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Regulation of the Dlx3 Homeobox Gene upon Differentiation of Mouse Keratinocytes

Regulation of the Dlx3 Homeobox Gene upon Differentiation of Mouse Keratinocytes THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 37, Issue of September 10, pp. 26599 –26608, 1999 Printed in U.S.A. Regulation of the Dlx3 Homeobox Gene upon Differentiation of Mouse Keratinocytes* (Received for publication, March 18, 1999, and in revised form, May 25, 1999) Geon Tae Park and Maria I. Morasso‡ From the Laboratory of Skin Biology, NIAMS, National Institutes of Health, Bethesda, Maryland 20892 The Distal-less Dlx3 homeodomain gene is expressed sential role of PKC signaling in the late stages of epidermal differentiation. Activation of PKC has been shown to be neces- in terminally differentiated murine epidermal cells, and there is evidence to support an essential role as a tran- sary for expression of late differentiation markers loricrin and scriptional regulator of the terminal differentiation profilaggrin and for the suppression of the spinous-specific process in these cells. In an attempt to determine the markers K1 and K10 (6). factors that induce Dlx3 gene expression, we have Dlx33, a murine ortholog of the Drosophila Distal-less home- cloned the 1.2-kilobase pair proximal region of murine odomain protein (7), is a member of the Dlx vertebrate family. gene and analyzed its cis-regulatory elements and po- This family comprises to date six genes identified both in tential trans-acting factors. The proximal region of the mouse and human and found to be organized as three conver- Dlx3 gene has a canonical TATA box and CCAAT box, gently transcribed pairs, each closely linked to one of the four and the transcription start site was located 205 base mammalian Hox clusters (8 –13). Disruption of the DLX3 cod- pairs upstream from the initiation of translation site. ing sequence has been associated with a human disorder, Tri- Serial deletion analysis showed that the region between cho-Dento-Osseous syndrome. This inherited autosomal domi- 284 and 234 confers the maximal promoter activity both nant disorder is characterized by defects in ectodermal in undifferentiated and differentiated primary mouse derivatives such as hair and teeth and craniofacial bone abnor- keratinocytes. Gel retardation assays and mutational malities (14). Dlx3 is expressed in the granular layer of the analysis demonstrated that the transcriptional regula- epidermis and in the hair matrix cells of the hair follicle (15, tor NF-Y (also referred to as CBF) binds to a CCAAT box 16), and there is evidence strongly supporting the critical role motif within this region and is responsible for the ma- of the Dlx3 homeoprotein in the regulation of expression of late jority of the Dlx3 promoter activity. In addition, an Sp1- epidermal differentiation genes (15). In vitro studies have binding site was located immediately upstream of tran- shown that Dlx3 binds to an AT-rich region and acts as a scription start site that acts as a positive regulatory positive transcriptional regulator (17), which is activated dur- element of the Dlx3 promoter, independent of the CCAAT box motif. Importantly, elements residing be- ing the Ca shift in keratinocytes induced to differentiate in tween 130 to 160 of the Dlx3 gene are responsible for culture (18). In transgenic mice, ectopic expression of Dlx3 in -dependent induction of Dlx3 during keratino- the Ca basal cells is accompanied by the cessation of cell proliferation cyte differentiation. and the up-regulation of expression of late epidermal differen- tiation structural genes including profilaggrin (15). A potential binding site for Dlx3 has been identified in the profilaggrin During epidermal differentiation, mitotically active basal gene, suggesting that the observed up-regulation of this gene in keratinocytes cease to proliferate, detach from the basement the transgenic mice may result from a direct effect of Dlx3 (15). membrane, and migrate through the spinous and granular Altogether, these data strongly support a role for Dlx3 as a layers to the outermost terminally differentiated cornified determinant factor in the activation of expression of granular layer of the skin. This cornification process is tightly associated markers during the terminal differentiation of keratinocytes. with a stepwise program of transcriptional regulation and is During the process of terminal epidermal differentiation, concurrent with the sequential induction and repression of many genes expressed in the keratinocyte are regulated at the structural and enzymatic differentiation-specific markers (1). transcriptional level (1). The transcription factors AP1 and AP2 This process can be achieved in mouse keratinocytes cultivated have been characterized as primary regulatory factors of kera- in vitro by increasing the Ca concentration from 0.05 to 0.12 tinocyte gene expression (19 –24). PKC is an upstream compo- mM in the culture medium (2), which produces a situation that nent of the pathway that regulates AP1 in many systems and mimics the endogenous Ca gradient present in the skin (3). may play a role in the epidermal differentiation expression of The Ca signaling differentiation pathway is associated with K5, K1, loricrin, profilaggrin, and involucrin (19, 21–24). Mem- increased phospholipase C activity (4) and activation of protein bers of the POU family of transcription factors such as Oct1, kinase C (PKC) (5). Previous work has demonstrated an es- Oct2, Oct6, Skn1a, and Skn1i have also been implicated as regulators of epidermal genes (25–28). In order to elucidate the role of Dlx3 in the cascade of tran- * The costs of publication of this article were defrayed in part by the scriptional events that ultimately leads to terminal differenti- payment of page charges. This article must therefore be hereby marked ation, we have cloned and characterized the mouse Dlx3 pro- “advertisement” in accordance with 18 U.S.C. Section 1734 solely to moter. Deletional promoter analysis was utilized to delineate indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted the sequences that regulate the transcription of Dlx3 in differ- TM to the GenBank /EBI Data Bank with accession number(s) AF167581. entiated and undifferentiated keratinocytes. In turn, these cis- ‡ To whom all correspondence should be addressed: Laboratory of Skin Biology, Bldg. 6, Rm. 134, NIAMS, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-402-2888; Fax: 301-402-2886; E-mail: [email protected]. phenicol acetyltransferase; PCR, polymerase chain reaction; bp, base The abbreviations used are: PKC, protein kinase C; CAT, chloram- pair. This paper is available on line at http://www.jbc.org 26599 This is an Open Access article under the CC BY license. 26600 Regulation of the Dlx3 Homeobox Gene CGGGAACTGGACTTTGAGA-39, for 21213 to 21189). The lowercase acting elements were used to identify the transcription factors and underlined letters correspond to restriction enzyme sites for SalI that regulate the Dlx3 promoter. Importantly, we have identi- and XbaI, and lowercase letters are the modified nucleotides. Each PCR fied a region residing between 110 and 160 that responds to product was digested with SalI and XbaI and inserted into the pCAT- the Ca shift used to differentiate the keratinocytes in basic vector. culture. Cell Culture—Primary mouse keratinocytes were isolated from BALB/c newborn mouse skins and grown in Eagle’s minimal essential MATERIALS AND METHODS 21 medium lacking Ca , with 8% Chelex-treated fetal bovine serum (2, Cloning of Upstream Regulatory Region of the Mouse Dlx3 Gene—To 18). Ca concentrations were determined by analysis in an atomic clone the upstream sequence of the Dlx3 gene we used the Genome- absorption spectrophotometer. Unless otherwise indicated, the Ca Walker Kit (CLONTECH). It is designed to amplify a specific genomic concentration of the medium was adjusted to 0.05 mM to maintain a DNA using an adapter sequence attached to the genomic DNA and basal cell-like population of undifferentiated cells. specific oligomers based on known sequence of the gene of interest. We Transfection and CAT Assays—Primary mouse keratinocytes were made gene-specific oligomers located 45 bp upstream of the translation transfected using the Lipofectin reagent (Life Technologies, Inc.). Typ- start site and used an oligomer located 30 bp further upstream as a ically, 1 mg of each CAT construct was used to transfect cells plated and nested primer. After a second round of PCR, a 754-bp DNA fragment cultured in 6-well plates coated with rat tail collagen (0.1 mg/ml). After was obtained and sequenced. To obtain a larger 59-regulatory sequence, 4 h of incubation, cells were treated with 15% glycerol in KSF medium genome walking was performed using the oligomers based on the newly (Life Technologies, Inc.) for 3.5 min and then maintained either in cloned DNA upstream sequence for the Dlx3 gene. After PCR, we medium with Ca concentration of 0.05 or 1.4 mM. CAT activities were obtained a 617-bp DNA fragment. The two overlapping DNA fragments determined 48 h after transfection and normalized to protein value at were cloned into the XbaI and SalI sites of the pCAT-basic vector A . CAT activities were measured by the fluor diffusion CAT assay (Promega). using Econofluor premixed scintillation fluid and [ H]acetyl coenzyme Plasmids Construction—The 21213 to 1160 upstream DNA frag- A (NEN Life Science Products) in the linear range of the assay. Results ment of the Dlx3 gene was inserted into a promoterless CAT plasmid are expressed as the counts/min transferred to the organic solvent (pCAT-basic; Promega) and designated as p1213CAT. All 59-deleted phase. Each transfection was done in duplicate, and the experiment clones were constructed by PCR using the oligonucleotides DEL1 (59- was repeated at least three times. cgcgtcgacCACAGGTCGGTCATTCAGGAC-39; 21029 to 21009), DEL2 Mapping of the Transcriptional Start Site—To determine the 11 site, (59-cgcgtcgacGACTTCCTGAAGAACACAGAA-39; 2827 to 2807), DEL3 we used the rapid amplification of cDNA ends method, as described by (59-cgcgtcgacTCCAGTAGGGACTTGCAGGCC-39; 2692 to 2672), Frohman et al. (29). The steps of reverse transcription, tailing, and PCR DEL4 (59-cgcgtcgacCGGAGAAACCCTGTCTCAAAAA-39; 2572 to were performed using the 59-rapid amplification of cDNA ends system 2552), DEL5 (59-cgcgtcgacGCCACTTTCT GTCTGTCATTT-39; 2437 to (Life Technologies, Inc.). The gene-specific oligomer was located at 2417), DEL6 (59-cgcgtcgacCTCCGCACAGCCAACCCCTCC-39; 2284 to 1124 bp from the translation initiation site of Dlx3. The final PCR 2264), DEL7 (59-cgcgtcgacTTAGGGGTAACAACAAAGAGG-39; 2162 products were cloned and sequenced to determine the extreme 59 mRNA to 2142), DEL8 (59-cgcgtcgacAGACTTGCAGCCAATCAGCGC-39; 284 sequence. to 264), DEL9 (59-cgcgtcgacTGAGTCTATAACCGGCTGGCC-39; 234 to Preparation of Nuclear Extracts—Nuclear extracts were prepared 214), and 160R (59-cgctctagaAACGGGCGGAGGAGCCCAGGT-39; from undifferentiated and differentiated primary mouse keratinocyte 1160 to 1140) with the p1213CAT vector as the template. The lower- cultures as described by Andrews and Faller (30). All steps were carried case letters correspond to extensions to the Dlx3-specific sequence with out at 4 °C. The cells (1 3 10 ) were harvested, washed three times with the underlined letters corresponding to restriction enzyme sites for SalI ice-cold phosphate-buffered saline, and pelleted. The keratinocytes and XbaI. Each PCR product was digested with SalI and XbaI and were resuspended in 0.4 