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. 2000 May 15;348(Pt 1):45–53.

Identification of an involucrin promoter transcriptional response element with activity restricted to keratinocytes.

M A Phillips 1, Q Qin 1, R H Rice 1
PMCID: PMC1221034  PMID: 10794712

Abstract

The involucrin proximal promoter was examined for response elements that confer cell-type specificity. Using a segment spanning positions -157 to +41, three possible response elements were identified by their protein-binding activity using DNase I footprinting. From distal to proximal, they were: an activator protein-1 (AP-1) site (previously identified) overlapping an Ets-like site; a second Ets-like site located 13 bp more proximally; and an extended region designated footprinted site A (FPA). Mutation of the distal Ets-like site had essentially no effect on the transcriptional activity in transfections, while mutation of the proximal site reduced the activity by half. FPA was shown by electrophoretic mobility-shift assay (EMSA) to be comprised of two separable binding sites, FPA1 (distal) and FPA2 (proximal). While mutation of FPA2 had only a modest effect on transcriptional activity in transient transfections, mutation of FPA1 reduced transcriptional activity to approx. 20% of that obtained with the intact promoter. Additional mutations of FPA1 indicated that the active region comprises positions -85 to -73 (GTGGTGAAACCTGT). The molecular masses of the major proteins binding to this site were shown by UV cross-linking to be approx. 40 and 50 kDa, while minor bands were observed at 80 and 110 kDa. Since the involucrin promoter exhibits much higher transcriptional activity in keratinocytes than in other cell types in transfection assays (indicating that cell type specificity of expression is retained), the comparative influence of FPA1 was examined. While mutation of the AP-1 site affected transcriptional activity similarly in all cell lines tested, mutation of FPA1 decreased activity substantially in keratinocytes, but not in NIH-3T3 and HeLa cells, evidence for a contribution to cell-type specificity of expression. Furthermore, a correlation between the sensitivity to FPA1 mutation and amount of involucrin expression in different keratinocyte cell lines was evident. EMSA showed that NIH-3T3 and HeLa cells lacked the same FPA1 DNA-protein complex as keratinocytes. However, the amount of complex formed with nuclear extracts from several keratinocyte lines did not correlate well with the level of involucrin expression. Other factors, such as differences in post-translational modification or co-activators, must account for varied transcriptional response mediated by this site among keratinocyte lines.

