|Molecular Vision 1999;
Received 9 April 1999 | Accepted 13 July 1999 | Published 15 July 1999
The transcription factor Sp3 interacts with promoter elements of the lens specific MIP gene
Sunghee Kim, Hong Ge, Chiaki Ohtaka-Maruyama,
Ana B. Chepelinsky
Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, MD, 20892
Correspondence to: Ana B. Chepelinsky, National Institutes of Health, Bldg
6, Rm 211, 6 Center Drive, MSC 2730, Bethesda, MD, 20892-2730; Phone:
(301) 496-9615; Fax: (301) 480-7933; Email:
Dr. Kim is now at the National Institute of Child and Human Development, National Institutes of Health. Bldg. 49, Room 5A38, Bethesda, MD, 20892.
Dr. Ohtaka-Maruyama is now at the Cellular Physiology Laboratory, the Institute for Chemical and Physical Science (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-01, JAPAN.
Purpose: To characterize the cis regulatory elements and their interaction with transcription factors responsible for the lens specific expression of the MIP gene, which encodes the Major Intrinsic Protein of the lens fiber membranes.
Methods: Study interaction of factors present in newborn mouse lens nuclear extracts with DNA fragments corresponding to mouse MIP gene 5' flanking sequence by electrophoresis mobility shift assay (EMSA) and DNase I footprinting.
Results: We found a high degree of identity in the first 100 bp of 5' flanking sequence of mice and humans, however, a lower degree of conservation is observed further upstream. We have found by DNase I footprinting analysis that lens specific factors may interact with the first 100 bp of 5' flanking sequence. A domain containing an E box, conserved in mouse and human, may interact with a lens specific factor. However, general factors may interact with a NF-1 binding site. An overlapping GC and CT box is present in the mouse MIP gene. In the human MIP gene GC and CT boxes are found in different domains of the MIP gene promoter. Both CT boxes interact with factors present in lens nuclear extracts including Sp3. They are able to interact with purified Sp1but not with Sp1 present in mouse lens nuclear extracts.
Conclusions: The transcription factor Sp3 may play an important role in regulating MIP gene expression in the lens.
Major Intrinsic Protein (MIP) is the most abundant protein of the ocular lens fiber membrane and is a member of an ancient family of membrane channel proteins . The MIP gene is specifically expressed in the lens fiber cells, which arise by differentiation of the lens epithelia, and starts being expressed in the primary lens fibers . MIP may play an important role in maintaining lens transparency by reducing the interfiber space, as suggested by its ability to function as a weak water channel [3-6] and possibly as an adhesion molecule . Mutations in the MIP gene have been linked to the mouse genetic cataracts Fraser mutation (CatFr) and lens opacity mutation (lop) .
The molecular mechanisms underlying the regulation of the lens-specific expression of the MIP gene are largely unknown. To investigate the regulation of the MIP gene, we have previously cloned and characterized the human MIP gene. Our results indicated that the human MIP gene sequence -253/+42 contained an active promoter in primary cultures of lens cells but was inactive in non-lens cells . We also characterized a cis regulatory element in a domain proximal to the TATA box. This element was required for MIP gene promoter activity in lens cells and contained overlapping Sp1 and AP2 binding sites .
Sp1 is an ubiquitous transcription factor which regulates the tissue-specific expression of a variety of genes [11-14]. This factor is developmentally regulated [15,16] and essential for embryonic mouse development . Genes encoding Sp1 related proteins, Sp2, Sp3 and Sp4, have been characterized, indicating the existence of a Sp family of transcription factors [18-20]. A three-zinc-finger DNA-binding domain is highly conserved in the members of this family, resulting in similar binding affinities to GGCGGG, CCTCCC and CCACCC motifs, also known as GC,CT and CA boxes, respectively. Sp1 and Sp3 may function either as an activator or as a repressor. Sp1 and Sp3 differ in their activation domains; they both contain two glutamine rich domains, but differ in their serine/threonine rich domains [18-24].
