Sequence-specific transcription factors (TFs) regulate gene expression by binding to cis-regulatory elements in promoter and enhancer DNA. While studies of TF–DNA binding have focused on TFs' intrinsic preferences for primary nucleotide sequence motifs, recent studies have elucidated additional layers of complexity that modulate TF–DNA binding. In this review, we discuss technological developments for identifying TF binding preferences and highlight recent discoveries that elaborate how TF interactions, local DNA structure, and genomic features influence TF–DNA binding. We highlight novel approaches for characterizing functional binding site motifs that promise to inform our understanding of how TF binding controls gene expression and ultimately contributes to phenotype.

Sequencing of exomes and genomes has revealed abundant genetic variation affecting the coding sequences of human transcription factors (TFs), but the consequences of such variation remain largely unexplored. We developed a computational, structure-based approach to evaluate TF variants for their impact on DNA-binding activity and used universal protein binding microarrays to assay sequence-specific DNA-binding activity across 41 reference and 117 variant alleles found in individuals of diverse ancestries and families with Mendelian diseases. We found 77 variants in 28 genes that affect DNA-binding affinity or specificity and identified thousands of rare alleles likely to alter the DNA-binding activity of human sequence-specific TFs. Our results suggest that most individuals have unique repertoires of TF DNA-binding activities, which may contribute to phenotypic variation.

substantial fraction of worldwide mortality is attributed to erythrocyte-related disorders 1-7. Variation in RBC traits is linked to mortality rates not related to primary hematologic disease 1,3,6. Genome-wide association studies (GWAS) have identified numerous variable genomic regions associated with human traits and diseases, including RBC traits 8-21. RBC-trait-associated single-nucleotide polymorphisms (SNPs) rarely affect DNA binding of MTFs, such as GATA2 and GATA1, even though they are often in close proximity to MTF target sequences 15,22,23. Additional mechanisms by which RBC SNPs result in the phenotypic variability of human genetic traits remain to be discovered. Environmental factors contribute to the phenotypic manifestation of complex human genetic traits 1,3,6. Under stress conditions, growth factors and small molecules activate signaling pathways 24-26 that converge on signal-induced effector transcription factors (STFs) to control gene expression. By coordinating with MTFs, the same STFs may be active in multiple cell types but exert tissue-specific functions 27,28. Hence, alterations in STF target sequences may lead to aberrant responses to various signals. Here, we observed that human erythroid-trait-associated non-coding SNPs are enriched in a small subset of enhancers co-bound by MTFs and STFs, which we named transcriptional sig