The Human Involucrin Gene Contains Spatially Distinct Regulatory Elements That Regulate Expression During Early Versus Late Epidermal Di€Erentiation

The human involucrin gene contains spatially distinct regulatory elements that regulate expression during early versus late epidermal dierentiation

James F Crish1, Frederic Bone1, Eric B Banks1 and Richard L Eckert*,1,2,3,4,5

1Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, 2109 Adelbert Road, Cleveland, Ohio, OH 44106-4970, USA; 2Department of Dermatology, Case Western Reserve University School of Medicine, 2109 Adelbert Road, Cleveland, Ohio, OH 44106-4970, USA; 3Department of Reproductive Biology, Case Western Reserve University School of Medicine, 2109 Adelbert Road, Cleveland, Ohio, OH 44106-4970, USA; 4Department of Biochemistry, Case Western Reserve University School of Medicine, 2109 Adelbert Road, Cleveland, Ohio, OH 44106-4970, USA; 5Department of Oncology, Case Western Reserve University School of Medicine, 2109 Adelbert Road, Cleveland, Ohio, OH 44106-4970, USA

Human involucrin (hINV) is a keratinocyte protein that Keywords: transgenic mice; activator protein-1; AP1; is expressed in the suprabasal compartment of the jun; fos; Sp1; gene regulation; epidermal keratinocyte; epidermis and other stratifying surface epithelia. In- keratinocyte dierentiation; tissue speci®c volucrin gene expression is initiated early in the dierentiation process and is maintained until terminal cell death. The distal regulatory region (DRR) is a Introduction segment of the hINV promoter (nucleotides 72473/ 71953) that accurately recapitulates the normal pattern The major cell type, and the cell type responsible for of suprabasal (spinous and granular layer) expression in the morphological characteristics of the epidermis, is transgenic mouse epithelia. To identify sequences that the epidermal keratinocyte (Green, 1980). Basal mediate expression at speci®c stages of dierentiation, keratinocytes give rise to cells that dierentiate to we divided the DRR into two segments, a 376 nucleotide produce the suprabasal epidermal layers. This involves upstream region (DRR72473/72100) and a 147 nucleotide a highly coordinated series of changes in cell downstream region (DRR72100/71953), and evaluated the morphology and biochemistry (Eckert et al., 1997a; ability of these sequences to drive expression in Green, 1980). The terminal dierentiated cell, or transgenic mice. The DRR72473/72100 segment drives corneocyte, is a ¯attened dead cell that consists of a expression at a level comparable to that observed for the network of cytokeratin ®laments surrounded by an DRR, but expression is restricted to the upper granular insoluble envelope of heavily crosslinked protein. The layers (i.e., no spinous layer expression). In contrast, the envelope precursors and transglutaminase, the enzyme DRR72100/71953 segment does not drive expression. activity responsible for assembly of the envelope (Kim However, reassembling the DRR restores the complete et al., 1995; Steinert, 1995; Thacher and Rice, 1985; range of expression. These results suggest that two Rice and Green, 1979), must be expressed at the distinct, spatially-separate elements are required to correct time and level during the dierentiation process specify the complete dierentiation-dependent program for proper envelope formation. Involucrin, an early of involucrin gene expression. To identify speci®c marker of keratinocyte dierentiation, and a compo- transcription factor binding sites involved in this nent of the corni®ed envelope, is speci®cally expressed regulation, we mutated an activator protein-1 binding in the suprabasal layers of the epidermis (Murthy et site, AP1-5, located within DRR72473/-2100 segment. al., 1993; Crish et al., 1993; Murphy et al., 1984). It This site binds AP1 transcription factors present in accumulates in the spinous and granular layers as a mouse epidermal extracts, and its mutation eliminates non-crosslinked precursor and becomes covalently appropriate hINV expression. This result suggests that crosslinked to other proteins and lipids to form the AP1 factors participate as components of a multi- corni®ed envelope scaolding during the ®nal stages of component transcription factor complex that is required keratinocyte dierentiation (Steinert and Marekov, for regulation. 1997; Rice and Green, 1977). Oncogene (2002) 21, 738 ± 747. DOI: 10.1038/sj/onc/ Previous studies in cultured keratinocytes indicate 1205038 that the distal regulatory region (DRR, nucleotides 72473/71953) (Banks et al., 1998, 1999; Welter et al., 1995) accounts for one-half of the activity of the hINV promoter (Welter et al., 1995). This region contains *Correspondence: L Eckert, Department of Physiology and weak and strong activator elements (Banks et al., Biophysics, Room E532, Case Western Reserve University School 1998). The weak activator element enhances the of Medicine, 2109 Adelbert Road, Cleveland, Ohio 44106-4970, USA; activity of the strong activator element. The strong E-mail: [email protected] Received 14 February 2001; revised 25 September 2001; accepted 9 activator element, nucleotides 72140/72088, plays a October 2001 key role in regulation of hINV gene expression. This Human involucrin in transgenic mice JF Crish et al 739 region encodes an AP1 (AP1-5) binding site. AP1 factors junB, junD and Fra-1 interact with the AP1-5 binding site (Banks et al., 1998; Welter et al., 1995). Mutation of the AP1-5 site results in a complete loss of promoter activity when the isolated distal regulatory region is tested in cultured human epidermal keratinocytes (Banks et al., 1998). hINV promoter function has also been studied in transgenic mice. Transgenic studies indicate that the full-length involucrin promoter drives tissue-speci®c and dierentiation-appropriate expression in surface epithelial cells (Crish et al., 1993; Carroll et al., 1993), and show that this expression pattern can be replicated by the isolated DRR (Crish et al., 1998). However, no knowledge is available regarding speci®c DRR regions that function to drive expression in particular epidermal layers, nor whether particular DNA binding sites are important in this context. Our present studies provide evidence that the DRR contains discrete, spatially distinct, DNA sequence elements that are required for expression at speci®c stages during epidermal dierentiation. We further identify AP1 factors as part of this regulatory complex.