In the present study we analyzed the regulatory elements conserved in the mouse and human MIP gene and their possible interaction with members of the Sp family of transcripton factors.
Oligonucleotides were synthesized in a PE Biosystems DNA synthesizer (Foster City, CA) and purified either by G-25 Sephadex columns or by urea-acrylamide gel electrophoresis.
Antibodies to Sp1, Sp2, Sp3 and Sp4 were from Santa Cruz Biotechnology, Inc, Santa Cruz, CA. Sp1 was a monoclonal antibody corresponding to amino acids CKDSEGRGSGDPGKKKQHI , Sp2 is a polyclonal antibody to amino acids KGTRSNANIQYQAVPQIQAS [18,19], Sp3 is a polyclonal antibody to amino acids DILTNTEIPLQLVTVSGNET [18,19], Sp4 is a polyclonal antibody to amino acids VTVAAISQDSNPATPNVSTN .
One- to three-day old CD-1 mice were obtained from Charles River Laboratory (Raleigh, NC) and handled according to the US Public Health Service Policy on Human Care and Use of Laboratory Animals.
Lens nuclear extracts were prepared from newborn mice. The intact nuclei from approximately 600 newborn (1-3 day-old) mouse lenses were prepared using a 2 M sucrose cushion centrifugation as described . The nuclei were resuspended in a buffer containing 20 mM Hepes (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, and the nuclear protein was extracted using small scale preparation as described by Schreiber et al . [alpha]TN4, a lens cell line transformed by SV40  was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and nuclear extracts were prepared as described . The nuclear protein concentration was determined using a commercial assay kit (Bio-Rad Laboratories, Hercules, CA). The nuclear extracts were aliquoted and stored at -80° C. HeLa and NIH3T3 cell nuclear extracts were purchased from Promega Corp.(Madison, WI) and Santa Cruz, respectively.
DNase I Footprinting
The EcoR1/ApaI DNA fragment corresponding to mouse MIP gene sequence -461 to +150 was isolated from the pMOMIP plasmid containing the mouse MIP genomic DNA (Ohtaka-Maruyama and Chepelinsky, unpublished) and purified by low-melting point agarose gel eletrophoresis. This ApaI/EcoRI DNA fragment was ligated into ApaI/EcoRI-digested pBluescript II SK+ vector (Stratagene, LaJolla, CA). The plasmid containing the ApaI/EcoRI fragment was prepared using CsCl gradient centrifugation, and the plasmid was digested with NcoI/EcoNI. The NcoI/EcoNI correspond to nucleotides -215 and +71, respectively. The NcoI/EcoNI fragment was purified using low-melting point agarose gel eletrophoresis. The NcoI site DNA fragment was end-labeled with [[alpha]-32P]dCTP (3000 Ci/mmol, Amersham, Arlington Heights, IL) by Klenow DNA polymerase (New England BioLabs, Beverly, MA). The end-labeled probe was purified using Sephadex G-50 chromatography (Amersham Pharmacia Biotech, Piscataway, NJ). The preparation of G+A Maxam-Gilbert sequencing ladder and the entire procedure of the footprinting were carried out using a Suretrack footprinting kit (Amersham Pharmacia), according to the manufacturer's instructions. Incubation of the labeled probe (15,000 cpm) with the nuclear extract (15 µg) was followed by digestion with DNase I, ranging from 0.3 to 3 U. The DNase I digested probe was resolved on a 8% polyacrylamide/urea gel. The gel was dried and bands were visualized by autoradiography.
