Abstract
In response to environmental stress, cells trigger a number of molecular mechanisms to control their survival and growth. These include changes in gene expression with corresponding Post-translational modifications to critical transcriptional-control proteins. Transcription is a highly-regulated process that is impacted by a large number of ubiquitous and specific factors. In order to determine how gene expression is regulated in response to environmental cues, it is necessary to correlate modifications to specific transcription proteins with an accurate assessment of the transcriptional response. This chapter details quantitative Real Time PCR (qPCR) and Luciferase assay protocols to illustrate, both in vivo and in vitro, the role of the USF-1 transcription factor in the UV-dependant regulation of pigmentation genes (POMC and MC1R). The procedures have been optimized for the USF-1 transcription factor and the regulation of specific target genes in response to physiological UV doses.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Lam FH, Steger DJ, O’Shea EK (2008) Chromatin decouples promoter threshold from dynamic range. Nature 453:246–250
Urnov FD, Wolffe AP (2001) Chromatin remodeling and transcriptional activation: the cast (in order of appearance). Oncogene 20:2991–3006
Corre S, Galibert MD (2005) Upstream stimulating factors: highly versatile stress-responsive transcription factors. Pigment Cell Res 18:337–348
Ferre-D’Amare AR, Pognonec P, Roeder RG, Burley SK (1994) Structure and function of the b/HLH/Z domain of USF. EMBO J 13:180–189
Bungert J, Kober I, During F, Seifart KH (1992) Transcription factor eUSF is an essential component of isolated transcription complexes on the duck histone H5 gene and it mediates the interaction of TFIID with a TATA-deficient promoter. J Mol Biol 223:885–898
Chiang CM, Roeder RG (1995) Cloning of an intrinsic human TFIID subunit that interacts with multiple transcriptional activators. Science 267:531–536
Meisterernst M, Horikoshi M, Roeder RG (1990) Recombinant yeast TFIID, a general transcription factor, mediates activation by the gene-specific factor USF in a chromatin assembly assay. Proc Natl Acad Sci U S A 87:9153–9157
Chang LA, Smith T, Pognonec P, Roeder RG, Murialdo H (1992) Identification of USF as the ubiquitous murine factor that binds to and stimulates transcription from the immunoglobulin lambda 2-chain promoter. Nucleic Acids Res 20:287–293
Carter RS, Ordentlich P, Kadesch T (1997) Selective utilization of basic helix-loop-helix-leucine zipper proteins at the immunoglobulin heavy-chain enhancer. Mol Cell Biol 17:18–23
Trepicchio WL, Krontiris TG (1993) IGH minisatellite suppression of USF-binding-site- and E mu-mediated transcriptional activation of the adenovirus major late promoter. Nucleic Acids Res 21:977–985
Peter M, Herskowitz I (1994) Joining the complex: cyclin-dependent kinase inhibitory proteins and the cell cycle. Cell 79:181–184
North S, Espanel X, Bantignies F et al (1999) Regulation of cdc2 gene expression by the upstream stimulatory factors (USFs). Oncogene 18:1945–1955
Cogswell JP, Godlevski MM, Bonham M, Bisi J, Babiss L (1995) Upstream stimulatory factor regulates expression of the cell cycle-dependent cyclin B1 gene promoter. Mol Cell Biol 15:2782–2790
Jung HS, Kim KS, Chung YJ et al (2007) USF inhibits cell proliferation through delay in G2/M phase in FRTL-5 cells. Endocr J 54:275–285
van Deursen D, Jansen H, Verhoeven AJ (2008) Glucose increases hepatic lipase expression in HepG2 liver cells through upregulation of upstream stimulatory factors 1 and 2. Diabetologia 51:2078–2087
Nowak M, Helleboid-Chapman A, Jakel H et al (2008) Glucose regulates the expression of the apolipoprotein A5 gene. J Mol Biol 380:789–798
Corre S, Primot A, Sviderskaya E et al (2004) UV-induced expression of key component of the tanning process, the POMC and MC1R genes, is dependent on the p-38 activated upstream stimulating factor-1 (USF-1). J Biol Chem 279:51226–51233
Galibert MD, Carreira S, Goding CR (2001) The Usf-1 transcription factor is a novel target for the stress-responsive p38 kinase and mediates UV-induced Tyrosinase expression. EMBO J 20:5022–5031
Corre S, Mekideche K, Adamski H, Mosser J, Watier E, Galibert MD (2006) In vivo and ex vivo UV-induced analysis of pigmentation gene expressions. J Invest Dermatol 126:916–918
Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res 6:986–994
Colosimo A, Goncz KK, Holmes AR et al (2000) Transfer and expression of foreign genes in mammalian cells. Biotechniques 29:314–318, 320–2, 324 passim
de Wet JR, Wood KV, DeLuca M, Helinski DR, Subramani S (1987) Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7:725–737
Acknowledgment
This work was supported over the years by the LNCC – “Comités Départementaux du Grand Ouest” and the ARC cancer care fundings. We would also like to thank the CNRS, University of Rennes and Brittany Region for their support.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Galibert, MD., Corre, S. (2010). In Vivo and In Vitro Tools to Identify and Study Transcriptional Regulation of USF-1 Target Genes. In: Higgins, P. (eds) Transcription Factors. Methods in Molecular Biology, vol 647. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-738-9_21
Download citation
DOI: https://doi.org/10.1007/978-1-60761-738-9_21
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-737-2
Online ISBN: 978-1-60761-738-9
eBook Packages: Springer Protocols