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Selected References

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  1. Agarwal C., Efimova T., Welter J. F., Crish J. F., Eckert R. L. CCAAT/enhancer-binding proteins. A role in regulation of human involucrin promoter response to phorbol ester. J Biol Chem. 1999 Mar 5;274(10):6190–6194. doi: 10.1074/jbc.274.10.6190. [DOI] [PubMed] [Google Scholar]
  2. Andreoli J. M., Jang S. I., Chung E., Coticchia C. M., Steinert P. M., Markova N. G. The expression of a novel, epithelium-specific ets transcription factor is restricted to the most differentiated layers in the epidermis. Nucleic Acids Res. 1997 Nov 1;25(21):4287–4295. doi: 10.1093/nar/25.21.4287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Banks E. B., Crish J. F., Welter J. F., Eckert R. L. Characterization of human involucrin promoter distal regulatory region transcriptional activator elements-a role for Sp1 and AP1 binding sites. Biochem J. 1998 Apr 1;331(Pt 1):61–68. doi: 10.1042/bj3310061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Butler R. N., August P., Ferdinand K. C., Phillips R. A., Roccella E. J. Hypertension: setting new goals for lower readings. Geriatrics. 1999 Apr;54(4):20-1, 25-6, 29-30 passim. [PubMed] [Google Scholar]
  5. Carroll J. M., Albers K. M., Garlick J. A., Harrington R., Taichman L. B. Tissue- and stratum-specific expression of the human involucrin promoter in transgenic mice. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10270–10274. doi: 10.1073/pnas.90.21.10270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carroll J. M., Taichman L. B. Characterization of the human involucrin promoter using a transient beta-galactosidase assay. J Cell Sci. 1992 Dec;103(Pt 4):925–930. doi: 10.1242/jcs.103.4.925. [DOI] [PubMed] [Google Scholar]
  7. Crish J. F., Howard J. M., Zaim T. M., Murthy S., Eckert R. L. Tissue-specific and differentiation-appropriate expression of the human involucrin gene in transgenic mice: an abnormal epidermal phenotype. Differentiation. 1993 Jul;53(3):191–200. doi: 10.1111/j.1432-0436.1993.tb00708.x. [DOI] [PubMed] [Google Scholar]
  8. Crish J. F., Zaim T. M., Eckert R. L. The distal regulatory region of the human involucrin promoter is required for expression in epidermis. J Biol Chem. 1998 Nov 13;273(46):30460–30465. doi: 10.1074/jbc.273.46.30460. [DOI] [PubMed] [Google Scholar]
  9. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Eckert R. L., Green H. Structure and evolution of the human involucrin gene. Cell. 1986 Aug 15;46(4):583–589. doi: 10.1016/0092-8674(86)90884-6. [DOI] [PubMed] [Google Scholar]
  11. Fischer D. F., Gibbs S., van De Putte P., Backendorf C. Interdependent transcription control elements regulate the expression of the SPRR2A gene during keratinocyte terminal differentiation. Mol Cell Biol. 1996 Oct;16(10):5365–5374. doi: 10.1128/mcb.16.10.5365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fuchs E. Epidermal differentiation: the bare essentials. J Cell Biol. 1990 Dec;111(6 Pt 2):2807–2814. doi: 10.1083/jcb.111.6.2807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gandarillas A., Watt F. M. The 5' noncoding region of the mouse involucrin gene: comparison with the human gene and genes encoding other cornified envelope precursors. Mamm Genome. 1995 Sep;6(9):680–682. doi: 10.1007/BF00352383. [DOI] [PubMed] [Google Scholar]
  14. Greenberg C. S., Birckbichler P. J., Rice R. H. Transglutaminases: multifunctional cross-linking enzymes that stabilize tissues. FASEB J. 1991 Dec;5(15):3071–3077. doi: 10.1096/fasebj.5.15.1683845. [DOI] [PubMed] [Google Scholar]
  15. Kachinskas D. J., Qin Q., Phillips M. A., Rice R. H. Arsenate suppression of human keratinocyte programming. Mutat Res. 1997 Jun;386(3):253–261. doi: 10.1016/s1383-5742(97)00015-x. [DOI] [PubMed] [Google Scholar]
  16. LaPres J. J., Hudson L. G. Identification of a functional determinant of differentiation-dependent expression in the involucrin gene. J Biol Chem. 1996 Sep 20;271(38):23154–23160. doi: 10.1074/jbc.271.38.23154. [DOI] [PubMed] [Google Scholar]
  17. Lee J. H., Jang S. I., Yang J. M., Markova N. G., Steinert P. M. The proximal promoter of the human transglutaminase 3 gene. Stratified squamous epithelial-specific expression in cultured cells is mediated by binding of Sp1 and ets transcription factors to a proximal promoter element. J Biol Chem. 1996 Feb 23;271(8):4561–4568. [PubMed] [Google Scholar]
