Search tips
Search criteria 


Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. 1995 April; 15(4): 2063–2070.
PMCID: PMC230433

Nuclear factor I and mammary gland factor (STAT5) play a critical role in regulating rat whey acidic protein gene expression in transgenic mice.


The rat whey acidic protein (WAP) gene contains a mammary gland-specific and hormonally regulated DNase I-hypersensitive site 830 to 720 bp 5' to the site of transcription initiation. We have reported previously that nuclear factor I (NFI) binding at a palindromic site and binding at a half-site are the major DNA-protein interactions detected within this tissue-specific nuclease-hypersensitive region. We now show that point mutations introduced into these NFI-binding sites dramatically affect WAP gene expression in transgenic mice. Transgene expression was totally abrogated when the palindromic NFI site or both binding sites were mutated, suggesting that NFI is a key regulator of WAP gene expression. In addition, a recognition site for mammary gland factor (STAT5), which mediates prolactin induction of milk protein gene expression, was also identified immediately proximal to the NFI-binding sites. Mutation of this site reduced transgene expression by approximately 90% per gene copy, but did not alter tissue specificity. These results suggest that regulation of WAP gene expression is determined by the cooperative interactions among several enhancers that constitute a composite response element.

Full Text

The Full Text of this article is available as a PDF (388K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Apt D, Liu Y, Bernard HU. Cloning and functional analysis of spliced isoforms of human nuclear factor I-X: interference with transcriptional activation by NFI/CTF in a cell-type specific manner. Nucleic Acids Res. 1994 Sep 25;22(19):3825–3833. [PMC free article] [PubMed]
  • Blum JL, Zeigler ME, Wicha MS. Regulation of rat mammary gene expression by extracellular matrix components. Exp Cell Res. 1987 Dec;173(2):322–340. [PubMed]
  • Burdon T, Sankaran L, Wall RJ, Spencer M, Hennighausen L. Expression of a whey acidic protein transgene during mammary development. Evidence for different mechanisms of regulation during pregnancy and lactation. J Biol Chem. 1991 Apr 15;266(11):6909–6914. [PubMed]
  • Burdon TG, Maitland KA, Clark AJ, Wallace R, Watson CJ. Regulation of the sheep beta-lactoglobulin gene by lactogenic hormones is mediated by a transcription factor that binds an interferon-gamma activation site-related element. Mol Endocrinol. 1994 Nov;8(11):1528–1536. [PubMed]
  • Cereghini S, Raymondjean M, Carranca AG, Herbomel P, Yaniv M. Factors involved in control of tissue-specific expression of albumin gene. Cell. 1987 Aug 14;50(4):627–638. [PubMed]
  • Chen LH, Bissell MJ. A novel regulatory mechanism for whey acidic protein gene expression. Cell Regul. 1989 Nov;1(1):45–54. [PMC free article] [PubMed]
  • Conaway RC, Bradsher JN, Conaway JW. Mechanism of assembly of the RNA polymerase II preinitiation complex. Evidence for a functional interaction between the carboxyl-terminal domain of the largest subunit of RNA polymerase II and a high molecular mass form of the TATA factor. J Biol Chem. 1992 Apr 25;267(12):8464–8467. [PubMed]
  • Dale TC, Krnacik MJ, Schmidhauser C, Yang CL, Bissell MJ, Rosen JM. High-level expression of the rat whey acidic protein gene is mediated by elements in the promoter and 3' untranslated region. Mol Cell Biol. 1992 Mar;12(3):905–914. [PMC free article] [PubMed]
  • Devinoy E, Maliénou-N'Gassa R, Thépot D, Puissant C, Houdebine LM. Hormone responsive elements within the upstream sequences of the rabbit whey acidic protein (WAP) gene direct chloramphenicol acetyl transferase (CAT) reporter gene expression in transfected rabbit mammary cells. Mol Cell Endocrinol. 1991 Oct;81(1-3):185–193. [PubMed]
