SnapShot: The Regulatory Genome

Graphical Abstract

Enhancers are key modulators of gene activity (Banerji et al., 1981) and the most pervasive constituents of the regulatory genome. We summarize their organization within an idealized interphase nucleus.

(1). Poised for Activation

Prior to activation, genes are primed—or “poised”—for efficient and reliable induction. There are two major mechanisms underlying this process: pioneer factors (Zaret and Carroll, 2011) and paused RNA polymerase II (Pol II) (Adelman and Lis, 2012). Pioneer factors are a class of sequence-specific DNA binding proteins that evict inhibitory nucleosomes at enhancers (orange rectangle) and promoters (black arrow). This process is not sufficient to trigger gene expression but instead fosters subsequent binding of activators (or repressors) and Pol II. Demethylation of CpG islands might also help destabilize nucleosomes in promoter regions.

(2). Transcriptional Activation

A typical enhancer is 100 bp–1 kb in length and contains binding sites for different classes of sequence-specific transcription factors, which can recruit Pol II to produce noncoding enhancer RNAs (eRNAs). Activators, co-activators, pioneer factors, and Pol II form higher-order assemblies that are sometimes called “transcriptional hubs” (yellow blob). Some of these proteins bind to specific DNA sequences, while others are recruited into the hub indirectly, through protein-protein or protein-RNA interactions. It has been suggested that these associations are mediated by liquid-liquid phase separation (Hnisz et al., 2017). The key concept here is that enhancers recruit clusters or condensates of Pol II (Cisse et al., 2013), consistent with the occurrence of transcriptional bursts (represented by pairs of elongating Pol II complexes separated by small gaps) (Tunnacliffe and Chubb, 2020). The hub model can further explain recent quantitative imaging measurements revealing large distances separating enhancers from their target promoters during activation (Benabdallah et al., 2019). Molecular crowding in the hub might not permit enhancers to come into close proximity with their promoters, as envisioned in classical models.

(3). Transcriptional Repression

Primed genes are poised for either activation or repression, depending on the identities of DNA binding proteins at the enhancer. In this example, sequence-specific repressors inhibit gene expression by forming a higher-order “repression hub” (orange blob).

(4). One Gene, Multiple Enhancers

Genes are often regulated by multiple enhancers, mediating expression in different cell types. Enhancers sometimes contain overlapping activities in the same cells, providing robustness of gene activation under stressful conditions (Osterwalder et al., 2018). The two enhancers shown here are located at opposite ends of the transcription unit, but they are sometimes organized in tandem arrays to form “superenhancers” (Hnisz et al., 2017).

(5). One Enhancer, Multiple Genes

Genes, often paralogs, sometimes share a common enhancer. The analysis of synthetic reporter genes suggests coactivation, producing coordinate bursts, possibly due to regulation by a common transcription hub (Fukaya et al., 2016). However, gene organization and orientation can influence differential regulation of linked genes by shared enhancers.

(6). Neighboring TADs and Intra-TAD Associations

Metazoan genomes are organized in a series of topologically associating domains (TADs) representing contiguous segments of the genome (~500–750 kb in vertebrates) that exhibit a high frequency of mutual contact (Dekker and Mirny, 2016). They are delineated by insulator sequences that often contain convergent binding sites for the “architectural” protein, CTCF (triangle), which blocks processivity of DNA translocation by cohesin (yellow rings). There is extensive evidence that these boundaries preclude interactions of enhancers with the wrong promoters. Tethering elements help mediate targeted association of an enhancer and promoter within the limits of the larger TAD (Calhoun and Levine, 2003).

(7). Polycomb Repression

Large blocks of the genome are stably silenced by Polycomb group proteins (Di Croce and Helin, 2013). In Drosophila, they are recruited to specific regions by sequence motifs called PREs. Insulators are thought to restrict the spread of Polycomb silencers and associated histone methylation (e.g., H3K27me).

(8). Heterochromatin

Centromeres, telomeres, and other regions of the genome often contain simple repetitive DNA sequences or transposable elements that are organized into highly compacted, transcriptionally silent heterochromatin. This is mediated by H3K9 trimethylation and association of the HP1 protein.