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Vertebrate rhodopsin promoters exhibit striking sequence identities proximal to the initiation site, suggesting that conserved transcription factors regulate rhodopsin expression in these animals. We identify and characterize two transcriptional activators of the Xenopus rhodopsin gene: homologs of the mammalian Crx and Nrl transcription factors, XOtx5 and XL-Nrl (originally named XL-maf), respectively. XOtx5 stimulated transcription approximately 10-fold in human 293 cells co-transfected with a plasmid containing the rhodopsin promoter (-508 to +41) upstream of luciferase, similar to the approximately 6-fold stimulation with human Crx. XL-Nrl stimulated transcription approximately 27-fold in mammalian 293 cells co-transfected with the rhodopsin luciferase reporter, slightly more than the approximately 17-fold stimulation with Nrl. Together, the Xenopus transcription factors synergistically activated the rhodopsin promoter (approximately 140-fold), as well as in combination with mammalian homologs. Deletion of the Nrl-response element, TGCTGA, eliminated the synergistic activation by both mammalian and Xenopus transcription factors. Deletion of the conserved ATTA sequences (Ret-1 or BAT-1), binding sites for Crx, did not significantly decrease activation by Crx/XOtx5. However, there was increased activation by Nrl/XL-Nrl and an increased synergy when the Ret-1 site was disrupted. These results illustrate conservation of mechanisms of retinal gene expression among vertebrates. In transgenic tadpoles, XOtx5 and XL-Nrl directed premature and ectopic expression from the Xenopus rhodopsin promoter-GFP transgene. Furthermore, activation of the endogenous rhodopsin gene was also observed in some animals, showing that XOtx5 and XL-Nrl can activate the promoter in native chromatin environment.
FIG. 1. XOtx5 and XL-Nrl activate the Xenopus rhodopsin promoter.
A, Xenopus transcription factors XOtx5 and XL-maf activate the
XOP promoter in a dose-dependent manner. 293 cells were transiently
co-transfected with 200 ng of XOP-GL2 and increasing amounts of
expression constructs, showing a dose-dependent activation of the promoter
with saturation at higher concentrations of transcription factor.
Error bars represent S.D. Data are presented as relative light units
(RLU). B, 293 cells were transiently co-transfected with 200 ng of
expression constructs and increasing amounts of XOP-GL2. Addition of
increasing amounts of promoter results in increased activity indicating
that the transcriptional machinery of the 293 cells is not saturated at
the concentrations used in C. Error bars represent S.D. C, XOP-GL2
was transfected into 293 cells with XOtx5 or Crx both individually and
in combination with Nrl. 200 ng of each construct was used in each
transfection. The luciferase activities are shown relative to the activity
observed by the XOP promoter alone. Data are presented as mean
S.E. D, same as A, with XL-Nrl instead of XOtx5.
FIG. 2. Phylogenetic relationships of large vertebrate Maf
genes. Representative phylogenetic tree calculated using maximum
parsimony. No root was assumed but for display purposes the tree is
shown with mammalian MafB roots. Species abbreviations are as follows:
Hs, Homo sapiens; Mm, Mus musculus; Rn, Rattus norvegicus; Xl,
X. laevis; Xt, X. tropicalis; Gg, Gallus gallus; Dn, Danio rerio; Cj,
Coturix japonica. Numbers indicate bootstrap values supporting each
node. XL-Nrl clusters with mammalian Nrl in all trees (brackets). The
scale bar represents the number of steps. Accession numbers are as
described under âExperimental Procedures.â
FIG. 3. XL-Nrl is expressed in the photoreceptor layer of immature and adult Xenopus tadpoles. In situ hybridizations were performed
on stage 48 (A and B) and adult (C and D) Xenopus sections. Full-length antisense (A and C) and sense (B and D) digoxigenin-labeled RNA probes
to XL-Nrl were used. BM purple was used to detect an alkaline peroxidase-conjugated digoxigenin antibody. Blue purple staining can be seen in
the outer nuclear layer of the photoreceptors. The light blue staining on the adult sections is 4,6-diamidino-2-phenylindole to visualize nuclei.
