A functional survey of the enhancer activity of conserved non-coding sequences from vertebrateIroquoiscluster gene deserts

Abstract

Recent studies of the genome architecture of vertebrates have uncovered two unforeseen aspects of its organization. First, large regions of the genome, called gene deserts, are devoid of protein-coding sequences and have no obvious biological role. Second, comparative genomics has highlighted the existence of an array of highly conserved non-coding regions (HCNRs) in all vertebrates. Most surprisingly, these structural features are strongly associated with genes that have essential functions during development. Among these, the vertebrate Iroquois (Irx) genes stand out on both fronts. Mammalian Irx genes are organized in two clusters (IrxA and IrxB) that span >1 Mb each with no other genes interspersed. Additionally, a large number of HCNRs exist within Irx clusters. We have systematically examined the enhancer activity of HCNRs from the IrxB cluster using transgenic Xenopus and zebrafish embryos. Most of these HCNRs are active in subdomains of endogenous Irx expression, and some are candidates to contain shared enhancers of neighboring genes, which could explain the evolutionary conservation of Irx clusters. Furthermore, HCNRs present in tetrapod IrxB but not in fish may be responsible for novel Irx expression domains that appeared after their divergence. Finally, we have performed a more detailed analysis on two IrxB ultraconserved non-coding regions (UCRs) duplicated in IrxA clusters in similar relative positions. These four regions share a core region highly conserved among all of them and drive expression in similar domains. However, inter-species conserved sequences surrounding the core, specific for each of these UCRs, are able to modulate their expression.

An evolutionarily conserved three-dimensional structure in the vertebrate Irx clusters facilitates enhancer sharing and coregulation

“…Of the 88 HCNRs, 16 (18 % ) activated expression in a robust and reproducible manner at the tail-bud stage ( Fig. 1 ), a proportion in line with our previous analysis of HCNRs from the IrxB cluster 3 . Th ese HCNRs drove reporter expression within sub-domains of the endogenous pattern of IrxA genes at this stage 14 , such as the midbrain, hindbrain, spinal cord, kidney and otic vesicle ( Fig.…”

“…One possibility is that the diff erent genes within a cluster share conserved intergenic regulatory elements. Consistent with this idea, we have identifi ed many highly conserved noncoding regions (HCNRs) that function as cis -regulatory elements and are distributed throughout the gene deserts of the IrxB cluster 3 . Th ese regulatory sequences drive the expression in sub-domains of the territories expressing the IrxB genes.…”

“…In these clusters, the Irx genes are separated by large intergenic regions, denoted gene deserts. Most vertebrates contain six Irx genes grouped in two paralog clusters of three genes each 2,3 . Th e IrxA cluster contains Irx1 , Irx2 and Irx4 , and the IrxB cluster contains Irx3 , Irx5 and Irx6 .…”

“…Th e expression of the third gene in each cluster, Irx4 or Irx6 , is usually more divergent. However, in some tissues, all the genes of a cluster, or even of both clusters, are identically expressed 3,7,10,14 .…”

Zebrafish enhancer detection (ZED) vector: A new tool to facilitate transgenesis and the functional analysis of cis‐regulatory regions in zebrafish

“…However, mouse as a model organism is best suited for small/medium-scale screens. Some of its limitations are overcome by using zebrafish, which allows for rapid, large-scale genomic screening, and has been shown to be a good model for efficient detection of enhancer activity in vivo (de la Calle-Mustienes et al, 2005;Allende et al, 2006;Navratilova et al, 2009). However, the genetic tools used in zebrafish for enhancer detection are far from optimized.…”