Limits...
Identification of genes that elicit disuse muscle atrophy via the transcription factors p50 and Bcl-3.

Wu CL, Kandarian SC, Jackman RW - PLoS ONE (2011)

Bottom Line: We do not know however, the genes targeted by NF-κB that produce the atrophied phenotype.Trim63 (MuRF1), Fbxo32 (MAFbx), Ubc, Ctsl, Runx1, Tnfrsf12a (Tweak receptor), and Cxcl10 (IP-10) showed increased Bcl-3 binding to κB sites in unloaded muscle and thus were direct targets of Bcl-3. p50 binding to the same sites on these genes either did not change or increased, supporting the idea of p50:Bcl-3 binding complexes. p65 binding to κB sites showed decreased or no binding to these genes with unloading.Fbxo9, Psma6, Psmc4, Psmg4, Foxo3, Ankrd1 (CARP), and Eif4ebp1 did not show changes in p65, p50, or Bcl-3 binding to κB sites, and so were considered indirect targets of p50 and Bcl-3.

View Article: PubMed Central - PubMed

Affiliation: Department of Health Sciences, Boston University, Boston, Massachusetts, United States of America.

ABSTRACT
Skeletal muscle atrophy is a debilitating condition associated with weakness, fatigue, and reduced functional capacity. Nuclear factor-kappaB (NF-κB) transcription factors play a critical role in atrophy. Knockout of genes encoding p50 or the NF-κB co-transactivator, Bcl-3, abolish disuse atrophy and thus they are NF-κB factors required for disuse atrophy. We do not know however, the genes targeted by NF-κB that produce the atrophied phenotype. Here we identify the genes required to produce disuse atrophy using gene expression profiling in wild type compared to Nfkb1 (gene encodes p50) and Bcl-3 deficient mice. There were 185 and 240 genes upregulated in wild type mice due to unloading, that were not upregulated in Nfkb1⁻/⁻ and Bcl-3⁻/⁻ mice, respectively, and so these genes were considered direct or indirect targets of p50 and Bcl-3. All of the p50 gene targets were contained in the Bcl-3 gene target list. Most genes were involved with protein degradation, signaling, translation, transcription, and transport. To identify direct targets of p50 and Bcl-3 we performed chromatin immunoprecipitation of selected genes previously shown to have roles in atrophy. Trim63 (MuRF1), Fbxo32 (MAFbx), Ubc, Ctsl, Runx1, Tnfrsf12a (Tweak receptor), and Cxcl10 (IP-10) showed increased Bcl-3 binding to κB sites in unloaded muscle and thus were direct targets of Bcl-3. p50 binding to the same sites on these genes either did not change or increased, supporting the idea of p50:Bcl-3 binding complexes. p65 binding to κB sites showed decreased or no binding to these genes with unloading. Fbxo9, Psma6, Psmc4, Psmg4, Foxo3, Ankrd1 (CARP), and Eif4ebp1 did not show changes in p65, p50, or Bcl-3 binding to κB sites, and so were considered indirect targets of p50 and Bcl-3. This work represents the first study to use a global approach to identify genes required to produce the atrophied phenotype with disuse.

Show MeSH

Related in: MedlinePlus

ChIP assays of Bcl-3, p50, and p65 protein binding to genes from Figure 1.ChIP assay was performed using gastrocnemius/plantaris muscle from weight bearing (WB) and unloaded (HU) mice. For each gene shown, binding of Bcl-3, p50 and p65 protein to a κB binding site was measured. ChIP showed an increase in Bcl-3 protein binding to 7 of the 14 genes studied, these were: A) Fbxo32, B) Trim63, C) Ubc, D) Ctsl, E) Runx1, F) Tnfrsf12a, and G) Cxcl10. There were also increases in p50 (B, E, G) and decreases in p65 (A, B, D, G) in these 7 genes. None of the proteasomal subunit genes showed changes in Bcl-3, p50, or p65 binding, Psmc4., Psmg4, or Psma6 (H–J). Fbxo9, Ankrd1, Foxo3, and Eif4ebp1 (K–N) genes also did not show changes in the binding of these proteins to κB sites. For each gene, ChIP assays were repeated using different samples from WB and HU groups. The PCR product size for all 14 ChIP assays performed was between 190 to 400bp.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3020958&req=5

pone-0016171-g002: ChIP assays of Bcl-3, p50, and p65 protein binding to genes from Figure 1.ChIP assay was performed using gastrocnemius/plantaris muscle from weight bearing (WB) and unloaded (HU) mice. For each gene shown, binding of Bcl-3, p50 and p65 protein to a κB binding site was measured. ChIP showed an increase in Bcl-3 protein binding to 7 of the 14 genes studied, these were: A) Fbxo32, B) Trim63, C) Ubc, D) Ctsl, E) Runx1, F) Tnfrsf12a, and G) Cxcl10. There were also increases in p50 (B, E, G) and decreases in p65 (A, B, D, G) in these 7 genes. None of the proteasomal subunit genes showed changes in Bcl-3, p50, or p65 binding, Psmc4., Psmg4, or Psma6 (H–J). Fbxo9, Ankrd1, Foxo3, and Eif4ebp1 (K–N) genes also did not show changes in the binding of these proteins to κB sites. For each gene, ChIP assays were repeated using different samples from WB and HU groups. The PCR product size for all 14 ChIP assays performed was between 190 to 400bp.