Results

Evidence for spatially separated regulatory regions Our previous studies show that the hINV distal regulatory region (DRR) is sucient to drive tissue- speci®c and dierentiation-appropriate gene expression in mouse surface epithelia (Crish et al., 1998). A goal of the present study is to identify speci®c regions within the DRR that are important for expression at various stages during dierentiation. In these studies, as previously reported (Crish et al., 1998), we use Figure 1 The hINV DRR region ± multiple elements control dierentiation-appropriate expression. (a) Construct P3.4B en- human involucrin as our reporter gene (LaCelle et al., codes the hINV minimal promoter. The arrow encompasses the 1998). This approach avoids problems associated with hINV gene transcribed region including the shaded small and dierences in turnover rate of involucrin as compared large rectangles denoting, respectively, the ®rst and second exons. to other possible reporter proteins, such as b- The scale at the top is in kilobases. In the other constructs, segments of the DRR (open boxes) are cloned adjacent the hINV galactosidase or luciferase, in epidermis. We began by minimal promoter. The shaded circle and box over the DRR testing the constructs shown in Figure 1a. The DRR- indicate, respectively, the AP1-5 and Sp1 sites (Banks et al., 1998, P3.4B construct encodes the complete DRR segment 1999). Nucleotide positions de®ning each DRR segment are presented relative to the transcription start site (Crish et al., 1998; linked to the minimal hINV promoter. DRR72473/ -P3.4B encodes nucleotides derived from the Eckert and Green, 1986). (b) To detect hINV transgene 72100 expression, equivalent quantities of epidermal total cell extract, upstream end of the DRR, and DRR72100/71953- prepared from non-transgenic (NT) animals or P3.4B (line 10), P3.4B encodes the nucleotides derived from the down- DRR-P3.4B (line 31), or DRR72473/72100-P3.4B (lines 4, 9, 14) stream end of the DRR. P3.4B encodes the hINV transgenic animals was electrophoresed on a 6% polyacrylamide minimal promoter. In Figure 1, we monitor expression denaturing and reducing gel, transferred to nitrocellulose, and hINV protein was detected using an hINV-speci®c antibody of these constructs in transgenic mouse epidermis. The (LaCelle et al., 1998). The lane marked rhINV contains P3.4B construct, which encodes the minimal hINV recombinant human involucrin, electrophoresed as a standard. promoter, does not drive expression in epidermis. The (c) The analysis is identical to that described in panel B, except three lines encoding the DRR -P3.4B con- that hINV expression was monitored in mice expressing the 72473/72100 DRR -P3.4B transgene (lines 23, 24, 35) struct express hINV at levels comparable to that 72100/71953 observed for DRR-P3.4B (Figure 1b). As expected, no expression was detected in non-transgenic (NT) mice. In contrast to the expression observed for We next utilized immunohistology to compare the DRR72473/72100, no expression was observed in each pattern of expression of the DRR, DRR72473/72100, of the three DRR72100/71953 transgenic lines (Figure and DRR72100/71953 constructs during dierentiation. 1c). Footpad is a tissue useful for histological study,

