Mapping of Cardiac Enhancers Reveals the Cooperative Regulation of Cardiac Transcription

Several studies have shown that tissue specific gene expression is controlled by distal genomic regulatory elements, termed enhancers. In the heart, gene expression is controlled by several transcription factors, but the relations between the different factors and the regulatory elements they bind are not completely understood.

Binding of transcription factors to enhancers requires local nucleosome eviction and the formation of “open chromatin”. Studies have shown that such “open chromatin” is an indicator of regulatory activity and can be used to identify regions of the genome bound by regulatory factors. We used Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq), combined with RNA-seq and chromosome conformation capture (4C-seq) for outlining such accessible genomic regions, in genome wide scale in cardiomyocytes and cardiac fibroblasts.

Using a differential approach, we identified cardiac and fibroblast specific regulatory elements, and show that these tissue-specific elements surround tissue specific genes. These regulatory elements are enhancers since they are located at a distance from the transcription start sites and not in proximal promoters. An unsupervised approach identified the main transcription factors that bind the tissue specific regulatory elements we mapped. This analysis shows that clusters of multiple cardiac transcription factors control most cardiac enhancers. These clusters contain different combination of factor binding sites, without specific order or motif grammar. Several enhancer regions containing different transcription factor binding sites converge on tissue specific genes to control their expression together.

In conclusion, we comprehensively mapped cardiac enhancers genome wide and identified the factors that control them. Our data support the ‘transcription factor collective’ model and show that cardiac transcription factors cooperate on three levels to control cardiac gene expression- by controlling the expression of each other, through cooperative binding of individual enhancers, and by converging on the same gene from multiple enhancers.