Fig. 4. Suppression of interphotoreceptor retinoid-binding protein (IRBP) transcription in nonphotoreceptor neuronal cells. A small specific conserved sequence between positions −156 and −140 in the mouse IRBP 5′ flanking region is utilized to repress IRBP transcription in neuronal cells that are not photoreceptors. The left four columns illustrate high-level expression of a reporter gene, chloramphenicol acetyl transferase (CAT), in photoreceptor-like WERI-Rb1 cells, which originated from a retinoblastoma tumor and exhibit several photoreceptor-like properties. The right four columns illustrate the corresponding expression levels in Neuro2a cells, which originated from a neuroblastoma tumor and exhibit numerous neuronal-like characteristics but none that are specific to photoreceptor or light-sensitive properties. In the absence of a promoter, the base expression vector, (labeled pSVOATCAT), shows low background levels of CAT activity in each of the two cell lines. An IRBP promoter fragment from −70 to +101 (labeled p70) exhibits high CAT expression in both cell lines, demonstrating that this fragment is sufficient to promote IRBP transcription in these two neuronal cell lines, and we further demonstrated in a transgenic frog [70] that this holds true for all neuronal cell lineages. A construct containing −140 to +101 (labeled p140) exhibited the same expression pattern in the WERI and Neuro2a; however, a construct containing −156 to +101 (labeled p156) was active in WERI cells but inactive in Neuro2a cells. This pair of constructs highlights the region between −140 and −156 as a critical sequence in the IRBP 5′ flanking region that is apparently essential to prevent expression in an incorrect type of neuronal cell. We proposed that an active suppression event occurs, such as the binding of a trans factor to this sequence, to repress an otherwise active promoter. Further, we proposed that this factor would be absent or lack repressive activity in photoreceptor cells. Subsequent work by Otteson et al. [88, 89] identified KLF15 as a factor with exactly these properties when tested with the human IRBP promoter and human rhodopsin promoter. The sequence numberings of the mouse and human IRBP promoters are slightly different because of small insertions/deletions across the two orthologous sequences.

TRANSCRIPTION FACTORS AND THEIR ROLE IN THE CONTROL OF IRBP EXPRESSION

Rx/rax Transcription Factor

The transcription factor Rx binds to the PCE I element in vitro. Transfection of HEK293 cells with the mIRBP-1783/CAT (chloramphenicol acetyl transferase) construct, and hRX expression plasmid shows that Rx transactivates CAT expression up to

Fig. 5. Footprint overlap comparison in the −70 to −45 region of the interphotoreceptor retinoid-binding protein (IRBP) 5′ flanking sequences. This sequence is identical in the human and mouse IRBP genes. Boxes represent the DNA sequence of the IRBP promoter protected in deoxyribonuclease (DNase) I footprinting experiments. Medium gray: This sequence is a portion of the entire footprint (bovine [74], human [86, 90]. Light gray [85]. The short, solid lines directly above or below the DNA sequence denote core consensus sites for nuclear factor binding. Dashed lines denote the sequence is on the reverse strand (1: [91]; 2: [74]; 3: [105]). The GATTAA invert repeat is in bold [71, 85].

14.8-fold in a concentration-dependent manner [92]. Rx-specific antibodies were used on immunoblot to show that Rx is present in adult mouse retina and iris but not liver, lens, or brain. Immunohistochemistry of adult rat retina shows Rx staining in ONL (nuclei of photoreceptors) (92). Rx/rax is a member of the paired-like homodomain family of transcription factors (93, 94). In the mouse, strong Rx/rax expression is found in the anterior neural plate of E8.5 embryos. By E10.5, Rx/rax expression is restricted to the developing eye and forebrain regions. At E15.5, Rx/rax is expressed uniformly in the neuroretina of the eye. In later stages of retinal development, there is a progressive reduction in Rx/rax expression in the retina and by P6.5 Rx/rax expression is found only in photoreceptor and inner nuclear layers. As cell proliferation ability decreases so does Rx expression. There is a correlation of expression of Rx/rax to the temporal and spatial patterns of mitotic activity in the retina (93, 94). By P13.5, in the mouse, Rx expression is undetectable in in situ hybridation experiments (94). However, northern blot analysis shows that Rax is expressed in the adult mouse retina (93). The differences seen in Rx/rax expression levels in the adult mouse may be due to the different probes that were used to detect Rx/rax expression. A 1.2-kb fragment that codes for almost the complete rax cDNA sequence was used in the northern blot experiments (93) while homeobox sequences were used in a 5’-RACE reaction to generate probes