ml of buffer A (10 mM HEPES, pH 7.9, 1.5 mM inserted into the pCAT-basic vector. The 39-deleted clones, p60(1)CAT MgCl ,10mM KCl, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl and p30(1)CAT, were constructed using the oligonucleotides 60R (59- fluoride), incubated on ice for 15 min, and then homogenized with a cgctctagaGCCGGCTGTGCCTCAGTCGCT-39; 160 to 140) and 30R (59- Dounce homogenizer B pestle for about 20 –25 strokes. The homogenate cgctctagaTCTCCGTGTCCCAAGCCACAG-39; 130 to 110), respec- was centrifuged at 14,000 rpm, and the nuclear pellet was resuspended tively, and DEL9. The shortened 39-deleted clone, p10(1)CAT, was in 0.2 ml of buffer B (20 mM HEPES, pH 7.9, 25% (v/v) glycerol, 0.42 M constructed using two oligonucleotides 10F (59-cgcgtcgacTGAGTC- NaCl, 1.5 mM MgCl , 0.2 mM EDTA, 1 mM dithiothreitol, and 1 mM TATAACCGGCTGGCCGGGCGGAGCTGGCAGCATTTGACT-39) and phenylmethylsulfonyl fluoride) and incubated on ice for 30 min. After 10R (59-cgctctagaAGTCAAATGCTGCCAGCTCCGCCCGGCCAGCCG- centrifugation at 14,000 rpm for 20 min at 4 °C, the supernatant frac- GTTATAGACTCA-39), which were annealed, digested with SalI and tion was aliquoted and stored at 270 °C. The protein concentration was XbaI, and inserted into the pCAT-basic vector. The other 39-deleted determined using the Bradford assay following the methods described clones, p1213(1)60CAT, p1213(1)30CAT, and p1213(1)10CAT, were for the Bio-Rad Protein Assay. constructed using the oligonucleotides 160R, 130R, 110R, and (59-cg- Gel Retardation Analysis—Gel retardation analysis was carried out cgtcgacATTACCCGGGAACTGGACTTTGAGA-39, 21213 to 21189), according to Ausubel et al. (31). P-End-labeled DNA fragments (about respectively. The general PCR conditions to generate the deletional 1 ng of DNA) were incubated for 15 min at 4 °C with nuclear extracts in fragments were 30 cycles of denaturation at 94 °C for 45 s, annealing at a total volume of 15 ml of binding buffer (10 mM HEPES, pH 7.9, 60 mM 55 °C for 45 s, and extension at 72 °C for 1 min. To make the KCl, 1 mM EDTA, 10% glycerol, 10 mM MgCl ) containing 2 mgof p84mSPCAT and p34mSPCAT constructs containing mutant Sp1-bind- poly(dI-dC). The products of the DNA-protein binding reaction were ing sites, site-directed mutagenesis was performed using the QuickC- separated by electrophoresis on a nondenaturing 6% polyacrylamide gel hange site-directed mutagenesis kit and the following conditions sugg- in low salt TBE buffer containing 44 mM Tris, 44 mM boric acid, and 1 ested in the protocol from the manufacturer (Stratagene). The two mM EDTA. DNA-protein complexes and unbound DNA probe were oligonucleotides DlxSpm5 (59-CCGGCTGGCCGatatGAGCTGGCAG- visualized in the gel by autoradiography on x-ray film. For the compe- 39), and DlxSpm3 (59-CTGCCAGCTcatatCGGCCAGCCGG-39) were tition experiments, various amounts of unlabeled DNA were added at designed and used for mutagenesis, with lowercase letters being the the beginning of the binding reaction. For gel mobility supershift anal- modified nucleotides. The CCAAT box mutant clone, p84mCAT, was ysis, the binding reactions were performed as described above, except constructed using the oligonucleotide M6 (59-AGACTTGCAtaaggaCA- that the nuclear extracts were incubated for 30 min at 4 °C with GCGCGCAGG-39) and 160R by PCR. The 39-deletion mutant constructs variable amounts of specific antisera prior to the addition of the radio- were made by PCR using oligonucleotides containing each mutant site labeled probe. The antisera raised against NF-1, C/EBP, Sp1, Sp2, Sp3, (59-cgctctagaGCCGGCTGTCGGTCAGTCGCTatacGCCTC-39) for M1C- Sp4, c-Jun, c-Fos, JunB, and JunD were purchased from Santa Cruz AT, (59-cgctctagaGCCGGCTGTCGGTCAGTCtagtCGTGCCTC-39) for Biotechnology, Inc.; the NF-YA antibody was purchased from Rockland M2CAT, (59-cgctctagaGCCGGCTGTCGGTCAactaCTGCGTGCCTC-39) Inc. for M3CAT, (59-cgctctagaGCCGGCTGTCGagacGTCGCTGCGTGCCT- RNA Blotting—Total RNA was isolated from keratinocytes cultured C-39) for M4CAT, (59-cgctctagaGCCGGCTtgatGTCAGTCGCTGCGTG- in medium containing 0.05, 0.12, or 1.4 mM Ca using Trizol following CCTC-39) for M5CAT, (59-cgctctagaGCCGaagtTCGGTCAGTCGCTGC- the instructions provided by the manufacturer (Life Technologies, Inc.). GTGCCTC-39) for M6CAT, (59-cgctctagaaattGCTGTCGGTCAGTCGC- The RNA samples (2 mg) were separated in a 1.2% agarose/methylmer- TGCGTGCCTC-39) for M7CAT, (59-cgctctagaGCCGGCTGTCGagacact- cury hydroxide gel (32), transferred to a nylon membrane, and hybrid- aCTGCGTGCCTC-39) for M34CAT, (59-cgctctagaGCCGaagtTCGGTCA- ized according to Church and Gilbert (33). The blot was hybridized with actaCTGCGTGCCTC-39) for M36CAT, (59-cgctctagaGCCGaagtTCGag- a 1.1-kilobase pair mouse Dlx3 cDNA probe (16), and a rat glyceralde- acGTCGCTGCGTGCCTC-39) for M46CAT, and (59-cgcgtcgacATTACC- hyde-3-phosphate dehydrogenase probe (34) was used as control, la- Regulation of the Dlx3 Homeobox Gene 26601 FIG.1. Dlx3 expression is up-regulated during differentiation induced by Ca . Northern blot analysis of 2 mg of total RNA from primary mouse keratinocytes cultured in 0.05, 0.12, and 1.4 mM Ca containing medium for the indicated times (in hours after Ca addi- tion). A 1.1-kilobase pair Dlx3 cDNA fragment was used as a hybrid- ization probe. The same RNA samples were hybridized with a glycer- aldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe for normalization. beled with P by random priming (Stratagene), and washed at high stringency (0.13 SSC, 0.1% SDS at 65 °C). The hybridizing bands were visualized by autoradiography on x-ray film and quantitated with a TD932 Macbeth densitometer using linear exposure. UV Cross-linking Assay—The 59-end-labeled probe was incubated with primary mouse keratinocyte nuclear extract under standard mo- bility shift assay conditions in the presence of 500 ng of poly(dI-dC) in a 30-ml binding reaction at room temperature. The reaction mixture was irradiated with an UV transilluminator (254 nm, 2400 microwatts/ cm ) for 20 min at 4 °C, and the complex was loaded on a 10% SDS- polyacrylamide gel. The gel was dried, and the proteins directly in- volved in the DNA interaction were detected by autoradiography. RESULTS FIG.2. Upstream sequence of the mouse Dlx3 gene. The major transcription start site is indicated with a bold arrow. The canonical Induction of Dlx3 Expression by Ca in Mouse Keratinocytes CCAAT box and TATA box are boxed. The putative transcription factor Cultured in Vitro—Northern blot analysis of total RNA from binding sites are underlined. The translation start site is in italics. The mouse keratinocytes cultured in 0.05 mM Ca medium re- numbers on the left indicate the nucleotide number with respect to the vealed very low levels of Dlx3 transcript (Fig. 1). However, after transcription start site. 12 h, Dlx3 expression increased 3.5-fold in keratinocytes in- 21 21 21 duced to differentiate by Ca increase (to 0.12 mM). As shown Ca ) or in cells differentiated in vitro by addition of Ca (1.4 in Fig. 1, the expression increased in a time-dependent manner mM Ca ) (Fig. 3). An increase in activity of all constructs was in 0.12 mM Ca medium, and by 12 h an 8-fold increased observed upon differentiation. This increase is coincident with expression of Dlx3 was observed in keratinocytes cultured at the results obtained for the endogenous gene shown in Fig. 1 by 1.4 mM. Northern blot analysis. In 0.05 mM Ca , the p84CAT construct Cloning and Analysis of the Upstream Sequence of the Mu- showed the highest activity, almost 3-fold increased activity rine Dlx3 Gene—We cloned two overlapping genomic DNA se- when compared with the p162CAT. The p34CAT construct quences of the Dlx3 upstream regulatory region (754 bp and containing the TATA box and putative Sp1-binding site showed 617 bp, see “Materials and Methods”), which combined were a 3-fold decrease in CAT activity when compared with the cloned as 1416 bp of upstream sequence from the translation p84CAT, even though the activity is still higher than that of the start site (Fig. 2). The transcription start site was determined full-length promoter (p1213CAT). These results indicated that using the rapid amplification of cDNA ends procedure (29) and there are, at least, one negative regulatory and one positive was located 205 bp upstream from the translation start site. regulatory region in the upstream sequence of Dlx3 gene, which Genomic DNA sequence analysis revealed that the upstream are located between 2162/284 and 284/234, respectively. The region of Dlx3 gene contains canonical CCAAT and TATA p1213CAT construct showed about 3.5-fold increased tran- boxes. Comparison of the proximal promoters of the Xenopus, scriptional activity by Ca addition. Similar induction by human, and mouse Dlx3 genes showed a striking degree of Ca addition was measured for each deletion construct, and as conservation. Particularly the mouse and human upstream shown in Fig. 3, the activity of the p34CAT construct is still at 21 21. regions exhibit about 91% homology in the region located be- least 2-fold higher in 1.4 mM Ca than in 0.05 mM Ca tween 2104 and 161 (data not shown), suggesting the possi- The Transcription Factor NF-Y Binds to the CCAAT Box and bility of conserved mechanisms of regulation for Dlx3 orthologs Plays an Important Role in the Dlx3 Promoter—By deletional during evolution. analysis, we showed that the region between 284 and 234 Deletion Analysis of the Dlx3 Promoter—Comparing the se- played a major regulatory role in the activity of the Dlx3 quence of the 21213 to 1160 fragment of the Dlx3 gene with promoter. To examine if a protein bound to this region and to the data base for consensus transcription factor binding se- define the nucleotides required for the formation of the com- quences, putative sites for AP1, GRE, OCT1, NFkB, and Sp1 plex, the 50-bp oligonucleotide (284 to 234; d oligonucleotide) were identified (Fig. 2). This information was taken into con- was used as a probe in gel shift analysis, and three overlapping sideration when serial deletion plasmids were constructed by oligomers (d1– d3) were used as competitors (Fig. 4A). One PCR in order to identify the regulatory elements with a func- major complex was found, and by competition analysis, binding tional role in Dlx3 gene expression. These constructs were was located to the proximal part of the region (between 284 transfected into primary mouse keratinocytes, and promoter and 259) (Fig. 4B). Better resolution of the specific nucleotides activity was determined by measuring CAT activity in cells required for formation of the complex was obtained using oli- maintained in an undifferentiated state (cultured in 0.05 mM gonucleotides with specific 3-bp mutations (Fig. 4C). Competi- 26602 Regulation of the Dlx3 Homeobox Gene FIG.3. Effect of 5*-deletions on the transcriptional activity of the mouse Dlx3 promoter. Primary mouse keratinocytes were transfected with each indicated deletion construct. The transfected cells were incubated with growth medium containing 0.05 or 1.4 mM Ca concentration, and the CAT activity of each plate of cells was measured. CAT activity was normalized to the protein concentration measured by the Bradford assay. The bars represent the average normalized CAT activity of duplicate plates from three experiments for each construct. FIG.4. Delineation of a binding site within the region between 284 and 234 that binds nuclear factors from keratinocytes. A, the DNA sequence between 284 and 234 (oligomer d) and oligomers used in the competitive gel shift assay (d1– d3); the CCAAT box is underlined. B, the d oligomer (284/234) was labeled and used as