We do know that the transcription factors p50 and Bcl-3 are essential for atrophy during disuse, so in the present study we first identified the genes being targeted by p50 and Bcl-3, thereby identifying genes that are required to produce the atrophied phenotype in each case. This work represents the first study to use a global approach to identify the genes required to produce the atrophied phenotype due to muscle unloading. Identification of genes that are NF-kB targets is a first step in the discovery of how these transcription factors produce the atrophied phenotype during skeletal muscle disuse. This study is the first to identify on a global scale, direct or indirect target genes of two transcription factors previously shown to be required for muscle disuse atrophy, p50 and Bcl-3. This was done using gene expression profiling of unloaded skeletal muscle from wild type mice compared to unloaded muscle from mice deficient for the gene encoding each of these transcription factors. Genes in wild type mice upregulated by hind limb unloading that are not upregulated in unloading of knockout mice are either direct or indirect targets of the gene knocked out. Since our previous work shows a complete atrophy inhibition when the genes encoding p50 and Bcl-3 are knocked out, it is not surprising to have a significant number of genes that are targets of these transcription factors. In combination with these data, we performed ChIP assays on kB sites in 14 of the kB -upregulated genes to assess whether they were direct targets of p50 and Bcl-3. We focused on upregulated genes because disuse elicits an MK-0683 increase in NF-kB binding to DNA and a robust increase in NF-kB dependent transcription in unloaded muscle from rats and mice. It was interesting however, that three of the nine proteasomal genes shown to be targets of p50 and Bcl-3, and chosen for further study, were indirect rather than direct kB target genes because there was no change in p65, p50, or Bcl-3 binding with unloading. This supports the idea that proteasomal subunit upregulation with atrophy is directly regulated by transcription factors other than NF-kB, although NF-kB may be regulating these unknown factors based on the gene expression data in the knockout mice. Another direct target gene of NF-kB Z-VAD-FMK Caspase inhibitor proteins is Runx1, previously shown to moderate myofibrillar stabilization and autophagy during disuse atrophy. It is a transcription factor with 29 putative gene targets in atrophying muscle. These targets appear to be mostly structural genes, distinct from the structural unloading-induced kB target genes. While kB regulates Runx1, Runx1 activity does not seem to involve activation of FoxO or NFkB- mediated transcription during disuse muscle atrophy. We show that Bcl-3 and p50 have structural gene targets and some of these share the same functional category as targets of Runx1 but none were the same genes as described by Wang et al.. The Tnfrsf12a gene was unique among the 14 genes studied for ChIP as it showed an increase in Bcl-3 binding, a moderate increase in p50 binding, and increased p65 binding. The lack of increased p65 binding to putative kB sites in all but one of the 14 genes studied by ChIP is consistent with previous work suggesting a lack of primary involvement of p65 in unloading atrophy. On the other hand, dominant negative overexpression of either IkBa SR, IKKa, or IKKb inhibited unloading atrophy by,50% suggesting that p65 may be involved in regulating some atrophy genes. In the case of Tnfrsf12a, regulation via kB proteins may involve a complex of p65:p50:Bcl-3 binding as previously described in response to TNFa. Tnfrsf12a, found to be a direct kB target is a receptor for a member of the TNF superfamily also known as TWEAK. This cytokine receptor is of interest as it has recently been shown to be increased with denervation atrophy. In addition, TWEAK is known to induce muscle wasting in whole muscle and it was required for denervation atrophy.