Reconstruction and Analysis of Transcriptional Regulatory Networks with TReNA Seth A. Ament Institute for Systems Biology Seattle, Washington Genes influence phenotypes through a network of networks Social Network Brain Connectivity Network Individual Neuronal Network Molecular Network DNA Transcriptional Regulatory Network Analysis (TReNA) Sequence Motifs DNase footprints Epigenomic States Evolutionary Conservation JASPAR ENCODE ROADMAP/FANTOM phastCons FootprintFinder Tissue-Specific TF Binding Sites Software Availability: https://github.com/PriceLab/TReNA Transcriptional Regulatory Network Analysis (TReNA) Sequence Motifs DNase footprints Epigenomic States Evolutionary Conservation JASPAR ENCODE ROADMAP/FANTOM phastCons FootprintFinder Tissue-Specific Tissue-Specific Transcriptome Profiles TF Binding Sites GTEx/GEO fitTRN Tissue-Specific Transcriptional Regulatory Network (TF-Target Gene Interactions) Software Availability: https://github.com/PriceLab/TReNA Combining diverse annotations improves prediction of TF binding sites 1.0 TRUE/FALSE classes: USF1 DNase footprints with/without USF1 ChIP-seq 0.8 peaks 0.6 All USF1 footprints: 79% sensitivity Sensitivity 31% specificity 0.4 USF1 footprints with 0.2 modeled probability > 50%: FIMO + Wellington + ChromHMM + phastCons 55% sensitivity FIMO p−value 70% specificity Wellington p−value 0.0 1.0 0.8 0.6 0.4 0.2 0.0 Specificity Combining TF binding sites and gene co-expression improves prediction of TFs’ functional target genes Co-Expression TF Binding Sites Ensemble 1.0 0.8 *** *** 0.6 OC R U 0.4 A 0.2 0.0 ble LASSO ARACNe Ensem Random Forest Combined TFBS Mutual Information Pearson Correlation TFBS count (0-1 kb) TFBS count (1-10TFBS kb) z-score (0-1 kb) TFBS z-score (1-10 kb) TFBS count (10-100 kb) TFBS count (100kb-1Mb)TFBS z-score (10-100 kb) TFBS z-scoreCombined (100kb-1Mb) Co−Expression shRNA-microarray profiling of 25 TFs in lymohoblasts Expression patterns of TFs accurately predict the expression patterns of thousands of genes in each tissue ) 2 (R Explained Explained Training Set Te s t S e t ( 5 -fold CV) Variance Variance Genes Ranked by R2 Prediction of brain gene expression with fitTRN Genome-scale TRN model for the human brain Input data • 4.6M predicted human brain TFBSs • 2,756 gene expression profiles from the Allen Brain Atlas Summary Statistics • 745 TFs • 11,093 target genes • 201,218 interactions (Ament et al., in prep.) (Ament et al, unpublished) TReNA reveals master regulator TFs and regulatory genetic variants in psychiatric disorders Master Regulator TFs (Stahl et al., in prep) Risk-associated SNP in PGC2-BD GWASPGC_BIP32b_mds7a.0.chr2 BD SCZ MDD p-value 1 0.8 0.6 0.4 0.2 0.1 1 . rs13026414 : Epilepsy(2E−9) snp / p / or / maf / info / directions a predicted POU3F2 SOX9 2 . rs2312147 : Schizophrenia(3E−7) a . rs57681866 / 5.00e−08 / 0.85 / 0.06 / 0.969 / 5−27−0 1e−28 3 . rs2717068 : Epilepsy(4E−7) 8 p = 5.0e−08 a ● ● SOX2 ● ●● ● ● binding site ● ● ● rs13384219 40 ● ● ● ● ● 1e−6 ● ● FOXJ1 6 ● logP) ● ● ● ● 6 