(Received for publication, July 21, 1994)
From the
Interphotoreceptor retinoid-binding protein (IRBP) is the major protein component of the interphotoreceptor matrix. IRBP has a highly restricted tissue-specific expression in retinal photoreceptor cells and in a subgroup of pinealocytes. With the purpose of understanding how transcriptional regulation contributes to the expression of human IRBP, we have studied a short promoter fragment (from -123 to +18, relative to the transcription start site). We demonstrate, by analysis of the expression of the lacZ reporter gene fused to this short promoter fragment in transgenic mice, that it is sufficient to confer tissue-specific expression in retinal photoreceptors and in pinealocytes. DNA/protein binding assays, performed to identify binding sites for tissue-specific trans-acting factors, have shown that an element between -45 and -58 binds a factor present only in nuclear extracts of retinoblastoma-derived cell lines, which express IRBP. An element further upstream, between -86 and -106, binds apparently ubiquitous factors. Site-directed mutagenesis was performed to disrupt a GATTAA motif included in the -45 to -58 binding site and a second inverted GATTAA motif present shortly upstream. In transgenic mice bearing the mutated version of the promoter fragment, the expression of the reporter gene was completely abolished, thus suggesting that this element is essential for tissue-specific expression. A GATTAA motif appears in the 5`-flanking regions of several photoreceptor-specific genes, suggesting that this could be the recognition site for a photoreceptor-specific factor.
Development in mammals is programed to define patterns of
differentiated cells in specialized tissues. During this developmental
process, tissue-specific genes become coordinately expressed due to
mechanisms of regulation, largely at the transcriptional level. The
interphotoreceptor retinoid-binding protein (IRBP) ()gene
represents a good model for investigating regulation of expression of
neural retina-specific genes. IRBP is a large lipoglycoprotein that
constitutes approximately 70% of the protein component of the
interphotoreceptor matrix(1, 2) . Although widely
distributed among the vertebrates, it has a highly restricted
tissue-specific expression and is found in the interphotoreceptor
matrix of the retina(3) . IRBP mRNA is present in photoreceptor
cells of the retina, prevalently in rod cells, and, at very low levels,
in a subgroup of pinealocytes(4) . IRBP is also expressed by
retinoblastoma-derived cell lines in vitro(5) , and
the level of IRBP expression can be altered by agents that affect
retinoblastoma cell differentiation(6) .
IRBP can bind a variety of retinoids(7) , and it has been suggested that it acts by actively transporting retinoids between photoreceptor cells and the retinal pigmented epithelium (8) and/or that it acts as a ``buffer'' protein for retinoids(9) . All of the bovine and human IRBP genes have been cloned(10, 11) ; the sequences of the human and murine 5`-flanking regions that are upstream from the transcription start site have been described(12) . The human IRBP gene has been mapped to the centromeric region of chromosome 10(13) .
The exquisite tissue-specific expression of IRBP indicates that there must be specific factors that control the expression of this gene. Initial investigations on the mechanism of tissue-specific expression of the IRBP gene have mainly been directed to the elucidation of transcriptional regulation. Hypomethylation of CpG islands in the fragment between -1578 and -1108 of the IRBP promoter and at the beginning of the first exon correlates with gene expression(14) . Mice that are transgenic for a reporter gene fused to a 1.3-kilobase pair human IRBP promoter segment show tissue-specific expression of the reporter gene(15) . A shorter 212-bp promoter fragment linked to a reporter gene showed tissue-specific expression(12) .
Here we report the results of a detailed analysis of a short IRBP promoter segment (-123 to +18). The ability to confer retina-specific expression of reporter gene has been studied in transgenic mice. By comparing the wild type 124-bp promoter fragment with a mutated version of the same fragment, we demonstrated that a cis-acting element that contains a repeated GATTAA motif is essential for tissue-specific expression. An analysis of binding sites for trans-acting factors contained in nuclear extracts of retinoblastoma IRBP-expressing cell lines compared with nuclear extracts of nonexpressing cell lines has been performed. We demonstrate that the same cis-acting element that was able to confer tissue-specific expression of the IRBP gene binds a factor present only in IRBP-expressing cells.
Figure 3: Nucleotide sequences reporting cis-acting elements, oligonucleotides used as labeled probes or competitors in DNA/protein binding assays, and substitutions introduced by mutagenesis. Lowercase letters indicate the nucleotide substitutions introduced by mutagenesis. Solid lines indicate DNase I protections in the promoter fragment and the GATTAA elements in the oligonucleotides. Dotted lines indicate mutated GATTAA elements in oligonucleotides.
Transgenic mice were produced by pronuclear injection into B6D2 F2 fertilized eggs (18) of 500 copies/ml DNA diluted in 10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA. Founder mice were identified by Southern blot analysis of EcoRI-digested tail DNA(19) .