Bright field (BM purple) and dark field (4,6-diamidino-2-phenylindole) images were merged in Adobe Photoshop. OS, outer segment; RPE, retina
pigment epithelia; GC, ganglion cell layer; white arrowhead, outer nuclear layer; open arrowhead, inner nuclear layer. Scale bars, 50 m (A and
C); 100 m (B and D).
FIG. 4. XOtx2 and MafB are able to activate the rhodopsin
promoter. A, 293 cells were transfected with XOP-GL2 either alone or
with various combinations of XOtx5, XOtx2, and human Nrl. The luciferase
activities from treatments are shown relative to the activity
observed by transfection with XOP-GL2 alone. Data are presented as
mean S.E. B, XOP-GL2 was transfected either alone or with various
combinations of XL-Nrl, XMafB, and human Crx (200 ng of each plasmid
used). The luciferase activities are shown relative to the activity
observed by transfection with the Xenopus rhodopsin promoter alone.
Data are presented as mean S.E.
FIG. 5. Comparison of transcriptional activities with rhodopsin-targeted
disruption constructs. A, the luciferase activity from
293 cells transfected with various targeted disruption constructs, human
Crx, and/or human Nrl are shown relative to the activity observed
by transfection of each promoter construct alone (n 2– 6). 200 ng of
each construct were used, and empty pMT was included when necessary
to make the total transfected DNA equivalent. Fold activities are
presented as mean S.E. B, same as A, using the Xenopus transcription
factors XOtx5 and XL-Nrl (n 3– 6). Deletion of the NRE stimulated
activity by Xotx5 (p 0.028), decreased activity stimulated by Nrl
or XL-Nrl (p 0.026, 0.029), and decreased activation stimulated by the
co-transfected transcription factors (Crx/Nrl p 0.029, XOtx5/XL-Nrl
p 0.002). Deletion of the Ret-1 element increased activity stimulated
by Nrl or XL-Nrl (p 0.004, 0.002), and increased activation stimulated
by the co-transfected transcription factors (Crx/Nrl p 0.019, XOtx5/
XL-Nrl p 0.005). Deletion of the BAT-1 element increased activity
stimulated by Nrl or XL-Nrl (p 0.001 for both), but did not significantly
change the activity stimulated by co-transfection of the transcription
factors (Crx/Nrl p 0.668, Otx5/XL-Nrl p 0.099).
FIG. 6. Activation of the rhodopsin promoter in transient
transfections of heads and trunks of Xenopus embryos. A, the
luciferase activity from embryo heads transfected with the XOP-GL2
and human Crx, human Nrl, or both are shown relative to the activity
observed from the XOP-GL2 (n 2– 4). Fold activities are presented as
mean S.E. Inset, representative results of a transfection experiment
comparing luciferase activity from the XOP promoter in heads and
trunks. Data are presented as mean S.D. RLU is relative light units.
B, the luciferase activity from embryo trunks transfected with the wild
type rhodopsin construct and human Crx, human Nrl, or both are
shown relative to the activity observed from the XOP promoter alone
(n 2–3). Fold activities are presented as mean S.E
FIG. 7. XOtx5 and XL-Nrl activate a rhodopsin promoter in
transgenic animals. Images of representative transgenic tadpoles
generated using XOP-GFP and either XL-Nrl (A and B), XOtx5 (C and
D), or an empty pMT expression vector (E and F). Panels A, C, and E
show expression at 2 days of development and panels B, D, and F show
expression at 5 days of development. Fluorescent images show widespread
expression throughout the body with addition of both XL-Nrl
and XOtx5.
FIG. 8. XL-Nrl can activate endogenous rhodopsin promoter in
transgenic animals. RNA from transgenic embryos produced as described
in the legend to Fig. 7 was subjected to RT-PCR using primers
to amplify GFP (top row) or the endogenous rhodopsin gene (bottom
row). The middle panel shows the samples prepared without reverse
transcription. The rhodopsin primers encompass an intron, further
ensuring that genomic DNA is not contributing to the band observed.
Endogenous rhodopsin expression is seen in two out of 15 animals
analyzed. The positive controls shown are from cDNA prepared from
the head of a 5-day-old tadpole positive for XOP-GFP (GFP expressed
only in the eye).