Oncogene Human involucrin in transgenic mice JF Crish et al 740 because the multiple layers make it easy to visualize (see Crish et al. (1993)). In contrast, the DRR72473/ dierentiation-dependent expression. As shown in 72100 drives expression only in the top few layers. As Figure 2, the intact DRR construct is expressed in all expected based on the immunoblot results presented in except the most basal footpad layers ± a pattern of Figure 1, the DRR72100/71953 does not drive any expression identical to that of the full-length promoter histologically detectable expression.

Role of the AP1-5 site The above studies indicate that sites required for dierentiation-appropriate expression are present at spatially distinct locations within the DRR. A major goal is to identify speci®c elements responsible for this regulation. One site that is required for appropriate hINV promoter activity in cultured keratinocytes is an AP1 site, AP1-5, located within the DRR (Banks et al., 1998, 1999; Crish et al., 1998; Welter et al., 1995). To determine whether this site is important for in vivo regulation, we generated and tested transgenes lacking a functional AP1-5 site (Figure 3a). Construct H6B includes the full-length (2473 nucleotide) promoter (Crish et al., 1993). In DRR-P3.4B, the 520 bp distal regulatory region, shown as an open box, is cloned adjacent the minimal promoter. Parallel constructs lacking a functional AP1.5 site, H6B(AP1 ± 5 mm) and DRR-P3.4B(AP1 ± 5 mm), were constructed encoding the mutant sequence (underlined in Figure 3b). We examined the ability of these constructs to drive expression in footpad epidermis. As shown in Figure 4a, H6B directs expression to the suprabasal (spinous and granular) footpad layers and DRR- P3.4B produces an essentially identical pattern of

Figure 3 Structure of hINV transgenes for characterization of AP1-1 site function. The structure of H6B and DRR-P3.4B, shown in (a), have been previously described (Crish et al., 1998). In H6B(AP1 ± 5 mm) and DRR-P3.4B(AP1 ± 5 mm) the AP1-5 site is inactivated by mutation. The open rectangle depicts the Figure 2 Multiple DRR regions control gene expression during hINV promoter distal regulatory region (DRR, nucleotides keratinocyte dierentiation. Sections of footpad epidermis from 72473/71953). The arrows encompass the transcribed region mice harboring the DRR, DRR72473/72100, and DRR72100/71953 including the shaded small and large rectangles denoting, transgenes were ®xed, sectioned, and stained sequentially with respectively, the ®rst and second exons. The shaded circle and hINV-speci®c antibody and horseradish peroxidase-conjugated box over the DRR indicate, respectively, the AP1-5 and Sp1 sites secondary antibody. Control sections, prepared in the absence of (Banks et al., 1998, 1999). Distances, in kilobases, are indicated as primary antibody, did not stain (not shown). Similar pro®les were positive downstream and negative upstream of the transcription observed in each of three transgenic lines for each construct. The start site. (b) Shows the sequence of the hINV promoter AP1-5 black arrows indicate the basal proliferative cells. The yellow 5'TGAGTCAG-3' and Sp1 5'-GGGCGGG-3' sites and surround- arrows indicate the involucrin-positive cells in DRR72473/72100- ing sequence. The altered nucleotides in the mutant AP1-5 site are EPI indicates the extent of the viable epidermis in each panel underlined