  • de Vries E, van Driel W, van den Heuvel SJ, van der Vliet PC. Contactpoint analysis of the HeLa nuclear factor I recognition site reveals symmetrical binding at one side of the DNA helix. EMBO J. 1987 Jan;6(1):161–168. [PubMed]
  • Ganguly R, Ganguly N, Mehta NM, Banerjee MR. Absolute requirement of glucocorticoid for expression of the casein gene in the presence of prolactin. Proc Natl Acad Sci U S A. 1980 Oct;77(10):6003–6006. [PubMed]
  • González-Crespo S, Levine M. Related target enhancers for dorsal and NF-kappa B signaling pathways. Science. 1994 Apr 8;264(5156):255–258. [PubMed]
  • Gouilleux F, Wakao H, Mundt M, Groner B. Prolactin induces phosphorylation of Tyr694 of Stat5 (MGF), a prerequisite for DNA binding and induction of transcription. EMBO J. 1994 Sep 15;13(18):4361–4369. [PubMed]
  • Goyal N, Knox J, Gronostajski RM. Analysis of multiple forms of nuclear factor I in human and murine cell lines. Mol Cell Biol. 1990 Mar;10(3):1041–1048. [PMC free article] [PubMed]
  • Graves RA, Tontonoz P, Ross SR, Spiegelman BM. Identification of a potent adipocyte-specific enhancer: involvement of an NF-1-like factor. Genes Dev. 1991 Mar;5(3):428–437. [PubMed]
  • Gronostajski RM. Analysis of nuclear factor I binding to DNA using degenerate oligonucleotides. Nucleic Acids Res. 1986 Nov 25;14(22):9117–9132. [PMC free article] [PubMed]
  • Gronostajski RM, Adhya S, Nagata K, Guggenheimer RA, Hurwitz J. Site-specific DNA binding of nuclear factor I: analyses of cellular binding sites. Mol Cell Biol. 1985 May;5(5):964–971. [PMC free article] [PubMed]
  • Hobbs AA, Richards DA, Kessler DJ, Rosen JM. Complex hormonal regulation of rat casein gene expression. J Biol Chem. 1982 Apr 10;257(7):3598–3605. [PubMed]
  • Ihle JN, Witthuhn BA, Quelle FW, Yamamoto K, Thierfelder WE, Kreider B, Silvennoinen O. Signaling by the cytokine receptor superfamily: JAKs and STATs. Trends Biochem Sci. 1994 May;19(5):222–227. [PubMed]
  • Jackson DA, Rowader KE, Stevens K, Jiang C, Milos P, Zaret KS. Modulation of liver-specific transcription by interactions between hepatocyte nuclear factor 3 and nuclear factor 1 binding DNA in close apposition. Mol Cell Biol. 1993 Apr;13(4):2401–2410. [PMC free article] [PubMed]
  • Jiang J, Levine M. Binding affinities and cooperative interactions with bHLH activators delimit threshold responses to the dorsal gradient morphogen. Cell. 1993 Mar 12;72(5):741–752. [PubMed]
  • Jones KA, Kadonaga JT, Rosenfeld PJ, Kelly TJ, Tjian R. A cellular DNA-binding protein that activates eukaryotic transcription and DNA replication. Cell. 1987 Jan 16;48(1):79–89. [PubMed]
  • Lee EY, Parry G, Bissell MJ. Modulation of secreted proteins of mouse mammary epithelial cells by the collagenous substrata. J Cell Biol. 1984 Jan;98(1):146–155. [PMC free article] [PubMed]
  • Lee KF, DeMayo FJ, Atiee SH, Rosen JM. Tissue-specific expression of the rat beta-casein gene in transgenic mice. Nucleic Acids Res. 1988 Feb 11;16(3):1027–1041. [PMC free article] [PubMed]
  • Levine JF, Stockdale FE. Cell-cell interactions promote mammary epithelial cell differentiation. J Cell Biol. 1985 May;100(5):1415–1422. [PMC free article] [PubMed]
  • Li S, Rosen JM. Distal regulatory elements required for rat whey acidic protein gene expression in transgenic mice. J Biol Chem. 1994 May 13;269(19):14235–14243. [PubMed]
  • Li S, Rosen JM. Glucocorticoid regulation of rat whey acidic protein gene expression involves hormone-induced alterations of chromatin structure in the distal promoter region. Mol Endocrinol. 1994 Oct;8(10):1328–1335. [PubMed]
  • Lichtsteiner S, Wuarin J, Schibler U. The interplay of DNA-binding proteins on the promoter of the mouse albumin gene. Cell. 1987 Dec 24;51(6):963–973. [PubMed]
  • Mink S, Härtig E, Jennewein P, Doppler W, Cato AC. A mammary cell-specific enhancer in mouse mammary tumor virus DNA is composed of multiple regulatory elements including binding sites for CTF/NFI and a novel transcription factor, mammary cell-activating factor. Mol Cell Biol. 1992 Nov;12(11):4906–4918. [PMC free article] [PubMed]
