Background: Only about 2% of the human genome is composed of protein-coding genes, and the biological functions of the remaining large number of non-coding sequences remain poorly understood. An enhancer regulatory element is a specific non-coding DNA sequence that regulates gene expression in cis by binding with transcription factors (TFs). Enhancers often have specific chromatin characteristics such as DNA sequence conservation, chromatin accessibility, histone modifications of H3K27ac/H3K4me1, and three-dimensional chromatin loop with the promoter. A large number (over 100,000 per cell type) of enhancers have been identified in the Encyclopedia of DNA Elements (ENCODE) and the Roadmap Epigenomics projects. However, it remains a major challenge to understand the function of these enhancers, such as predicting cell-type-specific enhancers, mapping their target genes, evaluating the regulatory role of enhancers on gene expression, and dissecting function of enhancers during development and disease occurrence.
Progress: To this end, numerous high-throughput techniques have been developed in the last decade, including multi-omics sequencing at both bulk and single-cell levels and genome editing. ATAC-seq measures chromatin accessibility, whereas ChIP-seq and CUT&RUN detect genome-wide profiles of TF binding on chromatin or histone modifications and Hi-C captures three-dimensional genome-wide chromatin organization. These assays have also been applied at the single-cell level, such as Paired-seq, SNARE-seq and SHARE-seq, which can simultaneously obtain chromatin accessibility and gene expression in individual cells. The CRISPR/Cas9-mediated techniques, such as CRISPR/Cas9 screen, Perturb-seq and CRISPRi-FlowFISH, have emerged to study enhancer function. Enhancers play an important role during development and disease occurrence. For example, the locus control region of human β-globin protein consists of several erythrocyte-lineage-specific enhancers that bind to the globin genes through chromatin loops at different developmental stages to promote gene-specific expression. Furthermore, during human hematopoietic stem cell differentiation, adult erythroid-specific genes are mainly regulated by Myb bound to distal enhancers, whereas embryonic erythroid-specific genes are mainly regulated by Gata1 occupancy in the proximal promoter region, reflecting a cell-specific regulation pattern. Many enhancers exist as clusters in the genome and control cell identity and disease genes, which provide an effective regulatory buffer for phenotypic robustness during development. Enhancer clusters drive or suppress cancer development in a variety of cancers such as breast, colorectal and acute lymphoid leukemia, and have important effects on tumor cell growth, drug resistance and immune escape. For example, abnormal regulation of the "blood enhancer cluster", located 1.7 Mb downstream of the MYC gene, dysregulates MYC gene expression and leads to the development of leukemia. In recent years, gene editing therapies targeting enhancer loci have been successfully applied to sickle cell anemia and β-thalassemia. Using CRISPR/Cas9 to cleave the GATA1 binding site on the patient's BCL11A enhancer specifically suppresses BCL11A expression in erythrocytes and restores the expression of γ-globin proteins that are suppressed after birth. The spatiotemporal specificity of enhancers makes it a new target for gene editing therapies with great potential in various diseases.
Perspective: Enhancers are key regulatory elements of gene transcription. Scientists have made great progress in studying the effects and mechanisms of enhancers. Functions of enhancers are closely related to the recruitment of transcription factors, cofactors and the formation of enhancer-promoter interactions. Recent developments in next-generation sequencing have also greatly expanded our knowledge and skills in the exploration of the composition of whole genomes. Despite the fact that 40 years have passed since the initial definition of enhancers, fascinating questions about enhancers still remain. How do enhancers select their target genes? How do multiple enhancers coordinate in regulatory networks, and how extensive is the redundancy in these enhancers? What are the mechanistic differences between enhancer and promoter activity? Is the three-dimensional loop chromatin structure a determinant of gene regulation? Finally, what is the precise mechanism by which enhancers activate the promoters of target genes? To answer these questions, more efforts are needed in developing new methods and consolidating data from different cell types and tissues to further understand the role and mechanism of enhancers in gene regulation and to provide more clues to the field.