Oncogene Human involucrin in transgenic mice JF Crish et al 741

Figure 4 Inactivation of AP1-5 results in a loss of footpad gene expression. (a) Sections of footpad epidermis from mice harboring the H6B, H6B(AP1 ± 5 mm), DRR or DRR(AP1 ± 5 mm) transgenes were ®xed, sectioned, and stained sequentially with hINV- speci®c antibody and horseradish peroxidase-conjugated secondary antibody. Control sections, prepared in the absence of primary antibody, were not stained (not shown). A similar pattern of staining was observed for three independent transgenic lines for each construct. EPI indicates the area of the section that encompasses living epidermis. (b) Immunoblot detection of hINV in transgenic mice. Equivalent quantities of epidermal total cell extract, prepared from non-transgenic H6B, H6B(AP1 ± 5 mm), DRR, and DRR(AP1 ± 5 mm) animals was electrophoresed on an 6% polyacrylamide denaturing and reducing gel, transferred to nitrocellulose, and hINV protein was detected using an hINV-speci®c antibody (LaCelle et al., 1998). The lane marked rhINV contains recombinant human involucrin, electrophoresed as a standard expression. However, expression is lost when AP1-5 is from footpad epidermis, back epidermis, and mutated in the context of the isolated DRR, DRR- esophagus, and measured hINV levels by immuno- P3.4B(AP1 ± 5 mm), or in the context of the full- blot. As displayed in Figure 6, H6B and DRR- length promoter, H6B(AP1 ± 5 mm). This loss of P3.4B, which contain the intact AP1-5 site (+), expression is con®rmed by immunoblot (Figure 4b). produce high-level hINV expression in each of these Thus, mutation of AP1-5, either in the context of the surface epithelia. In contrast, markedly reduced full-length promoter or in the isolated DRR, expression is observed in mice harboring the AP1-5 markedly reduces hINV expression in footpad site mutant constructs (7). Although the level of epidermis. expression is markedly reduced in these mice, the gene is not completely o. This is in contrast to the complete lack of expression in mice containing AP1-5 is required for expression in esophagus and cervix construct P3.4B, the minimal promoter construct Involucrin is known to be expressed in other stratifying (not shown) (Crish et al., 1993, 1998). surface epithelia (Murphy et al., 1984; Banks-Schlegel In previous reports, we described hINV transgene and Green, 1981; Rice and Green, 1979). To determine expression in the epithelia lining the proximal whether AP1-5 site is required for hINV expression in convoluted tubules of the kidney (Crish et al., 1993, these tissues, we measured hINV levels in esophagus 1998). This expression serves as a marker for integrity and cervix. As shown in Figure 5, both H6B and DRR of the transgene, as transgene-dependent expression is drive appropriate suprabasal expression in esophagus observed in kidney independent of surface epithelial (Figure 5a) and cervix (Figure 5b). In contrast, cell expression. As shown in Figure 6, kidney mutation of the AP1-5 site, in either the DRR or expression is observed for all of the constructs, full-length promoter contexts, reduces hINV expression whether the AP1-5 site is intact or mutated. The fact in both tissues. that the level of expression is maintained in the kidney argues that the reduced expression in the epithelia of AP1 ± 5 mm mice is not due to inappropriate integra- Mutation of AP1-5 results in reduced hINV protein levels tion and/or rearrangement of the transgene. These To con®rm the immunohistological data and com- results support the conclusion that the AP1-5 site is pare the level of hINV expression between mutant required for appropriate hINV expression in surface and intact constructs, we prepared tissue extracts epithelia.

