Figure 1.
Distribution of Bcl-3 binding peaks around transcription start sites (TSS) determined by Nebula/Galaxy.
Blue represents the plot of Bcl-3 peaks from unloaded muscle and gray represents the plot from peaks found in the input chromatin from unloaded muscle. The y-axis is the proportion of peaks relative to all genes in the genome. Peaks are plotted every 20 bases from −2500 to +2500 relative to the TSS.
Figure 2.
Plot of phylogenomic conservation for the 2,817 Bcl-3 peaks produced by unloading.
The peaks and surrounding genome regions (−1500 bp to +1500 bp) were compared to a database of Phastcon alignment scores for 31 placental mammals on the Galaxy/Cistrome server. Phastcon scores are higher for sequence similarity and are weighted higher for species farther removed from mice phylogenetically. A Phastcon score of 1.0 would reflect perfect identity in all 31 species. Conservation is highest at the center of the peaks indicating that the centers share sequence homology between species, a sign that the sites of Bcl-3 binding are important to function.
Figure 3.
Distribution of Bcl-3 peaks by location in genes.
(A) ChIPseeqer genomic annotation for the 2,817 peaks of increased Bcl-3 binding found in unloaded compared to control muscle. (B) ChIPseeqer genomic annotation for peaks found in the sequence alignments from the unloaded muscle input chromatin which was sheared and used to create a library without any further manipulation (no immunoprecipitation). The peak finder in ChIPseeqer was set to the same parameters as for the 2,817 Bcl-3 peaks in unloaded muscle and found 1,594 random peaks.
Figure 4.
GO terms enriched in genes with Bcl-3 peaks during unloading.
iPAGE analysis identified 23 GO terms over-represented (red bar) by genes with Bcl-3 peaks in promoters due to muscle unloading. Text labeling indicates the name of the GO term and the associated GO identification number.
Table 1.
The genes from iPAGE ontology analysis.
Figure 5.
Bcl-3 binding profile at Ubr1 and Ate1 genes.
(A) An assembly of ChIP-seq data for the Ubr1 (chromosome 2) and (B) Ate1 (chromosome 7) genes, visualized by IGV. In both A and B, the top line is a representation of genomic size and location of the region. Vertical ticks are 500 bp apart. The next rows are labeled as follows: Gene, the graphic for the name, location, and orientation for the gene nearest to the ChIP-seq alignment. The medium thick dark line is the 5′ utr of the gene and the thicker dark region is the first exon followed by a thin line with arrows which is intron 1; Conservation, the track of Phastcons for sequence similarity among placental mammals; ChIPseeqer peak, the black rectangular block is the location of the statistically-qualified peak of sequencing alignments called by the ChIPseeqer algorithm; HU Bcl-3, a representation of the.sam alignments for the Bcl-3 ChIPseq of the unloaded muscle; WB Bcl-3, a representation of the.sam alignments for the Bcl-3 ChIPseq of the weight bearing muscle; Input, a representation of the.sam alignments for non-ChIP unloaded chromatin; NF-κB site, location of a NF-κB consensus site identified by JASPAR.
Table 2.
qPCR of selected proteolysis genes with increased Bcl-3 promoter binding.
Figure 6.
Network of direct and indirect Bcl-3 target genes during unloading.
Display of ChIP-Array data from a comparison of 2,817 Bcl-3 binding peaks increased in unloading over controls vs. 3,334 genes with increased expression in unloaded muscles. A blue circle indicates the location of Bcl-3. Projections from Bcl-3 in yellow are the direct targets including 5 direct transcription factor target genes, which are indicated in pink circles. Projections from pink targets are indirect Bcl-3 targets indicated in gray. Thus, ChIP-Array found 241 direct targets, 5 direct targets with indirect targets (the transcription factors) and 305 indirect target genes of Bcl-3 in the gene expression array data. The direct targets are those from the Bcl-3 ChIP-seq list with some not identified because of gene name terminology differences in the ChIPArray format. However the direct targets with indirect targets yields important evidence for a hierarchy of gene regulation.
Figure 7.
Bcl-3 and p50 binding profile at MuRF1 locus.
An assembly of ChIP-seq data visualized by IGV for the Trim63/MuRF1 gene. The top line is a representation of the genomic size and location of the region of chromosome 4. Vertical ticks are 500 bp apart. The region of this gene (MuRF1) labeled is 3.1 kb 5′ of the TSS. The next rows are labeled as follows: Conservation, the track of Phastcons for sequence similarity among placental mammals; HU Bcl-3, a representation of the.sam alignments for the Bcl-3 ChIPseq of unloaded muscle; ChIPseeqer peak for Bcl-3, black horizontal bar indicates the location of the statistically-qualified peak of sequencing alignments called by the ChIPseeqer algorithm; HU p50, a representation of the.sam alignments for the p50 ChIPseq of the hindlimb unloaded muscle; ChIPseeqer peak for p50, black horizontal bar indicates the location of the statistically-qualified peak of sequencing alignments called by the ChIPseeqer algorithm; Input, a representation of the.sam alignments for the non-ChIP unloaded chromatin; NF-κB sites, location for the 3 JASPAR identified NF-κB consensus sites in this region and in our reporter construct.
Figure 8.
Luciferase activity of MuRF1 reporter constructs in weight bearing vs. unloaded muscle.
Reporter constructs were electroporated into rat soleus and then unloaded for 5 days. MuRF1 is 4.4 kb promoter region of mouse MuRF1 driving expression of luciferase. MuRF1 deletant is a plasmid containing the 4.4 kb of the MuRF1 promoter but the distal 2 kb of the promoter was excised (from the 4.4 kb promoter) thus removing all 3 κB sites, and MuRF1 3κBmut is a plasmid containing the 4.4 kb of the MuRF1 promoter but the 3 NF-kB binding sites were mutated. * indicates statistical difference compared to weight bearing (WB) (P<0.05).