The transcription factor Sp3 interacts with promoter ... · Animals: One- to three-day old CD-1 mice were obtained from Charles River Laboratory (Raleigh, NC) and handled according

Molecular Vision 1999; 5:12 <http://www.molvis.org/molvis/v5/p12>Received 9 April 1999 | Accepted 13 July 1999 | Published 15 July 1999

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:[email protected]. Kim is now at the National Institute of Child and Human Devel-opment, 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), 2-1Hirosawa, Wako, Saitama 351-01, JAPAN.

Major Intrinsic Protein (MIP) is the most abundant pro-tein of the ocular lens fiber membrane and is a member of anancient family of membrane channel proteins [1]. The MIPgene is specifically expressed in the lens fiber cells, whicharise by differentiation of the lens epithelia, and starts beingexpressed in the primary lens fibers [2]. MIP may play animportant role in maintaining lens transparency by reducingthe interfiber space, as suggested by its ability to function as aweak water channel [3-6] and possibly as an adhesion mol-ecule [7]. Mutations in the MIP gene have been linked to themouse genetic cataracts Fraser mutation (CatFr) and lens opac-ity mutation (lop) [8].

The molecular mechanisms underlying the regulation ofthe lens-specific expression of the MIP gene are largely un-known. To investigate the regulation of the MIP gene, we havepreviously cloned and characterized the human MIP gene. Ourresults indicated that the human MIP gene sequence -253/+42contained an active promoter in primary cultures of lens cellsbut was inactive in non-lens cells [9]. We also characterized acis regulatory element in a domain proximal to the TATA box.This element was required for MIP gene promoter activity inlens cells and contained overlapping Sp1 and AP2 bindingsites [10].

Sp1 is an ubiquitous transcription factor which regulatesthe tissue-specific expression of a variety of genes [11-14].This factor is developmentally regulated [15,16] and essential

for embryonic mouse development [17]. Genes encoding Sp1related 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 highlyconserved in the members of this family, resulting in similarbinding affinities to GGCGGG, CCTCCC and CCACCCmotifs, also known as GC,CT and CA boxes, respectively. Sp1and Sp3 may function either as an activator or as a repressor.Sp1 and Sp3 differ in their activation domains; they both con-tain two glutamine rich domains, but differ in their serine/threonine rich domains [18-24].

In the present study we analyzed the regulatory elementsconserved in the mouse and human MIP gene and their pos-sible interaction with members of the Sp family of transcriptonfactors.

METHODSOligonucleotides: Oligonucleotides were synthesized in a PEBiosystems DNA synthesizer (Foster City, CA) and purifiedeither by G-25 Sephadex columns or by urea-acrylamide gelelectrophoresis.

Antibodies: Antibodies to Sp1, Sp2, Sp3 and Sp4 werefrom Santa Cruz Biotechnology, Inc, Santa Cruz, CA. Sp1was a monoclonal antibody corresponding to amino acidsCKDSEGRGSGDPGKKKQHI [19], Sp2 is a polyclonal an-tibody to amino acids KGTRSNANIQYQAVPQIQAS [18,19],Sp3 is a polyclonal antibody to amino acidsDILTNTEIPLQLVTVSGNET [18,19], Sp4 is a polyclonalantibody to amino acids VTVAAISQDSNPATPNVSTN [18].

Animals: One- to three-day old CD-1 mice were obtainedfrom Charles River Laboratory (Raleigh, NC) and handledaccording to the US Public Health Service Policy on HumanCare and Use of Laboratory Animals.

Nuclear Extracts: Lens nuclear extracts were preparedfrom newborn mice. The intact nuclei from approximately 600newborn (1-3 day-old) mouse lenses were prepared using a 2

© Molecular Vision

The transcription factor Sp3 interacts with promoter elements ofthe 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

Purpose: To characterize the cis regulatory elements and their interaction with transcription factors responsible for thelens 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 correspond-ing 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, alower degree of conservation is observed further upstream. We have found by DNase I footprinting analysis that lensspecific factors may interact with the first 100 bp of 5' flanking sequence. A domain containing an E box, conserved inmouse and human, may interact with a lens specific factor. However, general factors may interact with a NF-1 bindingsite. An overlapping GC and CT box is present in the mouse MIP gene. In the human MIP gene GC and CT boxes arefound in different domains of the MIP gene promoter. Both CT boxes interact with factors present in lens nuclear extractsincluding 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.

