This step identifies regulatory TF whose expression associates with TF binding motif DNA methylation and it is carried out by function get.TFs.
When a group of enhancers is coordinately altered in a specific sample subset, this is often the result of an altered upstream master regulator transcription factor in the gene regulatory network. ELMER identifies master regulator TFs corresponding to each of the TF binding motifs enriched from the previous analysis step. For each enriched motif, ELMER takes the mean DNA methylation of all distal probes (in significant probe-gene pairs) that contain that motif occurrence (within a  ± 250bp region) and compares this mean DNA methylation to the expression of each gene annotated as a human TF.
In the Unsupervised mode, a statistical test is performed for each motif-TF pair, as follows. All samples are divided into two groups: the M group, which consists of the 20% of samples with the highest average methylation at all motif-adjacent probes, and the U group, which consisted of the 20% of samples with the lowest methylation. This step is performed by the get.TFs function, which takes minSubgroupFrac as an input parameter, again with a default of 20%. For each candidate motif-TF pair, the Mann-Whitney U test is used to test the null hypothesis that overall gene expression in group M is greater or equal than that in group U. This non-parametric test was used in order to minimize the effects of expression outliers, which can occur across a very wide dynamic range. For each motif tested, this results in a raw p-value (Pr) for each of the human TFs.
The new Supervised mode uses the same approach as described for the identification of putative target gene(s) step. The U and M groups are one of the the label group of samples and the minSubgroupFrac parameter is set to 100% to use all samples from both groups in the statistical test. This also can result in greater statistical power when using the Supervised mode.
Finally, all TFs were ranked by the  − log10(Pr), and those falling within the top 5% of this ranking were considered candidate upstream regulators. By default, the top 3 most anti-correlated TFs, and all TF classified by TFClass database in the same (sub)family are highlighted.