a probe in gel shift assay with nuclear extracts of keratinocytes (3 mg). In competition assays, the nuclear extracts were mixed with each competing oligonucleotide (100-fold molar excess) prior to addition of the P-labeled probe. The data presented in this figure are representative of electromobility shift assays that were performed at least three separate times with the same results. C, the DNA sequence for oligonucleotide d1 is shown, along with the location of the 3-bp mutations. The putative CCAAT motif is boxed. The serial 3-bp mutated sequences are underlined in each mutated oligomer (M1–M6). D, competition binding assay with P-labeled d1 oligomer used as a probe and competitor oligomers at 100-fold molar excess (d1 and M1–M6). Identical results were observed with multiple nuclear extract preparations. tion gel shift analysis with wild-type (d1) and mutant oligonu- been reported to bind selectively the CCAAT box binding pro- cleotides (M1–M6) showed that competitors M1, M2, and M3 teins were used as competitors (Fig. 5A). The CCAAT box bound to the specific complex (Fig. 4D). In contrast, oligonu- binding factors include NF-Y (also known as CBF), the C/EBP cleotides M4, M5, and M6 (which has the combined mutation of family, and NF-1 (35). As shown in Fig. 5B, the albumin C M4 and M5) failed to compete. These results indicated that the element, which is known to bind NF-Y (36), fully competed the complex was due to binding in the CCAAT region (Fig. 5B). To formation of the complex (Fig. 5B, left panel, 3rd lane). In determine the nature of the binding, oligonucleotides that have contrast, the consensus NF-1 and C/EBP oligonucleotides did Regulation of the Dlx3 Homeobox Gene 26603 FIG.6. Functional analysis of the Dlx3 promoter CCAAT box motif. A, the diagram illustrates the p84CAT and p34CAT deletion constructs and the sequence of the mutation substituting the CCAAT box motif. B, primary mouse keratinocytes were transfected with p84CAT, p84mCAT, and p34CAT and incubated with medium contain- ing 0.05 or 1.4 mM Ca . After 48 h, cells were harvested. The CAT activity was normalized to the protein concentration measured by the Bradford assay. The bars represent the average normalized CAT activ- FIG.5. Identification of the nuclear protein from undifferen- ity of three experiments done on duplicate plates for each construct. tiated and differentiated mouse keratinocytes that binds to the CCAAT box of Dlx3 promoter. A, DNA sequences used in gel shift analyses. The d1 sequence along with the albumin C element (NF-Y), NF-Y is a heterotrimeric transcription factor composed of C/EBP, and NF1 consensus sequences are shown. B, nuclear extract three subunits, A, B, and C (37– 43). The subunits A and C from undifferentiated (left panel) and differentiated (right panel) pri- 32 together form a heterodimer, which then interacts with the mary mouse keratinocytes (3 mg) were used to shift a P-labeled d1 subunit B to form the heterotrimeric NF-Y protein. This het- probe. The competitors, albumin C element (Alb C), consensus C/EBP, and NF-1 (Santa Cruz Biotechnology), were added at 100-fold molar erotrimeric NF-Y then binds to DNA to form an NF-Y-DNA excess. Top arrow indicates the protein-DNA complex formed between complex. To examine whether the complex consisted of all three nuclear extract and d1 probe. C, nuclear extracts from primary mouse subunits, we checked the molecular weight of the complex keratinocytes (3 mg) were incubated with 0.1, 0.2, or 0.4 mg of antibodies using the UV cross-linking assay (Fig. 5D). The complex (indi- against the NF-Y A-subunit, C/EBP, or NF-1 for 30 min on ice. P- Labeled d1 oligomer was added, and the preparation was separated by cated by arrow) which competed with self-oligonucleotide (d1) electrophoresis. The top arrow indicates supershifted complex when had an estimated molecular mass of ;100 kDa. This molecular using antibody against NF-Y. D, UV cross-linking assay was performed mass coincides with the approximate expected combined mo- to estimate the molecular weight of the protein(s) binding to the CCAAT lecular mass of subunits NF-YA, -B, and -C (35, 25, and ;40 box of the Dlx3 promoter (see “Materials and Methods”). For the com- petition analysis, the nonradioactive competitor oligonucleotides were kDa, respectively) (44). This result strongly supported that added at 100-fold molar excess. Arrow indicates the specific complex NF-Y was binding to the CCAAT box motif of Dlx3 promoter in formed. The numbers on the left are the molecular masses of protein its heterotrimeric form. standards. The function of the Dlx3 promoter CCAAT box was assessed by mutational analysis. Primary mouse keratinocytes were not compete for formation of the complex. The same pattern of transfected with the wild-type p84CAT construct, the mutant competition was found using nuclear extracts either from un- p84mCAT construct, and the p34CAT construct (Fig. 6A). The differentiated or differentiated keratinocytes (Fig. 5B, right CAT activity conferred by each construct was assayed in tran- panel). These results suggested that the nuclear protein that siently transfected cells grown in 0.05 and 1.4 mM Ca (Fig. bound to the CCAAT box motif in the Dlx3 promoter was NF-Y. 6B). The CAT activity of the mutant construct was reduced to To confirm this directly, supershift experiments were per- 40 and 25% as compared with the wild-type p84CAT construct formed with specific antisera (Fig. 5C). By using the labeled in the undifferentiated and differentiated cells, respectively. wild-type d1 oligonucleotide, either anti-NF-Y, anti-C/EBP, or These results underscore the functional importance of the anti-NF-1 antisera were added in increasing amounts to undif- CCAAT box for the activity of the Dlx3 promoter. ferentiated mouse primary keratinocyte nuclear extracts. Anti- Functional Sp1-binding Site in the Dlx3 Promoter—In addi- NF-Y antibody supershifted the complex with each amount, tion to CCAAT box sequence, analysis of the proximal promoter whereas the anti-C/EBP or anti-NF-1 antibody did not affect region revealed a putative recognition site for Sp1 transcription the mobility. These observations indicated that the specific factor, which was located between the TATA box and transcrip- complex contained the NF-Y transcription factor. tion start site (213 to 23). To examine this putative Sp1- 26604 Regulation of the Dlx3 Homeobox Gene ferentiated keratinocytes and to 62 and 72% of each respective wild-type in differentiated keratinocytes (Fig. 8). These results showed that the Sp1-binding site of Dlx3 promoter plays a positive regulatory role, and its function is not dependent on the CCAAT box-binding protein, NF-Y. Defining the Region of the Dlx3 Promoter Responsible for Ca Inducibility—Despite the finding that the binding sites for NF-Y and Sp1 play an important role in the transcriptional regulation of the Dlx3 gene, mutation of these sequences did not affect the activation of Dlx3 by Ca (Fig. 6 and 8). Serial 39-deletion constructs were made on the p34CAT construct, which contains the 160 bp of untranslated region (Fig. 9A). The constructs were made in an attempt to delineate the region responsible for the Ca inducibility. Each construct was trans- fected into primary mouse keratinocytes, and CAT activity was measured in 0.05 and 1.4 mM Ca conditions (Fig. 9A). The p60(1)CAT deletion retained the capability of induction by Ca , but the p10(1)CAT deletion showed no induction of CAT activity with Ca increase (Fig. 9A). Similar 39-deletions were made on the p1213CAT construct. These constructs were trans- fected into primary mouse keratinocytes (Fig. 9B), and the CAT activity indicated that the p1213(1)160CAT as well as the p1213(1)60CAT were induced 2.5-fold, whereas the p1213(1)30CAT was induced only 1.5-fold. In contrast, the p1213(1)10CAT deletion was not induced with Ca . Our find- ings indicated that the region located between 110 and 160 was indispensable for induction by Ca of the Dlx3 promoter. Characterization of the Ca -responsible Region of Dlx3 Pro- moter—Based on the results of the serial deletion analysis of the Dlx3 promoter (Fig. 9), the region between 130 and 160 has the element(s) that mediate the Ca induction. A muta- tional analysis was performed to determine the specific nucle- otides that conform the responsive element. Serial and double 4-bp mutations were made in the region between 130 and 160 FIG.7. Identification of nuclear proteins binding to the GC- in the context of the p1213(1)60CAT construct. These con- rich site of Dlx3 promoter. A, the sequences of oligonucleotides structs were transfected into primary mouse keratinocytes, containing the GC-rich putative Sp1-binding site (underlined), mutant cultured for 48 h in 0.05 or 1.4 mM Ca concentrations, and the Sp1-binding site (underlined), and consensus Sp1-binding site (under- lined) are shown. B,3 mg of nuclear extracts from undifferentiated (left CAT activity was measured. As shown in the Fig. 10A, the panel) or differentiated (right panel) keratinocytes were used to shift M1CAT, M2CAT, M5CAT, and M7CAT showed reduced pro- P-labeled dsp oligonucleotide as described. Nonradioactive competitor moter activity at 0.05 and 1.4 mM Ca , but the fold activation Sp1 consensus (Santa Cruz Biotechnology) and dspM oligonucleotides by Ca did not change significantly when compared with the were added at a 100-fold molar excess. Arrows indicate the protein- DNA complex formed between nuclear extract (3 mg) and the dsp probe. p1213(1)60CAT. In contrast, the M4CAT and M6CAT showed C, antibodies to Sp1, Sp2, Sp3, and Sp4 (Santa Cruz Biotechnology) increased promoter activity in 0.05 and 1.4 mM Ca and were used to test which members of Sp1 family are associated with the 21 slightly increased fold activation by Ca . These results sug- Sp1-binding site of Dlx3 promoter. The arrows indicate the specific gest that these sites mainly affect the basal promoter activity of complex formed and the supershifted complexes after addition of anti- bodies raised against Sp1 and Sp3 proteins. Dlx3. Interestingly, the M3CAT construct showed dramatic reduction in the fold activation by Ca , similar to that of p1213(1)30CAT. These results indicated that the nucleotides binding site, gel shift analysis was performed using an oligo- (142-CGAC-145) are crucial for the activation by Ca . Double nucleotide corresponding to 221 to 22 on the Dlx3 promoter mutant constructs (M34CAT, M36CAT, and M46CAT, Fig. (dsp), and a mutant form of this oligonucleotide (dspM). Nu- 10A) were made and assayed for CAT activity. The double clear extracts from undifferentiated and differentiated primary mutant constructs containing the M3 site (M34CAT and mouse keratinocytes were used in the gel shift analysis (Fig. M36CAT) showed reduced fold activation by Ca , but the 7A). As shown in Fig. 7B, the complexes formed were competed M46CAT mutation had no effect. These results again delin- with wild-type dsp and Sp1 consensus oligonucleotides but not eated the sequence (142-CGAC-145) as primarily responsible with the mutant form of dsp (dspM). In gel shift assays using for the Ca induction of Dlx3 promoter. antibodies corresponding to each of the Sp1-like family mem- We were interested in determining by gel shift analysis if bers, the Sp1 and Sp3 antisera supershifted the two complexes there were nuclear proteins that bound the 130/160 region formed between the Sp1-binding site of Dlx3 promoter and nuclear proteins (Fig. 7C). These results indicate that both of (CA30). The CA30 oligonucleotide was used as a probe in gel shift mobility assays with