Neurobiological functions of transcriptional enhancers

Abstract

Transcriptional enhancers are regulatory DNA elements that underlie the specificity and dynamic patterns of gene expression. Over the past decade, large-scale functional genomics projects have driven transformative progress in our understanding of enhancers. These data have relevance for identifying mechanisms of gene regulation in the CNS, elucidating the function of non-coding regulatory sequences in neurobiology and linking sequence variation within enhancers to genetic risk for neurological and psychiatric disorders. However, the sheer volume and complexity of genomic data presents a challenge to interpreting enhancer function in normal and pathogenic neurobiological processes. Here, to advance the application of genome-scale enhancer data, we offer a primer on current models of enhancer function in the CNS, we review how enhancers regulate gene expression across the neuronal lifespan, and we suggest how emerging findings regarding the role of non-coding sequence variation offer opportunities for understanding brain disorders and developing new technologies for neuroscience.

Fig. 1 |. Enhancer function is dependent on sequence and context.

a, Schematic of three neuronal cell types in the CNS illustrating a simple model of enhancer-mediated gene expression. Gene X is expressed in two of the three cell types and, in this simplified schematic expression, is mediated by one cell-type-specific enhancer in each cell. The activity of each enhancer is modulated by transcription factors that have cell-type-specific expression patterns. TF binding enables transcriptional activation via establishing enhancer–promoter physical interactions and recruiting co-factors and transcriptional complexes. b, TF binding is based on DNA-binding domains that have the ability to bind to specific sequence motifs. TF interaction is dependent on affinity to specific sequence motifs, with potential different regulatory impacts based on binding affinity. TF interaction can also be dependent on combinatorial binding or interactions between TFs; for example, one TF may not bind its cognitive recognition sequence unless a second TF is also bound. c, Context-dependent epigenetics impact enhancer activity, and enhancers can be identified by characteristic patterns of biochemical interaction and chromatin state. Left: an active enhancer, characterized by TF binding (blue triangles and circles), co-activators interaction (p300 shown as example), nucleosome-free open chromatin, characteristic histone post-translational modifications (H3K27ac and H3K4me1 are two of the most common marks), RNA Pol II complex recruitment and localized bidirectional eRNA transcription. Right: an enhancer that is epigenetically silenced, characterized by DNA methylation (black circles), histone PTMs such as H3K27me3, and corepressors such as polycomb repressor complex (PCR; orange square). d, The canonical definition of an enhancer is based on sequence-encoded function. Enhancers are capable of driving expression in a bidirectional, context-independent manner, generally as established via ectopic enhancer–reporter assays. In these assays, the candidate enhancer is cloned upstream of a minimal promoter and reporter gene (for example, GFP or RFP) and the construct is delivered to cells. In this example, when delivered to Cell B, Enhancer 2 drives expression of the reporter, whereas Enhancer 1 does not, due to which cell-type-specific TFs are present in Cell B. minP, minimal promoter.

Fig. 2 |. General models of CNS regulatory wiring.

Different general cis-regulatory structures exist across gene types in the CNS. Images represent simplified schematic of cis-regulatory structure and biophysical interactions mediating transcriptional state, with summary of cis-regulatory structure, gene type, steady-state mRNA concentration, transcription kinetics and biophysical components summarized in the table below.

Fig. 3 |. Enhancers across the neuronal lifespan.

Expression patterns of stage-dependent TFs are indicated over time. The effects of these TFs on enhancer accessibility and activity are indicated by the position of histones, the binding of TFs and modifications of DNA (lollipops; white, unmethylated; black, methylated) and histones (methylation, me1; acetylation, ac). a, Pluripotency TFs are rapidly downregulated when progenitors commit to the neuronal fate, and the enhancers they regulate are first decommissioned then permanently silenced. b, Identity TFs (ITF) for specific neuronal fates can act as pioneer factors (P1- and P2-TF) to open chromatin at enhancers that regulate neuronal cell-type-specific genes. Some of these TFs are downregulated after fate commitment, and other TFs will take their place. However, some identity TFs continue to be expressed and play a role either in fate maintenance and/or switch their targets to promote maturation. c, Constitutively expressed TFs can promote stage-specific gene expression through their signal-dependent modification and state-specific recruitment of transcriptional co-activators like p300–CBP.

Fig. 4 |. How disease-associated SNPs impact enhancer function.

a, Sequence variants may occur within the TF binding sites of an enhancer. In this case, the change in sequence can decrease or increase the binding affinity of the recruited TF (indicated by dotted lines), impacting the extent of enhancer-driven expression of coupled genes. b, SNPs may also occur between the coupled enhancer and its target gene promoter. In the example shown, a C is part of a binding site for an architectural factor (tan oval), and when the neighboring nucleotide changes to G from A, methylation (black lollipop) of the C blocks recruitment of the architectural factor. In this case the impact of the variation is likely to be structural, affecting the strength or specificity of promoter–enhancer looping, and reducing Pol II recruitment to coupled gene promoters. The thin arrows and single + represent low levels of transcription, whereas the thick arrows and +++ represent higher levels of transcription. c, Strategies for determining which non-coding sequence variants have consequences for disease phenotypes. The application of high-throughput functional validation of elements and CRISPR–Cas9 editing to the in vivo setting offers high potential for functionally meaningful validation. The red arrows highlight leading-edge advances that are bringing CRISPR–Cas9 and reporter assays toward the high-throughput in vivo category.