Identifying transcriptional cis‐regulatory modules in animal genomes

Abstract

Gene expression is regulated through the activity of transcription factors and chromatin modifying proteins acting on specific DNA sequences, referred to as cis-regulatory elements. These include promoters, located at the transcription initiation sites of genes, and a variety of distal cis-regulatory modules (CRMs), the most common of which are transcriptional enhancers. Because regulated gene expression is fundamental to cell differentiation and acquisition of new cell fates, identifying, characterizing, and understanding the mechanisms of action of CRMs is critical for understanding development. CRM discovery has historically been challenging, as CRMs can be located far from the genes they regulate, have few readily-identifiable sequence characteristics, and for many years were not amenable to high-throughput discovery methods. However, the recent availability of complete genome sequences and the development of next-generation sequencing methods has led to an explosion of both computational and empirical methods for CRM discovery in model and non-model organisms alike. Experimentally, CRMs can be identified through chromatin immunoprecipitation directed against transcription factors or histone post-translational modifications, identification of nucleosome-depleted “open” chromatin regions, or sequencing-based high-throughput functional screening. Computational methods include comparative genomics, clustering of known or predicted transcription factor binding sites, and supervised machine-learning approaches trained on known CRMs. All of these methods have proven effective for CRM discovery, but each has its own considerations and limitations, and each is subject to a greater or lesser number of false-positive identifications. Experimental confirmation of predictions is essential, although shortcomings in current methods suggest that additional means of validation need to be developed.

Perspectives on Gene Regulatory Network Evolution

“…Fortunately, methods for CRM discovery, both empirical and computational, have improved dramatically in the past several years [reviewed by 43], and examples of GRN evolution have now been observed in all of the common metazoan model species. Particularly strong examples are available from the echinoderms, due to the extensive cis -regulatory analysis that has been performed on sea urchin development [44], but several well-described vertebrate instances exist as well.…”

“…Chromatin profiling methods have revolutionized the field of CRM discovery over the past several years, allowing for high-quality predictions of CRM location in an unbiased fashion [reviewed by 43]. Applying these methods to profile CRMs active in liver tissue across 20 mammalian species found that the majority of CRM sequences are not conserved between species [45].…”

Using transgenic reporter assays to functionally characterize enhancers in animals

“…Recent development of high-throughput methods for mapping protein-DNA and DNA-protein-DNA interactions has provided a relatively straightforward way to obtain instant genome-wide pictures of potential regulatory regions in any tissue or cell type of an animal (reviewed in [11,24]). However, this has raised a challenge of how to interpret these data and, more specifically, whether most of the predicted regulatory regions have any biological significance [25][26][27] highlighting the…”

CRM Discovery Beyond Model Insects

“…They are typically a few hundred basepairs in length and can be located 5′ to, 3′ to, within introns of, or in some cases even within exons of, their target genes. Although characterizing CRMs has historically been a laborious and low-throughput activity, the advent of genome-wide approaches coupled with next-generation sequencing technologies has led to a renaissance of sorts in methods for CRM discovery (for review see [8, 12–14]). However, current methods are often problematic for insects, where issues of small organism size, lack of extensive tissue-specific cell lines, and lack of mature transgenic methods for most species make these methods technically challenging and often prohibitively expensive.…”