A Novel, Evolutionarily Conserved Enhancer of Cone Photoreceptor-specific Expression

Abstract

The ␣ subunit of cone transducin (T␣C) is expressed exclusively in cone photoreceptors of the eye and pineal. T␣C is a key phototransduction protein, and inherited mutations in T␣C cause total color blindness in humans. We use transgenic zebrafish to identify and characterize cone photoreceptor regulatory element 1 (CPRE-1) a novel 20-bp enhancer element in the T␣C promoter (T␣CP). CPRE-1 is located ϳ2.5 kb upstream of the translation start site and is necessary for strong cone photoreceptor-specific expression in vivo. CPRE-1 comprises of a modular arrangement of two 10-bp elements that have separate, but co-dependent transcriptional activities. In vitro, CPRE-1 specifically binds nuclear factors that are enriched in ocular tissue. Bioinformatic alignments reveal that CPRE-1 sites are evolutionarily conserved in the promoter regions of fish, rodent, and mammalian T␣C orthologues and identify a 5-CTGGAGTG(A/T)TGGA(G/A)G-CAGGG(G/C)T-3 consensus sequence.The vertebrate retina contains distinct cone and rod photoreceptors that mediate scotopic and photopic vision, respectively (1). Although cones and rods originate from the same population of retinal progenitor cells, they have unique gene expression profiles that account for their differential cell fate, morphology, and signal transduction mechanisms. For example, many components of the G protein-coupled receptor phototransduction cascade are encoded by cone-or rod-specific genes (1). The molecular genetics underpinning cone photoreceptor-specific gene expression remain poorly defined.Several transcription factors, including Crx, Nr2e3, Nrl, and Tr␤2, regulate photoreceptor-specific gene expression. Cone rod homeobox is expressed in photoreceptors, and cone rod homeobox depletion leads to a developmental loss of both cone and rod photoreceptors in mice and zebrafish and to blindness in humans (2-4). Nr2e3 and Nrl are expressed in rods where they repress expression of cone-specific genes (5-10). Mutations in Nr2e3 or Nrl lead to rods inappropriately expressing cone-specific markers and to the human retinal disease enhanced S-cone syndrome (5-12). Thyroid hormone receptor ␤2 is a nuclear receptor required for M-cone development (13,14). Targeted deletion of thyroid hormone receptor ␤2 leads to a loss of M-cone function with an increase in functional S-cones (13,14). Although these transcription factors are known to regulate photoreceptor genes, the molecular regulators of conespecific gene expression are not well defined.One method of deciphering the molecular mechanisms regulating cone-specific expression is to characterize the cis-elements in a cone-specific promoter. Several studies have identified large promoter regions sufficient to direct transgene expression in cones (15-23). However, many of these promoter regions have weak activity, exhibit ectopic, non-cone expression, or require a heterologous enhancer element (16 -19, 21-23). With the exception of a cone rod homeobox-binding element, none of these studies have characterized individual cis-elements that d...

G Proteins and Autocrine Signaling Differentially Regulate Gonadotropin Subunit Expression in Pituitary Gonadotrope

“…Several studies have suggested paracrine factors that influence gonadotropin expression (30 -34), [45][46][47][48][49][50]. We studied whether the G␣ s -mediated suppression of FSH might result from secreted autocrine factors.…”

“…Many nonexpressed G proteins are tissue-specific genes, thus the absence of their expression in gonadotropes is not surprising. For example, G␣ l is involved in odorant transduction and is exclusively expressed in the olfactory receptor neurons (47), and G␣ t2 and G␣ t3 are involved in light detection and found predominantly in photoreceptor cells of the eye (48). G␣ 15/16 expression is restricted to tissues rich in hematopoietic cell types such as spleen, thymus, and bone marrow (49,50).…”

Functional diversity of melanopsins and their global expression in the teleost retina

“…To complement the data obtained from bioinformatics as discussed above, a number of putative binding sites for retinal-specific transcription factors [46,48] that are not presently included in the NSITE or TRANSFAC transcription factor (http://www.generegulation.com/pub/databases/transfac/doc/toc.html) databases were investigated by manual comparison of both sense and antisense genomic regions with binding motif consensus sequences and applying a cutoff score value [0.7. These include: cone photoreceptor regulatory element-1 (cpre-1) (consensus sequence CTGGAGTGWT GGARGCAGGGST [49]); neural retina leucine zipper (nrl) [consensus sequence TGAN 6-8 GCA [50], where the N 6-8 core resembles the binding site for ap-1 (consensus sequence TGASTCA [45]) or a 3 0 -5 0 -cyclic adenosine monophosphate (cAMP) response element (TGAGCTCA [51,52])]; nuclear receptor subfamily 2 group E member 3 (nr2e3) (consensus sequence RAGRTCAAARRTCA [53])]; rar-related orphan receptor beta (ror b ) (consensus sequence WWAWBTAGGTCA [54]) (also employed in circadian gene regulation [55]); cone-rod homeobox (crx) (consensus sequence TAATCA [56]) and crx/orthodenticle homeobox-2 (crx/otx-2) (consensus sequence TYTAATCC [57]); retina-specific region-1/photoreceptor conserved element-1 (ret-1/pce-1) (consensus sequence CAATTAG [58]) and orthodenticle homeobox/bovine AT-rich sequence-1 (otx/bat-1) (consensus sequence TGATTAG [58]); retina-specific region-4 (ret-4) (consensus sequence GCTTAG [59]); glass-like binding motif (consensus sequence ACCCTTGAAATGCC [60]); and Caenorhabditis elegans homeobox-10 (ceh-10) containing homolog (chx10) (consensus sequence YTAATTRR [61]). Similarly, each upstream 3 kb melanopsin promoter region (sense and antisense strands) was investigated for the presence of putative d-box (consensus sequence RTTA YGTAAY [62] with a cutoff score value [0.75) and e-box (consensus sequences CACGTG [62] and CACGTT [62]) motifs known to be important in circadian regulation of rhythmic clock genes.…”

“…4). Significantly, a cone photoreceptor regulatory element-1 enhancer [49] was found in the proximal upstream region of opn4m-2, with a nuclear receptor subfamily 2 group E member 3-like binding motif [53] in the distal promoter region, which concomitantly may mediate the unique expression of this melanopsin gene in single cones, while repressing double cone expression. Collectively, all five melanopsin promoter regions contained binding sites for transcription factors that are involved at different hierarchical levels of ocular gene regulation, including the master regulator homeobox genes that influence eye development (e.g.…”

PRE-1, a cis element sufficient to enhance cone- and rod- specific expression in differentiating zebrafish photoreceptors

“…Previously, we identified ~3.2 and ~2.5 kb promoter fragments from the zebrafish TαC gene that initiate robust EGFP expression in the four morphological subtypes of differentiating cones at 3 dpf [24,25]. Subsequently, we characterised cone photoreceptor regulatory element 1 (CPRE-1), a 20 bp enhancer element ~ 2.5 kb upstream of the TαC promoter [25]. CPRE-1 is necessary for cone-specific expression from TαC promoter fragments, but is not sufficient to enhance activity from a heterologous UV opsin promoter [25].…”