M sucrose cushion centrifugation as described [25]. The nu-clei were resuspended in a buffer containing 20 mM Hepes(pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mMDTT, 1 mM PMSF, and the nuclear protein was extracted us-ing small scale preparation as described by Schreiber et al[26]. αTN4, a lens cell line transformed by SV40 [27] wasmaintained in Dulbecco’s modified Eagle’s medium (DMEM)supplemented with 10% fetal calf serum and nuclear extractswere prepared as described [26]. The nuclear protein concen-tration was determined using a commercial assay kit (Bio-Rad Laboratories, Hercules, CA). The nuclear extracts werealiquoted and stored at -80° C. HeLa and NIH3T3 cell nuclearextracts were purchased from Promega Corp.(Madison, WI)and Santa Cruz, respectively.

DNase I Footprinting: The EcoR1/ApaI DNA fragmentcorresponding to mouse MIP gene sequence -461 to +150 wasisolated from the pMOMIP plasmid containing the mouse MIPgenomic DNA (Ohtaka-Maruyama and Chepelinsky, unpub-lished) and purified by low-melting point agarose geleletrophoresis. This ApaI/EcoRI DNA fragment was ligatedinto ApaI/EcoRI-digested pBluescript II SK+ vector(Stratagene, LaJolla, CA). The plasmid containing the ApaI/EcoRI fragment was prepared using CsCl gradient centrifu-gation, and the plasmid was digested with NcoI/EcoNI. TheNcoI/EcoNI correspond to nucleotides -215 and +71, respec-tively. The NcoI/EcoNI fragment was purified using low-melt-ing point agarose gel eletrophoresis. The NcoI site DNA frag-ment was end-labeled with [α-32P]dCTP (3000 Ci/mmol,Amersham, Arlington Heights, IL) by Klenow DNA poly-merase (New England BioLabs, Beverly, MA). The end-la-beled probe was purified using Sephadex G-50 chromatogra-phy (Amersham Pharmacia Biotech, Piscataway, NJ). Thepreparation of G+A Maxam-Gilbert sequencing ladder and theentire procedure of the footprinting were carried out using aSuretrack footprinting kit (Amersham Pharmacia), accordingto the manufacturer’s instructions. Incubation of the labeledprobe (15,000 cpm) with the nuclear extract (15 µg) was fol-lowed by digestion with DNase I, ranging from 0.3 to 3 U.The DNase I digested probe was resolved on a 8% polyacry-lamide/urea gel. The gel was dried and bands were visualized

by autoradiography.Electrophoretic Mobility Shift Assays (EMSA): Single

stranded oligonucleotides were end-labeled with [γ-32P]ATP(5000 Ci/mmol, Amersham) and purified from free nucleotidesby Sephadex G-25 chromatography. Double-stranded probeswere prepared by annealing the end-labeled oligonucleotideswith a 1.2-fold molar excess of unlabeled correspondingcomplementary oligonucleotide. DNA-protein binding reac-tions were performed in a total volume 25 µl containing 2 µgnuclear proteins, 50 fmoles probes (20,000 cpm), and a bufferconsisting 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 µgpoly(dI):poly(dC). In experiments with purified Sp1, thenuclear extract was replaced by purified recombinant humanSp1 (one footprinting unit, Promega). For competition analy-

© Molecular VisionMolecular Vision 1999; 5:12 <http://www.molvis.org/molvis/v5/p12>

Figure 1. Alignment of mouse and human MIP 5'-flanking sequence.Mouse MIP sequence, -216/+47. Human MIP sequence, -214/+47[9]. Conserved sequences are indicated with yellow boxes. GC andCT boxes known to bind purified Sp1 in the human MIP gene [10]and the ones present in the mouse MIP gene, are indicated with solidline boxes. The E box conserved in mouse and human is shown as adotted line box.

