RESUMEN
Developmental patterning and tissue formation are regulated through complex gene regulatory networks (GRNs) driven through the action of transcription factors (TFs) converging on enhancer elements. Here, as a point of entry to dissect the poorly defined GRN underlying cardiomyocyte differentiation, we apply an integrated approach to identify active enhancers and TFs involved in Drosophila heart development. The Drosophila heart consists of 104 cardiomyocytes, representing less than 0.5% of all cells in the embryo. By modifying BiTS-ChIP for rare cells, we examined H3K4me3 and H3K27ac chromatin landscapes to identify active promoters and enhancers specifically in cardiomyocytes. These in vivo data were complemented by a machine learning approach and extensive in vivo validation in transgenic embryos, which identified many new heart enhancers and their associated TF motifs. Our results implicate many new TFs in late stages of heart development, including Bagpipe, an Nkx3.2 ortholog, which we show is essential for differentiated heart function.
Asunto(s)
Drosophila/embriología , Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica/genética , Corazón/embriología , Miocitos Cardíacos/citología , Organogénesis/genética , Regiones Promotoras Genéticas/genética , Animales , Animales Modificados Genéticamente , Diferenciación Celular/genética , Cromatina , Drosophila/genética , Redes Reguladoras de Genes/genética , Histonas/metabolismo , Organogénesis/fisiología , Factores de Transcripción/genéticaRESUMEN
Cardiac aging is a complex process, which is influenced by both environmental and genetic factors. Deciphering the mechanisms involved in heart senescence therefore requires identifying the molecular pathways that are affected by age in controlled environmental and genetic conditions. We describe a functional genomic investigation of the genetic control of cardiac senescence in Drosophila. Molecular signatures of heart aging were identified by differential transcriptome analysis followed by a detailed bio-informatic analysis. This approach implicated the JNK/dJun pathway and the transcription factor Vri/dNFIL3 in the transcription regulatory network involved in cardiac senescence and suggested the possible involvement of oxidative stress (OS) in the aging process. To validate these predictions, we developed a new in vivo assay to analyze heart performance in various contexts of adult heart-specific gene overexpression and inactivation. We demonstrate that, as in mammals, OS plays a central role in cardiac senescence, and we show that pharmacological interventions impinging on OS slow heart senescence. These observations strengthen the idea that cardiac aging is controlled by evolutionarily conserved mechanisms, further validating Drosophila as a model to study cardiac senescence. In addition, we demonstrate that Vri, the ortholog of the vertebrate NFIL3/E4B4 transcription factor, is a major genetic regulator of cardiac aging. Vri overexpression leads to major heart dysfunctions, but its loss of function significantly reduces age-related cardiac dysfunctions. Furthermore, we unambiguously show that the JNK/AP1 pathway, the role of which in cardiac aging in mammals is controversial, is activated during cardiac aging and has a detrimental effect on cardiac senescence. This data-driven functional genomic analysis therefore led to the identification of key components of the Gene Regulatory Network of cardiac aging in Drosophila and may prompt to investigate the involvement of their counterparts in the cardiac aging process in mammals.
Asunto(s)
Envejecimiento , Proteínas de Drosophila , Drosophila melanogaster , Corazón/fisiología , Sistema de Señalización de MAP Quinasas/genética , Factores de Transcripción , Envejecimiento/genética , Envejecimiento/metabolismo , Envejecimiento/fisiología , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Regulación de la Expresión Génica , Humanos , Estrés Oxidativo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Developmental transcriptional regulatory networks are circuits of transcription factors (TFs) and cis-acting DNA elements (Cis Regulatory Modules, CRMs) that dynamically control expression of downstream genes. Comprehensive knowledge of these networks is an essential step towards our understanding of developmental processes. However, this knowledge is mostly based on genome-wide mapping of transcription factor binding sites, and therefore requires prior knowledge regarding the TFs involved in the network. RESULTS: Focusing on how temporal control of gene expression is integrated within a developmental network, we applied an in silico approach to discover regulatory motifs and CRMs of co-expressed genes, with no prior knowledge about the involved TFs. Our aim was to identify regulatory motifs and potential trans-acting factors which regulate the temporal expression of co-expressed gene sets during a particular process of organogenesis, namely adult heart formation in Drosophila. Starting from whole genome tissue specific expression dynamics, we used an in silico method, cisTargetX, to predict TF binding motifs and CRMs. Potential Nuclear Receptor (NR) binding motifs were predicted to control the temporal expression profile of a gene set with increased expression levels during mid metamorphosis. The predicted CRMs and NR motifs were validated in vivo by reporter gene essays. In addition, we provide evidence that three NRs modulate CRM activity and behave as temporal regulators of target enhancers. CONCLUSIONS: Our approach was successful in identifying CRMs and potential TFs acting on the temporal regulation of target genes. In addition, our results suggest a modular architecture of the regulatory machinery, in which the temporal and spatial regulation can be uncoupled and encoded by distinct CRMs.