studies. Given this plethora of trans factors, it is not yet possible to predict when the story of IRBP transcription will be complete.

TRANSGENIC MICE

Transgenic mice were generated using the hIRBP promoter fragment −123 to +18 upstream of a reporter gene. When both the PCE I and CRX elements are mutated in transgenic mice, reporter gene activity is abolished in photoreceptor cells [99]. In vitro experiments show that mutation of either the Ret-1/PCE I or the CRX elements suppresses IRBP promoter activity [71]. The same fragment of the hIRBP promoter (−123 to +19) upstream of a reporter gene was used to make transgenic mice, and either the PCE I element or the CRX element was mutated [86]. Mutation of the CRX but not PCE I element abolishes reporter gene activity in photoreceptor cells. When the PCE I element but not the CRX element is mutated in the IRBP promoter, 9 of 17 lines of transgenic mice show photoreceptor-specific expression of the reporter gene [86]. These studies indicate that the CRX DNA element is required for photoreceptor-specific expression of IRBP. rtPCR experiments, using the different lines of transgenic mice, show that to have IRBP promoter activation, a protein interaction with the PCE I element is not required but is enhanced when the PCE I element is present [86].

REPRESSORS OF IRBP GENE EXPRESSION

A potential strong silencer was identified in the region between −206 and −66 bp of the mouse IRBP promoter in transient transfection experiments using nested deletions of the 5′-flanking region controlling expression of a reporter gene [90].

KLF15

Krüppel-like factor 15 (KLF15) was identified in a yeast one-hybrid assay using a bovine retinal cDNA library and a 29-bp fragment of the bovine rhodopsin promoter as bait. Transactivation experiments were performed in vitro to study the activity of KLF15 on the IRBP promoter since the rhodopsin and IRBP DNA sequences share some gene regulatory elements [88]. HEK293 cells were transiently transfected with a KLF15 expression plasmid and an IRBP promoter-reporter gene construct containing the proximal region of the bovine IRBP gene, −300 to +132 bp. Expression of KLF15 reduced reporter gene activity of the IRBP-reporter gene construct in a dose-dependent manner [88]. CRX expression is known to transactivate the IRBP promoter in vitro [74]. Reporter gene activity was also repressed by KLF15 expression when CRX or CRX and NRL were cotransfected with the IRBP-reporter gene construct and the KLF15 expression plasmid [88].

The zinc finger domains of the KLF15 transcription factor bind to the IRBP promoter [89]. DNA footprinting experiments using a KLF15 fusion protein identified three KLF15 binding sites in the bovine IRBP promoter. The same footprints are found on both positive and negative strands and are conserved among three species. DNA binding is zinc dependent, and there is evidence that retinal proteins bind to the KLF15 site. The footprinted site, K1-c, is next to the CpG dinucleotide (−115) that is hypomethylated specifically in retinal cells

[35, 77]. Perhaps KLF15 is regulating the methylation/chromatin structure of the IRBP 5′-flanking region for the repression of IRBP expression in the inner retina.

MOK2

MOK2 is a Krüppel/TFIIIA-related zinc finger protein. MOK2-binding sites are present in the IRBP gene, and experiments were performed to determine if IRBP is a potential MOK2 target gene [105]. In the IRBP promoter, 8 bp of the MOK2 core sequence is found (TTAAGGCT) in the reverse orientation, and this overlaps the CRX/ OTX2 site (Fig. 5). The IRBP DNA sequence defined as the MOK2 site is conserved between human, bovine, and mouse IRBP promoter sequences. A MOK2-binding site is also found in intron 2 of the human and bovine IRBP genes [105].

The IRBP promoter region binds to MOK2 recombinant protein in EMSAs, and the band is supershifted with an anti-MOK2 antibody showing that MOK2 protein binds to the IRBP promoter in vitro [105]. In transient transfection experiments of Weri-RB1 cells with an IRBP promoter-reporter construct alone or with MOK2 expression plasmids, a significant reduction in transcription activity was observed when the MOK2 protein was present, showing that MOK2 can act as a transcriptional repressor of the IRBP promoter activity in vitro. However, the sensitive technique of in situ rtPCR was required to detect a low level of Mok2 expression in a subset of nuclei in the ONL of 1-month-old mouse retinas [105].

Chx10

Chx10 gene expression is required for retinal progenitor cell proliferation and bipolar cell differentiation. Chx10 is required to block rod cell differentiation but is not essential for the proliferation of progenitor cells in the postnatal retina [106]. It contains a pairedlike homeodomain [107]. Chx10 represses transcription in vitro [108]. Chx10 knockdown inhibits the generation of bipolar cells and differentiation of rod photoreceptor cells. When overexpressed in newborn mouse retinas, Chx10 promotes the differentiation of bipolar cells without affecting cell division or survival [106].