Oncogene Human involucrin in transgenic mice JF Crish et al 742

Figure 5 Eect of AP1-5 site mutation on hINV expression in cervix and esophagus. Sections of cervix and esophagus were prepared from H6B, H6B(AP1 ± 5 mm), DRR or DRR(AP1 ± 5 mm) mice. The sections were prepared and incubated with primary and secondary antibodies as in Figure 2. Similar staining was observed for three independent transgenic lines for each construct. EPI indicates the extent of the viable epithelial layer. The arrowheads in each panel indicate the position of basal epithelial layer

Interaction of AP1 factors with the DRR AP1-5 site Discussion Finally, we used gel mobility shift analysis to con®rm The DRR contains elements that control AP1 factor binding at the AP1-5 site. As shown in differentiation-dependent expression Figure 7a, incubation of mouse epidermal nuclear extracts with the 32P-AP1-5, an oligonucleotide that Epidermal gene expression is activated in a precise encodes the AP1-5 site and ¯anking sequence, results temporal and spatial manner during keratinocyte in the appearance of several prominent shifted bands. dierentiation ± speci®c genes are activated during Incubation in the presence of a 10- or 100-fold molar early dierentiation, while other genes are expressed excess of AP1-5 oligonucleotide speci®cally eliminates late. This sequential activation is functionally impor- several bands. No competition is observed when tant. For example, proteins such as transglutaminase competition is with a non-speci®c oligonucleotide. and involucrin, which are involved in the early stages We next performed gel mobility supershift experiments of dierentiation, are expressed in the immediate to identify speci®c AP1 factors present in the shifted suprabasal layers (Murthy et al., 1993; Rice and complex. As shown in Figure 7b, our results suggest Green, 1979), while loricrin, which is utilized late in that pan-Fos (an antibody that detects c-Fos, FosB, dierentiation, is expressed later (Bickenbach et al., Fra-1 and Fra-2) results in a supershift, indicating the 1995; Hohl et al., 1991, 1993). In spite of the presence of Fos factors. Figure 7c shows that importance in normal skin biology, the mechanisms antibody speci®c for Fra-1 also produces a shift, that regulate gene expression during early versus late suggesting that it may be a Fos component of the dierentiation are not well understood. However, it can complex. A shifted band was also observed using a c- be assumed that this regulation will involve input from Jun speci®c antibody. JunD and JunB also produced a multiple transcription factors, and that the composition shift, although the bands were too faint to adequately of the regulatory complex may change during dier- reproduce. No shift was observed for Fra-2 or FosB entiation. Numerous studies indicate that transcription (not shown). factors and co-regulators function in complexes that, in

Oncogene Human involucrin in transgenic mice JF Crish et al 743 scription factor binding site(s) that is required, in conjunction with sites within DRR72473/72100,to confer a complete program of dierentiation-appropriate expression. A search of the DNA segment encoded in DRR72100/71953 reveals several possible candidate sites that could mediate this regulation, and we are presently performing transgenic studies to identify a speci®c element within this region.