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● − ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● FOXO1 1e−20 ● ●● 4 ●● (p-value) ● ● 4 ●● 20 PRRX1 Observed ( ● ● ● ● 10 ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●●● ● ● ● ●● ● ●● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● FOXN2 2 ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●●●●● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ●● ● ● ● ● ● ● ● 2 ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●●● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ●● ● ●● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ●● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ●● ● ● ● ●● ●● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●●●● ●● ●●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ●●● ● ●● ● ●● ●● ●●● ● ●●● ● ●● ● ● ●● ● ● ● ● ● ● -log ● ● ●● ● ● ● ● ● ● ● ● ● ●●● ●● ● ●● ●● ●● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ●●●● ● ● ●●●●● ● ● ● ● ● ● ●●●●●●●● ●● ● ●●●●●● ●● ● ● ● ● ● ●● ● ● ●●●● ●●● ●●●● ● ● ●● ●● ● ● ● ● ● ● ● ● ● 1●● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● HMBOX1 ● ● ● ●● 1● ● ● ● ● ● ● ●● ● ● ●● ● ●● ● ●●●●●●● ●● ● ●● ● ● ●● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ●●● ● ●2● ● ● ● ●● ● ●● ●● ● ● ● ● ● ● ● ● ● ●● ●● ● ●●● ●● ●●● ● ●● 2● ● ●● ● ● ● ● ●●●● ● ● ●● ●●● ● ● ● ●●● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ●●● ● ●●●●● ● ● ● ● ●●● ● ● ●●● ● ● ● ●● ● ● ● ● ●● ●● ● ●●● ●● ●● ● ● ●● ●● ● ●●●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●●●●●●●●●●●●●●●●●●● ● ● ●●● ● ● ● ● ●● ● ● ●● ● ●● ● ●● ● ● ●●●● ●● ●●● ●●●● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ●● ●●● ●● ● ●●● ●● ● ● ●● ● ●● ● ● ●●●●● ● ● ● ●●● ●●● ● ●●● ● ●● ●● ●● ●● 3● ● ● ● ● ● ●●●● ●●●● ● ●● ●●●● ● ● ● ●● ●● ● ●●● ● ●● ● ● ● ● ● ●●● ● ●● ●● ● ●●● ● ●● ●● ● ●●●● ●●● ●●●● ●●● ●● ●● ● ● ● ●●●●● ● ●● ●● ● ● ●●●●● ●● ●●●● ● ●● ●● ● ● ●● ● 3● ● ●● ● ● ● ● ●● ●● ●●● ● ●● ● ● ● ● ● ● ● ●● ● ● ●● ●● ● ●● ● ● ● ●●● ● ●● ● ● ●●●●●●●● ●●●● ● ●●●●●●●●● ●● ● ● ●●●● ●●● ●● ●● ● ●● ● ●● ●●● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ●● ●●●● ●● ●●●●●● ●●● ● ● ● ● ● ● ● ● ● ● ●●●● ● ●● ●●● ● ●● ● ● ● ● ●● ●●● ●●●●● ●● ●●● ● ● ●●●● ● ● ● ●● ● ● ●● ●●● ●●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●●●●● ●●● ● ●● ●●●●●●●●●●● ● ●● ● ● ●●●●● ● ●● ● ●●●●●●●●●●●●● ● ● ●● ● ●● ●● ● ● ● ●●● ● ● ● ● ● ●●●●● ●●●●● ●●●●● ●● ●● ●● ● ●●●●●●● ●●●●●●● ●●●● ●●● ●●●●●● ●●●●●●●●●●● ●●● ●● ●●● ● ● ●●● ● ● ● ● ● ● ● ●● ● ●●●●● ●●●● ● ● ●●●●●●●● ●● ●●● ●● ● ● ●●●● ●● ● ●●● ●● ●● ● ●● ● ● ● ● ●● ●●●● ● ● ●●● ● ● 0 0 (cM/Mb) Recombination rate TEAD1 0 VRK2 POU3F4 VRK2 RUNX1 VRK2 NPAS3 IRF9 rs1338421957900 allele58100 and POU3F258300 expression influence SREBF1 Chromosome 2 (kb) POU3F2 the activity of the VRK2 promoter 1.5 PPARA FOXO4 FOXN3 1.0 OTX1 SMAD1 SOX3 0.5 BD SCZ MDD p-value Activity Luciferase SOX9 1e−2 0.0 SOX2 1e−6 rs13384219 A G FOXJ1 POU3F2 (ng) FOXO1 1e−20 0 0.25 1 4 0 0.25 1 4 PRRX1 FOXN2 HMBOX1 TEAD1 POU3F4 RUNX1 NPAS3 IRF9 SREBF1 POU3F2 PPARA FOXO4 FOXN3 OTX1 SMAD1 SOX3.