Fig. 6. Footprint overlap comparison in the −351 to −1 region of the interphotoreceptor retinoid-binding protein (IRBP) 5′ flanking sequences. The mouse IRBP 5′ proximal flanking sequence is shown and marked with protected regions that are color coded according to each indicated study [59, 76, 86, 85, 74, 89, 90]. There is a degree of concordance in that about 200 nucleotides are not protected by any form of retina-specific protein or nuclear extract, and most of the sequences that are protected are confirmed by two independent experiments. This is despite the (1) differences in sequences across three species, (2) differences among the several different sources of proteins (some very crude nuclear extracts and others highly purified individual proteins), and (3) differences in techniques and stringencies in deoxyribonuclease (DNase) digestion conditions.

used in the in situ experiments (94). Rx/rax is required for eye formation from the early stages of eye development. The initial specification of retinal cells and their proliferation is likely regulated by the Rx/rax transcription factor.

NrL Transcription Factor

Neural retina leucine zipper (Nrl) is a transcription factor that is preferentially expressed in rod photoreceptor cells. It contains a basic motif–leucine zipper and has a synergistic effect with Crx on rhodopsin transcription regulation. In the mouse retina, Nrl transcripts are detected at E12 [93]. Ablation of the Nrl gene in gene-targeted mice results in a complete loss of rod photoreceptor function but enhanced cone-mediated activity that is attributed to S cones. At 5 weeks of age, Nrl null mice retinas contain

a similar number of nuclei in the ONL when compared to wild-type retinas; however, the rods have shorter outer segments with an abnormal disk morphology. Northern blot analysis shows that IRBP expression levels are unchanged between wild-type (+/+; 1.0), hemizygous (+/−; 1.7), and Nrl null (−/−; 1.5) mice at P10 [94]. This result is unexpected. In vitro transactivation assays measure the ability of a transcription factor to regulate the transcription of a reporter gene that is downstream of a promoter.

HEK293 cells were transfected with an IRBP promoter-reporter gene and plasmids that express transcription factors. Although no statistically significant differences were observed in transactivation activity when cells were transfected with or without an Nrl expression plasmid, a synergistic affect on transactivation activity was seen when both Nrl and Crx expression plasmids were used in these in vitro experiments [88]. Peng and Chen [95], however, showed that Nrl does transactivate the IRBP promoter in HEK293T cells. Both groups used the same IRBP-reporter gene plasmid, bRbp3-300-luc. The different results found by these two groups may be due to different levels of recombinant protein expression that is caused by different transfection efficiencies or the use of different expression plasmid vectors or different cell lines. Peng and Chen used the HEK293T cell line, while Otteson et al. [88] used HEK293 cells. HEK293T cells contain a stably integrated copy of the SV40 large T antigen [96] that could produce higher copy numbers of the expression plasmid in the transfected cells. A higher concentration of Nrl protein may be required to elicit detectable transactivation activity of the IRBP promoter.

Peng and Chen found that there is synergistic activity of Nrl with either Crx or Otx2 on the regulation by the IRBP promoter. Also, Nrl binds to a fragment of the IRBP promoter in ChIP assays, indicating that Nrl interacts with the IRBP promoter in vivo [95]. This interaction is not dependent on the Crx binding because ChIP assays show that Nrl binds to the IRBP promoter in retinas from Crx null mice [95].

Crx Transcription Factor

The cone rod homeobox gene (Crx) encodes a paired-like homeodomain protein, is a member of the otd/Otx gene family, and was first identified in the rhodopsin promoter [73, 74]. Northern blot analysis for Crx shows a single, abundant message in the adult mouse retina, and this expression was not seen in any other organ that was studied [73]. Crx expression is first detected in the mouse at E10.5 by rtPCR analysis [36] and at E12.5 by in situ hybridization experiments [73].

In human retinas, the onset of Crx expression is between Fwk 9.5 and 10.5 as shown by rtPCR analysis, and expression is maintained in the adult [36, 38]. The use of other techniques shows that Crx mRNA is first detected by in situ hybridization experiment at Fwk 13, while ICC experiments first detect Crx protein 2 weeks later at Fwk 15 [36]. The difference in timing is likely due to the difference in the threshold of sensitivity between the different techniques.

In vitro, Crx transactivates IRBP promoter-reporter gene constructs in a dose-dependent manner [74, 92]. Transactivation activity of the IRBP promoter is enhanced when both Crx and Nrl expression plasmids are used [95]. When the Crx site in the mouse IRBP promoter was mutated, there was a suppression of activity, but it was not abolished. The remaining transcription activation activity detected is likely due to the PCE I/Ret1 element [71]. ChIP assays show that Crx binds to the IRBP promoter in vivo [95].