Mechanisms of enhancer action: the known and the unknown

Abstract

Differential gene expression mechanisms ensure cellular differentiation and plasticity to shape ontogenetic and phylogenetic diversity of cell types. A key regulator of differential gene expression programs are the enhancers, the gene-distal cis-regulatory sequences that govern spatiotemporal and quantitative expression dynamics of target genes. Enhancers are widely believed to physically contact the target promoters to effect transcriptional activation. However, our understanding of the full complement of regulatory proteins and the definitive mechanics of enhancer action is incomplete. Here, we review recent findings to present some emerging concepts on enhancer action and also outline a set of outstanding questions.

Fig. 1

A simplistic schematic for enhancer-mediated transcription activation. A pioneer TF binds a nucleosome at an enhancer and nucleates the process of enhancer priming, facilitating the recruitment of other TFs (ERα) and chromatin remodeling and modification factors (CR-MFs). ERα recruits the coactivators SRC-3 and p300, plus other TFs and coactivators as the MegaTrans complex. The Mediator complex and other relevant coactivators with distinct enzyme activities are subsequently recruited and their conformational changes result in transfer of the enhancer-bound coactivators onto the promoter-bound RNAPII complexes, establishing “transient and direct” enhancer-promoter contact (EPC). Sequential phosphorylation of the RNAPII C-terminal domain (CTD) at S5 and S2 residues coordinates transcription initiation and transition to elongation. Once the RNAPII exits the promoter for productive transcript elongation (mRNA), the Mediator/coactivators can enter another round of recruitment to the enhancer and subsequent transfer to the promoter, aided by EPC formation and severance, completing another cycle of RICE (collective “RNAPII/coactivator recruitment ➔ transcription initiation➔ promoter clearance ➔productive elongation” events). Similar actions by other TFs at the enhancer can amplify TF action, resulting in synergistic transcription activation. Not shown for simplicity: GTFs, RNAPII and eRNA transcription at the enhancer, and other factors and CR-MFs at the promoter

Fig. 2

Cell-free assays to interrogate EPC and transcription. Four cell-free assays are envisaged where a construct with an enhancer and a promoter, or isolated enhancer and promoter fragments, or a BAC clone can be used as templates to interrogate EPC and transcription activation. Streptavidin-coated M280 magnetic beads are employed to capture EPCs on biotinylated fragments. These assays offer unique advantages not achievable through existing cell-based approaches, such as (1) same-source verification of EPC and transcription, (2) exploring mechanistic and/or causal link between EPC and transcription, (3) proteomics of EPC to identify protein complexes that (i) mediate looping and (ii) ensure transcription, and (4) biochemistry of EPC to elucidate requirements of non-proteinaceous ingredients (e.g., ATP, NTPs, coenzymes, and cofactors) in looping-coupled transcription activation. Arguably, any putative enhancer-promoter pair can be studied using the nuclear extract (NE) prepared from a relevant cell line. See (Panigrahi et al., 2018) for details

Fig. 3

A schematic for transcriptional coordination between the enhancer, promoter, and gene body. a Multiple protein complexes specializing in many structural and catalytic activities establish EPC. RNAPII recruitment and transcription initiation occur in a phase-separated condensate (a “transcription bubble”) where productive transcript elongation takes place. RNAPII remains within the EPC-encompassing transcription bubble, necessitating extrusion of the downstream template DNA into a loop behind—as elongation proceeds. Sequential recruitment of RNAPII represent transcription of the gene in multiple transcription units, each forming a DNA loop as in the petals of a sunflower. b Additionally, the chromatin landscape of a transcriptionally poised gene can exist in a sunflower arrangement where proteins assembled at intronic TFBS clusters hold the enhancer and promoter in proximity without direct EPC. Transcription initiation accompanies direct EPC. This model can supplement and co-operate with (a). c Multiple gene promoters can exist in physical proximity with a given enhancer within a phase-separated condensate, facilitating coordinated transcription activation. Likewise, a given promoter can also exist in association with multiple enhancers simultaneously