Genomic Views of Transcriptional Enhancers: Essential Determinants of Cellular Identity and Activity-Dependent Responses in the CNS

Abstract

Sprinkled throughout the genome are a million regulatory sequences called transcriptional enhancers that activate gene promoters in the right cells, at the right time. Enhancers endow the brain with its incredible diversity of cell types and also translate neural activity into gene induction. Thanks to rapid advances in genomic technologies, it is now possible to identify thousands of enhancers rapidly, test their transcriptional function en masse, and address their neurobiological functions via genome editing. Enhancers also promise to be a great technological opportunity for neuroscience, offering the potential for cell-type-specific genetic labeling and manipulation without the need for transgenesis. The objective of this review and the accompanying 2015 SfN mini-symposium is to highlight the use of new and emerging genomic technologies to probe enhancer function in the nervous system.

Significance statement: Transcriptional enhancers turn on genes in the right cells, at the right time. Enhancers are also the genomic sequences that encode the incredible diversity of cell types in the brain and enable the brain to turn genes on in response to new experiences. New technology enables enhancers to be found and manipulated. The study of enhancers promises to inform our understanding of brain development and function. The application of enhancer technology holds promise in accelerating basic neuroscience research and enabling gene therapies to be targeted to specific cell types in the brain.

Figure 1.

Enhancers versus promoters. Enhancers juxtapose with their specific target promoters via chromatin looping. Enhancers and promoters both recruit transcription factors and coactivators such as CBP (CREB-binding protein), as well as RNA polymerase II. Whereas enhancers initiate transcription of unspliced, nonpolyadenylated eRNAs, promoters initiate transcription of spliced, polyadenylated mRNAs or long, noncoding RNAs. eRNA transcription typically occurs bidirectionally. In addition to transcribing mRNAs, promoters also drive upstream antisense transcription of transcripts called uaRNAs, which exhibit properties similar to eRNAs. The differences between enhancers and promoters may arise entirely or in part due to the presence of the 5′ splice signal at the end of the first exon of mRNAs. For simplicity, nucleosomes are depicted as gray circles and DNA as a black line, but in reality, DNA is looped around nucleosomes.

Figure 2.

Detecting enhancer activity using reporter assays and massively parallel reporter assays (MPRAs). A, Promoter activity is detected by fusing a test sequence upstream of a reporter sequence (i.e., GFP) and introducing it into cells or embryos. B, Enhancer activity is detected by fusing a test sequence near a minimal promoter and reporter. Alternatively, enhancer activity may be detected with the test sequence in the 3′ untranslated region (UTR) of a reporter gene (the STARR-seq configuration; Arnold et al., 2013; Cotney et al., 2015). For high-throughput analysis of enhancer activity, identifying sequences (such as barcodes, BCs) are placed in the 3′ UTR to identify individual enhancers so that they may be combined for “massively parallel” or multiplex quantification. C, MPRAs rely on libraries of plasmids in which the enhancer activity of each test sequence is quantified through sequencing of the BCs (or for STARR-seq, the enhancer itself). For MPRAs in primary neurons or in vivo, enhancer libraries can be packaged into AAV and transduced.

Figure 3.

Spatial and temporal specificity of enhancers active in the developing forebrain. Developmental dynamics of the enhancer-associated histone mark H3K27ac at putative enhancers in mouse forebrain analyzed by ChIP-seq across seven stages of brain development. Most sites show temporally restricted H3K27ac marks. Each column of the heat map displays H3K27ac enrichment patterns from a collection of putative enhancers for a given developmental stage, which are arranged from constitutively active enhancers at the top to more developmental-stage-specific enhancers proceeding downward.

Figure 4.

A model for essential and redundant functions of an OR enhancer in cis and in trans, respectively. Schematic representations of different states in the olfactory sensory neuron nucleus (left) and corresponding transcriptional outputs (right) are shown. An OR gene (orange box) located proximal to an enhancer (orange circle) is repressed by H3K9me3 (red flag, A). The cis-proximal enhancer may facilitate derepression of the OR chromatin landscape (green flag, B), but is not sufficient for OR transcription. Multiple trans-interacting enhancers (colored circles) aggregate around the transcribed cis-proximal OR (orange box, C). This model of enhancer–promoter configuration can explain why some enhancers are required in cis but are redundant in trans.