Electrophoretic Mobility Shift Assays (EMSA)
Single stranded oligonucleotides were end-labeled with [[gamma]-32P]ATP (5000 Ci/mmol, Amersham) and purified from free nucleotides by Sephadex G-25 chromatography. Double-stranded probes were prepared by annealing the end-labeled oligonucleotides with a 1.2-fold molar excess of unlabeled corresponding complementary oligonucleotide. DNA-protein binding reactions were performed in a total volume 25 µl containing 2 µg nuclear proteins, 50 fmoles probes (20,000 cpm), and a buffer consisting of 5% glycerol, 0.1% Nonidet P-40, 0.5 mM EDTA, 50 mM NaCl, 10 mM Tris-Cl, at pH 7.5, with 1 µg poly(dI):poly(dC). In experiments with purified Sp1, the nuclear extract was replaced by purified recombinant human Sp1 (one footprinting unit, Promega). For competition analysis, the competitor probes were prepared by annealing equal molar complementary oligonucleotides and a 100-fold molar excess of unlabeled competitor probes was added to the binding reactions. For EMSA with antibodies, 1 µg of specific antibodies against Sp1, Sp2, Sp3 or Sp4 (Santa Cruz Biotechnology) was added to the respective binding reaction. After incubation at room temperature for 30 min, DNA-protein complexes were separated from free oligonucleotides by electrophoresis on a 5% polyacrylamide in a half ionic strength TBE buffer. DNA-protein complexes were visualized by autoradiography.
The 5'-flanking sequence of the mouse MIP gene
We had previously isolated the human MIP gene and characterized several cis regulatory elements [9,10,28]. We then turned to the isolation of the mouse MIP gene to be able to study the interaction of cis regulatory elements with lens transcription factors from the homologous species. After cloning the mouse MIP gene (Ohtaka-Maruyama and Chepelinsky, unpublished), we compared the mouse MIP 5'-flanking sequence with the human orthologous gene , using GCG DNA alignment programs, as shown in Figure 1. We found a high degree of identity in the 5'-flanking region proximal to the initiation site of transcription (90% identity in the -1/-106 sequence of mice and human). However, sequences located further upstream show a higher level of divergence (52% identity in the -106/-216 domain). In fact, a CT box present at position -49/-56, previously shown to be part of a regulatory element required for promoter activity in the lens , is conserved in the mouse and human MIP gene. The human MIP gene has an additional CT box and a GC box at positions -115/-121 and -147/-152, respectively. However, in the mouse MIP gene, the additional CT box and the GC box are found overlapping at position -163/-175 (see Figure 1).
We selected a DNA fragment corresponding to 213 bp of the mouse MIP gene 5'-flanking sequence and 71 bp of exon 1 to study its interaction with nuclear factors by DNase I footprinting. We compared the footprinting patterns obtained with nuclear extracts prepared from lens with those from HeLa cells. As shown in Figure 2, the region spanning approximately the first 120 bp of mouse MIP 5'-flanking sequence is not protected from DNase when incubated with HeLa nuclear extract (lanes 2-6). On the contrary, several domains in this region are protected by factors present in lens nuclear extracts (lanes 8-13). Some differences in the pattern of protection between both extracts are also noticed in the region approximately -150/-170.
We therefore synthesized several overlapping oligonucleotides, -90/-36, -128/-69, -186/-124, spanning the 5'-flanking sequence -36 to -186 of the mouse MIP gene to analyze their possible interaction with lens nuclear factors, and compared their interaction with nuclear extracts from two mouse cell lines. One of them, NIH3T3 is derived from mouse fibroblasts and the other one, [alpha]TN4 is an SV40 transformed lens cell line [27,29].
