mobility shift assays (EMSAs) [73], DNA footprinting assays, or numerous variants (with the chromatin immunoprecipitation [ChIP] assay a very important one). Other approaches include the yeast one-hybrid method to find proteins that interact with a putative DNA TFBS [74]. These measurements of binding interactions have been best once a TFBS has been shown to control the given promoter. The detection of legitimate, biologically significant interactions has been less successful when only a match to a consensus sequence motif has been detected. This may be explained by the relatively high backgrounds in EMSAs and too low a stringency employed in detecting matches to the consensus. Strategies to overcome these weaknesses include the use of sequence alignments among numerous species with known common visual capabilities, a strategy called phylogenetic footprinting [75]. This strategy will become much more powerful once the number of whole genomes that are completely sequenced becomes greater.

Gene expression is controlled by both genetic and epigenetic elements. The genetic elements include the DNA sequences (cis-acting controlling elements) and the protein complexes that bind to the specific DNA sequences. The proteins are called trans factors and are also known as transcription factors. Some transcription factors bind DNA, and others form protein–protein interactions to act in combination and regulate expression of an individual gene. Each transcription factor may contribute to the control of many genes. There are some genetic elements that are found in every cell for basal expression and the initiation of gene transcription. Other gene regulatory proteins are not ubiquitous and are found in specific cells to either activate or repress (gene silencer) gene expression. Celland tissue-specific transcription factors work to control and maintain terminal differentiation. Recent work has shown that noncoding RNAs (microRNAs) also have the ability to silence activated genes.

The epigenetic elements known to control gene expression include DNA methylation and chromatin structure. Histone deacetylases condense chromatin to make these regions inactive. Methylated DNA is found in transcriptionally silent regions of the genome. The IRBP gene methylation states were studied by Liou et al. [76] and Boatright et al. [77].

The study of the control of gene expression will lead to a better understanding of development, cancer, tissue-specific expression, and transcription factors. As an example, mutations in the Crx transcription factor are found in patients with retinal degeneration named cone-rod dystrophy (CORD2) [78, 79].

IDENTIFICATION OF DNA CIS-ACTING CONTROLLING ELEMENTS: IN VITRO AND IN VIVO EXPERIMENTS

Determining the sequence of the IRBP gene provided the information necessary to study the mechanisms that control the expression of the IRBP gene. The regulation of IRBP gene expression has been studied using IRBP promoter/reporter gene fusion constructs. These constructs have been tested in transient transfection assays and in transgenic mice. Transient transfection assays show that −204 bp of the IRBP promoter are sufficient for gene expression in Y79 retinoblastoma cells [80]. Transgenic mice have also been used to study the regulation of IRBP expression by its promoter.

A 1.3-kb fragment of the human IRBP promoter was shown to direct transgene expression to the retina and pineal gland in transgenic mice [81]. This same transgene expression profile was also seen using a 1.8-kb fragment of the mouse IRBP promoter [82]. In other studies, the 1.3-kb human IRBP fragment was shown to direct transgene expression to the photoreceptor cells of transgenic mice, but expression was heterogeneous throughout the retina [83]. The region required for tissue-specific expression was reduced to −212-bp of the human IRBP [84] and then to −123 bp [85, 86]. The critical region in the mouse IRBP promoter is similar in size [70]. A tissue-restricting factor is present between −156 and −70, and this region was refined further by Boatright et al. [87] to between −156 and −140 (Fig. 4 ), as illustrated by the differential expression of these promoters but not of the −70 promoter in Y79 and Neuro2a cells. This site appears to be the same one as described by Otteson et al. [88, 89] as the site to which Krüppel-like factor 15 (KLF15) binds in the human IRBP and opsin promoters.

Two sequences in the IRBP promoter were identified within the −123-bp fragment of the IRBP 5′-flanking region that are important for the regulation of IRBP gene expression. They consist of the GATTAA sequence and its inverted repeat, AATTAG, just upstream (Fig. 5; [71, 85]. The region containing these two sites is protected from digestion in in vitro deoxyribonuclease I (DNase I) protection experiments and has been identified by several groups (see Fig. 5; [74, 85, 86, 90]. These different groups used different protein pools in the DNase I footprinting assays yet achieved very similar results. Bobola et al. [85] used a 147-bp fragment of the human IRBP promoter and nuclear extracts from retinoblastoma cells to identify a 14-bp footprint in this region of the IRBP promoter (Fig. 5]. Other groups [74, 86, 90] identified larger footprints of the same region (Fig. 6). Fong and Fong [90], also using a fragment of human IRBP promoter sequence and nuclear extracts from retinoblastoma cells, identified the larger footprinted section from −38 to −73 bp of the IRBP promoter, and this region binds to the protein OTx2 in a yeast one-hybrid experiment. Fei et al. [86] used a fragment of the human IRBP promoter sequence and a nuclear extract isolated from bovine retinas to identify a retinal-specific footprint between −42 and −76 bp of the IRBP promoter. Chen et al. [74], using a fragment of the bovine IRBP promoter and a recombinant fusion protein containing the Crx homeodomain, footprinted the region −29 to −81 bp. It is likely that numerous proteins bind to the DNA located in the footprinted region between −45 and −75 bp of the IRBP promoter. Possible candidates for proteins that bind to this region include Rx, Otx2, Crx, and Mok2.

The AATTAG element had been previously identified in the mouse arrestin gene and named PCE I (photoreceptor conserved element I) because it binds retina-specific nuclear factors [91]. The PCE I core consensus sequence was determined to be CAATTAG. Closely related sequences are found in promoter regions of other vertebrate photoreceptor-specific genes, including the IRBP and opsin 5′-flanking regions. In the IRBP promoter, the PCE I sequence is conserved between the mouse, bovine, and human genomic sequences. The two regions in the mouse IRBP gene containing the PCE I are antisense strand between −1415 and −1397 and on sense strand between −74 and −55 upstream of the transcription start site [91].