Deciphering the multi-scale, quantitative cis-regulatory code

Abstract

Uncovering the cis-regulatory code that governs when and how much each gene is transcribed in a given genome and cellular state remains a central goal of biology. Here, we discuss major layers of regulation that influence how transcriptional outputs are encoded by DNA sequence and cellular context. We first discuss how transcription factors bind specific DNA sequences in a dosage-dependent and cooperative manner and then proceed to the cofactors that facilitate transcription factor function and mediate the activity of modular cis-regulatory elements such as enhancers, silencers, and promoters. We then consider the complex and poorly understood interplay of these diverse elements within regulatory landscapes and its relationships with chromatin states and nuclear organization. We propose that a mechanistically informed, quantitative model of transcriptional regulation that integrates these multiple regulatory layers will be the key to ultimately cracking the cis-regulatory code.

Keywords: RNA polymerase II; activation domain; chromatin; cis-regulatory code; cis-regulatory element; coactivator; cofactor; corepressor; enhancer; gene regulation; insulator; nucleosome; pioneer factor; promoter; regulatory domain; repression domain; silencer; topologically associating domain; transcription; transcription factor.

Figure 1.

Four layers of the cis-regulatory code. A. TF binding to DNA depends on the sequence recognition and ability to overcome the nucleosomal barrier through direct association with nucleosome and/or cooperative binding of multiple TFs in turn facilitating nucleosome eviction. TF occupancy is dependent on TF levels, posttranslational modifications, protein-protein interactions (which can either be structured or mediated by weak-affinity interactions among the intrinsically disordered regions, IDRs), and is regulated DNA-mediated cooperativity with other TFs, which is itself governed by cis-regulatory features such as motif arrangement, spacing, and affinity. B. TFs function at modular cis-regulatory elements by recruiting cofactors with diverse and sometimes competing functions such as coactivators (CoA) versus corepressors (CoR). Many interactions between TFs and coactivators are mediated by IDRs. C. Enhancers and other distal cis-regulatory elements selectively regulate promoters, depending on multiple features such as genomic distance, enhancer and promoter state, biochemical specificity between enhancer- and promoter-associated proteins and physical contacts. See also Figure 2. D. Diverse regulatory elements, including enhancers, promoters, silencers, insulators and tethering elements interact with each other physically and/or epistatically (e.g. redundancy or synergy) in the context of local chromosomal neighborhood and a spatially organized nucleus.

Figure 2.

Potential mechanisms underlying enhancer-promoter specificity. A. Enhancers tend to preferentially activate promoters at closer genomic distances. B. Enhancers typically activate promoters within the same topologically associating domain (TAD), though not exclusively and not necessarily to equal extents. This preference has been ascribed to the increased frequency of physical contacts of genomic regions within, as compared to between, TADs. In rare cases, focal enhancer-promoter loops are also observed on the contact frequency maps, especially at promoters and enhancers overlapping a CTCF binding site. C. Differences in relative enhancer activity levels (that can be estimated by quantitative levels of H3K27ac or other enhancer chromatin features) can create apparent promoter specificity as an enhancer must contribute a significant fraction of the total activation at a promoter to detectably regulate it. D. Promoters can be in repressed states unresponsive to enhancer activation. E. Promoters and enhancers can be grouped into classes (such as developmental and housekeeping) with biochemical specificity (albeit quantitative) for each other, resulting in preferential activation. F. The nonlinearity of transcription as a function of coactivator concentration can create apparent specificity, where weak promoters are more responsive to activation by enhancers.

Figure 3.

Enhancer epistasis. A. Examples of synergy, additivity, and redundancy between enhancers A and B, shown both from the perspective of enhancer addition (top) and enhancer removal (bottom) for the same examples. Dashed horizontal lines indicate the expected transcriptional output of adding both enhancers A and B (top) or of removing both enhancers (bottom). Bars indicate transcriptional output upon addition of enhancers while arrows indicate the change upon removal of enhancers. B. Illustration of how a single pair of enhancers, A and B, can exhibit multiple types of epistatic interaction across spatiotemporal positions (e.g. Drosophila embryonic anterior-posterior position as shown below x-axis, with dark blue color indicating one such position), simplified here as 2D axis.