Journal of Science 103, 925-930 (1992) 925 Printed in Great Britain © The Company of Biologists Limited 1992

Characterization of the involucrin promoter using a transient -galactosidase assay

JOSEPH M. CARROLL1 and LORNE B. TAICHMAN2,*

1Graduate Program in Cellular and Developmental Biology, 2Department of Oral Biology and Pathology, School of Dental Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794-8702, USA *Author for correspondence

Summary

Involucrin, a component of the cornified cell envelope, nascent RNA and suggested that sequences within the is expressed specifically in differentiating intron have regulatory activity. These results suggest of stratified squamous epithelia. To explore the regula- that the involucrin intron operates in vivo to regulate tion of involucrin expression, 3.7 kb of upstream expression in the . sequences of the human involucrin was cloned into a plasmid containing a -galactosidase reporter gene and transfected into early passage keratinocytes and a Abbreviations used: ADH, alcohol dehydrogenase; b-gal, b- galactosidase; DME, Dulbecco’s modified Eagle’s medium; variety of human cell types. The full-length construct DDAB, dimethyldioctyldecylammonium bromide; FCS, fetal calf gave maximal and tissue-specific expression. Deletion serum; kb, kilobase; ONPG, O-nitrophenyl b-D- analysis showed that sequences between 900 and 2500 galactopyranoside; PBS, phosphate buffered saline without bp upstream of the transcriptional start site and the calcium/magnesium salts; PCR, polymerase chain reaction; intron located between the transcriptional and transla- PtdEtn, dioleoyl-L-a -phosphatidylethanolamine; RSV, Rous tional start sites were required for maximal expression. sarcoma virus; SV40, simian virus 40. Further analysis of the intron indicated that its effects on expression were independent of it being present in Key words: involucrin, , keratinocytes.

Introduction pathway to terminal differentiation (Banks-Schlegel and Green, 1981; Watt and Green, 1981; Watt, 1983). The Keratinocytes of the epidermis undergo a progressive ter- appearance of involucrin and its mRNA specifically in the minal differentiation to form a stratified, squamous epithe- differentiating population of keratinocytes has made it an lium (for review, see Watt, 1988). During this process, a important marker of differentiation and indi- number of are specifically expressed in the suprabasal cates that a regulatory mechanism has evolved to restrict layers (Banks-Schlegel and Green, 1981; Moll et al. 1982; expression to this population of cells (Watt, 1983; Watt et Fleckman et al. 1985; Hohl et al. 1991). Included in this al. 1987). class of genes are those whose products are crosslinked, The genomic organization of the involucrin gene down- through the action of a reaction, to form stream of the transcriptional start site is relatively simple, the cornified cell envelope in the upper strata of the tissue consisting of a short non-coding exon (43 bp) followed by (for reviews, see Hohl, 1990; Polakowska and Goldsmith, an 1188 bp intron and a single coding exon (Eckert and 1991). A number of have been implicated as being Green, 1986). To gain an understanding of the genetic precursors to this highly insoluble, chemically resistant mechanisms governing involucrin expression in vitro, we structure (Rice and Green, 1979; Simon and Green, 1984; have linked segments of the upstream region to a b-galac- Zettergren et al. 1984; Ma and Sun, 1986; Richards et al. tosidase reporter gene and monitored transient expression 1988; Mehrel et al. 1990). One of the first proteins to be in primary human keratinocytes as well as other human cell identified as a component of the human cornified envelope types. Because of the importance of 5¢introns in gene reg- was involucrin (Rice and Green, 1979). The human involu- ulation (for review, see Kozak, 1991), we thought it impor- crin gene has been cloned and sequenced and found to tant to include this sequence in our analysis. The results of encode a 68,000 dalton with 39 repeats of 10 codons this study indicate a complex regulation involving promoter (Eckert and Green, 1986). Involucrin mRNA and protein sequences upstream of the transcriptional start site and the first appear in vitro and in vivo at some point after the ker- intron and imply mechanisms of gene regulation in common atinocyte has left the basal position and embarked on its with other eukaryotic genes. 926 J. M. Carroll and L. B. Taichman