  • Nagata K, Guggenheimer RA, Enomoto T, Lichy JH, Hurwitz J. Adenovirus DNA replication in vitro: identification of a host factor that stimulates synthesis of the preterminal protein-dCMP complex. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6438–6442. [PubMed]
  • Paonessa G, Gounari F, Frank R, Cortese R. Purification of a NF1-like DNA-binding protein from rat liver and cloning of the corresponding cDNA. EMBO J. 1988 Oct;7(10):3115–3123. [PubMed]
  • Peterson MG, Tjian R. Transcription. The tell-tail trigger. Nature. 1992 Aug 20;358(6388):620–621. [PubMed]
  • Puissant C, Houdebine LM. Cortisol induces rapid accumulation of whey acid protein mRNA but not of asl and b-casein mRNA in rabbit mammary explants. Cell Biol Int Rep. 1991 Feb;15(2):121–129. [PubMed]
  • Raught B, Khursheed B, Kazansky A, Rosen J. YY1 represses beta-casein gene expression by preventing the formation of a lactation-associated complex. Mol Cell Biol. 1994 Mar;14(3):1752–1763. [PMC free article] [PubMed]
  • Rossi P, Karsenty G, Roberts AB, Roche NS, Sporn MB, de Crombrugghe B. A nuclear factor 1 binding site mediates the transcriptional activation of a type I collagen promoter by transforming growth factor-beta. Cell. 1988 Feb 12;52(3):405–414. [PubMed]
  • Schmitt-Ney M, Doppler W, Ball RK, Groner B. Beta-casein gene promoter activity is regulated by the hormone-mediated relief of transcriptional repression and a mammary-gland-specific nuclear factor. Mol Cell Biol. 1991 Jul;11(7):3745–3755. [PMC free article] [PubMed]
  • Shaul Y, Ben-Levy R, De-Medina T. High affinity binding site for nuclear factor I next to the hepatitis B virus S gene promoter. EMBO J. 1986 Aug;5(8):1967–1971. [PubMed]
  • Tamura T, Miura M, Ikenaka K, Mikoshiba K. Analysis of transcription control elements of the mouse myelin basic protein gene in HeLa cell extracts: demonstration of a strong NFI-binding motif in the upstream region. Nucleic Acids Res. 1988 Dec 23;16(24):11441–11459. [PMC free article] [PubMed]
  • Topper YJ, Freeman CS. Multiple hormone interactions in the developmental biology of the mammary gland. Physiol Rev. 1980 Oct;60(4):1049–1106. [PubMed]
  • Wakao H, Gouilleux F, Groner B. Mammary gland factor (MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response. EMBO J. 1994 May 1;13(9):2182–2191. [PubMed]
  • Wakao H, Schmitt-Ney M, Groner B. Mammary gland-specific nuclear factor is present in lactating rodent and bovine mammary tissue and composed of a single polypeptide of 89 kDa. J Biol Chem. 1992 Aug 15;267(23):16365–16370. [PubMed]
  • Watson CJ, Gordon KE, Robertson M, Clark AJ. Interaction of DNA-binding proteins with a milk protein gene promoter in vitro: identification of a mammary gland-specific factor. Nucleic Acids Res. 1991 Dec 11;19(23):6603–6610. [PMC free article] [PubMed]
  • Welte T, Garimorth K, Philipp S, Doppler W. Prolactin-dependent activation of a tyrosine phosphorylated DNA binding factor in mouse mammary epithelial cells. Mol Endocrinol. 1994 Aug;8(8):1091–1102. [PubMed]
  • Welte T, Philipp S, Cairns C, Gustafsson JA, Doppler W. Glucocorticoid receptor binding sites in the promoter region of milk protein genes. J Steroid Biochem Mol Biol. 1993 Dec;47(1-6):75–81. [PubMed]
  • Wiens D, Park CS, Stockdale FE. Milk protein expression and ductal morphogenesis in the mammary gland in vitro: hormone-dependent and -independent phases of adipocyte-mammary epithelial cell interaction. Dev Biol. 1987 Mar;120(1):245–258. [PubMed]
  • Xiao H, Lis JT, Xiao H, Greenblatt J, Friesen JD. The upstream activator CTF/NF1 and RNA polymerase II share a common element involved in transcriptional activation. Nucleic Acids Res. 1994 Jun 11;22(11):1966–1973. [PMC free article] [PubMed]
  • Yao Z, Jones DH, Grose C. Site-directed mutagenesis of herpesvirus glycoprotein phosphorylation sites by recombination polymerase chain reaction. PCR Methods Appl. 1992 Feb;1(3):205–207. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)