The AP1 site appears to be required for expression throughout differentiation To identify transcription regulatory elements responsible for DRR72473/72100-dependent expression in late dierentiation, our strategy is to eliminate selected transcriptional elements within the upstream region of the DRR and then monitor the eects on expression of the transgene. AP1 factors are a family of transcription Figure 6 Detection of hINV protein. Equivalent quantities of factors that are expressed in a wide variety of tissues total cell extract, prepared from animals encoding the H6B (top panel) or DRR (bottom panel) transgenes containing intact (+) and cells that function as homodimers of two Jun or mutated (7) AP1-5 were prepared from back epidermis, family proteins or as heterodimers of one Jun and one footpad epidermis, esophageal epithelium, and kidney, were Fos family member. The Fos factors include c-Fos, electrophoresed on a 6% polyacrylamide gel under denaturing FosB, FosB2, Fra-1 and Fra-2, while the Jun factors conditions, and transferred to nitrocellulose membrane. hINV include c-Jun, JunB, and JunD (Suzuki et al., 1991; was detected by incubation with an hINV-speci®c primary antibody with secondary detection using a chemiluminescence. Nishina et al., 1990; Cohen and Curran, 1989; Cohen The arrows indicate hINV migration. The lane marked rhINV et al., 1989). All of the known AP1 factors are contains recombinant human involucrin, electrophoresed as a expressed in both human and mouse epidermis, each standard displaying a speci®c pattern of dierentiation-dependent expression (Rutberg et al., 1996; Welter and Eckert, 1995; Briata et al., 1993; Fisher et al., 1991). turn, interact with the basal transcription machinery Because the AP1-5 site has been speci®cally implicated (Xiao et al., 2000; Xu et al., 1999; Chiang et al., 1993). as an important regulator of involucrin promoter It could also be expected that multiple DNA binding activity in cultured cells (Takahashi et al., 1998; Welter sites may be involved, as this is frequently observed for et al., 1995; Takahashi and Iizuka, 1993; Banks et al., regulation during dierentiation, and has been ob- 1998, 1999; E®mova and Eckert, 2000; Ng et al., 2000; served for involucrin (Banks et al., 1999; Welter et al., E®mova et al., 1998), we chose to evaluate its role as a 1995, 1996). dierentiation regulator. Our results show that muta- Our previous studies identify a 520 nucleotide tion of the AP1-5 site results in markedly reduced (nucleotides 72473/71953) region of the hINV up- expression at all stages of dierentiation. In addition to stream regulatory region, the DRR, as being necessary epidermis, this site must also be functionally important and sucient to drive dierentiation-appropriate gene in other surface epithelia, as its inactivation results in a expression in epidermis (Crish et al., 1998). The present loss of expression in cervix and esophagus. Gel experiments were designed to begin identifying the mobility supershift analysis using nuclear extracts from DNA sequence elements within the DRR that are mouse epidermis shows that c-Jun, Fra-1, JunB, and required for expression at speci®c times during JunD bind to the hINV AP1-5 element, all AP1 family dierentiation. We approached this problem by members that are known to be expressed in mouse dividing the DRR region into two separate segments epidermis (Rutberg et al., 1996). In nuclear extracts and evaluating the ability of each to drive expression in prepared from cultured human keratinocytes, this transgenic mice. The results were revealing in that the DNA sequence binds JunD, JunB and Fra-1 (Welter upstream segment, DRR72473/72100, drives expression et al., 1995). This suggests that the regulation may be that is con®ned to the most highly dierentiated mediated by similar members of this family indepen- epidermal layers. In contrast, the downstream DRR dent of species. These results are consistent with segment, DRR72100/71953, does not drive transcription. previous reports indicating a role for AP1 factors in However, DRR72100/71953 clearly encodes DNA expression of genes during keratinocyte dierentiation. sequences that are necessary for appropriate expression Most of these studies have utilized in vitro cell culture during dierentiation, since eliminating this region models (DiSepio et al., 1995a; Casatorres et al., 1994; results in a loss of expression during early dierentia- Bernerd et al., 1993; Hu et al., 1993; Lu et al., 1994; tion. Moreover, when DRR72100/71953 is spliced in its Medvedev et al., 1999; Rothnagel et al., 1993; Yamada appropriate position downstream of DRR72473/72100, et al., 1997), although some have used transgenic mice expression during early dierentiation is restored. This (DiSepio et al., 1995b; reviewed in Eckert et al., result suggests that DRR72100/71953 contains a tran- 1997b).

Oncogene Human involucrin in transgenic mice JF Crish et al 744

Figure 7 Gel mobility shift analysis of the AP1-5 site. (a) Nuclear extracts were prepared from mouse epidermis. The extracts were then incubated with 32P-labeled double-stranded oligomer, 5'-TTAAGGCTCTTATTATGCCGTGAGTCAGAGGGCGGGAGG- CA-3', encoding the hINV AP1-5 binding site (bold) and ¯anking sequence. Speci®city was demonstrated by including a 10- and 100-fold molar excess of homologous oligomer during the binding reaction. No competition was observed using a 100-fold molar excess of non-speci®c oligonucleotide. Complexes were separated by electrophoresis and visualized by autoradiography. Migration of the free probe (FP) and AP1-speci®c bands is indicated. (b) Nuclear extracts were incubated with oligonucleotide as in (a) and incubated with no antibody, non-speci®c antibody (anti-IgG), or an antibody that detects all fos proteins added for 1 h prior to electrophoresis. Migration of the shifted radioactive probe was visualized by autoradiography. (c) Nuclear extracts were incubated as in (b), except using antibodies speci®c for anti-c-jun, anti-Fra-1, or anti-IgG (non-speci®c control)