Materials and methods RNA analysis Total cellular RNA was isolated from primary keratinocytes 40 h Preparation of expression constructs post-transfection by a modified guanidinium-isothiocyanate The plasmid pNAssbpL-2 was constructed to allow easy insertion method (Chomczynski and Sacchi, 1987). Poly(A)+ RNA was iso- of the involucrin promoter and its various derivatives (see Fig. 1, lated by one passage through a column of oligo(dT)-cellulose. For below). Polylinker sites were added to the plasmid pNAssb (cour- northern analysis of RNA, 2 mg of each respective RNA was run tesy G. MacGregor, Baylor University; MacGregor and Caskey, on a formaldehyde-denaturing 1% agarose gel for 16 h at 40 V 1989) and the final expression vector (called pNAssbpL-2) con- and transferred to nitrocellulose. Blots were probed with ribo- tains a polylinker region, a SV40 splice site, the translational probes prepared with the Gemini system (Promega). The ribo- initiation signal from the Drosophila melanogaster ADH gene probe constructs yielded a b-gal probe of 635 bp and an involu- fused to the bacterial lacZ gene, and a SV40 poly(A) signal. The crin probe of 440 bp, respectively. SV40 splice site includes the SV40 late gene 16s/19s splice donor and two splice acceptor sites (MacGregor and Caskey, 1989). To ensure correct transcriptional initiation of the involucrin con- Results structs, the 140 bp fragment (between the HII site and SD of the intron) that encompasses the transcriptional initiation site was Organization of the human involucrin promoter PCR-amplified from the plasmid H6B (kindly provided by H. For clarity, the upstream region of the involucrin gene has Green; Eckert and Green, 1986) and cloned into pUC19. Various been divided into a distal and a proximal region (- 2,500/ restriction fragments (derived from plasmid H6B) were then sub- - 900 and - 900/- 90, respectively), a TATAA region (- 90/ cloned in front of this segment and the entire respective involu- +40) and an intron (+40/+1240) (Fig. 1A). Restriction dele- crin clone was removed as a HIII(5¢)-EcoRI cassette to be inserted tions and rearrangements of these regions were inserted into into pNAssbpL-2. a b-gal expression vector and transfected into cultures of Cell culture early passage, newborn keratinocytes. All constructs con- Primary keratinocytes were obtained from human newborn fore- tained the 130 bp TATAA region. skins and grown in submerged cultures in the presence of irradi- ated 3T3 cells (Rheinwald and Green, 1975) using “KC medium”, Expression of constructs in primary keratinocytes as described by Wu et al. (1982) with 5% FCS. Keratinocytes A basal level of b-gal activity was defined by the pro- were commonly used at passage nos 4-8. Adult dermal fibroblasts moterless plasmid pL-2 and averaged 43.0 u (Fig. 1D). The (human explant), HeLa (ATCC) and MCF-7 cells (kindly provided values reported in Fig. 1 represent the average and the by Martha Stampfer) were all grown in DME with 10% FCS. s.e.m. of 5 independent experiments, each with duplicate samples. The TATAA region (- 90/+40) construct, contain- Cell transfections ing sequences 65 bp upstream of the TATAA Box gave All transfections were carried out using lipid-mediated gene trans- 61.8 u. The activity of proximal region was 179.5 u, a 3- fer as described by Rose et al. (1991) with slight modifications fold increase over the TATAA region construct. Addition 5 for keratinocytes. Keratinocytes were seeded at 6 ´ 10 cells in a of the distal region to the proximal region was without 60 mm dish and transfected two days later when 60-80% conflu- effect. The activity of the combined distal/proximal regions ent. All other cell types were seeded at approximately 5 ´ 105 the day before transfection. For transfections, 45 ml liposome mix was 158.7 u, as compared to 179.5 u of the proximal region (DDAB and PtdEtn) was added to 1.5 ml of serum-free KC alone. medium in a 15 ml tube and 2.3 ´ 10-6 moles of DNA were added Expression was greatly affected by the presence of the (this corresponded to 10 mg of pL-2). The tube was mixed gently involucrin intron. In its downstream (correct genomic) posi- and incubated at room temperature for 5-10 min prior to addition tion, the intron reduced expression of the proximal promoter to the cells. In the meantime, the cells were washed in serum-free from 179.5 u to 66.5 u (Fig. 1C). The intron was also noted KC medium. The lipid-DNA mix was added and the cells were to reduce activity of the TATAA region from 61.8 u to 35.0 then incubated at 37°C for 3 h (with hourly rocking to ensure even u. It would appear that the intron exerts a negative effect coverage). After this incubation, an equal volume 1.5 ml of KC on expression of the TATAA region as well as on the prox- medium (with serum) was added and the cells were incubated for imal region. an additional 3 h. Cells were then washed 2´ in KC medium, and incubated for 48-60 h before harvesting. Transfections were per- Addition of the intron 3¢to the combined distal/proximal formed on duplicate plates and at least one mock-transfected sister region construct resulted in the highest level of activity plate was used to determine the number of cells present per plate. seen, 586.4 u. It would appear that the intron is capable of increasing or decreasing expression, depending on whether -Galactosidase assay it is linked to the proximal or combined proximal/distal A modified ONPG assay was used to determine activity of pro- upstream regions. moter constructs (Norton and Coffin, 1985; Lim and Chae, 1989). Cells were lysed in PBS containing 0.5% Triton X-100. Lysates -Gal RNA analysis were centrifuged for 5 min in a microfuge at 4°C, ONPG (Sigma) Because one explanation for reduced activity in constructs was added to the supernatant at a concentration of 3.5 mM and where the intron is linked to the proximal region or the the mix was incubated at 37°C until a yellow color was observed. TATAA region is a failure of the intron to be removed The time of ONPG incubation was recorded and the reaction during splicing, the size of the b-gal mRNA was measured stopped by adding 1/5 volume of 1.5 M Na2CO3. The absorbance of the solution at 420 nm was then recorded. The total activity by electrophoresis and northern blot analysis. As seen in was determined using the formula: b-gal activity (u) = [109 ´ Fig. 2, RNA derived from constructs with (construct - 2500/ A420]/[cell no. (per p60) ´ time ONPG incubation (h)] (An et al. +i1240) and without (constructs - 900/+40 and - 2500/+40) 1982). the intron all contained a band at the expected size of 4 kb. Human involucrin promoter 927

