November 1, 2016;
Autoregulation of retinal homeobox (rax) gene promoter activity through a highly conserved genomic element.
The Retinal homeobox (rax
) gene is expressed in vertebrate retinal progenitor and stem cells and is essential for retinal development. In frogs, rax
is expressed in the ciliary marginal zone
(CMZ), a region containing retinal progenitor and stem cells at the anterior
of the eye
. Little is known regarding regulation of rax
transcription and regulation of transcription of rax
targets. We found that three ultra-conserved genomic elements (UCEs) flanking the rax
coding region regulate expression of a rax
promoter-GFP transgene in Xenopus tadpoles. One of these elements, UCE1, regulates expression of the transgene in the dorsal CMZ. UCE1 contains a Rax
binding site, PCE-1. We demonstrate that rax
regulates expression of the transgene through the PCE-1 site found in UCE1. Therefore, rax
transcription in the CMZ is controlled, in part, by autoregulatory mechanisms.
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References [+] :
UCE1 drives transgene expression in the dorsal CMZ. (a). Schematic diagram of the rax genomic locus (top) and the Tg(UCE1,rax:GFP)Hme transgene. (b) Addition of UCE1 to the Tg(rax:GFP)Hme transgene results in expression in the dorsal CMZ but not the ventral CMZ. (c, d) Addition of UCE1 to the X. laevis rax.S promoter drives expression in the dorsal CMZ. (c) The X. laevis Tg(rax.S:GFP)Mjam transgene is not expressed in the CMZ (open arrowheads). (d) Addition of UCE1 to the Tg(rax.S:GFP)Mjam transgene results in expression in the dorsal CMZ (filled arrowhead). Transgene expression was visualized by in situ hybridization using a GFP antisense riboprobe (blue color). L – lens. Sections are oriented so that the dorsal aspect of the embryo is toward the top of the figure.
Rax can bind to UCE1 in vitro and in vivo. (a) Schematic diagram of UCE1 showing the putative PCE-1 and Nkx sites. (b) Rax can bind the PCE-1 and Nkx sites in vitro. EMSA using an oligonucleotide spanning the UCE1 PCE-1 site as probe and competitor oligonucleotides spanning the same site (lanes 3 and 4), the mouse rhodopsin promoter PCE-1 site (lanes 5 and 6) or the UCE1 Nkx site (lanes 7 and 8). For each competitor we used a wild type version (lanes 3, 5, and 7) or a version in which the PCE-1 site was mutated (lanes 4, 6, and 8). Addition of in vitro translated Rax protein to the probe resulted in two bands of shifted mobility (S1, S2). (c) Rax can bind UCE1 in vivo. ChIP performed using embryos injected with RNA encoding myc-tagged Rax (MT-Rax) and immunoprecipitated using an antibody raised agains the myc tag. UCE1 is specifically precipitated by anti-myc antibody and not control IgG. UCE1 is specifically precipitated but UCE2 is not.
rax-dependent UCE1 activity is mediated through the PCE-1 site. (a–c) Expression of the Tg(UCE1,rax.S:GFP)Hme transgene containing mutations in the Nkx site (a), PCE-1 site (b), or both (c). (d) Expression of Tg(PCE-1,rax.S:GFP)Hme transgene. Expression is visualized by in situ hybridization of sectioned retinal tissue using a GFP antisense riboprobe. Open arrowheads indicate lack of expression in the CMZ; black arrowhead indicates expression in the CMZ. L – lens. Sections are oriented so that the dorsal aspect of the embryo is toward the top of the figure.
Adamska, Five Nkx5 genes show differential expression patterns in anlagen of sensory organs in medaka: insight into the evolution of the gene family. 2001, Pubmed