Oncogene Human involucrin in transgenic mice JF Crish et al 745 Based on these studies we propose a working model Materials and methods describing this regulation (Figure 8). A previous in vivo study indicates that an AP1/SP1 interaction is required Construction of hINV transgenes for optimal AP1 factor interaction at the AP1-5 site (Banks et al., 1998). The AP1-5 and Sp1 sites are The H6B transgene is a 6 kb HindIII/BamHI fragment containing the complete hINV gene that was derived by separated by a single nucleotide, and increasing this restricting Charon 4AlI-3 with HindIII/BamHI and subclon- spacing or mutating the Sp1 site results in decreased ing the resulting six kilobase fragment into HindIII/BamHI- AP1 factor binding at the AP1-5 site and decreased restricted pSP64 to yield pSP64lI-3 H6B (Eckert and Green, transcriptional activation (Banks et al., 1998). For this 1986). Constructs H6B, DRR-P3.4B and P3.4B were derived reason, and based on our present results, we suggest from this construct and have been previously described (Crish that Sp1 and AP1 factor binding within the DRR72473/ et al., 1998). To construct H6B(AP1 ± 5 mm), plasmid pINV- 2473(AP1 ± 5 mm/Sp1), which contains the AP1-5 mutant site 72100 is required for in vivo expression ± presumably these events are essential for expression during late (Banks et al., 1998; Welter et al., 1995), was digested with dierentiation, but are not adequate for expression HindIII/KpnI. This fragment was used to replace the early in dierentiation. For expression during early corresponding native sequence in pSP64lI-3 H6B to create H6B(AP1 ± 5 mm). The DRR-P3.4B(AP1 ± 5 mm) transgene dierentiation, we suggest the requirement for a second was constructed by HindIII/BglII digestion of H6B(AP1 ± element that is located within the DRR72100/71953 5 mm) and subsequent subcloning of the HindIII/BglII segment. This element, or elements, binds to a fragment containing the AP1-5 mutation into HindIII/BglII- presently unidenti®ed factor or complex that then digested DRR-P3.4B to create DRR-P3.4B(AP1 ± 5 mm). The participates, with the AP1/Sp1 complex, to drive the DRR72473/72100-P3.4B and DRR72100/71953-P3.4B trans- complete program of dierentiation-dependent expres- genes were constructed as follows. The DRR-P3.4B construct sion. An alternate model would suggest that the was digested with SphI/BglII, blunted with T4 DNA DRR element may function independently polymerase, and ligated to create DRR72473/72100-P3.4B. In 72100/71953 parallel, DRR-P3.4B was digested with SphI, blunted, and from the DRR72473/72100 region. However, if this were the case, we would expect to see the DRR digested with BglII. The released 147 bp hINV promoter 72100/71953 fragment (72100/71953) was cloned into a BglII/SmaI- transgene drive expression during early dierentiation, restricted pGL2-basic.. This plasmid, pGL2-DR72100/71953, and this is not observed in our transgenic studies. We was digested with HindIII/AccI and the released DRR further postulate that this transcriptional complex then 72100/ 71953 fragment was cloned to HindIII/AccI-digested P3.4B to interacts with the basic transcription machinery to create DRR72100/71953-P3.4B. For microinjection, the trans- regulate expression. Experiments are underway to test genes were released from plasmid sequences by restriction this model and to identify the transcriptional regula- with HindIII/BamHI. tory element(s) within the downstream DRR. Production and identification of hINV gene-positive mice Mouse embryos from a B6CBA6B6CBA mating were injected with each hINV gene construct and implanted into surrogate mothers as previously described (Crish et al., 1993, 1998). The ospring were characterized for the presence of the human involucrin (hINV) transgene by blotting of tail DNA.