  18. Lopez-Bayghen E., Vega A., Cadena A., Granados S. E., Jave L. F., Gariglio P., Alvarez-Salas L. M. Transcriptional analysis of the 5'-noncoding region of the human involucrin gene. J Biol Chem. 1996 Jan 5;271(1):512–520. doi: 10.1074/jbc.271.1.512. [DOI] [PubMed] [Google Scholar]
  19. Mack D. H., Laimins L. A. A keratinocyte-specific transcription factor, KRF-1, interacts with AP-1 to activate expression of human papillomavirus type 18 in squamous epithelial cells. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9102–9106. doi: 10.1073/pnas.88.20.9102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nakamaye K. L., Eckstein F. Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis. Nucleic Acids Res. 1986 Dec 22;14(24):9679–9698. doi: 10.1093/nar/14.24.9679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nemes Z., Steinert P. M. Bricks and mortar of the epidermal barrier. Exp Mol Med. 1999 Mar 31;31(1):5–19. doi: 10.1038/emm.1999.2. [DOI] [PubMed] [Google Scholar]
  22. Oettgen P., Alani R. M., Barcinski M. A., Brown L., Akbarali Y., Boltax J., Kunsch C., Munger K., Libermann T. A. Isolation and characterization of a novel epithelium-specific transcription factor, ESE-1, a member of the ets family. Mol Cell Biol. 1997 Aug;17(8):4419–4433. doi: 10.1128/mcb.17.8.4419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Phillips M. A., Rice R. H. Convergent differentiation in cultured rat cells from nonkeratinized epithelia: keratinocyte character and intrinsic differences. J Cell Biol. 1983 Sep;97(3):686–691. doi: 10.1083/jcb.97.3.686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Reddy S. P., Chuu Y. J., Lao P. N., Donn J., Ann D. K., Wu R. Expression of human squamous cell differentiation marker, SPR1, in tracheobronchial epithelium depends on JUN and TRE motifs. J Biol Chem. 1995 Nov 3;270(44):26451–26459. doi: 10.1074/jbc.270.44.26451. [DOI] [PubMed] [Google Scholar]
  25. Rheinwald J. G., Beckett M. A. Tumorigenic keratinocyte lines requiring anchorage and fibroblast support cultured from human squamous cell carcinomas. Cancer Res. 1981 May;41(5):1657–1663. [PubMed] [Google Scholar]
  26. Rheinwald J. G., Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell. 1975 Nov;6(3):331–343. doi: 10.1016/s0092-8674(75)80001-8. [DOI] [PubMed] [Google Scholar]
  27. Rice R. H., Green H. Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions. Cell. 1979 Nov;18(3):681–694. doi: 10.1016/0092-8674(79)90123-5. [DOI] [PubMed] [Google Scholar]
  28. Rice R. H., Steinmann K. E., deGraffenried L. A., Qin Q., Taylor N., Schlegel R. Elevation of cell cycle control proteins during spontaneous immortalization of human keratinocytes. Mol Biol Cell. 1993 Feb;4(2):185–194. doi: 10.1091/mbc.4.2.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sark M. W., Fischer D. F., de Meijer E., van de Putte P., Backendorf C. AP-1 and ets transcription factors regulate the expression of the human SPRR1A keratinocyte terminal differentiation marker. J Biol Chem. 1998 Sep 18;273(38):24683–24692. doi: 10.1074/jbc.273.38.24683. [DOI] [PubMed] [Google Scholar]
  30. Taylor J. W., Schmidt W., Cosstick R., Okruszek A., Eckstein F. The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8749–8764. doi: 10.1093/nar/13.24.8749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wang H., Liu K., Yuan F., Berdichevsky L., Taichman L. B., Auborn K. C/EBPbeta is a negative regulator of human papillomavirus type 11 in keratinocytes. J Virol. 1996 Jul;70(7):4839–4844. doi: 10.1128/jvi.70.7.4839-4844.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Watt F. M. Terminal differentiation of epidermal keratinocytes. Curr Opin Cell Biol. 1989 Dec;1(6):1107–1115. doi: 10.1016/s0955-0674(89)80058-4. [DOI] [PubMed] [Google Scholar]
  33. Welter J. F., Crish J. F., Agarwal C., Eckert R. L. Fos-related antigen (Fra-1), junB, and junD activate human involucrin promoter transcription by binding to proximal and distal AP1 sites to mediate phorbol ester effects on promoter activity. J Biol Chem. 1995 May 26;270(21):12614–12622. doi: 10.1074/jbc.270.21.12614. [DOI] [PubMed] [Google Scholar]

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