The expression of Chx10 is found early in retinal development in the mouse at E9.5 in the outermost region of the evaginating optic vesicle, the area that will form the neuroretina later in development [107]. At E16.5, when lamination of retina is starting and differentiated ganglion cells have been formed, Chx10 expression is detected in the neuroblast layer but not in the ganglion cell layer (GCL). Expression of Chx10 at P4 is further restricted to the future inner nuclear layer and is not found in the future ONL [107]. In the adult mouse retina, Chx10 expression is found in bipolar cells and a subset of Müller cells [109].

In the ocular retardation mouse mutant (Chx10or-J/or-J), ocular development is abnormal. The phenotype is characterized by blindness, small eyes (micropthalmia), abnormal photoreceptors, and absent optic nerve. Analysis of expression of other known transcription factors in the retina was performed to study the effect the absence of Chx10 has on these proteins. Unlike wild-type mice, Crx expression was not detected during retinal development in the embryo but was expressed after birth [110]. rtPCR experiments were performed to study the expression of proteins that are putative targets of Crx, and expression of many of these proteins was delayed in the Chx10or-J/or-J retina. The expression of

IRBP was not delayed in the Chx10or-J/or-J retina [110], further supporting the idea that Crx expression is not required for IRBP expression in the embryonic retina.

ChIP assays were used to identify potential gene targets of Chx10 in the in vivo chromatin context [111]. Mouse retina samples were obtained from mice at different ages and treated with formaldehyde to cross-link the proteins to their DNA targets and to each other. Sonication was performed to cut the DNA into an average size of about 1 kb. After incubation with anti-Chx-10 antibodies, the complexes were immunoprecipitated, purified, and the cross-links reversed. Three regions of the IRBP promoter were specifically chosen (targeted) for amplification by PCR to determine if Chx10 binds to the IRBP promoter at these sites. These sites were chosen because they are known to contain homeodomain-binding sites [91]. ChIP shows that Chx10 binds (in vivo) to one site in the IRBP promoter, upstream at −1.4 kb. This result was unexpected because the proximal IRBP promoter contains a PCE-1 element that is also present in the arrestin promoter. The PCE I element in the arrestin promoter does bind Chx10 both in vitro and in vivo, as shown by ChIP assays. The ChIP assays show that protein binding to DNA is dependent on chromatin context. Chx10 binds IRBP promotes in vivo at P0, P6, and P14. The consensus TAATtgac that they identify is part of the DNA fragment identified by ChIPeven though this sequence is outside of the fragment that was PCR amplified [111]. These ChIP experiments are not conclusive because Chx10 could be binding to regions of the IRBP promoter that were not identified and amplified by PCR.

Chx10 may be repressing IRBP gene expression in bipolar and Müller cells, but it is not acting by itself. Other factors are at work. IRBP gene expression is controlled by a combination of factors that include positive and negative regulators. The IRBP gene is not expressed in bipolar and Müller cells because repressors and not activators of IRBP expression are found in these cells.

Needless to say, nature can often switch a repressor to an activator, and vice versa, with posttranslational modifications. It is worth considering potentially analogous systems that use closely related or the same proteins. In muscle, myogenesis occurs when myoblasts differentiate into myotubes. An analogous set of proteins, including homeodomain proteins and basic helix-loop-helix (bHLH) proteins can form highly coordinated and synergistic complexes to activate transcription of genes needed to build myofibers. Figures 7 and 8, which show the IRBP 5′ flanking region, illustrate that matches to consensus trans factor binding sites are located upstream of the IRBP gene in numerous species. In particular, two such sites, a COMP1 and a Chx10 match, are found about 1,200 nucleotides upstream of the transcription initiator site in a well-conserved region (hereafter called the upstream conserved region). These sites are about 65 nucleotides apart. Chx10 is well known to the vision sciences community, but COMP1, which stands for COoperates with Myogenic Proteins 1, is a poorly understood cis element [112]. This element is known to bind trans factors cooperatively and to synergistically enhance transcription of a reporter gene when transfected into myoblasts that are differentiating into myotubes. Since this trans factor binding site was first recognized in 1992, it was found [113] that the COMP1 cis-element sequence TGATTGAC can be bound in a cooperative manner by a heterodimer of PBX/Meis1-Prep1 with another heterodimer consisting of E2a bound to MyoD, myogenin, Mrf-4, or Myf-5 (PBX/Meis1-Prep1 are homeodomain proteins whereas E2a, MyoD, myogenin, Mrf-4 and Myf-5 are bHLH proteins). While it is not clear that any of these proteins are available in the adult retina or actually bind to the