Transcriptional Regulation Analysis Protocol

Two-sided Student’s t-tests were performed to assign significance for cellular phenotypic assays and metabolite levels. Only reproducible binding sites identified by replicate ChIP-seq experiments were reported and used for downstream analyses. ChIP-seq peaks were identified using MACS peak caller [51 ], while motif analysis was performed using MEME [52 (link)] on the top 500 most enriched binding sites. For normalized ChIP-seq read-depth analyses, the number of reads were tabulated across binding site coordinates and normalized to the total number of aligned reads obtained for each ChIP-seq experiment. For mapping reads, we utilized a 100-bp sequence centered on the peak summit of each binding site. For ranking temporal changes in read depth, we generated a linear model comparing replicate ChIP-seq read enrichment values between two time points. Sites with altered enrichment were subsequently divided by slope and p value ranked. The top 50 % of p value-ranked sites from each category were used for cofactor motif analyses. For assigning TF motif fold enrichments, we compared the total number of motifs found across a set of binding sites with the number of motifs identified after scrambling binding site sequences. For RNAP2 read depth promoter analyses, sequences within 500 bp of transcription start sites were used as promoters. Connectivity maps were generated using Cytoscape (http://www.cytoscape.org/) and Enrichment map [53 (link)]. Kyoto Encyclopedia of Genes and Genomes (KEGG) gene pathway enrichments were generated using Gene Set Enrichment Analysis (GSEA; http://software.broadinstitute.org/gsea/msigdb/annotate.jsp). Wilcoxon rank sum tests were used to assign significance for H3K36me3 enrichment analyses. Differential gene expression was determined using DESeq [54 (link)]. High-confidence GW3965 + T0901317 responsive genes were assigned for all targets that passed significance and fold change cutoffs across both drug treatments. For comparison of gene expression with RNAP2 and H3K36me3 occupancy, if a gene harbored multiple promoters and/or transcripts, the promoter and/or gene body coordinate exhibiting the strongest read enrichment was used.