Figure 2. DNase I footprinting analysis of mouse MIP gene sequence-213/+71, with mouse and HeLa nuclear extract. A [32P] 5'-end-la-beled NcoI/EcoN1 DNA fragment corresponding to mouse MIP se-quence -215/+71 was incubated with 15 µg lens (lanes 8-13) or HeLa(lanes 2-6) nuclear extracts. The reaction mixtures were then digestedwith DNase I: 0.3 units [lanes 4,11], 1 unit [lanes 5,12], and 3 units[lanes 6 and 13]. Protected regions are indicated with empty boxes tothe right of each autoradiogram. M (lanes 1 and 7), G+A Maxam-Gilbert sequencing ladder. Mouse MIP gene 5'-flanking sequencepresent in the corresponding DNA fragments are indicated to the left.

sis, the competitor probes were prepared by annealing equalmolar complementary oligonucleotides and a 100-fold molarexcess of unlabeled competitor probes was added to the bind-ing reactions. For EMSA with antibodies, 1 µg of specificantibodies against Sp1, Sp2, Sp3 or Sp4 (Santa Cruz Biotech-nology) was added to the respective binding reaction. Afterincubation at room temperature for 30 min, DNA-protein com-plexes were separated from free oligonucleotides by electro-phoresis on a 5% polyacrylamide in a half ionic strength TBEbuffer. DNA-protein complexes were visualized by autorad-iography.

RESULTSThe 5'-flanking sequence of the mouse MIP gene: We hadpreviously isolated the human MIP gene and characterizedseveral cis regulatory elements [9,10,28]. We then turned tothe isolation of the mouse MIP gene to be able to study theinteraction of cis regulatory elements with lens transcriptionfactors from the homologous species. After cloning the mouseMIP gene (Ohtaka-Maruyama and Chepelinsky, unpublished),we compared the mouse MIP 5'-flanking sequence with thehuman orthologous gene [9], using GCG DNA alignment pro-grams, as shown in Figure 1. We found a high degree of iden-tity in the 5'-flanking region proximal to the initiation site of

transcription (90% identity in the -1/-106 sequence of miceand human). However, sequences located further upstreamshow 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 requiredfor promoter activity in the lens [10], is conserved in the mouseand human MIP gene. The human MIP gene has an additionalCT box and a GC box at positions -115/-121 and -147/-152,respectively. However, in the mouse MIP gene, the additionalCT box and the GC box are found overlapping at position -163/-175 (see Figure 1).

We selected a DNA fragment corresponding to 213 bp ofthe mouse MIP gene 5'-flanking sequence and 71 bp of exon 1to study its interaction with nuclear factors by DNase Ifootprinting. We compared the footprinting patterns obtainedwith nuclear extracts prepared from lens with those from HeLacells. As shown in Figure 2, the region spanning approximatelythe first 120 bp of mouse MIP 5'-flanking sequence is not pro-tected from DNase when incubated with HeLa nuclear extract(lanes 2-6). On the contrary, several domains in this region


Figure 3. EMSA with double stranded oligonucleotides correspond-ing to mouse MIP sequences -186/-124, -90/-36, -128/-69 incubatedwith mouse lens, αTN4 (SV), and NIH3T3 (3T3) nuclear extracts.EMSA was performed with the indicated [32P]-end-labeled probeswith 2 µg lens (lanes 2,6,10), αTN4 (lanes 3,7,11) or NIH3T3 (lanes4,8,12) nuclear extracts. DNA-protein complexes formed only withlens nuclear extracts are indicated with arrows as C1, C3, and C4. F:free probe. Lower panel shows DNA sequence of probes correspond-ing to mouse MIP 5'-flanking sequences.

