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1.
Figure 3

Figure 3. From: Extensive characterization of NF-?B binding uncovers non-canonical motifs and advances the interpretation of genetic functional traits.

One round of enrichment was sufficient with NF-kB p52p52. (a) 10-mer sequences enriched after one, two and three rounds of selection with NF-kB p52p52 during EMSA-Seq. (b) Ranked affinities of 11,065 10-mers that were continually enriched throughout the three rounds of SELEX with p52p52. The correlations of ranked affinities for these sequences throughout the process are shown (Pearson correlation test).

Daniel Wong, et al. Genome Biol. 2011;12(7):R70-R70.
2.
Figure 4

Figure 4. From: Extensive characterization of NF-?B binding uncovers non-canonical motifs and advances the interpretation of genetic functional traits.

EMSA-Seq profiling of the NF-κB RELA-containing dimers. (a) Grouping of 11-mer sequences bound by the homodimer RELARELA and the heterodimers RELAp50 and RELAp52 during EMSA-Seq. In parentheses are proportions out of all possible 2,097,152 11-mer sequences. (b) De novo motif identification was performed on the 50 and 1,000 top-scoring 11-mer sequences from each experiment using the Priority algorithm [51]. No priors were used for motif identification and logos were generated using the enoLOGOS web tool [52]. For every dimer, the percentage proportion of sequences that are non-canonical (MATCH < 0.75) and that have contributed towards construction of the motif has been indicated.

Daniel Wong, et al. Genome Biol. 2011;12(7):R70-R70.
3.
Figure 6

Figure 6. From: Extensive characterization of NF-?B binding uncovers non-canonical motifs and advances the interpretation of genetic functional traits.

Direct positive correlation of binding potential and in vivo binding. Presence of dimer-specific 11-mer sequences (colored boxes) enriched during EMSA-Seq within a 300-bp region (BRS) inside a binding region (BR) that was isolated during immunoprecipitation of RELA. Boxes are 11-mer sequences shown with an overlap of 10 bp for adjacent boxes. The gradient of coloration within boxes corresponds to relative binding affinities as determined by EMSA-Seq. A non-colored box represents an 11-mer sequence that was not bound by a RELA-containing dimer. Sequences that are also known NF-κB binders are indicated (filled triangles). Arrows indicate the positions of sequence polymorphisms between the two individuals.

Daniel Wong, et al. Genome Biol. 2011;12(7):R70-R70.
4.
Figure 7

Figure 7. From: Extensive characterization of NF-?B binding uncovers non-canonical motifs and advances the interpretation of genetic functional traits.

Binding potential and risk alleles of disease. (a) The trait/disease-associated (TAS) SNP rs2205960 (box, red outline) is associated with systemic lupus erythematosus. The 300-bp region (BRS) of individual NA12878, who is a carrier of the risk allele (T), contains a single 11-mer that was enriched during EMSA-Seq with RELAp50 whereas NA12891, who carries the normal allele, does not. This has resulted in NA12878 having a higher NF-κB binding potential that is directly correlated to higher in vivo binding. (b) The TAS rs68065278 (box, red outline) is associated with celiac disease. It is in linkage disequilibrium with another polymorphism, rs6776243, present within the BRS (box, green outline). The BRS of individual NA12878, who is a carrier of the C allele for rs6776243, contains more 11-mers that were enriched during EMSA-Seq with RELAp50 than that of the other individual. This has resulted in NA12878 having a higher NF-κB binding potential that is likewise directly correlated to higher in vivo binding.

Daniel Wong, et al. Genome Biol. 2011;12(7):R70-R70.
5.
Figure 2

Figure 2. From: Extensive characterization of NF-?B binding uncovers non-canonical motifs and advances the interpretation of genetic functional traits.