nuclear extracts from mouse primary these Sp1-like factors are able to bind the Sp1 site on the Dlx3 promoter. keratinocytes. One major complex I and two weak bands II and III were identified (Fig. 10B). These complexes were competed DNA constructs containing the mutant Sp1-binding site (Spm) in the p84CAT and p34CAT clones were transfected into with 100-fold excess of unlabeled cold CA30 oligomer. To ex- mouse keratinocytes to examine the function of this region (Fig. amine the relationship between the sequences affecting the 8). The CAT activity of p84spmCAT and p34spmCAT was re- Dlx3 promoter activity and these complexes, the oligomers duced to 55 and 66% of the wild types, respectively, in undif- containing each mutant site were used as competitors. The M3 Regulation of the Dlx3 Homeobox Gene 26605 FIG.8. Mutational analysis of the Sp1-binding site in the Dlx3 promoter. The diagram (left) illustrates the p84CAT, p34CAT, p84spmCAT, and p34spmCAT deletion and mutant constructs. The sequence of the mutation of the Sp1-binding site is the same shown in Fig. 7A. Primary mouse keratinocytes were transfected with p84CAT, p34CAT, p84spmCAT, or p34spmCAT, incubated with growth medium containing 0.05 or 1.4 mM Ca , and harvested after 48 h. The CAT activity was normalized to the protein concentration measured by the Bradford assay. The bars represent the average normalized CAT activity of duplicate plates for each construct (right panel). All experiments in this figure were repeated three times with similar results. FIG.9. Deletion analysis and func- tion of sequence residing down- stream of the transcription initiation site. A, p34CAT constructs with decreas- ing downstream promoter deletions were transfected into primary mouse keratino- cytes and incubated with growth medium containing either 0.05 or 1.4 mM Ca . CAT activity was assayed 48 h after transfection. B, p1213CAT constructs with decreasing downstream promoter deletions were transfected into keratino- cytes and assayed as described above. The bars represent the average normalized CAT activity of duplicate plates for each construct. All experiments in the figure were repeated in triplicate with similar results. DISCUSSION oligomer competed the complexes II and III, but did not com- pletely compete the major complex I. The M4, M6, and M34 The cascade of events that leads to terminal differentiation oligomers showed similar results for the complex I but did not can be triggered in primary mouse keratinocytes by the eleva- compete the complexes II and III. In contrast, none of the tion of extracellular Ca . The expression of the Dlx3 homeobox complexes were competed by the M36 oligomer that contains gene is restricted to the suprabasal layer of the epidermis (18), mutations in the M3 and M6 sites. These results indicated that and this expression is dramatically increased in primary mouse keratinocytes induced to differentiate by Ca in vitro. Evi- the M3 and M6 sites are important in the binding of the major dence obtained in transgenic mice suggests the role of Dlx3 as complex I and that the M4 site is partially involved in the a positive transcriptional activator of differentiation-specific formation of this complex. epidermal structural genes. In an attempt to decipher the The region between 130 and 160 contains an element (146- different stages of the pathway that culminate in terminal TGACCGA-152) with sequence homology to a consensus AP1- differentiation, we have cloned and characterized the upstream binding site. It has been suggested that the AP1 transcription 21 promoter sequence of the murine Dlx3 gene, and we deter- factor is involved in the Ca inducibility of epidermal differ- mined the regulatory elements necessary for expression of the entiation-specific markers (45). To determine the possible par- gene in keratinocytes. By sequence comparison we have found ticipation of AP1 in the protein-DNA complex formation with that the region close to the transcription start site of the mouse CA30, the AP1 consensus oligonucleotide and antibodies Dlx3 gene (2110 to 161) has striking homology with that of the against AP1 family members were used in competition and Xenopus and human dlx3 genes. In transgenic mice, 470 bp of supershift assays. As shown in Fig. 10C, the complexes formed the Xenopus Dlx3 promoter conferred an expression pattern to by CA30 were not competed by AP1 consensus oligonucleotide, the b-galactosidase reporter that was indistinguishable from and the supershift assays corroborated that AP1 family mem- that of the endogenous gene, including the Ca response (46). bers (c-Jun, c-Fos, JunB, and JunD) do not bind to this region. Thus, it could be hypothesized that the cis- and trans-regula- Therefore, the sequence (142-CGAC-145) present in the CA30 tory elements important in the regulation of the Dlx3 orthologs region is responsible for the transcriptional up-regulation of have been conserved through evolution and are contained Dlx3 promoter by Ca , through the binding of nuclear factors within the proximal promoter region. that are not members of the AP1 family of transcription factors. To understand the mechanism(s) that control the regulation 26606 Regulation of the Dlx3 Homeobox Gene FIG. 10. Characterization of Ca -responsible region of Dlx3 promoter. A, the region between 130 and 160 of Dlx3 promoter was mutated with serial or double 4-bp modified bases by the PCR method in the p1213(1)60CAT. Each mutated construct, p1213(1)60CAT, p1213(1)30CAT, and p1213(1)10CAT, was transfected into primary mouse keratinocytes. Underlined lowercase letters indicate the modified nucleotides. CAT assay was performed the same as in Fig. 3. The bars represent the average normalized CAT activity of duplicate plates for each construct. All experiments in the figure were repeated in triplicate with similar results. B, gel shift assay was performed using the oligonucleotide corresponding to the region between 130 and 160 (CA30) as a probe and 3 mg of nuclear extract of primary mouse keratinocytes. Incubations were carried out in the presence or absence of a 100-fold excess of unlabeled wild-type and mutant oligonucleotides. Arrows indicate the protein-DNA complexes, and roman numbers indicate each individual complex (I–III). C, supershift analysis was carried out using the antibodies against the AP1 family. Nuclear extracts were preincubated with 2 mg of each antibody as indicated, followed by incubation with labeled CA30 probe. The 3rd lane corresponds to the gel shift analysis performed in the presence of 100-fold excess unlabeled AP1 consensus oligonucleotide. All experiments in this figure were repeated at least three times with similar results. Regulation of the Dlx3 Homeobox Gene 26607 and expression of the Dlx3 promoter during epidermal differ- From the family of Sp1-related transcription factors, studies entiation, our studies examined the expression of Dlx3 promot- have shown that Sp3 acts as an antagonist to Sp1. The human er/CAT constructs in primary mouse keratinocytes to identify transcobalamin II gene is controlled by the relative ratios of putative regulatory elements that are required for Dlx3 expres- Sp1 and Sp3 (64), and activation of the human papilloma virus type 16 promoter correlates with the ratios of Sp1/Sp3 during sion. The data presented here demonstrate the existence of one positive regulatory region in the Dlx3 promoter located be- epithelial differentiation (65). We have shown that the Sp1 consensus motif on the Dlx3 promoter can bind specifically Sp1 tween 284 and 234. We determined that the CCAAT box in and Sp3, providing the possibility of antagonistic effects on this region exerts a significant positive influence on the Dlx3 transcription depending on the specific levels of each of these promoter activity in undifferentiated and differentiated kera- factors throughout the terminal differentiation process. tinocytes. Competition gel shift analysis and supershift analy- An essential aspect of the transcriptional regulation of the sis further demonstrated that the transcription factor NF-Y is Dlx3 promoter is the elucidation of the mechanism for inducing the protein that binds specifically to the CCAAT box motif. its expression by increases in external Ca during differenti- Mutation of this motif dramatically reduced transcription of ation. Until now, the prevailing evidence points to the involve- the Dlx3 promoter. Several genes also contain canonical sites ment of PKC isozymes in the induction of keratinocyte differ- for CCAAT-binding proteins that have been described to be entiation markers by Ca , although the exact mechanism is important in early functions of preinitiation complex formation unclear. In this study we show that the region located between (36). CCAAT boxes are typically located within 100 bp of the 110 and 160 is important for Ca inducibility of the Dlx3 transcription start sites, and CCAAT box binding factors in- gene. By mutational and gel shift analysis of the 130/160 clude NF-Y (also known as CBF), C/EBP family members, and sequence, we found that the crucial element responsible for NF-1 (35). NF-Y was originally identified as a ubiquitously Ca inducibility is located between 142 and 145 (CGAC) and expressed protein that binds to the Y box motif, defined as an that nuclear factor(s) are involved in the up-regulation of Dlx3 inverted CCAAT box motif in all major histocompatibility com- expression by Ca . It has been reported that the AP1 and Ets plex class II genes (47). NF-Y is a heterotrimeric transcription transcription factors are involved in the regulation of the hu- factor composed of three subunits (37– 43). Formation of a man SPRR1A keratinocyte terminal differentiation marker complex between the A and C subunits is required to bind the (45). However, in the case of the Dlx3 gene, the AP1 transcrip- B subunit, and together, the heterotrimeric complex binds tion factor does not bind to the 142/145 or 146/152 sequences, DNA (48). The activation domains of both B and C subunits, which strongly supports that it is not the determinant for the which are rich in glutamine and hydrophobic residues, share Ca inducibility of Dlx3. Future work will determine the spe- protein sequence homology with each other and with the glu- cific nature of the nuclear factor(s) involved in the binding to tamine-rich activation domain of the transcription factor Sp1 this regulatory element. The identification of a Ca response (48). element in the Dlx3 gene is the first link between the extracel- NF-Y has been demonstrated to act as both a positive (49 – lular signal and the transcriptional control of a regulatory gene 51) and a negative (52) regulator of transcription. Several stud- involved in keratinocyte differentiation, and represents an im- ies suggest that NF-Y acts by stabilizing the binding of addi- portant step in the elucidation of the molecular mechanisms tional factors to adjacent regulatory elements, such as RFX in underlying this developmental program. the major histocompatibility complex class II promoter (53, 54). NF-Y also interacts with transcription factors binding up- Acknowledgments—We thank Dr. Ulrike Lichti and members of the Laboratory of Skin Carcinogenesis for the Ca determination and for stream elements and basal transcription machinery, such as providing reagents. We are grateful to Drs. Peter Steinert, Janine HNF4 and TAF100 (42, 55). Recently it was shown that the Bryan, Andres Buonanno, and Thomas Sargent for reading and provid- interaction of NF-Y with cAMP-response element-binding pro- ing comments on the manuscript. 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Regulation of the Dlx3 Homeobox Gene upon Differentiation of Mouse Keratinocytes

