Abstract
BackgroundIn vertebrates, rod photoreceptor-specific gene expression is regulated by the large Maf and Pax-like transcription factors, Nrl/LNrl and Crx/Otx5. The ubiquitous occurrence of their target DNA binding sites throughout rod-specific gene promoters suggests that multiple transcription factor interactions within the promoter are functionally important. Cooperative action by these transcription factors activates rod-specific genes such as rhodopsin. However, a quantitative mechanistic explanation of transcriptional rate determinants is lacking.ResultsWe investigated the contributions of various paired-like transcription factors and their cognate cis-elements to rhodopsin gene activation using cultured cells to quantify activity. The Xenopus rhodopsin promoter (XOP) has a bipartite structure, with ~200 bp proximal to the start site (RPP) coordinating cooperative activation by Nrl/LNrl-Crx/Otx5 and the adjacent 5300 bp upstream sequence increasing the overall expression level. The synergistic activation by Nrl/LNrl-Crx/Otx5 also occurred when XOP was stably integrated into the genome. We determined that Crx/Otx5 synergistically activated transcription independently and additively through the two Pax-like cis-elements, BAT1 and Ret4, but not through Ret1. Other Pax-like family members, Rax1 and Rax2, do not synergistically activate XOP transcription with Nrl/LNrl and/or Crx/Otx5; rather they act as co-activators via the Ret1 cis-element.ConclusionsWe have provided a quantitative model of cooperative transcriptional activation of the rhodopsin promoter through interaction of Crx/Otx5 with Nrl/LNrl at two paired-like cis-elements proximal to the NRE and TATA binding site. Further, we have shown that Rax genes act in cooperation with Crx/Otx5 with Nrl/LNrl as co-activators of rhodopsin transcription.
Highlights
In vertebrates, rod photoreceptor-specific gene expression is regulated by the large Maf and Pax-like transcription factors, Nrl/Xenopus laevis neural retinal leucine zipper (LNrl) and Cone rod homeobox (Crx)/Orthodenticle-related homeobox (Otx5)
The activity in the absence of added transcription factors for either the promoter-less or a Xenopus rhodopsin promoter (XOP, -503/+41) containing plasmid was 1.5-fold higher in HEK293 cells compared to HEK293T cells (Figure 2A)
The rhodopsin proximal promoter (RPP) is solely responsible for synergistic activation, the 5′5′ upstream sequence (US) contributes to overall transcriptional activity
Summary
Rod photoreceptor-specific gene expression is regulated by the large Maf and Pax-like transcription factors, Nrl/LNrl and Crx/Otx. The ubiquitous occurrence of their target DNA binding sites throughout rod-specific gene promoters suggests that multiple transcription factor interactions within the promoter are functionally important. NRE, a Maf family recognition element (MARE) site forty nucleotides upstream of the TATA box, is a target for Nrl (neural retina-specific leucine zipper protein), a transcription factor expressed exclusively in rods [6]. Flanking the NRE are three highly conserved paired-like homeodomain binding sites, Ret, BAT1 and Ret1 These are target sites for several paired-like homeodomain transcription factors, most notably Crx and Otx and Rax [7,8,9,10,11] Crx (cone rod homeobox protein) is expressed in both rods and cones. A mechanistic basis for the combined transcriptional activity of Nrl and Crx (and their modulators) is yet to be established
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