MPRA for Characterization of transcription factor function and patterns fo gene regulation in HepG2 cells

Transcription factors (TFs) are trans-acting proteins that bind cis-regulatory elements (CREs) in DNA to control gene expression. Here, we analyze the genomic localization profiles of 529 sequence-specific TFs and 151 cofactors and chromatin regulators in the cancer cell line HepG2, for a total of 680 broadly-termed DNA-Associated Proteins (DAPs). We use this deep collection to model each TF's impact on gene expression, including identifying a cohort of 26 candidate transcriptional repressors. We examine High Occupancy Target (HOT) sites in the context of three-dimensional genome organization and show biased motif placement in enhancer-promoter connections involving HOT sites. We also find a substantial number of closed chromatin regions with multiple DAPs bound and explore their properties, finding that a MAFF/MAFK TF pair correlates with transcriptional repression. Altogether, these analyses provide novel insights into the regulatory logic of the human cell line HepG2 genome and demonstrate the usefulness of large genomic analyses for elucidation of individual TF functions. We tested hypotheses regarding ChIP-seq signal and TF binding by testing 170 bp DNA elements cloned upstream of a minimal promoter and expressed barcode sequence in a lentiviral MPRA in HepG2 cells.