Park, Geon Tae; Morasso, Maria I.
Journal of Biological Chemistry – Sep 1, 1999
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 37, Issue of September 10, pp. 26599 –26608, 1999 Printed in U.S.A. Regulation of the Dlx3 Homeobox Gene upon Differentiation of Mouse Keratinocytes* (Received for publication, March 18, 1999, and in revised form, May 25, 1999) Geon Tae Park and Maria I. Morasso‡ From the Laboratory of Skin Biology, NIAMS, National Institutes of Health, Bethesda, Maryland 20892 The Distal-less Dlx3 homeodomain gene is expressed sential role of PKC signaling in the late stages of epidermal differentiation. Activation of PKC has been shown to be neces- in terminally differentiated murine epidermal cells, and there is evidence to support an essential role as a tran- sary for expression of late differentiation markers loricrin and scriptional regulator of the terminal differentiation profilaggrin and for the suppression of the spinous-specific process in these cells. In an attempt to determine the markers K1 and K10 (6). factors that induce Dlx3 gene expression, we have Dlx33, a murine ortholog of the Drosophila Distal-less home- cloned the 1.2-kilobase pair proximal region of murine odomain protein (7), is a member of the Dlx vertebrate family. gene and analyzed its cis-regulatory elements and po- This family comprises to date six genes identified both in tential trans-acting factors. The proximal region of the mouse and human and found to be organized as three conver- Dlx3 gene has a canonical TATA box and CCAAT box, gently transcribed pairs, each closely linked to one of the four and the transcription start site was located 205 base mammalian Hox clusters (8 –13). Disruption of the DLX3 cod- pairs upstream from the initiation of translation site. ing sequence has been associated with a human disorder, Tri- Serial deletion analysis showed that the region between cho-Dento-Osseous syndrome. This inherited autosomal domi- 284 and 234 confers the maximal promoter activity both nant disorder is characterized by defects in ectodermal in undifferentiated and differentiated primary mouse derivatives such as hair and teeth and craniofacial bone abnor- keratinocytes. Gel retardation assays and mutational malities (14). Dlx3 is expressed in the granular layer of the analysis demonstrated that the transcriptional regula- epidermis and in the hair matrix cells of the hair follicle (15, tor NF-Y (also referred to as CBF) binds to a CCAAT box 16), and there is evidence strongly supporting the critical role motif within this region and is responsible for the ma- of the Dlx3 homeoprotein in the regulation of expression of late jority of the Dlx3 promoter activity. In addition, an Sp1- epidermal differentiation genes (15). In vitro studies have binding site was located immediately upstream of tran- shown that Dlx3 binds to an AT-rich region and acts as a scription start site that acts as a positive regulatory positive transcriptional regulator (17), which is activated dur- element of the Dlx3 promoter, independent of the CCAAT box motif. Importantly, elements residing be- ing the Ca shift in keratinocytes induced to differentiate in tween 130 to 160 of the Dlx3 gene are responsible for culture (18). In transgenic mice, ectopic expression of Dlx3 in -dependent induction of Dlx3 during keratino- the Ca basal cells is accompanied by the cessation of cell proliferation cyte differentiation. and the up-regulation of expression of late epidermal differen- tiation structural genes including profilaggrin (15). A potential binding site for Dlx3 has been identified in the profilaggrin During epidermal differentiation, mitotically active basal gene, suggesting that the observed up-regulation of this gene in keratinocytes cease to proliferate, detach from the basement the transgenic mice may result from a direct effect of Dlx3 (15). membrane, and migrate through the spinous and granular Altogether, these data strongly support a role for Dlx3 as a layers to the outermost terminally differentiated cornified determinant factor in the activation of expression of granular layer of the skin. This cornification process is tightly associated markers during the terminal differentiation of keratinocytes. with a stepwise program of transcriptional regulation and is During the process of terminal epidermal differentiation, concurrent with the sequential induction and repression of many genes expressed in the keratinocyte are regulated at the structural and enzymatic differentiation-specific markers (1). transcriptional level (1). The transcription factors AP1 and AP2 This process can be achieved in mouse keratinocytes cultivated have been characterized as primary regulatory factors of kera- in vitro by increasing the Ca concentration from 0.05 to 0.12 tinocyte gene expression (19 –24). PKC is an upstream compo- mM in the culture medium (2), which produces a situation that nent of the pathway that regulates AP1 in many systems and mimics the endogenous Ca gradient present in the skin (3). may play a role in the epidermal differentiation expression of The Ca signaling differentiation pathway is associated with K5, K1, loricrin, profilaggrin, and involucrin (19, 21–24). Mem- increased phospholipase C activity (4) and activation of protein bers of the POU family of transcription factors such as Oct1, kinase C (PKC) (5). Previous work has demonstrated an es- Oct2, Oct6, Skn1a, and Skn1i have also been implicated as regulators of epidermal genes (25–28). In order to elucidate the role of Dlx3 in the cascade of tran- * The costs of publication of this article were defrayed in part by the scriptional events that ultimately leads to terminal differenti- payment of page charges. This article must therefore be hereby marked ation, we have cloned and characterized the mouse Dlx3 pro- “advertisement” in accordance with 18 U.S.C. Section 1734 solely to moter. Deletional promoter analysis was utilized to delineate indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted the sequences that regulate the transcription of Dlx3 in differ- TM to the GenBank /EBI Data Bank with accession number(s) AF167581. entiated and undifferentiated keratinocytes. In turn, these cis- ‡ To whom all correspondence should be addressed: Laboratory of Skin Biology, Bldg. 6, Rm. 134, NIAMS, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-402-2888; Fax: 301-402-2886; E-mail: [email protected]. phenicol acetyltransferase; PCR, polymerase chain reaction; bp, base The abbreviations used are: PKC, protein kinase C; CAT, chloram- pair. This paper is available on line at http://www.jbc.org 26599 This is an Open Access article under the CC BY license. 26600 Regulation of the Dlx3 Homeobox Gene CGGGAACTGGACTTTGAGA-39, for 21213 to 21189). The lowercase acting elements were used to identify the transcription factors and underlined letters correspond to restriction enzyme sites for SalI that regulate the Dlx3 promoter. Importantly, we have identi- and XbaI, and lowercase letters are the modified nucleotides. Each PCR fied a region residing between 110 and 160 that responds to product was digested with SalI and XbaI and inserted into the pCAT- the Ca shift used to differentiate the keratinocytes in basic vector. culture. Cell Culture—Primary mouse keratinocytes were isolated from BALB/c newborn mouse skins and grown in Eagle’s minimal essential MATERIALS AND METHODS 21 medium lacking Ca , with 8% Chelex-treated fetal bovine serum (2, Cloning of Upstream Regulatory Region of the Mouse Dlx3 Gene—To 18). Ca concentrations were determined by analysis in an atomic clone the upstream sequence of the Dlx3 gene we used the Genome- absorption spectrophotometer. Unless otherwise indicated, the Ca Walker Kit (CLONTECH). It is designed to amplify a specific genomic concentration of the medium was adjusted to 0.05 mM to maintain a DNA using an adapter sequence attached to the genomic DNA and basal cell-like population of undifferentiated cells. specific oligomers based on known sequence of the gene of interest. We Transfection and CAT Assays—Primary mouse keratinocytes were made gene-specific oligomers located 45 bp upstream of the translation transfected using the Lipofectin reagent (Life Technologies, Inc.). Typ- start site and used an oligomer located 30 bp further upstream as a ically, 1 mg of each CAT construct was used to transfect cells plated and nested primer. After a second round of PCR, a 754-bp DNA fragment cultured in 6-well plates coated with rat tail collagen (0.1 mg/ml). After was obtained and sequenced. To obtain a larger 59-regulatory sequence, 4 h of incubation, cells were treated with 15% glycerol in KSF medium genome walking was performed using the oligomers based on the newly (Life Technologies, Inc.) for 3.5 min and then maintained either in cloned DNA upstream sequence for the Dlx3 gene. After PCR, we medium with Ca concentration of 0.05 or 1.4 mM. CAT activities were obtained a 617-bp DNA fragment. The two overlapping DNA fragments determined 48 h after transfection and normalized to protein value at were cloned into the XbaI and SalI sites of the pCAT-basic vector A . CAT activities were measured by the fluor diffusion CAT assay (Promega). using Econofluor premixed scintillation fluid and [ H]acetyl coenzyme Plasmids Construction—The 21213 to 1160 upstream DNA frag- A (NEN Life Science Products) in the linear range of the assay. Results ment of the Dlx3 gene was inserted into a promoterless CAT plasmid are expressed as the counts/min transferred to the organic solvent (pCAT-basic; Promega) and designated as p1213CAT. All 59-deleted phase. Each transfection was done in duplicate, and the experiment clones were constructed by PCR using the oligonucleotides DEL1 (59- was repeated at least three times. cgcgtcgacCACAGGTCGGTCATTCAGGAC-39; 21029 to 21009), DEL2 Mapping of the Transcriptional Start Site—To determine the 11 site, (59-cgcgtcgacGACTTCCTGAAGAACACAGAA-39; 2827 to 2807), DEL3 we used the rapid amplification of cDNA ends method, as described by (59-cgcgtcgacTCCAGTAGGGACTTGCAGGCC-39; 2692 to 2672), Frohman et al. (29). The steps of reverse transcription, tailing, and PCR DEL4 (59-cgcgtcgacCGGAGAAACCCTGTCTCAAAAA-39; 2572 to were performed using the 59-rapid amplification of cDNA ends system 2552), DEL5 (59-cgcgtcgacGCCACTTTCT GTCTGTCATTT-39; 2437 to (Life Technologies, Inc.). The gene-specific oligomer was located at 2417), DEL6 (59-cgcgtcgacCTCCGCACAGCCAACCCCTCC-39; 2284 to 1124 bp from the translation initiation site of Dlx3. The final PCR 2264), DEL7 (59-cgcgtcgacTTAGGGGTAACAACAAAGAGG-39; 2162 products were cloned and sequenced to determine the extreme 59 mRNA to 2142), DEL8 (59-cgcgtcgacAGACTTGCAGCCAATCAGCGC-39; 284 sequence. to 264), DEL9 (59-cgcgtcgacTGAGTCTATAACCGGCTGGCC-39; 234 to Preparation of Nuclear Extracts—Nuclear extracts were prepared 214), and 160R (59-cgctctagaAACGGGCGGAGGAGCCCAGGT-39; from undifferentiated and differentiated primary mouse keratinocyte 1160 to 1140) with the p1213CAT vector as the template. The lower- cultures as described by Andrews and Faller (30). All steps were carried case letters correspond to extensions to the Dlx3-specific sequence with out at 4 °C. The cells (1 3 10 ) were harvested, washed three times with the underlined letters corresponding to restriction enzyme sites for SalI ice-cold phosphate-buffered saline, and pelleted. The keratinocytes and XbaI. Each PCR product was digested with SalI and XbaI and were resuspended in 0.4 ml of buffer A (10 mM HEPES, pH 