Cis Regulatory Elements

As described on the Cis Regulatory Codes page, transcriptional regulation in metazoans is complex and regulatory elements can be far from a gene's transcription start site. In metazoans, gene expression is regulated in a tissue/cell-type-specific manner predominantly via stretches of noncoding sequence referred to as cis regulatory modules (CRMs). CRMs contain 1 or more DNA binding sites for 1 or more sequence-specific, regulatory transcription factors that function to modulate the expression of target gene(s). CRMs that activate gene expression are typically referred to as enhancers while those that repress gene expression are referred to as silencers. Transcriptional enhancers activate gene expression in a tissue-specific manner in development and also in adult cells in response to cellular or environmental stimuli.

We have developed computational algorithms that predict CRMs in the noncoding sequences flanking genes of interest. We previously developed an algorithm called PhylCRM (Warner, Philippakis, Jaeger et al., Nature Methods. 2008, 5(4):347-353), which combines data for individual motif occurrences scored on an alignment into a single CRM prediction. PhylCRM can scan very long genomic sequences for candidate CRMs by quantifying both motif clustering and conservation across arbitrarily many genomes using an evolutionary model consistent with the phylogeny of the genomes. In our study of cis regulatory elements involved in human myogenic differentiation, we examined 75 kb around the transcription start sites of genes, and utilized the phylogenetic tree containing all 8 sequenced mammalian genomes (human, chimp, macaque, mouse, rat, dog, cow, and opossum). Significantly scoring candidate CRMs of varying lengths, ranging from 20 to 500 bp, were identified and scored.

We have applied these approaches to various systems, both in mammals and Drosophila. The results allowed us to successfully identify novel, functional transcriptional enhancers. This approach is general and can be applied readily to any metazoan genome of interest.

Even though transcriptional enhancers have been the focus of many computational and experimental studies, identification of tissue/cell-type-specific enhancers in metazoans remains a significant challenge.

Previously, we developed 'enhancer-FACS-Seq' (eFS) technology (see figure to the left) for highly parallel identification of active, tissue-specific enhancers in Drosophila embryos. Analysis of enhancers identified by eFS as being active in mesodermal tissues revealed enriched DNA binding site motifs of known and putative, novel mesodermal transcription factors (TFs). Naïve Bayes classifiers using TF binding site motifs accurately predicted mesodermal enhancer activity. We are developing eFS further to enable quantitative analysis of enhancer activity. Application of eFS and related technologies to other cell types and organisms should accelerate the cataloging of enhancers and understanding how transcriptional regulation is encoded within them.

Like enhancers, silencers can function in a cell-type-specific manner. Indeed, silencers may contribute a crucial role in the specification of precise gene expression patterns, thus enabling the establishment of sharp expression domains, such as during development. Numerous genomic and computational studies have focused primarily on predicting and characterizing enhancers. In contrast to enhancers, silencers are much less well understood. Few transcriptional silencers have been described, and there have been no large-scale efforts to catalog silencers nor to identify predictive features of silencers, such as chromatin marks. In ongoing research, we are working to identify and quantify the activities of tissue-specific silencers using novel high-throughput assays. We also aim to identify the chromatin signatures of silencers, to identify silencer-associated DNA sequence motifs, and to elucidate the regulatory roles of the trans regulatory factors that bind them and that distinguish their functions as repressive elements. We anticipate that the features and chromatin signatures of silencers identified in this project will be evolutionarily conserved across metazoans.