Figure 4. EMSA with double stranded oligonucleotides correspond-ing to mouse MIP sequences -90/-36 and -128/-69 incubated withmouse lens nuclear extract. Competition experiments were performedin the presence of 100-fold molar excess of the indicated unlabeledcompetitors (lanes 3-5, 8-13). C1, complex formed with -90/-36 probe;C4, complex formed with -128/-69 probe; C5, complex formed with-90/-36 and -128/-69 probes. F: free probe. Lower panel shows DNAsequence of probes corresponding to mouse MIP 5'-flanking se-quences. E box is indicated as pink box. CT box is indicated as yel-low box.

are protected by factors present in lens nuclear extracts (lanes8-13). Some differences in the pattern of protection betweenboth extracts are also noticed in the region approximately -150/-170.

We therefore synthesized several overlapping oligonucle-otides, -90/-36, -128/-69, -186/-124, spanning the 5'-flankingsequence -36 to -186 of the mouse MIP gene to analyze theirpossible interaction with lens nuclear factors, and comparedtheir interaction with nuclear extracts from two mouse celllines. One of them, NIH3T3 is derived from mouse fibroblastsand the other one, αTN4 is an SV40 transformed lens cell line[27,29].

Double stranded oligonucleotides corresponding to se-quence -90/-36, -128/-69 and -186/-124 interact with factorspresent in lens nuclear extracts, as shown in Figure 3 (lanes2,6,10). Complex C1 was observed when the probes corre-sponding to sequences -186/-124 and -90/-36 were incubatedwith the lens nuclear extract (lanes 2,6). However, this com-plex was not formed with nuclear extracts prepared fromNIH3T3 (lanes 4,8) nor from αTN4 (lanes 3,7) cell lines.Complex C3 was observed when the probe corresponding tosequence -186/-124 was incubated with the lens nuclear ex-tract (lane 2) but not with nuclear extracts prepared fromNIH3T3 or αTN4 cell lines (lanes 3,4 respectively). The probecorresponding to sequence -128/-69 formed complex C4 when

incubated with lens nuclear extract (lane 10), but not withnuclear extracts prepared from NIH3T3 (lane 12) or αTN4(lane 11) cell lines. As complexes C1, C3 and C4 may be dueto the interaction with lens specific factors, we decided to fur-ther characterize these complexes formed by interaction ofDNA fragments corresponding to the mouse MIP gene 5'-flank-ing sequences with factors present in lens nuclear extracts.

Mouse MIP gene 5'-flanking sequence -106/-90, contain-ing an E box, interacts with lens nuclear factors: When adouble stranded oligonucleotide corresponding to sequence -128/-69 is incubated with lens nuclear extracts, it forms twocomplexes that are competed by the unlabeled probe (C4 and


Figure 5. EMSA with double stranded oligonucleotides correspond-ing to mouse MIP sequences -186/-124 and -90/-36 incubated withmouse lens nuclear extract. Competition experiments were performedin the presence of 100-fold molar excess of the indicated unlabeledcompetitors (lanes 3-6, 9-12). C1, complex formed with -90/-36 and-186/-124 probes; C2 and C3, complexes formed with -186/-124probe; C5, complex formed with -90/-36 probe. F: free probe. CTbox is indicated as yellow box. GC box overlapping with CT box isindicated as blue.

Figure 6. EMSA with double stranded oligonucleotides correspond-ing to wild type mouse MIP sequence -186/-148 and mutants M1 toM5, incubated with mouse lens nuclear extract. Lanes 1,3,5,7,9,11,incubation without nuclear extract. Lower panel shows the wild type(WT) -186/-148 sequence and the corresponding mutations intro-duced in M1 to M5. (*): Mutated nucleotides. Mutant probes areindicated as M1, M2, M3, M4, and M5. The complexes are indicatedas C1, C2, and C3. Mutants M2 (lane 6) and M3 (lane 8) do not formcomplexes C1, C2 and C3. Mutants M1 (lane 4), M4 (lane 10) andM5 (lane 12) form complexes C1, C2 and C3 similar to wild type(lane 2). Only mutations in the CCCCTCCC motif, shown as yellowbox, resulted in complete disappearance of the complexes.

