Dichotomy in redundant enhancers points to presence of initiators of gene regulation

Abstract

Background: The regulatory landscape of a gene locus often consists of several functionally redundant enhancers establishing phenotypic robustness and evolutionary stability of its regulatory program. However, it is unclear what mechanisms are employed by redundant enhancers to cooperatively orchestrate gene expression.

Results: By comparing redundant enhancers to single enhancers (enhancers present in a single copy in a gene locus), we observed that the DNA sequence encryption differs between these two classes of enhancers, suggesting a difference in their regulatory mechanisms. Initiator enhancers, which are a subset of redundant enhancers and show similar sequence encryption to single enhancers, differ from the rest of redundant enhancers in their sequence encryption, evolutionary conservation and proximity to target genes. Genes hosting initiator enhancers in their loci feature elevated levels of expression. Initiator enhancers show a high level of 3D chromatin contacts with both transcription start sites and regular enhancers, suggesting their roles as primary activators and intermediate catalysts of gene expression, through which the regulatory signals of redundant enhancers are propagated to the target genes. In addition, GWAS and eQTLs variants are significantly enriched in initiator enhancers compared to redundant enhancers, suggesting a key functional role these sequences play in gene regulation.

Conclusions: The specific characteristics and widespread abundance of initiator enhancers advocate for a possible universal hierarchical mechanism of tissue-specific gene regulation involving multiple redundant enhancers acting through initiator enhancers.

Keywords: Gene regulation; Redundant enhancers.

Fig. 1

Enrichment of TFBSs in single enhancers compared to redundant enhancers, in (a) HepG2 and (b) GM12878 cell lines. Blue color shows TFBSs enriched in single enhancers, while red color shows TFBSs enriched in redundant enhancers. P-value was calculated using the Fisher’s exact test. Only TFBSs with the p-value < 0.05 and enrichment fold > 1.5 are shown

Fig. 2

Classification accuracy for single enhancers vs randomly sampled human genomic sequences. a The receiver operating characteristic (ROC) and (b) the precision recall (PRC) curves for nine tissues

Fig. 3

a Distribution of distances between enhancers and their nearest TSSs. b Distribution of phastCons conservation scores for different classes of enhancers. Single and initiator enhancers are more evolutionarily conserved than regular enhancers. The plot shows the analysis performed for the right ventricle (E105) tissue. Grey color means background value. (* - p-value < 0.01). P-value was calculated using the Wilcoxon rank-sum test

Fig. 4

a Distribution of number of contacts between three classes of enhancers and TSSs/other enhancers with 1 Mb cutoff distance for interactions. b Distribution of number of contacts between enhancers and TSSs according to their distances to the nearest TSS (near half, distant half and total enhancers) with 1 Mb cutoff distance for interactions. The plot shows the analysis performed for the GM12878 (E116) cell line. (* - p-value < 1 × 10^− 7). P-value was calculated using the Wilcoxon rank-sum test

Fig. 5

Density of (a) GWAS and (b) eQTLs variants in three classes of enhancers for different tissues. (* - p-value < 0.05,** - p-value < 1 × 10^− 4). P-value was calculated using the Binomial test