2.3. Selection of Candidates

Five TFs out of the significant ones in the enrichment analysis were selected for biological validation of our strategy based on the previous literature and the repetition of the terms in at least three different modules. For each TF, we selected up to three target genes from the DCGs sets with enrichment for that particular TF, prioritizing those with conserved binding sites and/or positive chromatin immunoprecipitation-sequencing (ChIP-seq) data at promoters and/or H3K27Ac-rich regions, according to the ENCODE data from the University of California Santa Cruz (UCSC) genome browser (https://genome.ucsc.edu/). Revision of the literature helped to further prioritize those genes that could be more relevant to CD pathogenesis, and CISD2, HDAC4 and WDR43 were chosen for IFN regulatory factor-1 (IRF1); AKTIP, NAMPT and TPK1 for CAMP Responsive Element Binding Protein 1 (CREB1); CRTAM, PLLP and RFX5 for ETS domain-containing protein (ELK1), and WNT11 for NFKB1. Additionally, target genes that were not present in the DCGs sets but are well defined targets of the selected TFs were also included. Putative targets were sorted according to the number of TFs that are controlling them (based on the human/mouse/rat conserved binding sites from the UCSC genome browser tables utility), and the most specific target genes for the four candidate TFs were selected, namely CXCL11 and BATF2 for IRF1; ISG15 and HIST1H4C for CREB1; NKG7 and RAB17 for ELK1; and GSTA4, TFF1 and PLAUR for NFKB1.