RESUMEN
Development of the Drosophila visceral muscle depends on Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) signaling, which specifies founder cells (FCs) in the circular visceral mesoderm (VM). Although Alk activation by its ligand Jelly Belly (Jeb) is well characterized, few target molecules have been identified. Here, we used targeted DamID (TaDa) to identify Alk targets in embryos overexpressing Jeb versus embryos with abrogated Alk activity, revealing differentially expressed genes, including the Snail/Scratch family transcription factor Kahuli (Kah). We confirmed Kah mRNA and protein expression in the VM, and identified midgut constriction defects in Kah mutants similar to those of pointed (pnt). ChIP and RNA-Seq data analysis defined a Kah target-binding site similar to that of Snail, and identified a set of common target genes putatively regulated by Kah and Pnt during midgut constriction. Taken together, we report a rich dataset of Alk-responsive loci in the embryonic VM and functionally characterize the role of Kah in the regulation of embryonic midgut morphogenesis.
Asunto(s)
Quinasa de Linfoma Anaplásico , Proteínas de Unión al ADN , Proteínas de Drosophila , Desarrollo Embrionario , Proteínas del Tejido Nervioso , Proteínas Proto-Oncogénicas , Factores de Transcripción , Animales , Quinasa de Linfoma Anaplásico/genética , Diferenciación Celular/genética , Proteínas de Unión al ADN/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Proteínas de Drosophila/genética , Desarrollo Embrionario/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Mesodermo/crecimiento & desarrollo , Mesodermo/metabolismo , Desarrollo de Músculos/genética , Músculos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , RNA-Seq , Transducción de Señal/genética , Análisis de la Célula Individual , Factores de Transcripción/genéticaRESUMEN
In Drosophila, the male-specific lethal (MSL) complex specifically targets the male X chromosome and participates in a twofold increase in expression output leading to functional dosage compensation. The complex includes five proteins and two non-coding RNAs (ncRNAs). A number of additional associated factors have also been identified. However, the components' roles and interactions have not been fully elucidated. The in situ proximity ligation assay (PLA) provides a sensitive means to determine whether proteins and other factors have bound to chromosomes in close proximity to each other, and thus may interact. Thus, we modified, tested, and applied the assay to probe interactions of MSL complex components on polytene chromosomes. We show that in situ PLA can detect and map both protein-protein and protein-ncRNA interactions on polytene chromosomes at high resolution. We further show that all five protein components of the MSL complex are in close proximity to each other, and the ncRNAs roX1 and roX2 bind the complex in close proximity to MLE. Our results also indicate that JIL1, a histone H3 Ser10 kinase enriched on the male X chromosome, interacts with MSL1 and MSL2, but not MSL3 of the MSL complex. In addition, we corroborate proposed interactions of the MSL complex with both CLAMP and TopoII.
Asunto(s)
Cromosomas de Insectos , Drosophila melanogaster/genética , Genes Letales , Proteínas de Insectos/metabolismo , ARN/metabolismo , Animales , MasculinoRESUMEN
Transposable elements constitute a substantial portion of most eukaryotic genomes and their activity can lead to developmental and neuronal defects. In the germline, transposon activity is antagonized by the PIWI-interacting RNA pathway tasked with repression of transposon transcription and degrading transcripts that have already been produced. However, most of the genes required for transposon control are not expressed outside the germline, prompting the question: what causes deleterious transposons activity in the soma and how is it managed? Here, we show that disruptions of the Histone 3 lysine 36 methylation machinery led to increased transposon transcription in Drosophila melanogaster brains and that there is division of labour for the repression of transposable elements between the different methyltransferases Set2, NSD, and Ash1. Furthermore, we show that disruption of methylation leads to somatic activation of key genes in the PIWI-interacting RNA pathway and the preferential production of RNA from dual-strand piRNA clusters.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Animales , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Histonas/genética , Histonas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Elementos Transponibles de ADN/genética , Metilación , Lisina/genética , Lisina/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/genética , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismoRESUMEN
In Drosophila, two chromosomes require special mechanisms to balance their transcriptional output to the rest of the genome. These are the male-specific lethal complex targeting the male X chromosome and Painting of fourth targeting chromosome 4. Here, we explore the role of histone H3 methylated at lysine-36 (H3K36) and the associated methyltransferasesSet2, NSD, and Ash1in these two chromosome-specific systems. We show that the loss of Set2 impairs the MSL complexmediated dosage compensation; however, the effect is not recapitulated by H3K36 replacement and indicates an alternative target of Set2. Unexpectedly, balanced transcriptional output from the fourth chromosome requires intact H3K36 and depends on the additive functions of NSD and Ash1. We conclude that H3K36 methylation and the associated methyltransferases are important factors to balance transcriptional output of the male X chromosome and the fourth chromosome. Furthermore, our study highlights the pleiotropic effects of these enzymes.