Double stranded oligonucleotides corresponding to sequence -90/-36, -128/-69 and -186/-124 interact with factors present in lens nuclear extracts, as shown in Figure 3 (lanes 2,6,10). Complex C1 was observed when the probes corresponding to sequences -186/-124 and -90/-36 were incubated with the lens nuclear extract (lanes 2,6). However, this complex was not formed with nuclear extracts prepared from NIH3T3 (lanes 4,8) nor from [alpha]TN4 (lanes 3,7) cell lines. Complex C3 was observed when the probe corresponding to sequence -186/-124 was incubated with the lens nuclear extract (lane 2) but not with nuclear extracts prepared from NIH3T3 or [alpha]TN4 cell lines (lanes 3,4 respectively). The probe corresponding to sequence -128/-69 formed complex C4 when incubated with lens nuclear extract (lane 10), but not with nuclear extracts prepared from NIH3T3 (lane 12) or [alpha]TN4 (lane 11) cell lines. As complexes C1, C3 and C4 may be due to the interaction with lens specific factors, we decided to further characterize these complexes formed by interaction of DNA fragments corresponding to the mouse MIP gene 5'-flanking sequences with factors present in lens nuclear extracts.
Mouse MIP gene 5'-flanking sequence -106/-90, containing an E box, interacts with lens nuclear factors.
When a double stranded oligonucleotide corresponding to sequence -128/-69 is incubated with lens nuclear extracts, it forms two complexes that are competed by the unlabeled probe (C4 and C5, Figure 4 lanes 6,7). Complex C4 appears to be formed by the interaction of an element present in the region -106/-90, as this complex is competed by unlabeled competitor -112/-83 and -119/-83 but not by -130/-106 or -90/-36 (Figure 4 lanes 10,11,9,12-13 respectively). The -106/-90 sequence contains an E box, CAGCTG [30-33], at position -95/-100. C4 is formed with factor/s present in the lens but not in other cells (see also Figure 3). Therefore, these results suggest that the E box may be an element interacting with a lens specific factor.
The complex C5, which is not lens specific, is competed by unlabeled oligos -130/-106 and -90/-36 (Figure 4, lanes 9,12 respectively). Complex C5 is also formed by the labeled probe -90/-36 and is competed by unlabeled oligo -90/-36 and -128/-69 but not by -63/-43 (Figure 4, lanes 2-5). Therefore, this complex may be due to the interaction of a nuclear factor with an element present in the region -69/-90. Whether the same factor, or a different one, forms a complex with an element present in the region -128/-106, migrating with the same mobility as C5, requires further studies.
Mouse MIP gene 5'-flanking sequence -186/-160, containing a CT box, interacts with lens nuclear factors.
When a double stranded oligonucleotide corresponding to sequence -186/-124 is incubated with lens nuclear extracts, three complexes are observed, C1, C2 and C3. These three complexes are competed by the homologous unlabeled probe and by the -186/-160 unlabeled competitor (Figure 5, lanes 2-4). The unlabeled competitor -90/-36 competes with complexes C1 and C3 but not with complex C2 (Figure 5, lane 5). When the double stranded oligonucleotide corresponding to sequence -90/-36 is incubated with lens nuclear extracts it forms two complexes, C1 and C5 (Figure 4, lane 2; Figure 5, lane 8). As mentioned above, C5 may be formed with an element present in the region -69/-90 (see Figure 5, lane 11 and Figure 4, lane 12). However, C1 is also competed by -186/-124 (Figure 5, lane 10). As both domains share a CT box, one present at position -49/-56 and the other at position -163/-170, they both may be responsible for the formation of complex C1. However, the formation of complexes C2 and C3 may require other sequences surrounding the CT box at position -163/-170.
To address this question, we introduced mutations in the region -186/-148. The results shown in Figure 6 indicate that mutations introduced in the CT box present in the probe corresponding to mouse MIP sequence -186/-148 (mutants M2 and M3) abolish the ability to form complexes C1, C2 and C3 (Figure 6, lanes 5-8). However, mutants M1, M4 or M5, with mutations outside the CT box, do not affect complex formation (Figure 6, lanes 3, 4,9-12). These results suggest that the CT box present in the mouse MIP sequence -163/-170 is involved in the formation of complexes C1, C2 and C3.
The CT box located in the mouse MIP gene 5'-flanking sequence -186/-148 interacts with Sp3.