Mentions: Of the 14 genes on which a ChIP assay was performed, 11 had conserved κB sites. The other 3 genes contained κB sites, but none were conserved (Fbxo9, Psmg4, Eif4ebp1); not surprisingly, these 3 genes did not show changes in binding of the 3 κB factors studied, due to unloading. The most frequent and robust change in protein binding to κB sites of the 3 transcription factors studied was Bcl-3. There was an increase in Bcl-3 binding to conserved κB sites in 7 of the 14 genes studied. These were Fbxo32 (the site 550bp upstream of the TSS), Trim63 (the site in the third intron 4.8 kb downstream of the TSS), Ubc (the site 1.4 kb upstream of the TSS), Ctsl (the site 5.5 kb upstream of the TSS), Runx1 (the site 1.7 kb upstream of the TSS), Tnfrsf12a (the site 5.7 kb upstream of the TSS), and Cxcl10 (the two sites 157 bp upstream of the TSS) (Figure 2A–G). Of these 7 genes, p50 binding to the same κB site was either unchanged or, it was increased in Trim63, Runx1, and Cxcl10. In all 7 genes, the increased Bcl-3 binding to sites where p50 was bound suggests increased formation of p50-Bcl-3 complexes with unloading. For p65 binding there were marked decreases in 4 genes, Fbxo32, Trim63, Ctsl, and Cxcl10 (Figure 2A, B, D, G) and no detectable binding in control or unloaded muscle in Ubc or Runx1 (Figure 2C, E). The fact that no κB site showed marked p65 binding with unloading supports earlier work suggesting that p65 is not a major player in disuse muscle atrophy [8]. There was a moderate increase in p65 binding in one gene, Tnfrsf12a (Figure 2F). Each immunoprecipitation was repeated with different muscles from weight bearing and unloaded groups.


Identification of genes that elicit disuse muscle atrophy via the transcription factors p50 and Bcl-3.

Wu CL, Kandarian SC, Jackman RW - PLoS ONE (2011)

ChIP assays of Bcl-3, p50, and p65 protein binding to genes from Figure 1.ChIP assay was performed using gastrocnemius/plantaris muscle from weight bearing (WB) and unloaded (HU) mice. For each gene shown, binding of Bcl-3, p50 and p65 protein to a κB binding site was measured. ChIP showed an increase in Bcl-3 protein binding to 7 of the 14 genes studied, these were: A) Fbxo32, B) Trim63, C) Ubc, D) Ctsl, E) Runx1, F) Tnfrsf12a, and G) Cxcl10. There were also increases in p50 (B, E, G) and decreases in p65 (A, B, D, G) in these 7 genes. None of the proteasomal subunit genes showed changes in Bcl-3, p50, or p65 binding, Psmc4., Psmg4, or Psma6 (H–J). Fbxo9, Ankrd1, Foxo3, and Eif4ebp1 (K–N) genes also did not show changes in the binding of these proteins to κB sites. For each gene, ChIP assays were repeated using different samples from WB and HU groups. The PCR product size for all 14 ChIP assays performed was between 190 to 400bp.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3020958&req=5