7.9, 1.5 mM inserted into the pCAT-basic vector. The 39-deleted clones, p60(1)CAT MgCl ,10mM KCl, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl and p30(1)CAT, were constructed using the oligonucleotides 60R (59- fluoride), incubated on ice for 15 min, and then homogenized with a cgctctagaGCCGGCTGTGCCTCAGTCGCT-39; 160 to 140) and 30R (59- Dounce homogenizer B pestle for about 20 –25 strokes. The homogenate cgctctagaTCTCCGTGTCCCAAGCCACAG-39; 130 to 110), respec- was centrifuged at 14,000 rpm, and the nuclear pellet was resuspended tively, and DEL9. The shortened 39-deleted clone, p10(1)CAT, was in 0.2 ml of buffer B (20 mM HEPES, pH 7.9, 25% (v/v) glycerol, 0.42 M constructed using two oligonucleotides 10F (59-cgcgtcgacTGAGTC- NaCl, 1.5 mM MgCl , 0.2 mM EDTA, 1 mM dithiothreitol, and 1 mM TATAACCGGCTGGCCGGGCGGAGCTGGCAGCATTTGACT-39) and phenylmethylsulfonyl fluoride) and incubated on ice for 30 min. After 10R (59-cgctctagaAGTCAAATGCTGCCAGCTCCGCCCGGCCAGCCG- centrifugation at 14,000 rpm for 20 min at 4 °C, the supernatant frac- GTTATAGACTCA-39), which were annealed, digested with SalI and tion was aliquoted and stored at 270 °C. The protein concentration was XbaI, and inserted into the pCAT-basic vector. The other 39-deleted determined using the Bradford assay following the methods described clones, p1213(1)60CAT, p1213(1)30CAT, and p1213(1)10CAT, were for the Bio-Rad Protein Assay. constructed using the oligonucleotides 160R, 130R, 110R, and (59-cg- Gel Retardation Analysis—Gel retardation analysis was carried out cgtcgacATTACCCGGGAACTGGACTTTGAGA-39, 21213 to 21189), according to Ausubel et al. (31). P-End-labeled DNA fragments (about respectively. The general PCR conditions to generate the deletional 1 ng of DNA) were incubated for 15 min at 4 °C with nuclear extracts in fragments were 30 cycles of denaturation at 94 °C for 45 s, annealing at a total volume of 15 ml of binding buffer (10 mM HEPES, pH 7.9, 60 mM 55 °C for 45 s, and extension at 72 °C for 1 min. To make the KCl, 1 mM EDTA, 10% glycerol, 10 mM MgCl ) containing 2 mgof p84mSPCAT and p34mSPCAT constructs containing mutant Sp1-bind- poly(dI-dC). The products of the DNA-protein binding reaction were ing sites, site-directed mutagenesis was performed using the QuickC- separated by electrophoresis on a nondenaturing 6% polyacrylamide gel hange site-directed mutagenesis kit and the following conditions sugg- in low salt TBE buffer containing 44 mM Tris, 44 mM boric acid, and 1 ested in the protocol from the manufacturer (Stratagene). The two mM EDTA. DNA-protein complexes and unbound DNA probe were oligonucleotides DlxSpm5 (59-CCGGCTGGCCGatatGAGCTGGCAG- visualized in the gel by autoradiography on x-ray film. For the compe- 39), and DlxSpm3 (59-CTGCCAGCTcatatCGGCCAGCCGG-39) were tition experiments, various amounts of unlabeled DNA were added at designed and used for mutagenesis, with lowercase letters being the the beginning of the binding reaction. For gel mobility supershift anal- modified nucleotides. The CCAAT box mutant clone, p84mCAT, was ysis, the binding reactions were performed as described above, except constructed using the oligonucleotide M6 (59-AGACTTGCAtaaggaCA- that the nuclear extracts were incubated for 30 min at 4 °C with GCGCGCAGG-39) and 160R by PCR. The 39-deletion mutant constructs variable amounts of specific antisera prior to the addition of the radio- were made by PCR using oligonucleotides containing each mutant site labeled probe. The antisera raised against NF-1, C/EBP, Sp1, Sp2, Sp3, (59-cgctctagaGCCGGCTGTCGGTCAGTCGCTatacGCCTC-39) for M1C- Sp4, c-Jun, c-Fos, JunB, and JunD were purchased from Santa Cruz AT, (59-cgctctagaGCCGGCTGTCGGTCAGTCtagtCGTGCCTC-39) for Biotechnology, Inc.; the NF-YA antibody was purchased from Rockland M2CAT, (59-cgctctagaGCCGGCTGTCGGTCAactaCTGCGTGCCTC-39) Inc. for M3CAT, (59-cgctctagaGCCGGCTGTCGagacGTCGCTGCGTGCCT- RNA Blotting—Total RNA was isolated from keratinocytes cultured C-39) for M4CAT, (59-cgctctagaGCCGGCTtgatGTCAGTCGCTGCGTG- in medium containing 0.05, 0.12, or 1.4 mM Ca using Trizol following CCTC-39) for M5CAT, (59-cgctctagaGCCGaagtTCGGTCAGTCGCTGC- the instructions provided by the manufacturer (Life Technologies, Inc.). GTGCCTC-39) for M6CAT, (59-cgctctagaaattGCTGTCGGTCAGTCGC- The RNA samples (2 mg) were separated in a 1.2% agarose/methylmer- TGCGTGCCTC-39) for M7CAT, (59-cgctctagaGCCGGCTGTCGagacact- cury hydroxide gel (32), transferred to a nylon membrane, and hybrid- aCTGCGTGCCTC-39) for M34CAT, (59-cgctctagaGCCGaagtTCGGTCA- ized according to Church and Gilbert (33). The blot was hybridized with actaCTGCGTGCCTC-39) for M36CAT, (59-cgctctagaGCCGaagtTCGag- a 1.1-kilobase pair mouse Dlx3 cDNA probe (16), and a rat glyceralde- acGTCGCTGCGTGCCTC-39) for M46CAT, and (59-cgcgtcgacATTACC- hyde-3-phosphate dehydrogenase probe (34) was used as control, la- Regulation of the Dlx3 Homeobox Gene 26601 FIG.1. Dlx3 expression is up-regulated during differentiation induced by Ca . Northern blot analysis of 2 mg of total RNA from primary mouse keratinocytes cultured in 0.05, 0.12, and 1.4 mM Ca containing medium for the indicated times (in hours after Ca addi- tion). A 1.1-kilobase pair Dlx3 cDNA fragment was used as a hybrid- ization probe. The same RNA samples were hybridized with a glycer- aldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe for normalization. beled with P by random priming (Stratagene), and washed at high stringency (0.13 SSC, 0.1% SDS at 65 °C). The hybridizing bands were visualized by autoradiography on x-ray film and quantitated with a TD932 Macbeth densitometer using linear exposure. UV Cross-linking Assay—The 59-end-labeled probe was incubated with primary mouse keratinocyte nuclear extract under standard mo- bility shift assay conditions in the presence of 500 ng of poly(dI-dC) in a 30-ml binding reaction at room temperature. The reaction mixture was irradiated with an UV transilluminator (254 nm, 2400 microwatts/ cm ) for 20 min at 4 °C, and the complex was loaded on a 10% SDS- polyacrylamide gel. The gel was dried, and the proteins directly in- volved in the DNA interaction were detected by autoradiography. RESULTS FIG.2. Upstream sequence of the mouse Dlx3 gene. The major transcription start site is indicated with a bold arrow. The canonical Induction of Dlx3 Expression by Ca in Mouse Keratinocytes CCAAT box and TATA box are boxed. The putative transcription factor Cultured in Vitro—Northern blot analysis of total RNA from binding sites are underlined. The translation start site is in italics. The mouse keratinocytes cultured in 0.05 mM Ca medium re- numbers on the left indicate the nucleotide number with respect to the vealed very low levels of Dlx3 transcript (Fig. 1). However, after transcription start site. 12 h, Dlx3 expression increased 3.5-fold in keratinocytes in- 21 21 21 duced to differentiate by Ca increase (to 0.12 mM). As shown Ca ) or in cells differentiated in vitro by addition of Ca (1.4 in Fig. 1, the expression increased in a time-dependent manner mM Ca ) (Fig. 3). An increase in activity of all constructs was in 0.12 mM Ca medium, and by 12 h an 8-fold increased observed upon differentiation. This increase is coincident with expression of Dlx3 was observed in keratinocytes cultured at the results obtained for the endogenous gene shown in Fig. 1 by 1.4 mM. Northern blot analysis. In 0.05 mM Ca , the p84CAT construct Cloning and Analysis of the Upstream Sequence of the Mu- showed the highest activity, almost 3-fold increased activity rine Dlx3 Gene—We cloned two overlapping genomic DNA se- when compared with the p162CAT. The p34CAT construct quences of the Dlx3 upstream regulatory region (754 bp and containing the TATA box and putative Sp1-binding site showed 617 bp, see “Materials and Methods”), which combined were a 3-fold decrease in CAT activity when compared with the cloned as 1416 bp of upstream sequence from the translation p84CAT, even though the activity is still higher than that of the start site (Fig. 2). The transcription start site was determined full-length promoter (p1213CAT). These results indicated that using the rapid amplification of cDNA ends procedure (29) and there are, at least, one negative regulatory and one positive was located 205 bp upstream from the translation start site. regulatory region in the upstream sequence of Dlx3 gene, which Genomic DNA sequence analysis revealed that the upstream are located between 2162/284 and 284/234, respectively. The region of Dlx3 gene contains canonical CCAAT and TATA p1213CAT construct showed about 3.5-fold increased tran- boxes. Comparison of the proximal promoters of the Xenopus, scriptional activity by Ca addition. Similar induction by human, and mouse Dlx3 genes showed a striking degree of Ca addition was measured for each deletion construct, and as conservation. Particularly the mouse and human upstream shown in Fig. 3, the activity of the p34CAT construct is still at 21 21. regions exhibit about 91% homology in the region located be- least 2-fold higher in 1.4 mM Ca than in 0.05 mM Ca tween 2104 and 161 (data not shown), suggesting the possi- The Transcription Factor NF-Y Binds to the CCAAT Box and bility of conserved mechanisms of regulation for Dlx3 orthologs Plays an Important Role in the Dlx3 Promoter—By deletional during evolution. analysis, we showed that the region between 284 and 234 Deletion Analysis of the Dlx3 Promoter—Comparing the se- played a major regulatory role in the activity of the Dlx3 quence of the 21213 to 1160 fragment of the Dlx3 gene with promoter. To examine if a protein bound to this region and to the data base for consensus transcription factor binding se- define the nucleotides required for the formation of the com- quences, putative sites for AP1, GRE, OCT1, NFkB, and Sp1 plex, the 50-bp oligonucleotide (284 to 234; d oligonucleotide) were identified (Fig. 2). This information was taken into con- was used as a probe in gel shift analysis, and three overlapping sideration when serial deletion plasmids were constructed by oligomers (d1– d3) were used as competitors (Fig. 4A). One PCR in order to identify the regulatory elements with a func- major complex was found, and by competition analysis, binding tional role in Dlx3 gene expression. These constructs were was located to the proximal part of the region (between 284 transfected into primary mouse keratinocytes, and promoter and 259) (Fig. 4B). Better resolution of the specific nucleotides activity was determined by measuring CAT activity in cells required for formation of the complex was obtained using oli- maintained in an undifferentiated state (cultured in 0.05 mM gonucleotides with specific 3-bp mutations (Fig. 4C). Competi- 26602 Regulation of the Dlx3 Homeobox Gene FIG.3. Effect of 5*-deletions on the transcriptional activity of the mouse Dlx3 promoter. Primary mouse keratinocytes were transfected with each indicated deletion construct. The transfected cells were incubated with growth medium containing 0.05 or 1.4 mM Ca concentration, and the CAT activity of each plate of cells was measured. CAT activity was normalized to the protein concentration measured by the Bradford assay. The bars represent the average normalized CAT activity of duplicate plates from three experiments for each construct. FIG.4. Delineation of a binding site within the region between 284 and 234 that binds nuclear factors from keratinocytes. A, the DNA sequence between 284 and 234 (oligomer d) and oligomers used in the competitive gel shift assay (d1– d3); the CCAAT box is underlined. B, the d oligomer (284/234) was labeled and used as a probe in gel shift assay