As several members of the Sp family interact with GC and CT boxes [18,19], we analyzed whether antibodies to members of the Sp family would affect the formation of the complexes formed with the CT box. Figure 7 shows the results obtained when the probe -186/-148 was incubated with lens nuclear extracts in the presence of Sp1, Sp2, Sp3 or Sp4 antibodies. Complex C2 is not affected by any of these four antibodies, suggesting that this complex may be due to the interaction with a factor that is not a member of the Sp family. Neither Sp1, Sp2 nor Sp4 antibodies affect the formation of the complexes C1 and C3 (lanes 2,4,6,10). However, Sp3 antibody abolishes the formation of complexes C1 and C3 (lanes 2,8), suggesting that Sp3 is the only member of the Sp family present in lens nuclear extracts that interacts with the CT box located in the mouse MIP sequence -163/-170.
Purified Sp1 interacts with the overlapping CT/GC boxes located in the mouse MIP gene 5'-flanking sequence -163/-175.
We previously showed that a GC box present in the human MIP gene (-160/-129) interacts with Sp1 present in mouse lens nuclear extract, indicating the presence of functional Sp1 in this extract . Therefore, it was important to determine whether purified Sp1 was able to interact with the GC/CT boxes located in the mouse MIP sequence -186/-148. As shown in Figure 8, lanes 1-3, purified Sp1 is able to interact with an element present in this domain. When the GC box is mutated and only the CT box is present (mutant M1), purified Sp1 is still able to form a complex (Figure 8, lane 5). When the CT box is mutated and only the GC box is present (mutant M3), purified Sp1 is still able to form a complex (lane 9). The interaction with purified Sp1 is only abolished in mutant M2, containing mutations that disrupt at the same time the overlapping GC and CT boxes (Figure 8, lane 7). On the contrary, the interaction with factors present in lens nuclear extracts is only affected by mutations in the CT box (mutants M2 and M3; see Figure 6, lanes 6 and 8). Taken together, these results suggest that even though purified Sp1 is able to interact with the overlapping GC and CT boxes located in the mouse MIP sequence -163/-175, Sp1 present in lens nuclear extracts is not able to interact with them. One possible interpretation of the results could be that Sp3 present in lens nuclear extracts prevents Sp1 binding to the -170 region. However, it is also possible that the sequences surrounding the GC box may also play a role, by binding additional transcription factors, which in turn may prevent Sp1 binding.
Cloning the MIP gene from human and mouse allowed us to analyze their non-coding sequence for the presence of possible regulatory elements evolutionarily conserved and responsible for the specific expression of this gene in lens fibers. We found a high degree of identity in the 5'- flanking region proximal to the initiation site of transcription but a higher level of divergence in the sequences located further upstream. The isolation of the MIP gene from mouse and the feasibility of preparing nuclear extracts from newborn mouse lenses provided us with the tools to analyze the interaction of putative regulatory elements of the MIP gene with nuclear extracts from the homologous species.
We previously characterized a regulatory element of the human MIP gene, located at position -37/-65, required for promoter activity in lens cells . This element contains a CT box which interacts with the transcription factor Sp1. We mapped six additional Sp1 binding sites in the -200/+47 region of the human MIP gene . The presence of multiple Sp1 binding sites in the same gene results in DNA looping when the Sp1 molecules bound to those sites interact with each other, establishing in this way interactions between promoters and distant regulatory elements and among different transcription factors [34-40]. As Sp1 is involved in the tissue specificity of a variety of genes in many tissues [11-14], it may also be involved in the lens-specific expression of the MIP gene. The proximal CT box, at position -49/-56, located in an element required for promoter activity in lens cells, is conserved in the mouse and human MIP gene. The human MIP gene contains a second CT box at position-115/-121 and a GC box at position-147/-152. The mouse MIP gene also contains two CT boxes and one GC box. However, in the mouse the second CT box, at position -163/-170, overlaps with the GC box located at position -168/-175. Those GC and CT boxes are able to interact with purified Sp1. However, their interaction with factors present in lens nuclear extracts differs. Both Sp1 and Sp3 genes are expressed in the mouse lens (Ge and Chepelinsky, unpublished). The GC box in the human MIP gene is able to interact with Sp1 present in mouse lens nuclear extracts . However, even though the proximal CT box conserved in the human and mouse gene promoter interacts with purified Sp1, it is not able to interact with Sp1 present in lens nuclear extracts, interacting instead with Sp3 . The overlapping CT/GC boxes in the mouse MIP gene, are both able to interact with purified Sp1 but not with Sp1 present in lens nuclear extracts. Instead, only the CT box interacts with Sp3 present in the lens nuclear extracts. The different sequences flanking the 5'- and 3'- ends of the GC box in the human and mouse MIP gene, adenines or cytidines, respectively, may prevent Sp1 binding to the CT box of the mouse gene by interacting with additional transcription factors. Alternatively, Sp1 may be in a non-active form or with low binding affinities due to post-translational modifications and/or other protein-protein interactions.
