Transcription factor-DNA binding: beyond binding site motifs

Abstract

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.

Figure 1. Numerous features of TFs or DNA binding sites beyond primary nucleotide sequence motifs can modulate transcription factor (TF)–DNA recognition

TF-level features: (A) Several TFs can display binding specificity for multiple, distinct nucleotide sequence motifs. Motifs shown are examples of two motifs bound by the bispecific human forkhead TF FOXN2: FHL (red box) and FkhP (blue box) binding site motifs; motifs were obtained from UniPROBE (Accession Number UP00521) [45]. Interactions between (B) TFs and (C) TFs and non-DNA-binding cofactors [51] can specify distinct binding site motifs from the monomeric TF motif. DNA-level features: (D) DNA modifications, such as 5-methylcytosine (left), can modulate TF binding. (E) Numerous TFs use DNA shape readout, such as minor groove width (depicted by red arrows), and rotational parameters such as helix twist, propeller twist, and roll, as part of TF–DNA recognition. (F) Sequences and features outside of the binding site motif (depicted by blue box), such as GC content and / or DNA shape, can modulate TF– DNA binding. These features may immediately flank the core binding site, or may extend more distally from the motif. (G) Genetic variation in either the TF protein sequence (depicted by orange star, middle) or the DNA binding site (depicted by X, right) can alter TF–DNA binding.

Figure 2. Combinations of in vitro and in vivo approaches allow identification and investigation of functional TF–DNA binding sites

(A) Techniques such as B1H, PBMs, and SELEX enable one to determine the intrinsic binding specificities of TFs. (B) Motif catalogs aid in identification of putative TF binding site motifs. Results from technologies in (A) can then be integrated with (C) in vivo genomic approaches, such as ChIP-seq, RNA-seq, and chromatin accessibility profiling methods (e.g., DNase-seq, ATAC-seq) to identify or infer direct versus indirect DNA binding sites in vivo and regulatory roles of the TFs. Data from (D) investigations of natural genetic variation, such as in genome-wide association studies, which allow investigators to identify signals associated with TFs or TF binding sites implicated in particular traits or as expression quantitative trait loci (eQTL); (E) enhancer or promoter activity reporter assays; and (F) experimental perturbation approaches (e.g., genome editing), are used in assessing the contributions of motifs to gene expression and phenotypes.