RESUMO
Mammalian Hox gene clusters contain a range of CTCF binding sites. In addition to their importance in organizing a TAD border, which isolates the most posterior genes from the rest of the cluster, the positions and orientations of these sites suggest that CTCF may be instrumental in the selection of various subsets of contiguous genes, which are targets of distinct remote enhancers located in the flanking regulatory landscapes. We examined this possibility by producing an allelic series of cumulative in cis mutations in these sites, up to the abrogation of CTCF binding in the five sites located on one side of the TAD border. In the most impactful alleles, the global chromatin architecture of the locus was modified, yet not drastically, illustrating that CTCF sites located on one side of a strong TAD border are sufficient to organize at least part of this insulation. Spatial colinearity in the expression of these genes along the major body axis was nevertheless maintained, despite abnormal expression boundaries. In contrast, strong effects were scored in the selection of target genes responding to particular enhancers, leading to the misregulation of Hoxd genes in specific structures. Altogether, while most enhancer-promoter interactions can occur in the absence of this series of CTCF sites, the binding of CTCF in the Hox cluster is required to properly transform a rather unprecise process into a highly discriminative mechanism of interactions, which is translated into various patterns of transcription accompanied by the distinctive chromatin topology found at this locus. Our allelic series also allowed us to reveal the distinct functional contributions for CTCF sites within this Hox cluster, some acting as insulator elements, others being necessary to anchor or stabilize enhancer-promoter interactions, and some doing both, whereas they all together contribute to the formation of a TAD border. This variety of tasks may explain the amazing evolutionary conservation in the distribution of these sites among paralogous Hox clusters or between various vertebrates.
Assuntos
Cromatina , Elementos Facilitadores Genéticos , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Cromatina/genética , Elementos Facilitadores Genéticos/genética , Genes Homeobox/genética , Mamíferos/genética , Camundongos , MutagêneseRESUMO
Despite the crucial importance of Hox genes functions during animal development, the mechanisms that control their transcription in time and space are not yet fully understood. In this context, it was proposed that Hotair, a lncRNA transcribed from within the HoxC cluster regulates Hoxd gene expression in trans, through the targeting of Polycomb and consecutive transcriptional repression. This activity was recently supported by the skeletal phenotype of mice lacking Hotair function. However, other loss of function alleles at this locus did not elicit the same effects. Here, we re-analyze the molecular and phenotypic consequences of deleting the Hotair locus in vivo. In contrast with previous findings, we show that deleting Hotair has no detectable effect on Hoxd genes expression in vivo. In addition, we were unable to observe any significant morphological alteration in mice lacking the Hotair transcript. However, we find a subtle impact of deleting the Hotair locus upon the expression of the neighboring Hoxc11 and Hoxc12 genes in cis. Our results do not support any substantial role for Hotair during mammalian development in vivo. Instead, they argue in favor of a DNA-dependent effect of the Hotair deletion upon the transcriptional landscape in cis.
Assuntos
Desenvolvimento Embrionário/genética , Proteínas de Homeodomínio/genética , RNA Longo não Codificante/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/biossíntese , Humanos , Camundongos , Camundongos Knockout , Proteínas do Grupo Polycomb/genéticaRESUMO
E-cadherin plays a pivotal role in epithelial cell polarity, cell signalling and tumour suppression. However, how E-cadherin dysfunction promotes tumour progression is poorly understood. Here we show that the actin-capping protein heterodimer, which regulates actin filament polymerization, has a dual function on DE-cadherin in restricted Drosophila epithelia. Knocking down capping protein in the distal wing disc epithelium disrupts DE-cadherin and Armadillo localization at adherens junctions and upregulates DE-cadherin transcription. In turn, DE-cadherin provides an active signal, which prevents Wingless signalling and promotes JNK-mediated apoptosis. However, when cells are kept alive with the Caspase inhibitor P35, the activity of the JNK pathway and of the Yorkie oncogene trigger massive proliferation of cells that fail to stably retain associations with their neighbours. Moreover, loss of capping protein cooperates with the Ras oncogene to induce massive tissue overgrowth. Taken together, our findings argue that in some epithelia, the dual effect of capping protein loss on DE-cadherin triggers the elimination of mutant cells, preventing them from proliferating. However, the appearance of a second mutation that blocks cell death may allow for the development of some epithelial tumours.
Assuntos
Proteínas de Capeamento de Actina/metabolismo , Caderinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Junções Aderentes/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose , Proteínas do Domínio Armadillo/metabolismo , Polaridade Celular , Proliferação de Células , Sobrevivência Celular , Drosophila/citologia , Drosophila/genética , Proteínas de Drosophila/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Genes de Insetos , Genes ras , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Mutação , Neoplasias/etiologia , Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Asas de Animais/citologia , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismo , Proteínas de Sinalização YAPRESUMO
Developmental genes are often controlled by large regulatory landscapes matching topologically associating domains (TADs). In various contexts, the associated chromatin backbone is modified by specific enhancer-enhancer and enhancer-promoter interactions. We used a TAD flanking the mouse HoxD cluster to study how these regulatory architectures are formed and deconstructed once their function achieved. We describe this TAD as a functional unit, with several regulatory sequences acting together to elicit a transcriptional response. With one exception, deletion of these sequences didn't modify the transcriptional outcome, a result at odds with a conventional view of enhancer function. The deletion and inversion of a CTCF site located near these regulatory sequences did not affect transcription of the target gene. Slight modifications were nevertheless observed, in agreement with the loop extrusion model. We discuss these unexpected results considering both conventional and alternative explanations relying on the accumulation of poorly specific factors within the TAD backbone.
Assuntos
Cromatina/metabolismo , Elementos Facilitadores Genéticos/genética , Genitália/metabolismo , Mamíferos/genética , Animais , Fator de Ligação a CCCTC/metabolismo , Genes Homeobox/genética , Camundongos , Família Multigênica/genética , Regiões Promotoras GenéticasRESUMO
The actin-Capping Protein heterodimer, composed of the α and ß subunits, is a master F-actin regulator. In addition to its role in many cellular processes, Capping Protein acts as a main tumor suppressor module in Drosophila and in humans, in part, by restricting the activity of Yorkie/YAP/TAZ oncogenes. We aimed in this report to understand how both subunits regulate each other in vivo. We show that the levels and capping activities of both subunits must be tightly regulated to control F-actin levels and consequently growth of the Drosophila wing. Overexpressing capping protein α and ß decreases both F-actin levels and tissue growth, while expressing forms of Capping Protein that have dominant negative effects on F-actin promote tissue growth. Both subunits regulate each other's protein levels. In addition, overexpressing one of the subunit in tissues knocked-down for the other increases the mRNA and protein levels of the subunit knocked-down and compensates for its loss. We propose that the ability of the α and ß subunits to control each other's levels assures that a pool of functional heterodimer is produced in sufficient quantities to restrict the development of tumor but not in excess to sustain normal tissue growth.