Abstract
NF-kappaB transcriptional activation is required for skeletal muscle disuse atrophy. We are continuing to study how the activation of NF-kB regulates the genes that encode the protein products that cause atrophy. Using ChIP-sequencing we found that Bcl-3, an NF-kB transcriptional activator required for atrophy, binds to the promoters of a number of genes whose collective function describes two major aspects of muscle wasting. By means of bioinformatics analysis of ChIP-sequencing data we found Bcl-3 to be directing transcription networks of proteolysis and energy metabolism. The proteolytic arm of the Bcl-3 networks includes many E3 ligases associated with proteasomal protein degradation, including that of the N-end rule pathway. The metabolic arm appears to be involved in organizing the change from oxidative phosphorylation to glycolysis in atrophying muscle. For one gene, MuRF1, ChIP-sequencing data identified the location of Bcl-3 and p50 binding in the promoter region which directed the creation of deletant and base-substitution mutations of MuRF1 promoter constructs to determine the effect on gene transcription. The results provide the first direct confirmation that the NF-kB binding site is involved in the muscle unloading regulation of MuRF1. Finally, we have combined the ChIP-sequencing results with gene expression microarray data from unloaded muscle to map several direct targets of Bcl-3 that are transcription factors whose own targets describe a set of indirect targets for NF-kB in atrophy. ChIP-sequencing provides the first molecular explanation for the finding that Bcl3 knockout mice are resistant to disuse muscle atrophy. Mapping the transcriptional regulation of muscle atrophy requires an unbiased analysis of the whole genome, which we show is now possible with ChIP-sequencing.
Highlights
Skeletal muscle atrophy is the result of a metabolic shift that increases the rate of proteolysis and/or decreases the rate protein synthesis in the cells that make up muscle
We focused on finding the direct target genes of nuclear factor-kappaB (NF-kB) transcription factors during muscle unloading in order to identify the genes producing atrophy
Using antibodies for p50 and Bcl-3 to immunoprecipitate the muscle chromatin followed by highthroughput sequencing and high-resolution genome mapping, we identified the genes that are being directly targeted by these NF-kB transcription factors in unloaded muscle
Summary
Skeletal muscle atrophy is the result of a metabolic shift that increases the rate of proteolysis and/or decreases the rate protein synthesis in the cells that make up muscle. To identify the genes regulated by p50 or Bcl-3 that produce the atrophied phenotype, global gene expression analysis was used to compare wild type and the two knockout strains of mice in response to unloading [10]. The genes upregulated in wild type mice that were not upregulated in knockout mice due to unloading were from several muscle atrophy gene functional groups including proteolysis. This analysis cannot distinguish direct vs indirect target genes
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