Binding profiles of the different NF-κB dimers. Heat map illustration of binding profiles obtained from microarray analysis of dimers. Within the heat map, probes that contain the 803 11-mer sequences and represent 'k-mer' space given by the consensus RGGRNNHHYYB can be found as rows whilst the nine NF-κB dimers have been organized into columns. A graded color scheme has been used to represent the ranked affinities of a dimer for a probe. From lightest to darkest this corresponds to decreasing affinity. Hierarchical clustering was used to describe relationships between binding profiles of the different dimers (Euclidean distance correlation; complete linkage analysis). The profile of RELARELA was largely distinct from those of the other eight dimers. On the whole, homodimers also have binding profiles that render these TFs to be less alike as a class. This is in contrast to the higher degree of similarity found between profiles within the heterodimer class. Two groups of sequences that contribute to similarities and differences between RELARELA and the other dimers have been used to construct representative binding models.

Daniel Wong, et al. Genome Biol. 2011;12(7):R70-R70.
6.
Figure 1

Figure 1. From: Extensive characterization of NF-?B binding uncovers non-canonical motifs and advances the interpretation of genetic functional traits.

Outline of the dual platform approach used to profile NF-κB family dimers. Double-purified, His-tagged NF-κB dimers interact with DNA-probes (microarray) or DNA-ligands (electrophoretic mobility shift assay-sequencing (EMSA-Seq)). Two separate stains are available for the visualization of DNA and protein on EMSA-gels. SYBR Green highlights both DNA bound by the dimer ('bound DNA') and also unbound DNA ('free DNA'). The SYPRO Ruby stain identifies proteins such as those within a dimer-DNA complex ('complex'). Both microarray and EMSA-Seq platforms generate data that provide binding affinities for individual sequences that interact with a dimer. Profiles of nine different dimers illustrating their binding affinities for 803 sequences were constructed using microarrays. In addition, RELARELA, RELAp50 and RELAp52 were also profiled using EMSA-Seq. Deep sequencing revealed dimer-specific binding affinities for distinctive groups of 11-mer sequences. Two classes of these sequences, formed on the basis of similarity to a reference NF-κB binding-model, were used as targets for a UV footprinting experiment. Finally, differences for in vitro binding potential as determined using binding affinities from EMSA-Seq and differences for in vivo binding as established by a ChIP-Seq study were then co-examined across 7,762 comparisons of paired individuals.

Daniel Wong, et al. Genome Biol. 2011;12(7):R70-R70.
7.
Figure 5

Figure 5. From: Extensive characterization of NF-?B binding uncovers non-canonical motifs and advances the interpretation of genetic functional traits.

Specific interaction of NF-κB dimers with canonical and non-canonical sequences. (a) Interaction of four NF-κB dimers, p50p50, RELARELA, RELAp50 and RELAp52, with canonical sequences containing either a H-2 binding site (lanes 1 to 5), or a HIV recognition site (lanes 6 to 10). These were profiled using EMSA (top panel), UV laser (middle panel) and DNAse I (bottom panel) footprinting techniques (with interactor regions demarcated with vertical black lines). For example, RELA dimer-DNA complexes were detected with EMSA (lanes 3 and 8; red arrows). Furthermore, a 'UV footprint' in the form of lower intensity banding observed within the interactor region (relative to controls in lanes 1 and 6) indicates specific interactions of varying affinities between the dimer and DNA. (b) Interaction of RELARELA with the non-canonical sequences was non-specific. With both sequences, distinct dimer-DNA complexes were observed by EMSA with all dimers except RELARELA, for which a smear was obtained (lane 4: RELARELA). No footprint was observed with RELARELA, whilst for the other dimers a stronger footprint was obtained with AGGGGAAGTTA compared to CTGGGGATTTA. (c) Median enrichment of 11-mers bound by the three RELA-containing dimers in EMSA-Seq. Five groupings of sequences were formed on the basis of MATCH similarity (Grp1 ≤ 0.20, 0.201 ≥ Grp2 ≤ 0.40, 0.401 ≥ Grp3 ≤ 0.60, 0.601 ≥ Grp4 ≤ 0.80 and Grp5 ≥ 0.801). There is a trend of enrichment increasing alongside MATCH similarity. Also shown are the average enrichment values and corresponding similarities to the reference for the six 11-mer sequences that were footprinted (crosses with sequence indicated).

Daniel Wong, et al. Genome Biol. 2011;12(7):R70-R70.

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