state while simultaneously protecting this accessible region against aberrant and/or ectopic transcription initiation. We find that loss of NF-Y binding in mammalian cells disrupts the promoter chromatin landscape, leading to nucleosomal encroachment over the canonical TSS. Importantly, this chromatin rearrangement is accompanied by upstream relocation of the transcription pre-initiation complex and ectopic transcription initiation. Further, this phenomenon generates aberrant extended transcripts that undergo translation, disrupting gene expression profiles. These results suggest NF-Y is a central player in TSS selection in metazoans and highlight the deleterious consequences of inaccurate transcription initiation.
Asunto(s)
Factor de Unión a CCAAT/metabolismo , Nucleosomas/metabolismo , Sitio de Iniciación de la Transcripción , Iniciación de la Transcripción Genética , Animales , Factor de Unión a CCAAT/genética , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Células Madre Embrionarias , Técnicas de Silenciamiento del Gen , Ratones , Nucleosomas/genética , Regiones Promotoras Genéticas/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Mammary stem/progenitor cells (MaSCs) maintain self-renewal of the mammary epithelium during puberty and pregnancy. DNA methylation provides a potential epigenetic mechanism for maintaining cellular memory during self-renewal. Although DNA methyltransferases (DNMTs) are dispensable for embryonic stem cell maintenance, their role in maintaining MaSCs and cancer stem cells (CSCs) in constantly replenishing mammary epithelium is unclear. Here we show that DNMT1 is indispensable for MaSC maintenance. Furthermore, we find that DNMT1 expression is elevated in mammary tumours, and mammary gland-specific DNMT1 deletion protects mice from mammary tumorigenesis by limiting the CSC pool. Through genome-scale methylation studies, we identify ISL1 as a direct DNMT1 target, hypermethylated and downregulated in mammary tumours and CSCs. DNMT inhibition or ISL1 expression in breast cancer cells limits CSC population. Altogether, our studies uncover an essential role for DNMT1 in MaSC and CSC maintenance and identify DNMT1-ISL1 axis as a potential therapeutic target for breast cancer treatment.
Asunto(s)
Neoplasias de la Mama/genética , Carcinogénesis/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Proteínas con Homeodominio LIM/genética , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Experimentales/genética , Células Madre Neoplásicas/metabolismo , Factores de Transcripción/genética , Animales , Western Blotting , Neoplasias de la Mama/metabolismo , Línea Celular , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Regulación hacia Abajo , Femenino , Humanos , Proteínas con Homeodominio LIM/metabolismo , Células MCF-7 , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/crecimiento & desarrollo , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Microscopía Fluorescente , Células Madre Neoplásicas/citología , Células Madre/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Helicase-like transcription factor (HLTF) is a member of the SWI/SNF (mating type switching/sucrose non-fermenting) family of ATPases/helicases and also has a RING-finger motif characteristic of ubiquitin ligase proteins. These features have led to suggestions that HLTF functions like yeast Rad5, which promotes replication through DNA lesions via a post-replication repair pathway. However, the function of HLTF in higher eukaryotes is still unknown. Herein, we found the overexpression of HLTF in radiation recurrent human uterine cervical carcinoma tissues when compared to disease free survived patients tissues. In this study, we used RNA interference techniques to investigate the potential function of HLTF in cervical cancer cell line HeLa and found that the cell proliferation was reduced by knockdown (KD) of HLTF. A host-cell reactivation assay showed that the capacity for repair to DNA damage induced by X-ray irradiation was reduced in HLTF KD cells. X-rays also increased apoptosis in HLTF KD cells. These results suggest that HLTF is involved in DNA repair and apoptosis in cancer cells, which might represent a target for gene therapies of human cancer.