Fig. 1. Organization and promoter activity of involucrin upstream sequences. (A) The genomic structure of the involucrin upstream region was divided into 3 regions: the distal and proximal regions, as well as the intron (1188 bp). A TATAA box was also present. The broken line refers to the intron including SD and SA sites. (B) Numbers over stated restriction sites are given in relation to the transcriptional initiation site (wavy arrow). The transcriptional initiation site was confirmed by primer extension (data not shown). The SD site is located at +40 with respect to the transcriptional initiation site. Involucrin segments were cloned into the b-gal expression vector pNAssb-pL2 as described in Materials and methods. (C) Restriction deletion constructs were transfected (lipid- mediated) into early passage keratinocytes and b-gal activity determined 48 h later. Constructs are named based on their sequence distance upstream (- ) and downstream (+) of the transcriptional initiation site. Constructs without the involucrin intron end 2 bp before the SD site. All constructs contain the SV40 16s/19s intron present in pNAssb. b-gal activities reported here represent the average of five independent experiments with two independent plasmid preparations of each construct (± s.e.m.).

Therefore the effects seen on activity here are not due to an attempt was made to distinguish between these possi- any aberrant splicing patterns detectable by northern analy- bilities. sis. Positional dependence of the intron’s regulatory effect Involucrin intron The intron was placed upstream of the proximal and prox- imal/distal regions where it would not be transcribed, It should be mentioned at this point that the involucrin thereby eliminating any possible co- or post-transcriptional intron contains a number of interesting features, including regulatory effect. As seen in Fig. 3 (constructs 3, 4 and 5), a consensus “Blessing” sequence (bp 618-625 of intron) the negative effect of the intron on the proximal region was (Blessing et al. 1987; Blessing et al. 1989) and a stem-loop even more pronounced when placed in this upstream posi- structure (bp 712-732 of intron). Such stem-loop structures tion. This effect was also independent of orientation. have been found to be important in cotranscriptional regu- Whereas the intron exerted a strong positive effect down- lation of various genes (Eick and Bornkamm, 1986; Rama- stream of the combined distal/proximal promoter (Fig. 3, murthy et al. 1990; Mechti et al. 1991). The stem-loop construct 6), when placed upstream it exerted a negative structure has a 7 bp stem, a 5 bp loop and a DG of - 9 kcal. effect in the correct orientation and a slight positive effect Because the negative regulatory effect of the intron could in the opposite orientation (constructs 7 and 8). These be functioning at the pre-, co- or post-transcriptional levels, results indicate that the intron can still exert regulatory effects in the upstream position where it is not present in Fig. 2. b-gal RNA of the RNA and suggest that the repressive effect observed on transfected keratinocytes. expression by the intron operates at the pretranscriptional Poly(A)+ RNA was extracted level. from keratinocytes 40 h post- transfection and 1 mg was run Effect of the intron on a heterologous promoter on a formaldehyde-1% To determine if regulatory effects of the intron were specific agarose denaturing gel. After for the involucrin promoter, the involucrin intron was transfer to nitrocellulose the inserted into a heterologous promoter (RSV) construct blot was hybridized to a 635 between the transcriptional and translational start sites bp b-gal riboprobe overnight (RSVib-gal), as in the involucrin gene. As seen in Fig. 4, and washed. All samples were run on one gel but the presence of the intron decreased RSV-derived activity specific lanes were rearranged to 50% of its original level in primary keratinocytes and to for clarity. Lane 1, - 900/+40. 17% of its original level in MCF-7 cells. No effect of the Lane 2, - 2500/+40. Lane 3, - 2500/+i1240. Lane 4, RSVb-gal. intron on RSV expression was noted in dermal fibroblasts The expected size of this transcript is 4.0 kb and is included as a or HeLa cells. However, in these two cell types control size standard. RSV expression was low. 928 J. M. Carroll and L. B. Taichman