with nuclear extracts of keratinocytes (3 mg). In competition assays, the nuclear extracts were mixed with each competing oligonucleotide (100-fold molar excess) prior to addition of the P-labeled probe. The data presented in this figure are representative of electromobility shift assays that were performed at least three separate times with the same results. C, the DNA sequence for oligonucleotide d1 is shown, along with the location of the 3-bp mutations. The putative CCAAT motif is boxed. The serial 3-bp mutated sequences are underlined in each mutated oligomer (M1–M6). D, competition binding assay with P-labeled d1 oligomer used as a probe and competitor oligomers at 100-fold molar excess (d1 and M1–M6). Identical results were observed with multiple nuclear extract preparations. tion gel shift analysis with wild-type (d1) and mutant oligonu- been reported to bind selectively the CCAAT box binding pro- cleotides (M1–M6) showed that competitors M1, M2, and M3 teins were used as competitors (Fig. 5A). The CCAAT box bound to the specific complex (Fig. 4D). In contrast, oligonu- binding factors include NF-Y (also known as CBF), the C/EBP cleotides M4, M5, and M6 (which has the combined mutation of family, and NF-1 (35). As shown in Fig. 5B, the albumin C M4 and M5) failed to compete. These results indicated that the element, which is known to bind NF-Y (36), fully competed the complex was due to binding in the CCAAT region (Fig. 5B). To formation of the complex (Fig. 5B, left panel, 3rd lane). In determine the nature of the binding, oligonucleotides that have contrast, the consensus NF-1 and C/EBP oligonucleotides did Regulation of the Dlx3 Homeobox Gene 26603 FIG.6. Functional analysis of the Dlx3 promoter CCAAT box motif. A, the diagram illustrates the p84CAT and p34CAT deletion constructs and the sequence of the mutation substituting the CCAAT box motif. B, primary mouse keratinocytes were transfected with p84CAT, p84mCAT, and p34CAT and incubated with medium contain- ing 0.05 or 1.4 mM Ca . After 48 h, cells were harvested. The CAT activity was normalized to the protein concentration measured by the Bradford assay. The bars represent the average normalized CAT activ- FIG.5. Identification of the nuclear protein from undifferen- ity of three experiments done on duplicate plates for each construct. tiated and differentiated mouse keratinocytes that binds to the CCAAT box of Dlx3 promoter. A, DNA sequences used in gel shift analyses. The d1 sequence along with the albumin C element (NF-Y), NF-Y is a heterotrimeric transcription factor composed of C/EBP, and NF1 consensus sequences are shown. B, nuclear extract three subunits, A, B, and C (37– 43). The subunits A and C from undifferentiated (left panel) and differentiated (right panel) pri- 32 together form a heterodimer, which then interacts with the mary mouse keratinocytes (3 mg) were used to shift a P-labeled d1 subunit B to form the heterotrimeric NF-Y protein. This het- probe. The competitors, albumin C element (Alb C), consensus C/EBP, and NF-1 (Santa Cruz Biotechnology), were added at 100-fold molar erotrimeric NF-Y then binds to DNA to form an NF-Y-DNA excess. Top arrow indicates the protein-DNA complex formed between complex. To examine whether the complex consisted of all three nuclear extract and d1 probe. C, nuclear extracts from primary mouse subunits, we checked the molecular weight of the complex keratinocytes (3 mg) were incubated with 0.1, 0.2, or 0.4 mg of antibodies using the UV cross-linking assay (Fig. 5D). The complex (indi- against the NF-Y A-subunit, C/EBP, or NF-1 for 30 min on ice. P- Labeled d1 oligomer was added, and the preparation was separated by cated by arrow) which competed with self-oligonucleotide (d1) electrophoresis. The top arrow indicates supershifted complex when had an estimated molecular mass of ;100 kDa. This molecular using antibody against NF-Y. D, UV cross-linking assay was performed mass coincides with the approximate expected combined mo- to estimate the molecular weight of the protein(s) binding to the CCAAT lecular mass of subunits NF-YA, -B, and -C (35, 25, and ;40 box of the Dlx3 promoter (see “Materials and Methods”). For the com- petition analysis, the nonradioactive competitor oligonucleotides were kDa, respectively) (44). This result strongly supported that added at 100-fold molar excess. Arrow indicates the specific complex NF-Y was binding to the CCAAT box motif of Dlx3 promoter in formed. The numbers on the left are the molecular masses of protein its heterotrimeric form. standards. The function of the Dlx3 promoter CCAAT box was assessed by mutational analysis. Primary mouse keratinocytes were not compete for formation of the complex. The same pattern of transfected with the wild-type p84CAT construct, the mutant competition was found using nuclear extracts either from un- p84mCAT construct, and the p34CAT construct (Fig. 6A). The differentiated or differentiated keratinocytes (Fig. 5B, right CAT activity conferred by each construct was assayed in tran- panel). These results suggested that the nuclear protein that siently transfected cells grown in 0.05 and 1.4 mM Ca (Fig. bound to the CCAAT box motif in the Dlx3 promoter was NF-Y. 6B). The CAT activity of the mutant construct was reduced to To confirm this directly, supershift experiments were per- 40 and 25% as compared with the wild-type p84CAT construct formed with specific antisera (Fig. 5C). By using the labeled in the undifferentiated and differentiated cells, respectively. wild-type d1 oligonucleotide, either anti-NF-Y, anti-C/EBP, or These results underscore the functional importance of the anti-NF-1 antisera were added in increasing amounts to undif- CCAAT box for the activity of the Dlx3 promoter. ferentiated mouse primary keratinocyte nuclear extracts. Anti- Functional Sp1-binding Site in the Dlx3 Promoter—In addi- NF-Y antibody supershifted the complex with each amount, tion to CCAAT box sequence, analysis of the proximal promoter whereas the anti-C/EBP or anti-NF-1 antibody did not affect region revealed a putative recognition site for Sp1 transcription the mobility. These observations indicated that the specific factor, which was located between the TATA box and transcrip- complex contained the NF-Y transcription factor. tion start site (213 to 23). To examine this putative Sp1- 26604 Regulation of the Dlx3 Homeobox Gene ferentiated keratinocytes and to 62 and 72% of each respective wild-type in differentiated keratinocytes (Fig. 8). These results showed that the Sp1-binding site of Dlx3 promoter plays a positive regulatory role, and its function is not dependent on the CCAAT box-binding protein, NF-Y. Defining the Region of the Dlx3 Promoter Responsible for Ca Inducibility—Despite the finding that the binding sites for NF-Y and Sp1 play an important role in the transcriptional regulation of the Dlx3 gene, mutation of these sequences did not affect the activation of Dlx3 by Ca (Fig. 6 and 8). Serial 39-deletion constructs were made on the p34CAT construct, which contains the 160 bp of untranslated region (Fig. 9A). The constructs were made in an attempt to delineate the region responsible for the Ca inducibility. Each construct was trans- fected into primary mouse keratinocytes, and CAT activity was measured in 0.05 and 1.4 mM Ca conditions (Fig. 9A). The p60(1)CAT deletion retained the capability of induction by Ca , but the p10(1)CAT deletion showed no induction of CAT activity with Ca increase (Fig. 9A). Similar 39-deletions were made on the p1213CAT construct. These constructs were trans- fected into primary mouse keratinocytes (Fig. 9B), and the CAT activity indicated that the p1213(1)160CAT as well as the p1213(1)60CAT were induced 2.5-fold, whereas the p1213(1)30CAT was induced only 1.5-fold. In contrast, the p1213(1)10CAT deletion was not induced with Ca . Our find- ings indicated that the region located between 110 and 160 was indispensable for induction by Ca of the Dlx3 promoter. Characterization of the Ca -responsible Region of Dlx3 Pro- moter—Based on the results of the serial deletion analysis of the Dlx3 promoter (Fig. 9), the region between 130 and 160 has the element(s) that mediate the Ca induction. A muta- tional analysis was performed to determine the specific nucle- otides that conform the responsive element. Serial and double 4-bp mutations were made in the region between 130 and 160 FIG.7. Identification of nuclear proteins binding to the GC- in the context of the p1213(1)60CAT construct. These con- rich site of Dlx3 promoter. A, the sequences of oligonucleotides structs were transfected into primary mouse keratinocytes, containing the GC-rich putative Sp1-binding site (underlined), mutant cultured for 48 h in 0.05 or 1.4 mM Ca concentrations, and the Sp1-binding site (underlined), and consensus Sp1-binding site (under- lined) are shown. B,3 mg of nuclear extracts from undifferentiated (left CAT activity was measured. As shown in the Fig. 10A, the panel) or differentiated (right panel) keratinocytes were used to shift M1CAT, M2CAT, M5CAT, and M7CAT showed reduced pro- P-labeled dsp oligonucleotide as described. Nonradioactive competitor moter activity at 0.05 and 1.4 mM Ca , but the fold activation Sp1 consensus (Santa Cruz Biotechnology) and dspM oligonucleotides by Ca did not change significantly when compared with the were added at a 100-fold molar excess. Arrows indicate the protein- DNA complex formed between nuclear extract (3 mg) and the dsp probe. p1213(1)60CAT. In contrast, the M4CAT and M6CAT showed C, antibodies to Sp1, Sp2, Sp3, and Sp4 (Santa Cruz Biotechnology) increased promoter activity in 0.05 and 1.4 mM Ca and were used to test which members of Sp1 family are associated with the 21 slightly increased fold activation by Ca . These results sug- Sp1-binding site of Dlx3 promoter. The arrows indicate the specific gest that these sites mainly affect the basal promoter activity of complex formed and the supershifted complexes after addition of anti- bodies raised against Sp1 and Sp3 proteins. Dlx3. Interestingly, the M3CAT construct showed dramatic reduction in the fold activation by Ca , similar to that of p1213(1)30CAT. These results indicated that the nucleotides binding site, gel shift analysis was performed using an oligo- (142-CGAC-145) are crucial for the activation by Ca . Double nucleotide corresponding to 221 to 22 on the Dlx3 promoter mutant constructs (M34CAT, M36CAT, and M46CAT, Fig. (dsp), and a mutant form of this oligonucleotide (dspM). Nu- 10A) were made and assayed for CAT activity. The double clear extracts from undifferentiated and differentiated primary mutant constructs containing the M3 site (M34CAT and mouse keratinocytes were used in the gel shift analysis (Fig. M36CAT) showed reduced fold activation by Ca , but the 7A). As shown in Fig. 7B, the complexes formed were competed M46CAT mutation had no effect. These results again delin- with wild-type dsp and Sp1 consensus oligonucleotides but not eated the sequence (142-CGAC-145) as primarily responsible with the mutant form of dsp (dspM). In gel shift assays using for the Ca induction of Dlx3 promoter. antibodies corresponding to each of the Sp1-like family mem- We were interested in determining by gel shift analysis if bers, the Sp1 and Sp3 antisera supershifted the two complexes there were nuclear proteins that bound the 130/160 region formed between the Sp1-binding site of Dlx3 promoter and nuclear proteins (Fig. 7C). These results indicate that both of (CA30). The CA30 oligonucleotide was used as a probe in gel shift mobility assays with nuclear extracts from mouse