Molecular Biology Reports

, Volume 41, Issue 9, pp 6089–6096 | Cite as

Transcriptional regulation analysis and the potential transcription regulator site in the extended KAP6.1 promoter in sheep



The high glycine/tyrosine type II keratin protein 6.1 (KAP6.1) is a member of the keratin-associated protein family, which is restricted to cells in hair follicles and is associated with fiber diameter and fiber curvature in Merino sheep. In this study, we obtained a series of progressive 5′-deletion fragments of the KAP6.1 gene promoter from ovine genomic DNA. The KAP6.1 5′-upstream region was fused to luciferase and transfected into sheep fetal fibroblast cells (sFFCs). We demonstrated that the sequence from −1,523 to −1 bp (taking the A of the initiator methionine ATG as the +1 nt position) gave rise to a higher luciferase activity comparing to the published region from −1,042 to −1 bp. Whereas, decreased transcriptional activity of the KAP6.1 promoter was observed when the sequence extended to −3,699 bp. To identify the DNA elements that are important for transcriptional activity, we performed structural analysis and electrophoretic mobility shift assay (EMSA). Structural analysis of the KAP6.1 promoter showed that transcription factors NF-kappa-B, AP-1, and C/EBP-alpha synergistically activate KAP6.1 promoter, while POU2F1 might function as a strong negative regulator. The EMSA showed that NF-kappa-B element bound to the nuclear protein extracted from sFFCs. We conclude that NF-kappa-B binding site is an enhancer element of KAP6.1 promoter in vitro. The results are potentially useful for elucidating the regulator mechanisms of KAP6.1.


Ovine KAP6.1 Transcriptional regulation Electrophoretic mobility shift assay 



This work was supported by the Major Project for Cultivation Technology of New Varieties of Genetically Modified Organisms of the Ministry of Agriculture (2013ZX08008-001, 2011ZX08008-001).