C5, Figure 4 lanes 6,7). Complex C4 appears to be formed bythe interaction of an element present in the region -106/-90, asthis complex is competed by unlabeled competitor -112/-83and -119/-83 but not by -130/-106 or -90/-36 (Figure 4 lanes10,11,9,12-13 respectively). The -106/-90 sequence containsan E box, CAGCTG [30-33], at position -95/-100. C4 is formedwith factor/s present in the lens but not in other cells (see alsoFigure 3). Therefore, these results suggest that the E box maybe an element interacting with a lens specific factor.

The complex C5, which is not lens specific, is competedby unlabeled oligos -130/-106 and -90/-36 (Figure 4, lanes9,12 respectively). Complex C5 is also formed by the labeledprobe -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 factorwith an element present in the region -69/-90. Whether thesame factor, or a different one, forms a complex with an ele-ment present in the region -128/-106, migrating with the samemobility as C5, requires further studies.

Mouse MIP gene 5'-flanking sequence -186/-160, con-taining a CT box, interacts with lens nuclear factors: When adouble stranded oligonucleotide corresponding to sequence -186/-124 is incubated with lens nuclear extracts, three com-plexes 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). Theunlabeled competitor -90/-36 competes with complexes C1and C3 but not with complex C2 (Figure 5, lane 5). When thedouble stranded oligonucleotide corresponding to sequence -90/-36 is incubated with lens nuclear extracts it forms twocomplexes, C1 and C5 (Figure 4, lane 2; Figure 5, lane 8). Asmentioned above, C5 may be formed with an element presentin the region -69/-90 (see Figure 5, lane 11 and Figure 4, lane12). However, C1 is also competed by -186/-124 (Figure 5,lane 10). As both domains share a CT box, one present at po-sition -49/-56 and the other at position -163/-170, they both


Figure 7. Sp3 present in lens nuclear extracts interacts with the CTbox. The [32P]-end-labeled probe -186/-148 was incubated with lensnuclear extract in the absence or presence of antibodies against Sp1(lane 4), Sp2 (lane 6), Sp3 (lane 8) or Sp4 (lane 10). The complexesare indicated as C1, C2, and C3. Only the antibody against Sp3 pro-tein completely abolished the formation of C1 and C3 complexes.

Figure 8. Purified Sp1 interacts with the overlapping GC and CTboxes. Lanes 2,3,5,7 and 9 incubated with purified human Sp1. Lanes1-3, WT oligo-186/-148; lanes 4,5, mutant M1; lanes 6,7, mutantM2; lanes 8,9, mutant M3. C: retarded band; ss: supershifted bandwith Sp1 antibody. Only the mutation M2, that disrupts the overlap-ping GC and CT boxes, shown as blue boxes, resulted in completedisappearance of complex C. Disruption of either the GC or CT boxesalone (M1 and M3) does not affect the formation of complex C.

may be responsible for the formation of complex C1. How-ever, the formation of complexes C2 and C3 may require othersequences surrounding the CT box at position -163/-170.

To address this question, we introduced mutations in theregion -186/-148. The results shown in Figure 6 indicate thatmutations introduced in the CT box present in the probe cor-responding to mouse MIP sequence -186/-148 (mutants M2and M3) abolish the ability to form complexes C1, C2 and C3(Figure 6, lanes 5-8). However, mutants M1, M4 or M5, withmutations outside the CT box, do not affect complex forma-tion (Figure 6, lanes 3, 4,9-12). These results suggest that theCT box present in the mouse MIP sequence -163/-170 is in-volved in the formation of complexes C1, C2 and C3.

