Decoding transcriptional enhancers: Evolving from annotation to functional interpretation

Abstract

Deciphering the intricate molecular processes that orchestrate the spatial and temporal regulation of genes has become an increasingly major focus of biological research. The differential expression of genes by diverse cell types with a common genome is a hallmark of complex cellular functions, as well as the basis for multicellular life. Importantly, a more coherent understanding of gene regulation is critical for defining developmental processes, evolutionary principles and disease etiologies. Here we present our current understanding of gene regulation by focusing on the role of enhancer elements in these complex processes. Although functional genomic methods have provided considerable advances to our understanding of gene regulation, these assays, which are usually performed on a genome-wide scale, typically provide correlative observations that lack functional interpretation. Recent innovations in genome editing technologies have placed gene regulatory studies at an exciting crossroads, as systematic, functional evaluation of enhancers and other transcriptional regulatory elements can now be performed in a coordinated, high-throughput manner across the entire genome. This review provides insights on transcriptional enhancer function, their role in development and disease, and catalogues experimental tools commonly used to study these elements. Additionally, we discuss the crucial role of novel techniques in deciphering the complex gene regulatory landscape and how these studies will shape future research.

Keywords: CRISPR genome editing; Development; Disease; Enhancers; Functional genomics; Next-generation sequencing; Transcription factors.

Figure 1. Multiple levels of genome architecture and gene regulation

A schematic of distinct levels of gene regulatory control and nuclear architecture is given. Structural features of the nucleus including lamina associated domains (LADs) and transcription factories are shown. A topologically associated domain (TAD) within a transcription factory is given below. Intra-TAD enhancer-promoter contacts are displayed. A closer look at enhancer looping between intra-TAD enhancer-promoter interactions is given in the last panel. Chromatin state near active regulatory elements and promoter sequences are altered through histone modifications and correlate with enhancer state as well as gene expression levels. Enhancer elements expressing eRNAs and bound by transcription factors (TF) and associated transcriptional cofactors and chromatin remodeling enzymes (COF) associate with distal promoter sequences through long-range looping interactions, leading to increased expression of target genes.

Figure 2. Next-generation sequencing based disease studies

Experimental approaches that can be applied for assessing the role of enhancers or other non-coding, regulatory variants in diseases are shown. (A) A schematic of patient-derived iPSC models is given. Reprogrammed patient iPSCs into relevant cell types can be used for functional genomic studies and cellular phenotypic analyses. These functional genomic studies can identify key elements using an integrative approach that incorporates complementary information, including DNA methylation (DNA Methyl), DNA-binding protein occupancy (TF), regions of open chromatin (Chrom), histone modifications (Histone), eRNA production (eRNA), long range interactions (3D) and RNA expression (mRNA) to identify regulatory elements (1). Subsequent CRISPR genome editing of key regulatory elements is used to validate target genes (2), whereas regulatory element swapping can be used to predict the functional effect of regulatory sequence variants (3). (B) A high-throughput reporter assay screen is shown. After construction and transfection of a complex pool of reporter plasmids harboring hundreds of enhancer sequences from case and control populations, NGS can identify both rare and common variants within enhancer sequences that lead to functional effects on gene expression. (P = Promoter; GFP = Green Fluorescent Protein).