Fig. 3. Positional dependence of the involucrin intron. The entire intron was cloned in either orientation or in a single orientation upstream of the TATAA, proximal and distal segments. b-gal activities were determined as described in Fig. 1.

Tissue-specific nature of expression tory effects in some epithelia as well as primary ker- To ascertain the tissue-specific nature of expression, all con- atinocytes. structs were transfected into HeLa cells, MCF-7 cells and dermal fibroblasts. HeLa cells and MCF-7 cells are epithe- Discussion lial in origin (endocervix and mammary epithelium, respec- tively), and are not known to express involucrin. The lack of involucrin mRNA was confirmed in these cell types (Fig. The major conclusions of this study are that the involucrin 5A). The results of transfecting the involucrin promoter intron exerts a strong silencing effect on both endogenous constructs into these cells are noted in Fig. 5B. There was and heterologous promoter sequences and that high levels virtually no involucrin promoter-derived b-gal activity in of expression in primary keratinocytes require both the HeLa cells or dermal fibroblasts. The lower level of RSV intron and sequences within the distal region. In other sys- expression in these cell types may be due to either a lower tems the addition of an intron, even a heterologous intron, transfection efficiency or a lower level of RSV-promoter to various gene promoters alters gene expression without activity in these cells (Gorman et al. 1982). In MCF-7 cells, regard for the specific intron added (Callis et al. 1987; one construct - 2500/+i1240 produced 105.1 u. Although Nordqvist and Akusjärvi, 1990; Palmiter et al. 1991). The consistently observed, the activity was 6-fold lower than specific effects of the involucrin intron observed in this the activity seen in primary keratinocytes (Fig. 1). Hence, study cannot be explained as simply the result of having an all involucrin promoter-derived constructs exhibited back- intron present because all constructs tested contained a ground, or near-background, levels of expression in 3 cell SV40 intron. RNA derived from different constructs con- types which do not normally express involucrin. The find- tained different 5¢ ends depending on the presence or ing that the intron had such a potent negative effect on absence of the intron and, therefore, some of the effects the heterologous promoter in MCF-7 cells (Fig. 4) is some- seen may have been due to RNA stability or processing. what surprising, yet it suggests that the intron has regula- However, the negative regulatory effects of the intron were still observed when the intron was moved upstream of the promoter and was therefore no longer present in the RNA. In addition, because the involucrin intron had varying Fig. 4. Intron effect on heterologous effects depending on the upstream sequences present, such promoter. The 1188 a generalized, non-specific effect is unlikely. bp intron was The repressive effects of the intron on expression are placed in the SmaI independent of its position and orientation (Figs 1 and 3). site of RSVb-gal These are characteristics seen in cis-acting repressive ele- (construct RSVib- ments present in other genes (Muglia and Rothman-Denes, gal). This position 1986; Bentley and Groudine, 1988; Herbst et al. 1990). The is between the silencing activity of the involucrin intron on a heterologous transcriptional and promoter (RSV) was also noted. In addition to a silencing translational start effect, the intron also exerted a positive effect. Indeed, in sites, as in the involucrin gene. RSVb-gal activity for each cell type was order to obtain full expression it was necessary for the assigned a value of 100%; reductions for the RSVib-gal construct intron to be in its correct genomic position in the full-length are listed as % of full activity. KC, human foreskin keratinocytes; construct (Figs 1 and 3). Because in this position the intron DF, human dermal fibroblasts; HeLa and MCF-7 are defined in would be present in the 5¢ end of the nascent RNA, it is Materials and methods. All cells were transfected using lipid- possible that the positive intron effects operate at the co- mediated transfection. transcriptional or post-transcriptional level. Alternatively, it Human involucrin promoter 929