The CT box located in the mouse MIP gene 5'-flankingsequence -186/-148 interacts with Sp3: As several membersof the Sp family interact with GC and CT boxes [18,19], weanalyzed whether antibodies to members of the Sp familywould affect the formation of the complexes formed with theCT box. Figure 7 shows the results obtained when the probe -186/-148 was incubated with lens nuclear extracts in the pres-ence of Sp1, Sp2, Sp3 or Sp4 antibodies. Complex C2 is notaffected by any of these four antibodies, suggesting that thiscomplex may be due to the interaction with a factor that is nota member of the Sp family. Neither Sp1, Sp2 nor Sp4 antibod-ies affect the formation of the complexes C1 and C3 (lanes2,4,6,10). However, Sp3 antibody abolishes the formation ofcomplexes C1 and C3 (lanes 2,8), suggesting that Sp3 is theonly member of the Sp family present in lens nuclear extractsthat interacts with the CT box located in the mouse MIP se-quence -163/-170.

Purified Sp1 interacts with the overlapping CT/GC boxeslocated in the mouse MIP gene 5'-flanking sequence -163/-175: We previously showed that a GC box present in the hu-man MIP gene (-160/-129) interacts with Sp1 present in mouselens nuclear extract, indicating the presence of functional Sp1in this extract [10]. Therefore, it was important to determinewhether purified Sp1 was able to interact with the GC/CT boxeslocated in the mouse MIP sequence -186/-148. As shown inFigure 8, lanes 1-3, purified Sp1 is able to interact with anelement present in this domain. When the GC box is mutatedand only the CT box is present (mutant M1), purified Sp1 isstill able to form a complex (Figure 8, lane 5). When the CTbox is mutated and only the GC box is present (mutant M3),purified Sp1 is still able to form a complex (lane 9). The inter-action with purified Sp1 is only abolished in mutant M2, con-taining mutations that disrupt at the same time the overlap-ping GC and CT boxes (Figure 8, lane 7). On the contrary, theinteraction with factors present in lens nuclear extracts is onlyaffected by mutations in the CT box (mutants M2 and M3; seeFigure 6, lanes 6 and 8). Taken together, these results suggestthat even though purified Sp1 is able to interact with the over-lapping GC and CT boxes located in the mouse MIP sequence-163/-175, Sp1 present in lens nuclear extracts is not able tointeract with them. One possible interpretation of the resultscould be that Sp3 present in lens nuclear extracts preventsSp1 binding to the -170 region. However, it is also possiblethat the sequences surrounding the GC box may also play arole, by binding additional transcription factors, which in turnmay prevent Sp1 binding.

DISCUSSIONCloning the MIP gene from human and mouse allowed us toanalyze their non-coding sequence for the presence of pos-sible regulatory elements evolutionarily conserved and respon-sible for the specific expression of this gene in lens fibers. Wefound a high degree of identity in the 5'- flanking region proxi-mal to the initiation site of transcription but a higher level ofdivergence in the sequences located further upstream. The iso-lation of the MIP gene from mouse and the feasibility of pre-paring nuclear extracts from newborn mouse lenses providedus with the tools to analyze the interaction of putative regula-tory elements of the MIP gene with nuclear extracts from thehomologous species.

We previously characterized a regulatory element of thehuman MIP gene, located at position -37/-65, required for pro-moter activity in lens cells [10]. This element contains a CTbox which interacts with the transcription factor Sp1. Wemapped six additional Sp1 binding sites in the -200/+47 re-gion of the human MIP gene [10]. The presence of multipleSp1 binding sites in the same gene results in DNA loopingwhen the Sp1 molecules bound to those sites interact witheach other, establishing in this way interactions between pro-moters and distant regulatory elements and among differenttranscription factors [34-40]. As Sp1 is involved in the tissuespecificity of a variety of genes in many tissues [11-14], itmay also be involved in the lens-specific expression of theMIP gene. The proximal CT box, at position -49/-56, locatedin an element required for promoter activity in lens cells, isconserved in the mouse and human MIP gene. The humanMIP gene contains a second CT box at position-115/-121 anda GC box at position-147/-152. The mouse MIP gene alsocontains two CT boxes and one GC box. However, in the mousethe second CT box, at position -163/-170, overlaps with theGC box located at position -168/-175. Those GC and CT boxesare able to interact with purified Sp1. However, their interac-tion with factors present in lens nuclear extracts differs. BothSp1 and Sp3 genes are expressed in the mouse lens (Ge andChepelinsky, unpublished). The GC box in the human MIPgene is able to interact with Sp1 present in mouse lens nuclearextracts [10]. However, even though the proximal CT boxconserved in the human and mouse gene promoter interactswith purified Sp1, it is not able to interact with Sp1 present inlens nuclear extracts, interacting instead with Sp3 [10]. Theoverlapping CT/GC boxes in the mouse MIP gene, are bothable to interact with purified Sp1 but not with Sp1 present inlens nuclear extracts. Instead, only the CT box interacts withSp3 present in the lens nuclear extracts. The different sequencesflanking the 5'- and 3'- ends of the GC box in the human andmouse MIP gene, adenines or cytidines, respectively, may pre-vent Sp1 binding to the CT box of the mouse gene by interact-ing with additional transcription factors. Alternatively, Sp1 maybe in a non-active form or with low binding affinities due topost-translational modifications and/or other protein-proteininteractions.

