Transcriptomics as a tool to dissect copper homeostasis and COMMD protein function

Abstract

Gene expression profiling offers the possibility to objectively screen the complete genome for variations in gene expression in many different organisms in various defined environments. In the present work we aimed to gain novel insights in copper metabolism and in the function of COMMD (copper metabolism MURR1 domain) proteins by using gene expression profiles. Copper is a trace metal that is essential, but toxic in excess. This is illustrated by the severe phenotypes of the inherited disorders Menkes disease and Wilson disease in which patients suffer from copper deficiency or copper overload, respectively. Information about the transcriptional changes that are induced by copper overload in mammals could help to elucidate the pathophysiological mechanisms that underlie copper overload disorders. We therefore examined the hepatic transcriptional changes after copper exposure by genome-wide gene expression analysis and by a literature-based transcriptomics meta-analysis. Both analyses revealed that, genes involved in defined biological processes, including cholesterol synthesis, were differentially expressed after copper overload. In the second part of the present work, we used transcriptomics to investigate the function of a protein that was previously implicated in the regulation of copper homeostasis; COMMD1. Genome-wide gene expression studies in COMMD1 knockout mice and COMMD1 deficient cell lines revealed that COMMD1 regulates the expression of distinct genes involved in hypoxia and NF-?B (nuclear factor-kappa B) signalling. As COMMD1 is a member of a protein family that shares a homologous COMM domain, we investigated the possibility that this COMMD family of proteins could define transcriptional specificity. We investigated the genome-wide gene expression changes in cell lines deficient for COMMD1, COMMD6 or COMMD9 after incubation with the NF-ΚB stimulus TNF (tumor necrosis factor) and demonstrated COMMD-specific differential expression of TNF target genes compared to control cells. In addition, we identified two nuclear export signals (NESs) in the COMM domain of COMMD1 that were conserved among this COMMD family. The function of many proteins involved in NF-ΚB signaling is regulated though nucleocytoplasmic shuttling that is dominated by NESs and nuclear localization signals. We therefore investigated the functional activity of these NESs by studying COMMD1 localization and COMMD1 function when the COMMD1 NESs were mutated or when nuclear export was blocked with the chemical compound leptomycin B. These studies confirmed that COMMD1 can be actively transported out of the nucleus and that the localization of COMMD1 affects COMMD1 ubiquitination and COMMD1-mediated NF-ΚB signalling. In conclusion, our work has increased our knowledge on copper-dependent transcriptional changes in mammals and has contributed to a better characterization of the function of COMMD proteins and the mechanism of COMMD1-mediated transcriptional regulation in inflammation and hypoxia.