A survey of ancient conserved non-coding elements in the PAX6 locus reveals a landscape of interdigitated cis-regulatory archipelagos

Abstract

Biological differences between cell types and developmental processes are characterised by differences in gene expression profiles. Gene-distal enhancers are key components of the regulatory networks that specify the tissue-specific expression patterns driving embryonic development and cell fate decisions, and variations in their sequences are a major contributor to genetic disease and disease susceptibility. Despite advances in the methods for discovery of putative cis-regulatory sequences, characterisation of their spatio-temporal enhancer activities in a mammalian model system remains a major bottle-neck. We employed a strategy that combines gnathostome sequence conservation with transgenic mouse and zebrafish reporter assays to survey the genomic locus of the developmental control gene PAX6 for the presence of novel cis-regulatory elements. Sequence comparison between human and the cartilaginous elephant shark (Callorhinchus milii) revealed several ancient gnathostome conserved non-coding elements (agCNEs) dispersed widely throughout the PAX6 locus, extending the range of the known PAX6 cis-regulatory landscape to contain the full upstream PAX6-RCN1 intergenic region. Our data indicates that ancient conserved regulatory sequences can be tested effectively in transgenic zebrafish even when not conserved in zebrafish themselves. The strategy also allows efficient dissection of compound regulatory regions previously assessed in transgenic mice. Remarkable overlap in expression patterns driven by sets of agCNEs indicates that PAX6 resides in a landscape of multiple tissue-specific regulatory archipelagos.

The Gene Regulatory Network of Lens Induction Is Wired through Meis-Dependent Shadow Enhancers of Pax6

“…It will be interesting to see if other features of SIMO regulation, such as Six3 interaction, are maintained in zebrafish. No functional data exist for the zebrafish EE, although at the sequence level this regulatory element is evolutionarily conserved from human to fish [13, 15, 25]. It remains to be seen if the evolutionary strategy of maintaining lens "shadow enhancers" in the Pax6 locus is utilized in zebrafish, or the developmental robustness is achieved via Pax6 gene duplication giving rise to Pax6 .…”

“…Transcriptional control of Pax6 gene expression is very complex and different cells and tissues choose specific promoters and distal regulatory regions from an archipelago of enhancers scattered within the large Pax6 genomic region [13, 14]. The expression of Pax6 in lens ectoderm was initially shown to be driven by an ectoderm enhancer (EE) located approximately 4kb upstream of the Pax6 P0 promotor [15, 16].…”

Signaling and Gene Regulatory Networks in Mammalian Lens Development

“…Earlier studies have shown that the mouse Pax6 locus resides within a 420 kb region of chromosome 2 [102] and contains a landscape of interdigitated distal enhancers, many of them being highly conserved throughout vertebrate evolution [103]. To elucidate the onset of Pax6 expression in the aPPR, it is necessary to first identify the earliest active enhancer(s) and corresponding transcription factors that regulate Pax6 expression and link them with the extracellular signaling pathways described above.…”

Transcriptional and epigenetic mechanisms of early cortical development: An examination of how Pax6 coordinates cortical development

“…The first cis-regulatory elements controlling PAX6 expression were described in human patients with aniridia bearing chromosomal breakpoints and rearrangements downstream of Pax6 (Fantes et al, 1995; Lauderdale et al, 2000; Kleinjan et al, 2001). Several cis-regulatory elements that regulate Pax6 expression in the eye have since been described (Williams et al, 1998; Xu et al, 1999; Griffin et al, 2002; Kleinjan et al, 2004; 2008; McBride et al, 2011; Bhatia et al, 2013; 2014). An innovative approach recently identified two new cis-regulatory elements around the Pax6 locus by looking at the conservation of ancient conserved non-coding elements (aCNES) which are defined as evolutionarily conserved sequences found in multiple contemporary species that are evolutionarily very divergent (Bhatia et al, 2014).…”

“…Several cis-regulatory elements that regulate Pax6 expression in the eye have since been described (Williams et al, 1998; Xu et al, 1999; Griffin et al, 2002; Kleinjan et al, 2004; 2008; McBride et al, 2011; Bhatia et al, 2013; 2014). An innovative approach recently identified two new cis-regulatory elements around the Pax6 locus by looking at the conservation of ancient conserved non-coding elements (aCNES) which are defined as evolutionarily conserved sequences found in multiple contemporary species that are evolutionarily very divergent (Bhatia et al, 2014). In this study, the scientists compared the Pax6 locus of an elephant shark and that of humans and discovered new ocular enhancers for Pax6 .…”