Homotypic clusters of transcription factor binding sites are a key component of human promoters and enhancers

Abstract

Clustering of multiple transcription factor binding sites (TFBSs) for the same transcription factor (TF) is a common feature of cis-regulatory modules in invertebrate animals, but the occurrence of such homotypic clusters of TFBSs (HCTs) in the human genome has remained largely unknown. To explore whether HCTs are also common in human and other vertebrates, we used known binding motifs for vertebrate TFs and a hidden Markov model–based approach to detect HCTs in the human, mouse, chicken, and fugu genomes, and examined their association with cis-regulatory modules. We found that evolutionarily conserved HCTs occupy nearly 2% of the human genome, with experimental evidence for individual TFs supporting their binding to predicted HCTs. More than half of the promoters of human genes contain HCTs, with a distribution around the transcription start site in agreement with the experimental data from the ENCODE project. In addition, almost half of the 487 experimentally validated developmental enhancers contain them as well—a number more than 25-fold larger than expected by chance. We also found evidence of negative selection acting on TFBSs within HCTs, as the conservation of TFBSs is stronger than the conservation of sequences separating them. The important role of HCTs as components of developmental enhancers is additionally supported by a strong correlation between HCTs and the binding of the enhancer-associated coactivator protein Ep300 (also known as p300). Experimental validation of HCT-containing elements in both zebrafish and mouse suggest that HCTs could be used to predict both the presence of enhancers and their tissue specificity, and are thus a feature that can be effectively used in deciphering the gene regulatory code. In conclusion, our results indicate that HCTs are a pervasive feature of human cis-regulatory modules and suggest that they play an important role in gene regulation in the human and other vertebrate genomes.

Systematic dissection of genomic features determining transcription factor binding and enhancer function

“…Second, consistent with previous observations that functional enhancers often contain homotypic clusters of motif sites (62,63), the presence of additional PPARγ/RXR motif sites correlated strongly with enhancer activity. Candidate enhancers containing additional PPARγ/RXR motif sites showed nearly twofold higher enhancer activity than those with only a single motif site.…”

Cis-regulatory properties of medaka synexpression groups

“…This modular organisation of the over-represented motifs in SGs supports their regulatory potential, as transcription factors are well known to interact with other cofactors, likely aided by colocalisation into compact CRMs (Howard and Davidson, 2004;Wasserman and Sandelin, 2004). Moreover, the multiple occurrence of a de novo identified motif within the same CRM is in agreement with previous studies that have shown that local clustering of transcription factor binding sites is a feature of vertebrate enhancers (Gotea et al, 2010).…”

Identification of Biologically Relevant Enhancers in Human Erythroid Cells

“…Phylogenetic conservation has been used as an indicator for functional conservation of enhancers throughout evolution (16,(27)(28)(29)(30)(31)(32); however, varying degrees of sequence conservation between cell types in different species have been observed. In some human tissues, such as forebrain, enhancers are subject to stringent evolutionary constraint, whereas in others, such as heart, they are under weak evolutionary constraint (12,16,(33)(34)(35)(36)(37)(38). In some cell and tissue types, structural and functional conservation of enhancer elements is significantly lacking, even between species as close as mice and humans (34).…”