T targets, 5 direct targets with indirect targets (transcription factors) and 305 indirect target genes of Bcl-3. The indirect target genes, according to this analysis, are controlled by the direct Bcl-3 targeted transcription factors Max, Gepotidacin biological activity Zfp740, Nfic, Cux1 and Pou2f1. Max appears to regulate the largest number of indirect target genes.Gene Ontology Terms Identified by Genome-wide Increased Bcl-3 Binding to Promoter Regions in Unloaded MuscleTo find the important functional groups of genes that show increased Bcl-3 binding with muscle unloading, we evaluated the peaks found in unloaded compared to control muscle for gene ontology terms/pathways. To do this we used the iPAGE algorithm, a module of the ChIPseeqer set of computational programs (Figure 4). iPAGE was set up to restrict its analysis to the 845 peaks (out of the 2,817 total) which were found in promoters (24 kb to +2 kb relative to TSS). As with any gene ontology (GO) mapping algorithm, iPAGE identifies GO terms in which the peaks found are statistically over-represented relative to calculations for random distribution. The 23 GO terms that were found for genes containing Bcl-3 peaks in unloaded muscle were from three biological processes: protein catabolism, development/ differentiation and sugar/glucose metabolism. There were 24 genes found in the 23 GO pathways and these are presented in Table 1. The most abundant group with 14 genes in 11 GO pathways was for protein catabolism. The genes are ones that function in several aspects of catabolism in muscle including several E3 ligases of the ubiquitin proteasome pathway, and importantly, two genes that contribute to the cell catabolism driven by the N-end rule. Those genes are Ubr1/E3a, the N-end recognin E3 ligase, and Ate1, the arginyltransferase responsible for modifying several amino acid amino termini for Ubr1 recognition. The GLPG0187 site sequence alignments and locations for the peaks for these two genes have been visualized by use of Integrative Genomics Viewer (IGV) [22], (http://www.broadinstitute.org/igv/) and are shown in Figure 5. For both genes, a Bcl-3 peak due to unloading was identified at an evolutionary conserved region close to the TSS and was in close proximity to a JASPAR matrices defined NF-kB site. In addition, data for ChIP-seq with p50 antibodies showed p50 binding at or very near the peak sites of Bcl-3 binding (data not shown). Another E3 ligase found was Trim63/MuRF1, a muscle specific protein thought to target heavy myosin chains during atrophy [23,24].Testing a Bcl-3 Binding Region in Gene ActivationIn a previous paper, we found genes to be direct or indirect targets of Bcl-3 based on gene expression in unloaded muscle from wild type vs. Bcl3 knockout mice. We selected several of these genes for further study that were thought to be involved with the atrophy process. We identified NF-kB sites in these genes in silico and we found ChIP-PCR support for increased Bcl-3 binding [10]. One of these genes, MuRF1, had three in silico 1379592 NF-kB sites in the 4.4 kb region of the promoter that had already been cloned into a luciferase reporter [18]. The present study identified MuRF1 by iPAGE as being a Bcl-3 target in the GO categories of proteolysis. The data identified a peak at one of the in silico-identified NF-kB sites of the MuRF1 promoter. The alignments for the Bcl-3 binding site in the MuRF1 promoter are shown in Figure 7. Data for ChIP-seq with p50 antibodies are also presented, indicating the associated binding.T targets, 5 direct targets with indirect targets (transcription factors) and 305 indirect target genes of Bcl-3. The indirect target genes, according to this analysis, are controlled by the direct Bcl-3 targeted transcription factors Max, Zfp740, Nfic, Cux1 and Pou2f1. Max appears to regulate the largest number of indirect target genes.Gene Ontology Terms Identified by Genome-wide Increased Bcl-3 Binding to Promoter Regions in Unloaded MuscleTo find the important functional groups of genes that show increased Bcl-3 binding with muscle unloading, we evaluated the peaks found in unloaded compared to control muscle for gene ontology terms/pathways. To do this we used the iPAGE algorithm, a module of the ChIPseeqer set of computational programs (Figure 4). iPAGE was set up to restrict its analysis to the 845 peaks (out of the 2,817 total) which were found in promoters (24 kb to +2 kb relative to TSS). As with any gene ontology (GO) mapping algorithm, iPAGE identifies GO terms in which the peaks found are statistically over-represented relative to calculations for random distribution. The 23 GO terms that were found for genes containing Bcl-3 peaks in unloaded muscle were from three biological processes: protein catabolism, development/ differentiation and sugar/glucose metabolism. There were 24 genes found in the 23 GO pathways and these are presented in Table 1. The most abundant group with 14 genes in 11 GO pathways was for protein catabolism. The genes are ones that function in several aspects of catabolism in muscle including several E3 ligases of the ubiquitin proteasome pathway, and importantly, two genes that contribute to the cell catabolism driven by the N-end rule. Those genes are Ubr1/E3a, the N-end recognin E3 ligase, and Ate1, the arginyltransferase responsible for modifying several amino acid amino termini for Ubr1 recognition. The sequence alignments and locations for the peaks for these two genes have been visualized by use of Integrative Genomics Viewer (IGV) [22], (http://www.broadinstitute.org/igv/) and are shown in Figure 5. For both genes, a Bcl-3 peak due to unloading was identified at an evolutionary conserved region close to the TSS and was in close proximity to a JASPAR matrices defined NF-kB site. In addition, data for ChIP-seq with p50 antibodies showed p50 binding at or very near the peak sites of Bcl-3 binding (data not shown). Another E3 ligase found was Trim63/MuRF1, a muscle specific protein thought to target heavy myosin chains during atrophy [23,24].Testing a Bcl-3 Binding Region in Gene ActivationIn a previous paper, we found genes to be direct or indirect targets of Bcl-3 based on gene expression in unloaded muscle from wild type vs. Bcl3 knockout mice. We selected several of these genes for further study that were thought to be involved with the atrophy process. We identified NF-kB sites in these genes in silico and we found ChIP-PCR support for increased Bcl-3 binding [10]. One of these genes, MuRF1, had three in silico 1379592 NF-kB sites in the 4.4 kb region of the promoter that had already been cloned into a luciferase reporter [18]. The present study identified MuRF1 by iPAGE as being a Bcl-3 target in the GO categories of proteolysis. The data identified a peak at one of the in silico-identified NF-kB sites of the MuRF1 promoter. The alignments for the Bcl-3 binding site in the MuRF1 promoter are shown in Figure 7. Data for ChIP-seq with p50 antibodies are also presented, indicating the associated binding.
