Dissection of thousands of cell type-specific enhancers identifies dinucleotide repeat motifs as general enhancer features

Abstract

Gene expression is determined by genomic elements called enhancers, which contain short motifs bound by different transcription factors (TFs). However, how enhancer sequences and TF motifs relate to enhancer activity is unknown, and general sequence requirements for enhancers or comprehensive sets of important enhancer sequence elements have remained elusive. Here, we computationally dissect thousands of functional enhancer sequences from three different Drosophila cell lines. We find that the enhancers display distinct cis-regulatory sequence signatures, which are predictive of the enhancers' cell type-specific or broad activities. These signatures contain transcription factor motifs and a novel class of enhancer sequence elements, dinucleotide repeat motifs (DRMs). DRMs are highly enriched in enhancers, particularly in enhancers that are broadly active across different cell types. We experimentally validate the importance of the identified TF motifs and DRMs for enhancer function and show that they can be sufficient to create an active enhancer de novo from a nonfunctional sequence. The function of DRMs as a novel class of general enhancer features that are also enriched in human regulatory regions might explain their implication in several diseases and provides important insights into gene regulation.

Challenges and progress in interpretation of non-coding genetic variants associated with human disease

“…Several pioneering studies reported that TF binding motifs were predictors of enhancer activity and tissue specificity. For example, Yanez-Cuna et al 36 reported that GATA and E-box motifs were functionally important for Drosophila S2 cell-specific enhancer function, whereas Ahmad et al 37 found that Myb was crucial in activity of contractile cardial cells. On the other hand, Young's lab proposed the concept of superenhancers, which were characterized by densely clustered enhancers and occupied with high levels of mediator complex.…”

Multiplex enhancer-reporter assays uncover unsophisticated TP53 enhancer logic

“…Such enhancers are generally classified under the enhanceosome or billboard models, depending on whether or not the order and spacing of motifs is important (Arnosti and Kulkarni 2005). Other local features in the DNA sequence of an enhancer have also been shown to contribute to the discrimination of bound versus unbound sites, such as GC content (White et al 2013), preferential sequences for nucleosome positioning (Lidor Nili et al 2010), DNA shape features (Chiu et al 2015), and dinucleotide repeat motifs (Yáñez-Cuna et al 2014). However, to gain further insight into the cis-regulatory enhancer code and identify those sites that are truly bound and functional, validation assays are needed.…”

Comparative genomic analysis of upstream miRNA regulatory motifs in Caenorhabditis

“…Supporting this hypothesis, dinucleotide repeat motifs, including GA (and CT) motifs, are evolutionarily conserved and are enriched in enhancers active across multiple cell types, although they are also required in cell-specific enhancers (Yanez-Cuna et al 2014). This could suggest that motif m3 may provide broad regulatory activity across multiple tissues and/or during development, while motifs such as m1 and m2, which do not occur upstream of all miRNAs despite their strong conservation, could provide additional regulatory information in a cell-or temporally specific manner.…”