Journal of Cell Science 103, 925-930 (1992) 925 Printed in Great Britain © The Company of Biologists Limited 1992 Characterization of the human 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 keratinocytes 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 epidermis. sequences of the human involucrin gene 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, gene expression, 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 keratinocyte differentiation and indi- number of genes 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 transglutaminase 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 proteins 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 protein 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.