Sp1 and Sp3 share a highly conserved three-zinc-fingerDNA binding domain but their protein-protein interaction do-mains are less conserved. Both are able to function as activa-tors or repressors [18-24]. Sp1 has been shown to play a cen-


tral role in the activation of genes expressed in various tissues[41-46]. However, Sp1 and Sp3 may also function as negativeregulators. Negative regulatory elements which interact withSp1 and Sp3 transcription factors have been characterized inthe Id4 gene promoter [47]. Sp1 is a critical negative regula-tor of megakaryocyte-specific α

IIb gene [48]. Sp3 has been

shown to repress the transcriptional activation by Sp1 [49,50].Sp3 may repress Sp1-mediated transactivation through an in-hibitor domain [23,24]. However, Sp3 is also involved in theinduction of the p21 gene promoter during keratinocyte dif-ferentiation [51] and in the activation of the integrin CD11cand b genes in myelomonocytic cells [52]. Sp3 may encodemultiple proteins that activate or repress transcription [23].Both Sp1 and Sp3 appear to be involved in the regulation ofvarious genes [44,46,51]. Changes in Sp1/Sp3 ratios have beenobserved during differentiation of primary humankeratinocytes [53] and changes in GC box binding factors havebeen observed during differentiation of rat lens epithelia ex-plants [54]. Therefore, Sp3 and Sp1 may either play a role inactivating MIP gene expression in lens fibers or in repressingits expression in lens epithelia. Future studies will allow us todiscern between both possibilities.

The DNA-binding and transactivating properties of Sp1are triggered by cAMP-dependent protein kinase A (PKA).Sp1 is activated by PKA and thus Sp1-dependent genes maybe modulated through a cAMP-dependent PKA signaling path-way [13,42,55-58]. Changes in the phosphorylated forms ofSp1 occur during liver differentiation and may play a role inthe growth arrest that occurs during terminal differentiation[16]. Sp1 activity can be modulated by cell cycle-regulatorssuch as cyclins and members of the Rb and E2F families [59-64]. Rb may be directly or indirectly involved in Sp1-DNAbinding activity by liberating Sp1 from a Sp1-inhibitor thatalso interacts with Rb [62]. The retinoblastoma gene productRb interacts with Sp1 and Sp3 and synergistically activatesSp1 or Sp3 mediated transcription [61]. Rb plays a role inwithdrawal from the cell cycle in differentiating lens fiber cellsand MIP expression is markedly decreased in Rb -/- mice [65],suggesting the possibility that Rb interaction with Sp1 and/orSp3 may play a role in activating MIP gene expression in thelens fibers. Sp1 interacts with E2F1, -2 and 3 and both areable to activate transcription synergistically [63,64]. The in-teraction of Sp1 with E2F1 is cell cycle specific and occursduring mid- to late G1 [64]. Five members of the E2F familyare expressed in lens epithelia; however only E2F-1, -3 and -5 are expressed in the lens fibers[66]. There are multiple phos-phorylated forms of Rb in lens epithelia but predominantlythe hypophosphorylated form in lens fibers. pRb and p107seem to be the primary regulators of E2F activity in lens fi-bers [66,67]. Interaction of Rb with Sp1 and Sp3 may regu-late the interaction of Rb with E2F, suggesting a role for Sp1and 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, form-ing complexes C1 and C3, which may be lens-specific. How-ever, it also interacts with another factor, which is not a mem-ber of the Sp family and appears to be also expressed in othertissues, forming complex C2. Other transcription factors thatalso interact with CT boxes, like the zinc finger protein MAZ,