Sp1 and Sp3 share a highly conserved three-zinc-finger DNA binding domain but their protein-protein interaction domains are less conserved. Both are able to function as activators or repressors [18-24]. Sp1 has been shown to play a central role in the activation of genes expressed in various tissues [41-46]. However, Sp1 and Sp3 may also function as negative regulators. Negative regulatory elements which interact with Sp1 and Sp3 transcription factors have been characterized in the Id4 gene promoter . Sp1 is a critical negative regulator of megakaryocyte-specific [alpha]IIb gene . Sp3 has been shown to repress the transcriptional activation by Sp1 [49,50]. Sp3 may repress Sp1-mediated transactivation through an inhibitor domain [23,24]. However, Sp3 is also involved in the induction of the p21 gene promoter during keratinocyte differentiation  and in the activation of the integrin CD11c and b genes in myelomonocytic cells . Sp3 may encode multiple proteins that activate or repress transcription . Both Sp1 and Sp3 appear to be involved in the regulation of various genes [44,46,51]. Changes in Sp1/Sp3 ratios have been observed during differentiation of primary human keratinocytes  and changes in GC box binding factors have been observed during differentiation of rat lens epithelia explants . Therefore, Sp3 and Sp1 may either play a role in activating MIP gene expression in lens fibers or in repressing its expression in lens epithelia. Future studies will allow us to discern between both possibilities.
The DNA-binding and transactivating properties of Sp1 are triggered by cAMP-dependent protein kinase A (PKA). Sp1 is activated by PKA and thus Sp1-dependent genes may be modulated through a cAMP-dependent PKA signaling pathway [13,42,55-58]. Changes in the phosphorylated forms of Sp1 occur during liver differentiation and may play a role in the growth arrest that occurs during terminal differentiation . Sp1 activity can be modulated by cell cycle-regulators such as cyclins and members of the Rb and E2F families [59-64]. Rb may be directly or indirectly involved in Sp1-DNA binding activity by liberating Sp1 from a Sp1-inhibitor that also interacts with Rb . The retinoblastoma gene product Rb interacts with Sp1 and Sp3 and synergistically activates Sp1 or Sp3 mediated transcription . Rb plays a role in withdrawal from the cell cycle in differentiating lens fiber cells and MIP expression is markedly decreased in Rb -/- mice , suggesting the possibility that Rb interaction with Sp1 and/or Sp3 may play a role in activating MIP gene expression in the lens fibers. Sp1 interacts with E2F1, -2 and 3 and both are able to activate transcription synergistically [63,64]. The interaction of Sp1 with E2F1 is cell cycle specific and occurs during mid- to late G1 . Five members of the E2F family are expressed in lens epithelia; however only E2F-1, -3 and -5 are expressed in the lens fibers. There are multiple phosphorylated forms of Rb in lens epithelia but predominantly the hypophosphorylated form in lens fibers. pRb and p107 seem to be the primary regulators of E2F activity in lens fibers [66,67]. Interaction of Rb with Sp1 and Sp3 may regulate the interaction of Rb with E2F, suggesting a role for Sp1 and Sp3 in cell cycle regulation in the lens.
