Abstract
Cis-regulatory elements are regions of DNA that regulate the expression of genes located on that same molecule of DNA. Though these elements are important for gene expression regulation, the functions of cis elements remain largely unknown. To explore the mechanisms of gene activation by short sequence enhancers, we examined the enhancer activity of short sequence DNA and its derived sequences using a GFP expression system. We found that AA sequence (5′-GTGAAATAAATGCAAATAAAGT) induced strong GFP gene expression, while 7pieA (5′-GTGAAAAAAATGCAAAAAAAGT) did not. We mutated the five T bases of the AA sequence to A, C or G. Our findings indicated that sequences retaining the 7th and/or 17th Ts possessed strong enhancer activity. RT-PCR and RNA synthesis inhibition analysis using actinomycin D revealed that the enhanced GFP gene expression induced by the AA sequence occurred at the transcriptional level. To determine whether the AA sequence formed a secondary structure via atypical complementation, PAGE method was used, and the results showed that the AA sequence formed a secondary structure. Our results support previous evidence that AATAAA is an important composition of cis elements (enhancer/promoter), and suggest that the formation of an unstable stem-loop structure via atypical complementation may be a new mechanism of enhancer activity.
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Chou SH, Zhu L, Gao Z, Cheng JW, Reid BR (1996) Hairpin loops consisting of single adenine residues closed by sheared A–A and G–G pairs formed by the DNA triplets AAA and GAG: solution structure of the d (GTACAAAGTAC) hairpin. J Mol Biol 264:981–1001
Chou SH, Chin KH, Wang AH (2003) Unusual DNA duplex and hairpin motifs. Nucleic Acids Res 31:2461–2474
Frank R, Köster H (1979) DNA chain length markers and the influence of base composition on electrophoretic mobility of oligodeoxyribonucleotides in polyacrylamide-gels. Nucleic Acids Res 6:2069–2087
Gimmi ER, Soprano KJ, Rosenberg M, Reff ME (1988) Deletions in the SV40 late polyadenylation region downstream of the AATAAA mediate similar effects on expression in various mammalian cell lines. Nucleic Acids Res 16:8977–8997
Gong WF, Zhong JH, Xiang BD, Ma L, You XM, Zhang QM, Li LQ (2013) Single nucleotide polymorphism 8q24 rs13281615 and risk of breast cancer: meta-analysis of more than 100,000 cases. PLoS One 8:e60108
Hancks DC, Kazazian HH Jr (2012) Active human retrotransposons: variation and disease. Curr Opin Genet Dev 22:191–203
Holmquist GP (1992) Chromosome bands, their chromatin flavors, and their functional features. Am J Hum Genet 51:17–37
Italia K, Sawant P, Surve R, Wadia M, Nadkarni A, Ghosh K, Colah R (2012) Variable haematological and clinical presentation of β-thalassaemia carriers and homozygotes with the Poly A (T → C) mutation in the Indian population. Eur J Haematol 89:160–164
Kankia BI (2003) Binding of Mg2+ to single-stranded polynucleotides: hydration and optical studies. Biophys Chem 104:643–654
Kuzin A, Kundum M, Ross J, Koizumi K, Brody T, Odenwald WF (2012) The cis-regulatory dynamics of the Drosophila CNS determinant castor are controlled by multiple sub-pattern enhancers. Gene Expr Patterns 12:261–272
Li SP, Feng JJ, Wang HG, Wang XF, Lv ZJ (2012) The effects of SV40 PolyA sequence and its AATAAA signal on upstream GFP gene expression and transcription termination. Yi Chuan 34:113–119
Mees ST, Mardin WA, Schleicher C, Colombo-Benkmann M, Senninger N, Haier J (2011) Role of tumor microenvironment on gene expression in pancreatic cancer tumor models. J Surg Res 171:136–142
Mojtahedi Z, Erfani N, Malekzadeh M, Haghshenas MR, Ghaderi A, Samsami Dehaghani A (2013) HER2 Ile655Val single nucleotide polymorphism in patients with ovarian cancer. Iran Red Crescent Med J 15:1–3