primary these Sp1-like factors are able to bind the Sp1 site on the Dlx3 promoter. keratinocytes. One major complex I and two weak bands II and III were identified (Fig. 10B). These complexes were competed DNA constructs containing the mutant Sp1-binding site (Spm) in the p84CAT and p34CAT clones were transfected into with 100-fold excess of unlabeled cold CA30 oligomer. To ex- mouse keratinocytes to examine the function of this region (Fig. amine the relationship between the sequences affecting the 8). The CAT activity of p84spmCAT and p34spmCAT was re- Dlx3 promoter activity and these complexes, the oligomers duced to 55 and 66% of the wild types, respectively, in undif- containing each mutant site were used as competitors. The M3 Regulation of the Dlx3 Homeobox Gene 26605 FIG.8. Mutational analysis of the Sp1-binding site in the Dlx3 promoter. The diagram (left) illustrates the p84CAT, p34CAT, p84spmCAT, and p34spmCAT deletion and mutant constructs. The sequence of the mutation of the Sp1-binding site is the same shown in Fig. 7A. Primary mouse keratinocytes were transfected with p84CAT, p34CAT, p84spmCAT, or p34spmCAT, incubated with growth medium containing 0.05 or 1.4 mM Ca , and harvested after 48 h. The CAT activity was normalized to the protein concentration measured by the Bradford assay. The bars represent the average normalized CAT activity of duplicate plates for each construct (right panel). All experiments in this figure were repeated three times with similar results. FIG.9. Deletion analysis and func- tion of sequence residing down- stream of the transcription initiation site. A, p34CAT constructs with decreas- ing downstream promoter deletions were transfected into primary mouse keratino- cytes and incubated with growth medium containing either 0.05 or 1.4 mM Ca . CAT activity was assayed 48 h after transfection. B, p1213CAT constructs with decreasing downstream promoter deletions were transfected into keratino- cytes and assayed as described above. The bars represent the average normalized CAT activity of duplicate plates for each construct. All experiments in the figure were repeated in triplicate with similar results. DISCUSSION oligomer competed the complexes II and III, but did not com- pletely compete the major complex I. The M4, M6, and M34 The cascade of events that leads to terminal differentiation oligomers showed similar results for the complex I but did not can be triggered in primary mouse keratinocytes by the eleva- compete the complexes II and III. In contrast, none of the tion of extracellular Ca . The expression of the Dlx3 homeobox complexes were competed by the M36 oligomer that contains gene is restricted to the suprabasal layer of the epidermis (18), mutations in the M3 and M6 sites. These results indicated that and this expression is dramatically increased in primary mouse keratinocytes induced to differentiate by Ca in vitro. Evi- the M3 and M6 sites are important in the binding of the major dence obtained in transgenic mice suggests the role of Dlx3 as complex I and that the M4 site is partially involved in the a positive transcriptional activator of differentiation-specific formation of this complex. epidermal structural genes. In an attempt to decipher the The region between 130 and 160 contains an element (146- different stages of the pathway that culminate in terminal TGACCGA-152) with sequence homology to a consensus AP1- differentiation, we have cloned and characterized the upstream binding site. It has been suggested that the AP1 transcription 21 promoter sequence of the murine Dlx3 gene, and we deter- factor is involved in the Ca inducibility of epidermal differ- mined the regulatory elements necessary for expression of the entiation-specific markers (45). To determine the possible par- gene in keratinocytes. By sequence comparison we have found ticipation of AP1 in the protein-DNA complex formation with that the region close to the transcription start site of the mouse CA30, the AP1 consensus oligonucleotide and antibodies Dlx3 gene (2110 to 161) has striking homology with that of the against AP1 family members were used in competition and Xenopus and human dlx3 genes. In transgenic mice, 470 bp of supershift assays. As shown in Fig. 10C, the complexes formed the Xenopus Dlx3 promoter conferred an expression pattern to by CA30 were not competed by AP1 consensus oligonucleotide, the b-galactosidase reporter that was indistinguishable from and the supershift assays corroborated that AP1 family mem- that of the endogenous gene, including the Ca response (46). bers (c-Jun, c-Fos, JunB, and JunD) do not bind to this region. Thus, it could be hypothesized that the cis- and trans-regula- Therefore, the sequence (142-CGAC-145) present in the CA30 tory elements important in the regulation of the Dlx3 orthologs region is responsible for the transcriptional up-regulation of have been conserved through evolution and are contained Dlx3 promoter by Ca , through the binding of nuclear factors within the proximal promoter region. that are not members of the AP1 family of transcription factors. To understand the mechanism(s) that control the regulation 26606 Regulation of the Dlx3 Homeobox Gene FIG. 10. Characterization of Ca -responsible region of Dlx3 promoter. A, the region between 130 and 160 of Dlx3 promoter was mutated with serial or double 4-bp modified bases by the PCR method in the p1213(1)60CAT. Each mutated construct, p1213(1)60CAT, p1213(1)30CAT, and p1213(1)10CAT, was transfected into primary mouse keratinocytes. Underlined lowercase letters indicate the modified nucleotides. CAT assay was performed the same as in Fig. 3. The bars represent the average normalized CAT activity of duplicate plates for each construct. All experiments in the figure were repeated in triplicate with similar results. B, gel shift assay was performed using the oligonucleotide corresponding to the region between 130 and 160 (CA30) as a probe and 3 mg of nuclear extract of primary mouse keratinocytes. Incubations were carried out in the presence or absence of a 100-fold excess of unlabeled wild-type and mutant oligonucleotides. Arrows indicate the protein-DNA complexes, and roman numbers indicate each individual complex (I–III). C, supershift analysis was carried out using the antibodies against the AP1 family. Nuclear extracts were preincubated with 2 mg of each antibody as indicated, followed by incubation with labeled CA30 probe. The 3rd lane corresponds to the gel shift analysis performed in the presence of 100-fold excess unlabeled AP1 consensus oligonucleotide. All experiments in this figure were repeated at least three times with similar results. Regulation of the Dlx3 Homeobox Gene 26607 and expression of the Dlx3 promoter during epidermal differ- From the family of Sp1-related transcription factors, studies entiation, our studies examined the expression of Dlx3 promot- have shown that Sp3 acts as an antagonist to Sp1. The human er/CAT constructs in primary mouse keratinocytes to identify transcobalamin II gene is controlled by the relative ratios of putative regulatory elements that are required for Dlx3 expres- Sp1 and Sp3 (64), and activation of the human papilloma virus type 16 promoter correlates with the ratios of Sp1/Sp3 during sion. The data presented here demonstrate the existence of one positive regulatory region in the Dlx3 promoter located be- epithelial differentiation (65). We have shown that the Sp1 consensus motif on the Dlx3 promoter can bind specifically Sp1 tween 284 and 234. We determined that the CCAAT box in and Sp3, providing the possibility of antagonistic effects on this region exerts a significant positive influence on the Dlx3 transcription depending on the specific levels of each of these promoter activity in undifferentiated and differentiated kera- factors throughout the terminal differentiation process. tinocytes. Competition gel shift analysis and supershift analy- An essential aspect of the transcriptional regulation of the sis further demonstrated that the transcription factor NF-Y is Dlx3 promoter is the elucidation of the mechanism for inducing the protein that binds specifically to the CCAAT box motif. its expression by increases in external Ca during differenti- Mutation of this motif dramatically reduced transcription of ation. Until now, the prevailing evidence points to the involve- the Dlx3 promoter. Several genes also contain canonical sites ment of PKC isozymes in the induction of keratinocyte differ- for CCAAT-binding proteins that have been described to be entiation markers by Ca , although the exact mechanism is important in early functions of preinitiation complex formation unclear. In this study we show that the region located between (36). CCAAT boxes are typically located within 100 bp of the 110 and 160 is important for Ca inducibility of the Dlx3 transcription start sites, and CCAAT box binding factors in- gene. By mutational and gel shift analysis of the 130/160 clude NF-Y (also known as CBF), C/EBP family members, and sequence, we found that the crucial element responsible for NF-1 (35). NF-Y was originally identified as a ubiquitously Ca inducibility is located between 142 and 145 (CGAC) and expressed protein that binds to the Y box motif, defined as an that nuclear factor(s) are involved in the up-regulation of Dlx3 inverted CCAAT box motif in all major histocompatibility com- expression by Ca . It has been reported that the AP1 and Ets plex class II genes (47). NF-Y is a heterotrimeric transcription transcription factors are involved in the regulation of the hu- factor composed of three subunits (37– 43). Formation of a man SPRR1A keratinocyte terminal differentiation marker complex between the A and C subunits is required to bind the (45). However, in the case of the Dlx3 gene, the AP1 transcrip- B subunit, and together, the heterotrimeric complex binds tion factor does not bind to the 142/145 or 146/152 sequences, DNA (48). The activation domains of both B and C subunits, which strongly supports that it is not the determinant for the which are rich in glutamine and hydrophobic residues, share Ca inducibility of Dlx3. Future work will determine the spe- protein sequence homology with each other and with the glu- cific nature of the nuclear factor(s) involved in the binding to tamine-rich activation domain of the transcription factor Sp1 this regulatory element. The identification of a Ca response (48). element in the Dlx3 gene is the first link between the extracel- NF-Y has been demonstrated to act as both a positive (49 – lular signal and the transcriptional control of a regulatory gene 51) and a negative (52) regulator of transcription. Several stud- involved in keratinocyte differentiation, and represents an im- ies suggest that NF-Y acts by stabilizing the binding of addi- portant step in the elucidation of the molecular mechanisms tional factors to adjacent regulatory elements, such as RFX in underlying this developmental program. the major histocompatibility complex class II promoter (53, 54). NF-Y also interacts with transcription factors binding up- Acknowledgments—We thank Dr. Ulrike Lichti and members of the Laboratory of Skin Carcinogenesis for the Ca determination and for stream elements and basal transcription machinery, such as providing reagents. We are grateful to Drs. Peter Steinert, Janine HNF4 and TAF100 (42, 55). Recently it was shown that the Bryan, Andres Buonanno, and Thomas Sargent for reading and provid- interaction of NF-Y with cAMP-response element-binding pro- ing comments on the manuscript. 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