Inducible degradation of the Drosophila Mediator subunit Med19 reveals its role in regulating developmental but not constitutively-expressed genes

Abstract

The multi-subunit Mediator complex plays a critical role in gene expression by bridging enhancer-bound transcription factors and the RNA polymerase II machinery. Although experimental case studies suggest differential roles of Mediator subunits, a comprehensive view of the specific set of genes regulated by individual subunits in a developing tissue is still missing. Here we address this fundamental question by focusing on the Med19 subunit and using the Drosophila wing imaginal disc as a developmental model. By coupling auxin- inducible degradation of endogenous Med19 in vivo with RNA-seq, we got access to the early consequences of Med19 elimination on gene expression. Differential gene expression analysis reveals that Med19 is not globally required for mRNA transcription but specifically regulates positively or negatively less than a quarter of the expressed genes. By crossing our transcriptomic data with those of Drosophila gene expression profile database, we found that Med19-dependent genes are highly enriched with spatially-regulated genes while the expression of most constitutively expressed genes is not affected upon Med19 loss. Whereas globally downregulation does not exceed upregulation, we identified a functional class of genes encoding spatially-regulated transcription factors, and more generally developmental regulators, responding unidirectionally to Med19 loss with an expression collapse. Moreover, we show in vivo that the Notch-responsive wingless and the E(spl)-C genes require Med19 for their expression. Combined with experimental evidences suggesting that Med19 could function as a direct transcriptional effector of Notch signaling, our data support a model in which Med19 plays a critical role in the transcriptional activation of developmental genes in response to cell signaling pathways. Author summaryThe Mediator is a large evolutionarily conserved multisubunit complex that plays essential functions in gene expression by relaying cues emanating from enhancer-bound transcription factors to the RNA polymerase II machinery. The transcriptional landscapes regulated by each Mediator subunit, especially in vivo in the context of developmental processes, remains poorly characterized. We therefore sought to provide a comprehensive view of the genes directly regulated by Med19, an archetypal Mediator subunit, in the context of the development of the Drosophila wing. Our work has important methodological implications as to carry out our analysis in the best experimental conditions, we generated mutant flies in which we could trigger fast degradation of the Med19 subunit on demand in the tissues of the living animal. We found that only a small part of the genes expressed in the developing wing are controlled by Med19 showing that this Mediator component exercises specific functions. Another major finding is the strong involvement of Med19 in the regulation of the expression of the genes involved in developmental processes supporting the idea that Med19 is a Mediator component dedicated to highly regulated mode of gene expression. The most unanticipated discovery of our study is the fact that Med19 does not appear to play a role in the expression of the genes that are constitutively transcribed, among which the housekeeping genes. This raises the notion that in the context of simple --non-regulated-- mode of expression, part of the Mediator functionalities, among which Med19, otherwise dedicated to highly regulated type of transcription, may become dispensable.