RESUMEN
It is well established that tumours are not homogenous, but comprise cells with differing invasive, proliferative and tumour-initiating potential. A major challenge in cancer research is therefore to develop methods to characterize cell heterogeneity. In melanoma, proliferative and invasive cells are characterized by distinct gene expression profiles and accumulating evidence suggests that cells can alternate between these states through a process called phenotype switching. We have used microfluidic technology to isolate single melanoma cells grown in vitro as monolayers or melanospheres or in vivo as xenografted tumours and analyse the expression profiles of 114 genes that discriminate the proliferative and invasive states by quantitative PCR. Single-cell analysis accurately recapitulates the specific gene expression programmes of melanoma cell lines and defines subpopulations with distinct expression profiles. Cell heterogeneity is augmented when cells are grown as spheres and as xenografted tumours. Correlative analysis identifies gene-regulatory networks and changes in gene expression under different growth conditions. In tumours, subpopulations of cells that express specific invasion and drug resistance markers can be identified amongst which is the pluripotency factor POUF51 (OCT4) whose expression correlates with the tumorigenic potential. We therefore show that single-cell analysis can be used to define and quantify tumour heterogeneity based on detection of cells with specific gene expression profiles.
Asunto(s)
Perfilación de la Expresión Génica , Melanoma/genética , Melanoma/patología , Análisis de la Célula Individual , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica , Humanos , Melanoma/metabolismo , Ratones , Factor de Transcripción Asociado a Microftalmía/metabolismoRESUMEN
The TEAD (1-4) transcription factors comprise the conserved TEA/ATTS DNA-binding domain recognising the MCAT element in the promoters of muscle-specific genes. Despite extensive genetic analysis, the function of TEAD factors in muscle differentiation has proved elusive due to redundancy among the family members. Expression of the TEA/ATTS DNA-binding domain that acts as a dominant negative repressor of TEAD factors in C2C12 myoblasts inhibits their differentiation, whereas selective shRNA knockdown of TEAD4 results in abnormal differentiation characterised by the formation of shortened myotubes. Chromatin immunoprecipitation coupled to array hybridisation shows that TEAD4 occupies 867 promoters including those of myogenic miRNAs. We show that TEAD factors directly induce Myogenin, CDKN1A and Caveolin 3 expression to promote myoblast differentiation. RNA-seq identifies a set of genes whose expression is strongly reduced upon TEAD4 knockdown among which are structural and regulatory proteins and those required for the unfolded protein response. In contrast, TEAD4 represses expression of the growth factor CTGF (connective tissue growth factor) to promote differentiation. Together these results show that TEAD factor activity is essential for normal C2C12 cell differentiation and suggest a role for TEAD4 in regulating expression of the unfolded protein response genes.
Asunto(s)
Diferenciación Celular/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas Musculares/metabolismo , Miogenina/genética , Factores de Transcripción/metabolismo , Respuesta de Proteína Desplegada/genética , Animales , Secuencia de Bases , Fusión Celular , Línea Celular , Inmunoprecipitación de Cromatina , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Técnicas de Silenciamiento del Gen , Ratones , Datos de Secuencia Molecular , Desarrollo de Músculos/genética , Mioblastos/citología , Mioblastos/metabolismo , Miogenina/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica/genética , Proteínas Represoras/metabolismo , Factores de Transcripción de Dominio TEARESUMEN
Malignant melanoma is an aggressive cancer known for its notorious resistance to most current therapies. The basic helix-loop-helix microphthalmia transcription factor (MITF) is the master regulator determining the identity and properties of the melanocyte lineage, and is regarded as a lineage-specific 'oncogene' that has a critical role in the pathogenesis of melanoma. MITF promotes melanoma cell proliferation, whereas sustained supression of MITF expression leads to senescence. By combining chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-seq) and RNA sequencing analyses, we show that MITF directly regulates a set of genes required for DNA replication, repair and mitosis. Our results reveal how loss of MITF regulates mitotic fidelity, and through defective replication and repair induces DNA damage, ultimately ending in cellular senescence. These findings reveal a lineage-specific control of DNA replication and mitosis by MITF, providing new avenues for therapeutic intervention in melanoma. The identification of MITF-binding sites and gene-regulatory networks establish a framework for understanding oncogenic basic helix-loop-helix factors such as N-myc or TFE3 in other cancers.