the single strand CT-binding factors hnRNP K or CNBP, havebeen characterized [68,69]. The factor that forms complex C2may very well be one of these CT box-binding factor or anovel one. Further studies are needed to characterize this fac-tor, which appears to be expressed in other tissues besides thelens.

Another complex, C4, may be lens-specific and involvesthe E box located in the MIP gene at position -94/-99. Thiselement is conserved in the mouse and human orthologousgene. E-boxes contain the CANNTG motif and interact withproteins belonging to the basic helix-loop-helix (bHLH) fam-ily of transcription factors [30-33]. The bHLH proteins acti-vate the expression of various tissue specific genes by form-ing heterodimers between ubiquitous and cell-specific familymembers, such as MyoD in muscle, BETA2 in pancreas orCapsulin in epicardial progenitors and mesenchyme of vis-ceral organs [70-73]. The nucleotide sequence at the NN posi-tions in the E box determines the specificity of binding to dif-ferent members of the bHLH family of transcription factors.They either belong to the class A or class B subfamily. The Ebox present in the MIP gene, 5'-CACAGCTGTG-3', shows aperfect dyad of symmetry. The E box containing the motifCAGCTG interact with the class B of bHLH proteins.

The MyoD and AP4 helix-loop-helix proteins interact withthis same E box sequence [73,74]. Whether AP4 or anothermember of the class B of basic helix-loop-helix of transcrip-tion factors is expressed in the lens and interacts with the Ebox, present in the 5'-flanking region of the MIP gene to regu-late promoter expression in the lens, requires further studies.

Interestingly, a domain in the Na,K-ATPase alpha 2 sub-unit gene promoter has been characterized, containing GC,CT and E boxes. The E box functions as a negative regulatoryelement and the GC and CT boxes function as positive regula-tory elements [75]. Similarly, in the human Id4 gene promoter,an E box, GC and CT boxes have been characterized, wherebHLH-zip factor, Sp1 and Sp3 interact respectively, playing arole in activating or repressing this gene. Repression of Id genesoccurs during differentiation of many cell lineages [47]. Theseresults raise the possibility that the interaction of the bHLHfactor that interacts with the MIP gene E box, may also inter-act with Sp1 and/or Sp3 to regulate MIP gene expression inthe lens.

There is is a MARE motif (Maf regulatory element) over-lapping with the E box, located at position -93/-98. Severalmembers of the Maf family of transcription factors, L-maf,maf-1, maf-2, Nrl, have been found to be expressed in thelens and are involved in regulating gene expression in the lens[76-78]. Whether the MARE element is the one responsiblefor complex C4 and plays a role in regulating the MIP genepromoter requires further study.

CBP, NF1 or another factor binding CAT boxes may beresponsible for complex C5, binding to the element located atposition -69/-83 of the MIP gene, containing an NF1 elementand a CAT box [79-81].

Synergism between different transcription factors, someof them ubiquitous and some of them preferentially expressedin selected tissues, is required to achieve precise regulation oftissue-specific gene expression. In fact, non lens-specific tran-scription 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 transcrip-

tion of the MIP gene. Further investigation of the involve-ment of Sp1, Sp3, AP2 alpha [83] and other transcription fac-tors in regulating MIP gene expression will help us to under-stand the functional roles and synergism of general and tis-sue-selective factors in lens-specific gene expression.

ACKNOWLEDGEMENTSWe thank Devonne Parker-Wilson for assistance in isolatingmouse lenses.

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