Detection of hINV transgene expression To detect hINV expression in mouse tissues, total protein extracts were prepared from tissue samples in Laemmli sample buer. The samples were electrophoresed on acrylamide gels and transferred to nitrocellulose for antibody-dependent detection of hINV. The blot was incubated with a primary antibody prepared against recombinant human involucrin (LaCelle et al., 1998) diluted 1 : 600, followed by visualization using a chemiluminescent detection system. To localize hINV expression, immunohistology was Figure 8 Discrete elements are required for tissue-speci®c and dierentiation-appropriate hINV gene expression. The AP1-5 and performed as previously described (Crish et al., 1993). Sp1 binding sites are located at adjacent locations in the DRR. These sites are occupied, respectively, by AP1 factors (c-Jun, Fra- Nuclear extract preparation and gel mobility shift and 1, JunB, and JunD in mouse ± Figure 7; JunB, JunD, and Fra-1 supershift assays in humans (Welter et al., 1995)), and Sp1 (Banks et al., 1998). Based on the present study, we suggest that an additional Mouse epidermal extracts were prepared as previously unknown element (or elements) is located within the DRR72100/ described with slight modi®cation (Welter et al., 1995; Fei 71953 segment. We postulate that factors binding to this region et al., 1995). All procedures were carried out at 48C. The cooperate with the Sp1/AP1 factor complex to recruit additional back epidermis from an adult mouse was harvested by ®rst factors to form an active transcriptional complex which then interacts with the basal transcription machinery. We suggest that removing the hair and then scraping the epidermis from the this mechanism is responsible for activating appropriate expres- dermis. Minced epidermis (400 mg) was homogenized in 5 ml sion during keratinocyte dierentiation of Buer A (0.6% Nonidet P-40, 150 mM NaCl, 10 mM

Oncogene Human involucrin in transgenic mice JF Crish et al 746 HEPES pH 7.9, 1 mM EDTA, 0.5 mM PMSF) using type B room temperature and samples were immediately electro- pestle (Kontes). After 25 strokes the homogenate was phoresed at 250 V for 2.5 h on a 5% nondenaturing centrifuged for 30 s at 2000 r.p.m. to remove tissue clumps. acrylamide gel using 1/46TBE running buer. The gels were The supernatant was incubated for 5 min on ice and then then dried for autoradiography. For competition studies, centrifuged for 5 min at 5000 r.p.m. to collect the nuclei. The radioinert DNA competitor was added as a 10- or 100-fold pelleted nuclei were extracted in 500 ml of solution B (25% molar excess. For gel supershift assays, the complete gel glycerol, 20 mM HEPES pH 7.9, 420 mM NaCl, 1.2 mM mobility shift assay mixture, without the 32P-labeled MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, 2 mM benzamidine, oligonucleotide, was incubated at 48C for 2 h in the presence and 5 mg/ml of pepstatin, leupeptin, and aprotinin) on ice for of a rabbit polyclonal antibodies, obtained from Santa Cruz, 20 min. The lysed nuclei were transferred to a microcen- speci®c for pan-fos (sc-253), junD (sc-74), junB (sc-46), c-jun trifuge tube and cellular debris was pelleted by a 15 s (sc-1694), Fra-1 (sc-605), Fra-2 (sc-171), and FosB (sc-48). centrifugation. The supernatant containing the DNA-binding The labeled oligonucleotide was then added and the reaction proteins was then stored frozen in liquid nitrogen and stored mixture incubated and electrophoresed as above. at ± 808C. For gel mobility shift assay, the reaction (20 ml) contained 15% glycerol, 75 mM KCl, 0.375 mM dithiothreitol, Acknowledgments 0.375 mM phenylmethylsulfonyl ¯uoride, 12.5 mM NaCl, This work utilized the facilities of the Skin Diseases 0.1 mg/ml poly(dl-dC), 2.5 mg of nuclear extract, and 0.3 ng Research Center (AR39750) and was supported by grants radiolabeled DNA. The mixture was incubated for 15 min at from the National Institutes of Health (RL Eckert).

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