  1. 1.
    McLaren RJ et al (1997) Linkage mapping of wool keratin and keratin-associated protein genes in sheep. Mamm Genome 8(12):938–940CrossRefPubMedGoogle Scholar
  2. 2.
    Powell BC, Rogers GE (1997) The role of keratin proteins and their genes in the growth, structure and properties of hair. EXS 78:59–148PubMedGoogle Scholar
  3. 3.
    Rogers MA, Schweizer J (2005) Human KAP genes, only the half of it? Extensive size polymorphisms in hair keratin-associated protein genes. J Invest Dermatol 124:vii–ixCrossRefPubMedGoogle Scholar
  4. 4.
    Cai RL (1998) Human CART1, a paired-class homeodomain protein, activates transcription through palindromic binding sites. Biochem Biophys Res Commun 250(2):305–311CrossRefPubMedGoogle Scholar
  5. 5.
    Powell BC, Nesci A, Rogers GE (1991) Regulation of keratin gene expression in hair follicle differentiation. Ann N Y Acad Sci 642:1–20CrossRefPubMedGoogle Scholar
  6. 6.
    Plowman JE, Paton LN, Bryson WG (2007) The differential expression of proteins in the cortical cells of wool and hair fibres. Exp Dermatol 16(9):707–714CrossRefPubMedGoogle Scholar
  7. 7.
    Parsons YM, Cooper DW, Piper LR (1994) Evidence of linkage between high-glycine-tyrosine keratin gene loci and wool fibre diameter in a Merino half-sib family. Anim Genet 25(2):105–108CrossRefPubMedGoogle Scholar
  8. 8.
    Fratini A, Powell BC, Rogers GE (1993) Sequence, expression, and evolutionary conservation of a gene encoding a glycine/tyrosine-rich keratin-associated protein of hair. J Biol Chem 268(6):4511–4518PubMedGoogle Scholar
  9. 9.
    Adelson DL et al (2004) Gene expression in sheep skin and wool (hair). Genomics 83(1):95–105CrossRefPubMedGoogle Scholar
  10. 10.
    Plowman J E, B.W.G (2006) Wool keratins - the challenge ahead. Proceedings of the New Zealand Society of Animal Production 66Google Scholar
  11. 11.
    Dunn SM et al (1998) Regulation of a hair follicle keratin intermediate filament gene promoter. J Cell Sci 111(Pt 23):3487–3496PubMedGoogle Scholar
  12. 12.
    Rossi A et al (1998) Effect of AP1 transcription factors on the regulation of transcription in normal human epidermal keratinocytes. J Invest Dermatol 110(1):34–40CrossRefPubMedGoogle Scholar
  13. 13.
    Maytin EV et al (1999) Keratin 10 gene expression during differentiation of mouse epidermis requires transcription factors C/EBP and AP-2. Dev Biol 216(1):164–181CrossRefPubMedGoogle Scholar
  14. 14.
    Chen TT et al (1997) Regulation of K3 keratin gene transcription by Sp1 and AP-2 in differentiating rabbit corneal epithelial cells. Mol Cell Biol 17(6):3056–3064PubMedCentralPubMedGoogle Scholar
  15. 15.
    Sugihara TM et al (2001) The POU domain factor Skin-1a represses the keratin 14 promoter independent of DNA binding. A possible role for interactions between Skn-1a and CREB-binding protein/p300. J Biol Chem 276(35):33036–33044CrossRefPubMedGoogle Scholar
  16. 16.
    Andersen B et al (1997) Functions of the POU domain genes Skn-1a/i and Tst-1/Oct-6/SCIP in epidermal differentiation. Genes Dev 11(14):1873–1884CrossRefPubMedGoogle Scholar
  17. 17.
    Radoja N et al (2004) Thyroid hormones and gamma interferon specifically increase K15 keratin gene transcription. Mol Cell Biol 24(8):3168–3179PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Komine M et al (2001) Interleukin-1 induces transcription of keratin K6 in human epidermal keratinocytes. J Invest Dermatol 116(2):330–338CrossRefPubMedGoogle Scholar
  19. 19.
    Komine M et al (2000) Inflammatory versus proliferative processes in epidermis. Tumor necrosis factor alpha induces K6b keratin synthesis through a transcriptional complex containing NFkappa B and C/EBPbeta. J Biol Chem 275(41):32077–32088CrossRefPubMedGoogle Scholar
  20. 20.
    Ma S et al (1997) Transcriptional control of K5, K6, K14, and K17 keratin genes by AP-1 and NF-kappaB family members. Gene Expr 6(6):361–370PubMedGoogle Scholar
  21. 21.
    Radoja N et al (2000) Novel mechanism of steroid action in skin through glucocorticoid receptor monomers. Mol Cell Biol 20(12):4328–4339PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Dubchak I, Ryaboy DV (2006) VISTA family of computational tools for comparative analysis of DNA sequences and whole genomes. Methods Mol Biol 338:69–89PubMedGoogle Scholar
  23. 23.
    Brudno M et al (2003) LAGAN and Multi-LAGAN: efficient tools for large-scale multiple alignment of genomic DNA. Genome Res 13(4):721–731PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Liu LR et al (2005) T to C substitution at -175 or -173 of the gamma-globin promoter affects GATA-1 and Oct-1 binding in vitro differently but can independently reproduce the hereditary persistence of fetal hemoglobin phenotype in transgenic mice. J Biol Chem 280(9):7452–7459CrossRefPubMedGoogle Scholar
  25. 25.
    Liu Y et al (2004) Eukaryotic regulatory element conservation analysis and identification using comparative genomics. Genome Res 14(3):451–458PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Jiang CK et al (1993) Epidermal growth factor and transforming growth factor alpha specifically induce the activation- and hyperproliferation-associated keratins 6 and 16. Proc Natl Acad Sci USA 90(14):6786–6790PubMedCentralCrossRefPubMedGoogle Scholar
  27. 27.
    Gordon DF et al (1996) Human Cart-1: structural organization, chromosomal localization, and functional analysis of a cartilage-specific homeodomain cDNA. DNA Cell Biol 15(7):531–541CrossRefPubMedGoogle Scholar
  28. 28.
    Cai RL (1998) Human CART1, a paired-class homeodomain protein, activates transcription through palindromic binding sites. Biochem Biophys Res Commun 250(2):305–311CrossRefPubMedGoogle Scholar
  29. 29.
    Holmqvist E et al (2010) Two antisense RNAs target the transcriptional regulator CsgD to inhibit curli synthesis. EMBO J 29(11):1840–1850PubMedCentralCrossRefPubMedGoogle Scholar
  30. 30.
    Zhang F, Hinnebusch AG (2011) An upstream ORF with non-AUG start codon is translated in vivo but dispensable for translational control of GCN4 mRNA. Nucleic Acids Res 39(8):3128–3140PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  1. 1.Beijing Key Laboratory of Animal Genetic Improvement & Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and TechnologyChina Agricultural UniversityBeijingChina

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