Seven myths of how transcription factors read the cis-regulatory code

Abstract

Genomics data are now being generated at large quantities, of exquisite high resolution and from single cells. They offer a unique opportunity to develop powerful machine learning algorithms, including neural networks, to uncover the rules of the cis-regulatory code. However, current modeling assumptions are often not based on state-of-the-art knowledge of the cis-regulatory code from transcription, developmental genetics, imaging and structural studies. Here I aim to fill this gap by giving a brief historical overview of the field, describing common misconceptions and providing knowledge that might help to guide computational approaches. I will describe the principles and mechanisms involved in the combinatorial requirement of transcription factor binding motifs for enhancer activity, including the role of chromatin accessibility, repressors and low-affinity motifs in the cis-regulatory code. Deciphering the cis-regulatory code would unlock an enormous amount of regulatory information in the genome and would allow us to locate cis-regulatory genetic variants involved in development and disease.

Keywords: Transcription factors; chromatin accessibility; cis-regulatory code; cooperative binding; enhancer repression; low-affinity binding motif; motif syntax; transcriptional regulatory networks.

Figure 1:. The cis-regulatory code defines how DNA sequence regulates enhancer activity.

(A) TFs are regulated transcriptionally and by extracellular signals such that each cell type contains a unique set of active TFs. Dependent on the specific TF combination, different sets of enhancers become active in each cell type. (B) The cis-regulatory DNA sequence contains TF motifs in specific arrangements (syntax). Dependent on syntax, the motifs are bound by TFs cooperatively. TFs then recruit co-activators or co-repressors, which regulate the activity of the enhancer.

Figure 2:. TF motifs often function together in an AND logic.

(A) Mutating different motifs in an enhancer can each lead to a loss of enhancer activity. Such AND logic between motifs can occur through (B) cooperative TF binding to composite motifs, (C) cooperative binding to motifs spaced with helical periodicity (~10 bp x N), (D) one TF opening chromatin such that another TF can bind (assisted loading), or (E) synergistic co-activator function. (F) The resulting enhancer activity follows a sigmoidal curve with increasing concentrations of a TF.

Figure 3:. Chromatin accessibility is a readout of multiple TFs.

In the absence of appropriate TFs, nucleosomes maintain DNA in an inaccessible state (left). Pioneer TFs can bind their motifs in the presence of chromatin and make the region accessible (primed or poised enhancer, middle). The chromatin accessibility may be further increased by TFs both during the pioneering phase and during enhancer activation.

Figure 4:. Mechanisms by which repressors (A-C) or low-affinity TF binding motifs (D-F) regulate enhancer activity and specificity.

(A) When dedicated repressors bind to their motifs, they counteract the activity of TFs bound nearby. (B) Dual TFs may be weakly activating by themselves, but (C) have a repressing effect when they recruit a repressor to a nearby repressor motif. Low-affinity motifs (D) are likely bound with shorter dwell times and require higher TF concentration to mediate enhancer activation, (E) may discriminate between closely related TF family members, or (F) may be dependent on a partner TF for binding.