Figure 1.
Selection of clones for differential expression analysis.
The selection is divided into 2 phases, where the clones selected for Phase 2 are a subset of all clones tested in Phase 1. Phase 1 and Phase 2 samples are from different plasmid preparations.
Table 1.
List of genomic regions validated as driving muscle-specific expression.
Table 2.
Over-represented TFBS in the background regions of the validated set vs. the non-responding background regions (ranked by Fisher p-values).
Table 3.
Over-represented TFBSs in the non-background regions of the validated set vs. the non-responding non-background regions (ranked by Fisher p-values).
Table 4.
Over-represented TFBSs in all regions of the validated set vs. the non-responding regions (ranked by Fisher p-values).
Table 5.
Sequence composition characteristics of the responding regions vs. non-responding regions.
Figure 2.
Dinucleotide frequencies in responding regions vs. non-responding regions.
a) Muscle Regulatory Regions. Muscle Validated = 19 validated muscle regions in this study. Muscle Reference = 28 muscle reference regions from literature. Muscle Non-Responders = all regions that were tested in this study and found not to drive gene expression. b) Pleiades Curated Regulatory Regions. Pleiades Curated All = 1341 curated regulatory regions from all species. Pleiades Curated Human = 631 curated regulatory regions in humans only. Non-Responders = all regions that were tested and found not to drive gene expression.
Table 6.
Sequence composition characteristics of the MyoD ChIP-Seq peaks compared against the non-responding regions from this study.
Figure 3.
Muscle TFBS hits (threshold of 80%) and the phastCons conservation profile for the region are shown as well. When square brackets are shown, they indicate the original CRM prediction. a) Positive sequence from the muscle set. The muscle-specific TFBS are located in regions of high sequence conservation. b) Positive sequence from the non-muscle. This sequence showed the most consistent increase in reporter expression, with all 12 replicates determined as significantly up-regulated in muscle. c) Positive sequence from the background set. Despite the clear cluster of muscle-specific TFBS located in the region of high sequence conservation, none of the CRM prediction tools could classify this as a muscle CRM.
Table 7.
Sequence conservation based on phastCons scores (28-way Placental Mammals).
Figure 4.
Phylogenetic depth analysis of TFBSs in responding and non-responding regions using phyloP (46-way, hg19).
The x-axis grouping indicates the species used to calculate the phyloP scores. TFBSs were searched using all vertebrate profiles from the JASPAR CORE collection using the threshold of 0.8. TFs with at least 2-fold increase in phyloP scores (46wayAll) in the TFBS positions over the non-TFBS positions in the responding regions were identified. The score ratios for these TFs were compared among the three region sets. a) Average phyloP scores for the predicted TFBS positions and non-TFBS positions in each region set. b) Ratios of phyloP scores for the TFBS positions and non-TFBS positions in each region set.
Table 8.
Regions overlapping MyoD ChIP-Seq peaks in C2C12 cells.
Figure 5.
Histone modifications in the responding and non-responding regions.
Proportion of the regions that overlap with ChIP-Seq peaks from Asp et al. are displayed. (MB = Myoblasts, MT = Myotubes).