Phylogenetic footprinting is an evolutionary tool used for the identification of functional elements
in selected genomes. This method relies on the search for similarities among ancestrally related sequences
and has been widely used to identify cis-regulatory elements in non-coding genomic regions. Conserved
non-coding elements (CNEs) have been shown to be crucial for controlling gene transcription in vertebrate
genomes. Here we present practical guidelines for discovering new cis-regulatory elements using phylogenetic
footprinting and discuss pros and cons of different approaches for the functional characterization of
these elements. In addition, we present two case studies to provide practical examples of how this powerful
technique is used. The first case study illustrates the in silico identification of the Myostatin gene
promoter conserved in vertebrates and its biological validation. The second case study describes the search
for cis-regulatory elements of the Raldh2 gene and demonstrates the conserved biological function of an intronic
CNE responsible for modulating the gene activity in the embryonic dorsal spinal cord in vertebrates.
The comparison of these two case studies highlights the similarities and differences regarding bioinformatic
strategies and validation methods for the identification and functional characterization of promoters and distal
cis-regulatory elements (enhancers/silencers).
Keywords: Phylogenetic footprinting, Conserved non-coding elements(CNE), Enhancer, Vertebrate, Mouse, Chicken, Zebrafish,
Xenopus, Myostatin (Mstn), Retinaldehyde Dehydrogenase 2 (Raldh2), Orthologous Sequences, GFP, RFP.
