How to Identify Gene Promoters and Enhancers by ChIP-Seq

What is ChIP Sequencing?

Chromatin immunoprecipitation (ChIP) is an effective method for isolating DNA fragments bound to specific proteins in vivo. DNA-binding proteins can be transcription factors (TFs) or other chromatin-associated proteins, such as histones. With the rapid development of DNA sequencing technology, the direct sequencing of DNA fragments obtained in ChIP using high-throughput sequencing is called ChIP-seq. Due to its high resolution and coverage, ChIP-seq has become a popular method for analyzing chromatin modifications and TF binding sites in eukaryotic genomes.

What are Promoters and Enhancers?

The genome contains a large number of non-coding DNA regulatory elements, including silencers, insulators, promoters and enhancers, which play an important role in gene expression.

A promoter is a DNA sequence that is recognized, bound and initiated by RNA polymerase. It contains conserved sequences required for RNA polymerase-specific binding and transcription initiation, and is generally located upstream of the transcription start site.

Enhancers are DNA sequences that cause a significant increase in gene transcription frequency and are key regulatory elements that can affect gene transcription independent of their direction or distance, and enhancers can typically be thousands of base pairs away from their regulatory targets. Enhancers are usually located in open regions of chromatin and are enriched for transcription factors, cofactors (p300), histone modification marks (H3K4me1 and H3K27ac), etc., which are often used as markers of enhancers for their detection.

Enhancers differ from promoters in two ways: (1) enhancers are not fixed with respect to the position of the promoter, but can be highly variable; (2) they can interact in two directions. An enhancer is not limited to promoting the transcription of a particular promoter, it can stimulate any promoter in its vicinity.

ChIP-Seq Distinguishes Enhancers and Promoters by Analyzing Histone Modifications

Histone modifications generally affect transcriptional activity by affecting the affinity of histones to DNA duplexes, thereby altering the nucleosome structure and the sparse or condensed state of chromatin, or by affecting the affinity of other transcription factors to structural gene promoters to exert a regulatory effect on gene expression. Since histone modifications alter the sparse or condensed state of chromatin, the accessibility of chromatin is also altered.

Thus, histone modifications can predict the type of chromatin (heterochromatin or euchromatin), distinguish functional elements of the genome (promoters, enhancers, gene bodies) and detect the decision to have these elements in an active or repressed state. For example, H3K4me2 and H3K4me3 modifications are mostly enriched in promoters near the transcription start site to activate gene expression, while H3K27me2 and H3K27me3 are associated with gene repression.

Chromatin landscape for heterochromatin, poised and active enhancer regions (Ordoñez R et al. 2019)

Therefore, the distribution of histone modifications can be analyzed by ChIP-seq to find the promoter and enhancer regions of genes and whether they activate or repress gene expression.

Common Histone Modifications

Five core histone modifications are widely used for ChIP-seq analysis:

• H3 lysine 4 monomethylation (H3K4me1) or H3 lysine 27 acetylation (H3K27ac), associated with enhancer regions
• H3 lysine 4 trimethylation (H3K4me3), associated with the promoter region
• H3 lysine 36 trimethylation (H3K36me3), associated with the transcriptional region in the gene body
• H3 lysine 27 trimethylation (H3K27me3), associated with Polycomb repression
• H3 lysine 9 trimethylation (H3K9me3), associated with heterochromatin

Application of Super-Enhancer Research

When multiple enhancers are clustered in certain regions while being densely bound by a series of transcription factor proteins, they regulate the activation, high-level expression of a large number of cell type-determining associated genes, and this region is called super-enhancers (SEs). Super-enhancers can strongly drive the expression of genes controlling cell identity and can be used to explain cell type-specific expression patterns, showing great potential for application in the study of the pathogenesis of diseases such as developmental biology and cancer. Super enhancers are usually identified by ChIP-seq, and screening of key SEs and their regulatory genes is important for elucidating the regulatory mechanisms of key driver genes in diseases and tumors, anti-tumor function studies and drug screening.

Reference:

1. Ordoñez R, Martínez-Calle N, Agirre X, et al. DNA methylation of enhancer elements in myeloid neoplasms: think outside the promoters? Cancers, 2019, 11(10): 1424.