Fig. 5. Tissue-specific expression of involucrin-b-gal constructs. (A) Northern analysis of involucrin RNA in various human cell types. 10 mg total cellular RNA from these various cell types was electrophoresed and probed with the involucrin riboprobe described in Materials and methods. The uppermost band in all lanes is 28 S RNA and is a measure of the relative amounts of RNA loaded. The arrow indicates the position of involucrin RNA. (B) Three cell types were transfected with the constructs depicted in Fig. 1. Values represent the average and s.e.m. of five independent experiments. *b-gal activity of RSVb-gal in MCF-7 cells was 10,600 ± 642 u. is possible that cis-acting regulatory elements in the intron keratinocytes (Mehrel et al. 1990), also has an 1188 bp are responsible for this stimulatory effect. In fact, there is intron positioned between the transcriptional and transla- an AP2-like site present in the 5¢end of the intron (bp 83- tional start sites, with a very short non-coding first exon 90 of intron). Such sites have recently been shown to be and a single coding exon (D. Hohl, personal communica- necessary for high levels of transcription in the K14 gene tion). Additionally, the human profilaggrin gene, another and to be present in a number of other epidermal genes gene expressed exclusively in the differentiating ker- (Leask et al. 1990; Leask et al. 1991). However the intron atinocyte (Fleckman et al. 1985), and known to be a minor acts to regulate expression, full activity was seen only when component of the cornified envelope (Richards et al. 1988), the distal region was present in the construct. has an intron located between the transcriptional and trans- This study shows that both the intron and distal/proxi- lational start sites (Gan et al. 1990), although the size is mal regions are required for high levels of tissue-specific about one half of the involucrin intron. These three genes expression and that neither alone is sufficient for are also all linked on 1q21 (McKinley-Grant expression. Although the mechanisms of this complex reg- et al. 1989; Simon et al. 1989; Korge et al. 1992). The sim- ulation are not understood, the finding that maximal activity ilar genomic organization and genetic linkage of these three is seen only when the distal region and the intron are present genes raise the possibility that the presence of these 5¢ suggests that both segments are essential for correct involu- introns plays a role in regulating expression of differen- crin expression. In vivo and in vitro, involucrin is expressed tiation-specific genes including components of the cornified only in the suprabasal layers of stratified squamous epithe- cell envelope. lia. An interplay between the negative and positive com- ponents of the intron with sequences upstream in the distal We are very grateful to Bobbie Peters (Cold Spring Harbor region may result in the strata-specific pattern of involucrin Laboratory) for doing the structural DNA analysis; to Marcia expression observed in keratinocytes. Specifically by silenc- Simon (SUNY-Stony Brook) and Miki Blumenberg (NYU Med- ing expression in basal keratinocytes as well as in simple ical School) for critical reading of the manuscript; and to Jim Skill- man for his skilful rendition of our data. This research was funded epithelia cells, and by stimulating expression in differenti- by a grant from the National Institute of Dental Research (DE ating keratinocytes, it would be possible to effect this com- 04511). plex regulation. The similarity between the genomic structure of the involucrin intron and that of other keratinocyte genes must References be commented upon. , another protein component An, G., Hidaka, K., & Siminovitch, L. (1982). Expression of bacterial b- of the cornified cell envelope expressed in differentiating galactosidase in animal cells. Mol. Cell. Biol.2, 1628-1632. 930 J. M. Carroll and L. B. Taichman

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