Systematic human/zebrafish comparative identification of cis-regulatory activity around vertebrate developmental transcription factor genes

Abstract

Pan-vertebrate developmental cis-regulatory elements are discernible as highly conserved noncoding elements (HCNEs) and are often dispersed over large areas around the pleiotropic genes whose expression they control. On the loci of two developmental transcription factor genes, SOX3 and PAX6, we demonstrate that HCNEs conserved between human and zebrafish can be systematically and reliably tested for their regulatory function in multiple stable transgenes in zebrafish, and their genomic reach estimated with confidence using synteny conservation and HCNE density along these loci. HCNEs of both human and zebrafish function as specific developmental enhancers in zebrafish. We show that human HCNEs result in expression patterns in zebrafish equivalent to those in mouse, establishing zebrafish as a suitable model for large-scale testing of human developmental enhancers. Orthologous human and zebrafish enhancers underwent functional evolution within their sequence and often directed related but non-identical expression patterns. Despite an evolutionary distance of 450 million years, one pax6 HCNE drove expression in identical areas when comparing zebrafish vs. human HCNEs. HCNEs from the same area often drive overlapping patterns, suggesting that multiple regulatory inputs are required to achieve robust and precise complex expression patterns exhibited by developmental genes.

Conserved noncoding elements and the evolution of animal body plans

“…Experiments in both zebrafish (11,14,24,28) and mice (8,16) have confirmed this prediction ( Fig. 2A,B).…”

Long-range gene regulation links genomic type 2 diabetes and obesity risk regions to HHEX , SOX4 , and IRX3

“…The final enhancer test vector contained an HCNE in front of the zebrafish gata2 promoter coupled to the EGFP gene and an polyA signal, all flanked by Tol2 transposition sequences. Microinjection and screening were done as described (12). For description of bioinformatic methods, cloning of HCNEs, generation of transgenic mice, in situ hybridization, histology, and RT-PCR, see SI Materials and Methods.…”

“…GRBs often include unrelated genes termed bystander genes and can frequently be recognized through the conservation of synteny among all sequenced vertebrate genomes. Conserved synteny of bystander and target genes is maintained by the presence of enhancers within introns of the bystander genes that are specifically required for the regulation of the target gene (11,12). A consequence of this type of arrangement is that intronic variants in one gene can affect a target gene further away, potentially leading to erroneous disease-gene association.…”

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.…”