Transgene expression was detected on frozen
sections of fixed organs. Specimens were fixed for 2 h in 4%
paraformaldehyde in phosphate-buffered saline (10 mM phosphate
buffer, pH 7.4, 150 mM NaCl), incubated for 2 h in 30% sucrose
in phosphate-buffered saline for cryopreservation, embedded in OCT
compound (Miles), and frozen in liquid nitrogen. 10-µm-thick
sections were collected on gelatin-coated slides and stained for
anywhere from 2 h to overnight (depending on the transgene) in 1 mg/ml
X-gal (Sigma), 5 mM KFe(CN)
, 5 mM K
Fe(CN)
, and 2 mM MgCl
in phosphate-buffered saline with 0.001% Triton X-100. Nuclear
fast red was used for counterstaining.
Figure 1:
Sections of retina and epiphysis of
transgenic mice carrying the pNB21 construct. A section (10-µm
thick) from an adult eye stained for 2 h with X-gal is shown. A, -galactosidase activity is present only in the
photoreceptor cell layer. g, ganglion cell layer; i,
inner nuclear layer; p, photoreceptor cells nuclei (outer
nuclear layer); r, retinal pigment epithelium. Bar corresponds to 50 µm. B, lower magnification of the
same histological section shown in A. n, optic nerve. Bar corresponds to 50 µm. C, coronal section
(15-µm thick) at the epiphysis level of an adult brain stained with
X-gal.
-Galactosidase activity is present in some pinealocytes
located in the wall of the pineal stalk (arrowheads). e, lumen of the pineal peduncle. Bar corresponds to
100 µm.
The pNB20 plasmid, containing a 1.3-kilobase pair promoter
segment, showed the same tissue-specific expression, although
-galactosidase activity was detected only in a few photoreceptor
cells and not in pinealocytes, a pattern similar to that reported by
others for the same fragment(22) .
The specificity and strength of the 124-bp promoter addressed the search for binding sites of tissue-specific trans-acting factors inside its sequence.
Figure 2: DNase I footprinting of a segment of the IRBP promoter with nuclear extracts of different cell lines. A segment of plasmid pNB1 containing the sequence between -123 and +18 was labeled at its 3` end, incubated without or with nuclear extract (NE) from different cell lines, and treated with DNase I. Lanes 1 and 6, Maxam-Gilbert G + A sequencing reaction; lanes 2, 7, and 11, DNase I digestion pattern of the naked DNA incubated without cell nuclear extract; lanes 3-5, incubated with 28 µg of nuclear extract from Weri-RB1 cells without competitor (lane 3) or with competition by a 250-fold molar excess of a double-stranded A oligonucleotide (lane 4) or a double-stranded B oligonucleotide (lane 5); lanes 8-10, incubation with 30 µg of nuclear extract from HeLa cells, without (lane 8) or with competition by a 250-fold molar excess of either a double-stranded A oligonucleotide (lane 9) or a double-stranded B oligonucleotide (lane 10); lanes 12-14, incubation with 20 µg of nuclear extract from Y79, HeLa, and H9 cells, respectively, without competitor.
Taken together, these results suggest that nuclear factors present in cell lines that express IRBP bind two different elements in this short promoter sequence in a sequence-specific manner, and that one of these factors is contained only in nuclear extracts from cells that express IRBP. Moreover, the factors interacting with the A and B sites seem to interfere with each other for their respective binding.
Figure 4: Gel retardation assay with the A oligonucleotide and nuclear extracts from Weri-Rb1 and HeLa cell lines. A 5` end-labeled double-stranded A oligonucleotide was incubated with crude nuclear extract (NE) of Weri-RB1 (lanes 1 and 3-7) or HeLa cells (lane 2). The same unlabeled double-stranded oligonucleotide was included as a competitor (Comp.) at increasing molar excess with respect to the labeled probe (25-, 50-, 100-, and 200-fold molar excess in lanes 4-7, respectively).
With a labeled double-stranded oligonucleotide containing the B element, we observed a major retarded complex with nuclear extracts from both Weri-RB1 and HeLa cells (Fig. 5, square bracket). This complex could be specifically competed by the same unlabeled oligonucleotide (Fig. 5, lanes 4-7). A computer analysis of the B sequence, which is relatively GC-rich, indicated sequence similarity with binding sites of two already characterized transcription factors, SP1 and AP2. A double-stranded oligonucleotide containing the consensus SP1 recognition sequence (Promega, Madison, WI) competed the formation of the complex as efficiently as the oligonucleotide containing the B element (Fig. 5, lanes 8-11), whereas no competition was observed with an oligonucleotide containing the consensus AP2 recognition sequence (Promega) (data not shown). Some bands that migrate more quickly and that are present in low abundance appeared to be also due to sequence-specific binding, being competed by the B oligonucleotide but not by the SP1 or the AP2 oligonucleotides.
Figure 5: Gel retardation assay with the B oligonucleotide and nuclear extracts from Weri-Rb1 and HeLa cell lines. A 5` end-labeled double-stranded B oligonucleotide was incubated with crude nuclear extract (NE) of Weri-RB1 (lanes 1 and 3-11) or HeLa cells (lane 2). The same unlabeled double-stranded oligonucleotide was included as a competitor (Comp.) at increasing molar excess with respect to the labeled probe (25-, 50-, 100-, and 250-fold molar excess in lanes 4-7, respectively). A consensus SP1 oligonucleotide (Promega) was included as a competitor at increasing molar excess with respect to the labeled probe (25-, 50-, 100-, and 200-fold molar excess in lanes 8-11, respectively).