Mouchiroud D, Fichant G, Bernardi G (1987) Compositional compartmentalization and gene composition in the genome of vertebrates. J Mol Evol 26:198–204
Peyret N, Seneviratne PA, Allawi HT, SantaLucia J Jr (1999) Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A–A, C–C, G–G, and T–T mismatches. Biochemistry 38:3468–3477
Pochareddy S, Edenberg HJ (2010) Identification of a FOXA-dependent enhancer of human alcohol dehydrogenase 4 (ADH4). Gene 460:1–7
Schlientz NW, Schuster GB (2003) Long-distance radical cation migration in duplex DNA: the effect of contiguous A–A and T–T mismatches on efficiency and mechanism. J Am Chem Soc 125:15732–15733
Schmieder A, Michel J, Schönhaar K, Goerdt S, Schledzewski K (2012) Differentiation and gene expression profile of tumor-associated macrophages. Semin Cancer Biol 22:289–297
Schulpen SH, Robinson JF, Pennings JL, van Dartel DA, Piersma AH (2013) Dose response analysis of monophthalates in the murine embryonic stem cell test assessed by cardiomyocyte differentiation and gene expression. Reprod Toxicol 35:81–88
Semaan SJ, Li Y, Nickells RW (2010) A single nucleotide polymorphism in the Bax gene promoter affects transcription and influences retinal ganglion cell death. ASN Neuro 2:e00032
Tanaka Y, Mukaide M, Orito E, Yuen MF, Ito K, Kurbanov F, Sugauchi F, Asahina Y, Izumi N, Kato M (2006) Specific mutations in enhancer II/core promoter of hepatitis B virus subgenotypes C1/C2 increase the risk of hepatocellular carcinoma. J Hepatol 45:646–653
Tanaka C, Coling DE, Manohar S, Chen GD, Hu BH, Salvi R, Henderson D (2012) Expression pattern of oxidative stress and antioxidant defense-related genes in the ageing Fischer 344/NHsd rat cochlea. Neurobiol Aging 33:1842.e1–1842.e14. doi:10.1016/j.neurobiolaging.2011.12.027
Tasiopoulou M, Boussiou M, Sinopoulou K, Moraitis G, Loutradi-Anagnostou A, Karababa P (2008) Gγ-196 C → T, Aγ-201 C → T: two novel mutations in the promoter region of the γ-globin genes associated with nondeletional hereditary persistence of fetal hemoglobin in Greece. Blood Cells Mol Dis 40:320–322
Wang XF, Wang XY, Liu J, Feng JJ, Mu WL, Shi XJ, Yang QQ, Duan XC, Xie Y, Lu ZJ (2009) Alu tandem sequences inhibit GFP gene expression by triggering chromatin wrapping. Genes Genom 31:209–215
Wang HG, Wang XF, Jing XY, Li Z, Zhang Y, Lv ZJ (2011a) Effect of mutations in a simian virus 40 PolyA signal enhancer on green fluorescent protein reporter gene expression. Genet Mol Res 10:1866–1883
Wang HG, Sun WZ, Li Z, Wang XF, Lv ZJ (2011b) Identification and characterization of two critical sequences in SV40PolyA that activate the green fluorescent protein reporter gene. Genet Mol Biol 34:396–405
Wilson WD, Zuo ET, Jones RL, Zon GL, Baumstark BR (1987) Sequence dependent electrophoretic mobilities and melting temperatures for A–T containing oligodeoxyribonucleotides. Nucleic Acids Res 15:105–118
Yin K, Wang XF, Ma H, Xie Y, Feng JJ, Yang QQ, Lü ZJ (2010) Impact of copy number of distinct SV40PolyA segments on expression of a GFP reporter gene. Sci China Life Sci 53:606–612
Acknowledgments
We thank L. Z. Zhang for the technical help. This study was financially supported by the Hebei Province Natural Science Foundation of China (Grant Numbers H2013206101 and C2011206043), the Key Project of Hebei Province (Grant Number 08276101D-90) and Scientific Research Funding from the Department of Public Health of Hebei Province (Grant Number 20100242).
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Ma, Z., Jing, X., Cheng, J. et al. The effects of a short sequence enhancer (5′-GTGAAATAAATGCAAATAAAGT) and its derived sequences on green fluorescent protein expression. Genes Genom 36, 455–464 (2014). https://doi.org/10.1007/s13258-014-0180-2
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DOI: https://doi.org/10.1007/s13258-014-0180-2