pone-0016171-g002: ChIP assays of Bcl-3, p50, and p65 protein binding to genes from Figure 1.ChIP assay was performed using gastrocnemius/plantaris muscle from weight bearing (WB) and unloaded (HU) mice. For each gene shown, binding of Bcl-3, p50 and p65 protein to a κB binding site was measured. ChIP showed an increase in Bcl-3 protein binding to 7 of the 14 genes studied, these were: A) Fbxo32, B) Trim63, C) Ubc, D) Ctsl, E) Runx1, F) Tnfrsf12a, and G) Cxcl10. There were also increases in p50 (B, E, G) and decreases in p65 (A, B, D, G) in these 7 genes. None of the proteasomal subunit genes showed changes in Bcl-3, p50, or p65 binding, Psmc4., Psmg4, or Psma6 (H–J). Fbxo9, Ankrd1, Foxo3, and Eif4ebp1 (K–N) genes also did not show changes in the binding of these proteins to κB sites. For each gene, ChIP assays were repeated using different samples from WB and HU groups. The PCR product size for all 14 ChIP assays performed was between 190 to 400bp.
Mentions: Of the 14 genes on which a ChIP assay was performed, 11 had conserved κB sites. The other 3 genes contained κB sites, but none were conserved (Fbxo9, Psmg4, Eif4ebp1); not surprisingly, these 3 genes did not show changes in binding of the 3 κB factors studied, due to unloading. The most frequent and robust change in protein binding to κB sites of the 3 transcription factors studied was Bcl-3. There was an increase in Bcl-3 binding to conserved κB sites in 7 of the 14 genes studied. These were Fbxo32 (the site 550bp upstream of the TSS), Trim63 (the site in the third intron 4.8 kb downstream of the TSS), Ubc (the site 1.4 kb upstream of the TSS), Ctsl (the site 5.5 kb upstream of the TSS), Runx1 (the site 1.7 kb upstream of the TSS), Tnfrsf12a (the site 5.7 kb upstream of the TSS), and Cxcl10 (the two sites 157 bp upstream of the TSS) (Figure 2A–G). Of these 7 genes, p50 binding to the same κB site was either unchanged or, it was increased in Trim63, Runx1, and Cxcl10. In all 7 genes, the increased Bcl-3 binding to sites where p50 was bound suggests increased formation of p50-Bcl-3 complexes with unloading. For p65 binding there were marked decreases in 4 genes, Fbxo32, Trim63, Ctsl, and Cxcl10 (Figure 2A, B, D, G) and no detectable binding in control or unloaded muscle in Ubc or Runx1 (Figure 2C, E). The fact that no κB site showed marked p65 binding with unloading supports earlier work suggesting that p65 is not a major player in disuse muscle atrophy [8]. There was a moderate increase in p65 binding in one gene, Tnfrsf12a (Figure 2F). Each immunoprecipitation was repeated with different muscles from weight bearing and unloaded groups.

Bottom Line: We do not know however, the genes targeted by NF-κB that produce the atrophied phenotype.Trim63 (MuRF1), Fbxo32 (MAFbx), Ubc, Ctsl, Runx1, Tnfrsf12a (Tweak receptor), and Cxcl10 (IP-10) showed increased Bcl-3 binding to κB sites in unloaded muscle and thus were direct targets of Bcl-3. p50 binding to the same sites on these genes either did not change or increased, supporting the idea of p50:Bcl-3 binding complexes. p65 binding to κB sites showed decreased or no binding to these genes with unloading.Fbxo9, Psma6, Psmc4, Psmg4, Foxo3, Ankrd1 (CARP), and Eif4ebp1 did not show changes in p65, p50, or Bcl-3 binding to κB sites, and so were considered indirect targets of p50 and Bcl-3.

View Article: PubMed Central - PubMed

Affiliation: Department of Health Sciences, Boston University, Boston, Massachusetts, United States of America.

ABSTRACT
Skeletal muscle atrophy is a debilitating condition associated with weakness, fatigue, and reduced functional capacity. Nuclear factor-kappaB (NF-κB) transcription factors play a critical role in atrophy. Knockout of genes encoding p50 or the NF-κB co-transactivator, Bcl-3, abolish disuse atrophy and thus they are NF-κB factors required for disuse atrophy. We do not know however, the genes targeted by NF-κB that produce the atrophied phenotype. Here we identify the genes required to produce disuse atrophy using gene expression profiling in wild type compared to Nfkb1 (gene encodes p50) and Bcl-3 deficient mice. There were 185 and 240 genes upregulated in wild type mice due to unloading, that were not upregulated in Nfkb1⁻/⁻ and Bcl-3⁻/⁻ mice, respectively, and so these genes were considered direct or indirect targets of p50 and Bcl-3. All of the p50 gene targets were contained in the Bcl-3 gene target list. Most genes were involved with protein degradation, signaling, translation, transcription, and transport. To identify direct targets of p50 and Bcl-3 we performed chromatin immunoprecipitation of selected genes previously shown to have roles in atrophy. Trim63 (MuRF1), Fbxo32 (MAFbx), Ubc, Ctsl, Runx1, Tnfrsf12a (Tweak receptor), and Cxcl10 (IP-10) showed increased Bcl-3 binding to κB sites in unloaded muscle and thus were direct targets of Bcl-3. p50 binding to the same sites on these genes either did not change or increased, supporting the idea of p50:Bcl-3 binding complexes. p65 binding to κB sites showed decreased or no binding to these genes with unloading. Fbxo9, Psma6, Psmc4, Psmg4, Foxo3, Ankrd1 (CARP), and Eif4ebp1 did not show changes in p65, p50, or Bcl-3 binding to κB sites, and so were considered indirect targets of p50 and Bcl-3. This work represents the first study to use a global approach to identify genes required to produce the atrophied phenotype with disuse.

Show MeSH
Related in: MedlinePlus