From these observations we conclude that a factor, present in IRBP-expressing cells, binds a double-stranded oligonucleotide containing a GATTAA sequence. Furthermore, an ubiquitous factor, possibly SP1 according to the competition experiment, binds the B element.
Figure 6: Effect of different competitor oligonucleotides on binding of a tissue-specific factor to the A oligonucleotide. A 5` end-labeled double-stranded A oligonucleotide was incubated with crude nuclear extract of Weri-RB1 cells without competitor (Comp., lane 1) or with increasing molar excess (50-, 100-, and 200-fold molar excess with respect to the labeled probe for all competitor oligonucleotides) of the A oligonucleotide (lanes 2-4), AL oligonucleotide (lanes 5-7), 1M oligonucleotide (lanes 8-10), and AM oligonucleotide (lanes 11-13).
In this paper we show that a 124-bp fragment derived from the IRBP promoter fused to the lacZ reporter gene was able to drive expression of the reporter gene in transgenic mice. Moreover, we identify a cis-acting element located inside this fragment that is responsible for tissue-specific expression.
Comparison of 5`-flanking regions of the human and murine IRBP genes shows a high degree of sequence homology (76%) in the first 277 bp upstream of the transcription start site and with 79% homology in a second region between -1277 and -1526(12) , confirming the importance of the most proximal region in controlling IRBP gene. Our results demonstrate that the first 124 bp contain the elements that are necessary and sufficient for correct tissue localization of the gene product in the adult mouse. IRBP is a protein expressed early in embryonic development(25) , and our work is in progress to assess whether the 124-bp minimal promoter is also sufficient for correct temporal expression of the reporter gene during embryonic development.
Because it is known that IRBP gene transcription can be modulated by light (26) and by agents such as cAMP(6) , it is also interesting to evaluate whether this minimal promoter fragment might be able to modulate gene expression in response to different factors. Our preliminary sequence analysis in search of potential responsive cis-acting elements indicated that an AP1-like element and an ATF-like element overlap in a region centered around -115.
We have characterized the 124-bp promoter region sufficient for tissue-specific expression by DNase I footprinting and GRA using nuclear extracts prepared from IRBP-expressing cells (retinoblastoma cell lines) and nonexpressing cell lines. A previous report(32) , in which a characterization of DNA/protein interactions in the first 300 bp of the IRBP promoter was performed by GRA using retina extracts and nuclear extracts from Y79, shows that these extracts give rise to comparable retarded complexes, different from complexes formed when other tissue extracts were used.
Our
results showed that the 124-bp promoter contains two protein-binding
sites that are recognized by different factors. One, named B, binds
apparently ubiquitous factors, among which is SP1 or SP1-related
factor, and our data ()suggested that this B element
represses transcription. The second, called A, that contains a GATTAA
motif in its central part, binds a factor that is present only in
nuclear extracts of IRBP-expressing cells and seems to be the most
probable candidate for tissue-specific regulation of the gene. We
demonstrated, by analysis of transgenic mice carrying the reporter gene
fused to the 124-bp IRBP promoter, which had been modified in the A
element by site-directed mutagenesis, that this element is essential
for tissue-specific expression of the gene. In transgenic mice bearing
the mutated version of the promoter, the expression of the reporter
gene was completely abolished both in the retina and in the pineal
gland.
The GATTAA element, besides playing an essential role in the IRBP promoter, probably by binding a tissue-specific factor, appears to be highly conserved in the 5`-flanking region of several photoreceptor-specific genes in humans and in other species of vertebrates(27, 28, 29) . Moreover, in different species of Drosophila this same GATTAA element is also contained in the 5`-flanking region of a variety of photoreceptor-specific genes. Site-directed mutagenesis of a sequence containing the GATTAA element in rhodopsin genes of Drosophila melanogaster has been shown to cause the loss of reporter gene expression(30) , similar to what we have demonstrated for the IRBP promoter. Thus, the importance of the GATTAA element is indicated both by the high degree of conservation of this sequence in the 5`-flanking regions of different photoreceptor-specific genes even in very different species (e.g. humans and species of Drosophila) and by its localization inside regions that have been demonstrated to be involved in gene regulation(27, 30) .
The IRBP promoter lacks a
consensus TATA box. It is questionable whether the GATTAA motif
centered around -50 functions as a site of interaction of TFIID
components necessary for the assembly of the basal transcription
machinery. Some photoreceptor-specific genes contain both the GATTAA
motif and the TATA box. On the other hand, the sequence surrounding the
transcription start site in IRBP matches the consensus
PyPyA(T/A)PyPy, which is indicated as initiator element for
TATA-less promoters(31) .
The identification of the tissue-specific factor(s) interacting with the cis-acting element that we have described will contribute substantially toward understanding the mechanism of regulation of the IRBP gene and, quite likely, the regulation of many retinal/visual transduction genes.