The CT box present in the mouse MIP sequence -163/-170 interacts with Sp3 present in lens nuclear extracts, forming complexes C1 and C3, which may be lens-specific. However, it also interacts with another factor, which is not a member of the Sp family and appears to be also expressed in other tissues, forming complex C2. Other transcription factors that also interact with CT boxes, like the zinc finger protein MAZ, the single strand CT-binding factors hnRNP K or CNBP, have been characterized [68,69]. The factor that forms complex C2 may very well be one of these CT box-binding factor or a novel one. Further studies are needed to characterize this factor, which appears to be expressed in other tissues besides the lens.
Another complex, C4, may be lens-specific and involves the E box located in the MIP gene at position -94/-99. This element is conserved in the mouse and human orthologous gene. E-boxes contain the CANNTG motif and interact with proteins belonging to the basic helix-loop-helix (bHLH) family of transcription factors [30-33]. The bHLH proteins activate the expression of various tissue specific genes by forming heterodimers between ubiquitous and cell-specific family members, such as MyoD in muscle, BETA2 in pancreas or Capsulin in epicardial progenitors and mesenchyme of visceral organs [70-73]. The nucleotide sequence at the NN positions in the E box determines the specificity of binding to different members of the bHLH family of transcription factors. They either belong to the class A or class B subfamily. The E box present in the MIP gene, 5'-CACAGCTGTG-3', shows a perfect dyad of symmetry. The E box containing the motif CAGCTG interact with the class B of bHLH proteins.
The MyoD and AP4 helix-loop-helix proteins interact with this same E box sequence [73,74]. Whether AP4 or another member of the class B of basic helix-loop-helix of transcription factors is expressed in the lens and interacts with the E box, present in the 5'-flanking region of the MIP gene to regulate promoter expression in the lens, requires further studies.
Interestingly, a domain in the Na,K-ATPase alpha 2 subunit gene promoter has been characterized, containing GC, CT and E boxes. The E box functions as a negative regulatory element and the GC and CT boxes function as positive regulatory elements . Similarly, in the human Id4 gene promoter, an E box, GC and CT boxes have been characterized, where bHLH-zip factor, Sp1 and Sp3 interact respectively, playing a role in activating or repressing this gene. Repression of Id genes occurs during differentiation of many cell lineages . These results raise the possibility that the interaction of the bHLH factor that interacts with the MIP gene E box, may also interact with Sp1 and/or Sp3 to regulate MIP gene expression in the lens.
There is is a MARE motif (Maf regulatory element) overlapping with the E box, located at position -93/-98. Several members of the Maf family of transcription factors, L-maf, maf-1, maf-2, Nrl, have been found to be expressed in the lens and are involved in regulating gene expression in the lens [76-78]. Whether the MARE element is the one responsible for complex C4 and plays a role in regulating the MIP gene promoter requires further study.
CBP, NF1 or another factor binding CAT boxes may be responsible for complex C5, binding to the element located at position -69/-83 of the MIP gene, containing an NF1 element and a CAT box [79-81].
Synergism between different transcription factors, some of them ubiquitous and some of them preferentially expressed in selected tissues, is required to achieve precise regulation of tissue-specific gene expression. In fact, non lens-specific transcription factors such as Pax6, Sox1, Nrl are involved in the lens-specific expression of several crystallin genes [78,82].
Various transcription factors may regulate the transcription of the MIP gene. Further investigation of the involvement of Sp1, Sp3, AP2 alpha  and other transcription factors in regulating MIP gene expression will help us to understand the functional roles and synergism of general and tissue-selective factors in lens-specific gene expression.
We thank Devonne Parker-Wilson for assistance in isolating mouse lenses.
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