Oncogene (2002) 21, 738 ± 747 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc 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 keratino- cytes (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,