Murine Myocardial Transcriptome Analysis Reveals a Critical Role of COPS8 in the Gene Expression of Cullin-RING Ligase Substrate Receptors and Redox and Vesicle Trafficking Pathways.

Ammara Abdullah,Peng Xiao,Erliang Zeng,Kathleen M Eyster,Xuejun Wang,Travis Bjordahl

doi:10.3389/fphys.2017.00594

Abstract

Background: The COP9 signalosome (CSN) consisting of 8 unique protein subunits (COPS1 through COPS8) serves as the cullin deneddylase, regulating the catalytic dynamics of cullin RING ligases (CRLs), the largest family of ubiquitin ligases Background: The COP9 signalosome (CSN) consisting of 8 unique protein subunits (COPS1 through COPS8) serves as the cullin deneddylase, regulating the catalytic dynamics of cullin RING ligases (CRLs), the largest family of ubiquitin ligases. Supported primarily by the decrease of substrate receptor (SR) proteins of CRLs in cells deficient of a CSN subunit, CSN-mediated cullin deneddylation is believed to prevent autoubiquitination and self-destruction of the SR in active CRLs. However, it is unclear whether the decrease in SRs is solely due to protein destabilization. Moreover, our prior studies have demonstrated that cardiac specific knockout of Cops8 (Cops8-CKO) impairs autophagosome maturation and causes massive necrosis in cardiomyocytes but the underlying mechanism remains poorly understood. Given that Cops8 is nucleus-enriched and a prior report showed its binding to the promoter of several genes and association of its ablation with decreased mRNA levels of these genes, we sought to determine the dynamic changes of myocardial transcriptome in mice with perinatal Cops8-CKO and to explore their functional implications.Methods and Results: Myocardial transcriptomes of Cops8flox/flox, Cops8flox/+::Myh6-Cre, and Cops8flox/flox::Myh6-Cre littermate mice at postnatal 2 and 3 weeks were analyzed. The data were imported into an in-house analysis pipeline using Bioconductor for quantile normalization and statistical analysis. Differentially expressed genes (DEGs) between groups at each time point or between time points within the group were revealed by t-test. Genes with p < 0.05 after Benjamini and Hochberg false discovery rate correction for multiple hypothesis testing were considered as significant DEGs. We found that (1) the Ingenuity Pathway Analysis (IPA) revealed significant enrichment of DEGs in multiple pathways, especially those responding to oxidative stress, in homozygous Cops8-CKO hearts at both 2 and 3 weeks, corroborating the occurrence of massive cardiomyocyte necrosis at 3 weeks; (2) the decreases in multiple CRL SR proteins were associated with decreased transcript levels; and (3) enrichment of DEGs in the chromatin remodeling pathway and the microtubule motility and vesicle trafficking pathways.Conclusions: Our data are consistent with the notion that Cops8/CSN plays a role in the transcriptional regulation of CRL SRs and in the redox and vesicle trafficking pathways.

Highlights

Identified in mutant Arabidopsis that exhibits constitutive photomorphogenesis (Wei and Deng, 1992), the COP9 signalosome (CSN) is evolutionally conserved from yeast to humans, with its bona fide biochemical activity being cullin deneddylation responsible for the deconjugation of the ubiquitinlike protein NEDD8 from the cullin scaffold subunit of cullinRING ligases (CRLs) (Lyapina et al, 2001; Bosu and Kipreos, 2008; Sozen et al, 2015)
We have previously described generation of CSN8-CKO mice with the use of the Cre-loxP system in which transgenic Cre expression is driven by the mouse α-myosin heavy chain (Myh6) promoter (Su et al, 2011b)
Genes with p < 0.05 after Benjamini and Hochberg false discovery rate correction for multiple hypothesis testing were considered as significant Differentially expressed genes (DEGs)

Summary

Introduction

Identified in mutant Arabidopsis that exhibits constitutive photomorphogenesis (Wei and Deng, 1992), the COP9 signalosome (CSN) is evolutionally conserved from yeast to humans, with its bona fide biochemical activity being cullin deneddylation responsible for the deconjugation of the ubiquitinlike protein NEDD8 from the cullin scaffold subunit of cullinRING ligases (CRLs) (Lyapina et al, 2001; Bosu and Kipreos, 2008; Sozen et al, 2015). The assembly of a CRL is centered on a cullin scaffold which binds a substrate receptor (SR) module at its amino terminal end and a catalytic RING-containing protein (either RBX1 or RBX2) at its carboxyl terminal end. According to the current model, when the substrate for an active CRL runs low, the CSN binding and subsequent deneddylation of CRLs prevents the autoubiquitination and resultant destruction of the SR proteins of the CRL complex (Wang and Martin, 2015). Dissociation of the CSN from the unloaded and deneddylated CRL allows binding by CAND1 (Cullin-Associated and Neddylation-Dissociated 1) protein. The COP9 signalosome (CSN) consisting of 8 unique protein subunits (COPS1 through COPS8) serves as the cullin deneddylase, regulating the catalytic dynamics of cullin RING ligases (CRLs), the largest family of ubiquitin ligases. Given that Cops is nucleus-enriched and a prior report showed its binding to the promoter of several genes and association of its ablation with decreased mRNA levels of these genes, we sought to determine the dynamic changes of myocardial transcriptome in mice with perinatal

Methods

Results

Conclusion