Abstract
Steroid hormone receptors (SHR) are important transcription factors for regulating different physiological and pathological processes. Their altered expression has been strongly associated to cancer progression. Epigenetic marks such as DNA methylation have been proposed as one of the regulatory mechanisms for SHR expression in cancer. DNA methylation occurs at CpG dinucleotides, which form clusters known as CpG islands. These islands are mostly observed at promoter regions of housekeeping genes, and their aberrant methylation in cancer cells is associated with silencing of tumor-suppressor gene expression. SHR genes are characterized for presenting alternative promoters with different CpG island content, which are prone to be methylated. The method of choice for studying DNA methylation is bisulfite sequencing, since it provides information about the methylation pattern at single-nucleotide level. The method is based on the deamination of cytosine residues to uracil after treatment with sodium bisulfite. The converted DNA is amplified by a polymerase chain reaction, cloned, and sequenced. Here, we describe a protocol for bisulfite sequencing suitable for analyzing different CpG regions in SHR genes.
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References
Griekspoor A, Zwart W, Neefjes J et al (2007) Visualizing the action of steroid hormone receptors in living cells. Nucl Recept Signal 5:1–9
Ahmad N, Kumar R (2011) Steroid hormone receptors in cancer development: a target for cancer therapeutics. Cancer Lett 300:1–9
Green CD, Han JD (2011) Epigenetic regulation by nuclear receptors. Epigenomics 3:59–72
Mani SK, Mermelstein PG, Tetel MJ et al (2012) Convergence of multiple mechanisms of steroid hormone action. Horm Metab Res 44:569–576
Berdasco M, Esteller M (2010) Aberrant epigenetic landscape in cancer: how cellular identity goes awry. Dev Cell 19:698–711
Korlach J, Turner SW (2012) Going beyond five bases in DNA sequencing. Curr Opin Struct Biol 22:251–261
Portela A, Esteller M (2010) Epigenetic modifications and human disease. Nat Biotechnol 28:1057–1068
Deaton AM, Bird A (2011) CpG islands and the regulation of transcription. Genes Dev 25:1010–1022
Sandoval J, Esteller M (2012) Cancer epigenomics: beyond genomics. Curr Opin Genet Dev 22:50–55
Chatterjee R, Vinson C (2012) CpG methylation recruits sequence specific transcription factors essential for tissue specific gene expression. Biochim Biophys Acta 1819:763–770
Doi A, Park IH, Wen B et al (2009) Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts. Nat Genet 41:1350–1353
Nagae G, Isagawa T, Shiraki N et al (2011) Tissue-specific demethylation in CpG-poor promoters during cellular differentiation. Hum Mol Genet 20:2710–2721
Irizarry RA, Ladd-Acosta C, Wen B et al (2009) The human colon cancer methylome shows similar hypo- and hypermethylation at conserved tissue-specific CpG island shores. Nat Genet 41:178–186
Brait M, Sidransky D (2011) Cancer epigenetics: above and beyond. Toxicol Mech Methods 21:275–288
Hayatsu H (2008) Discovery of bisulfite-mediated cytosine conversion to uracil, the key reaction for DNA methylation analysis—a personal account. Proc Jpn Acad Ser B Phys Biol Sci 84:321–330
Pappas JJ, Toulouse A, Bradley WE (2009) A modified protocol for bisulfite genomic sequencing of difficult samples. Biol Proced Online 11:99–112
Zhang Y, Rohde C, Tierling S et al (2009) DNA methylation analysis by bisulfite conversion, cloning, and sequencing of individual clones. Methods Mol Biol 507:177–187
Hansberg-Pastor V, Gonzalez-Arenas A, Pena-Ortiz MA et al (2013) The role of DNA methylation and histone acetylation in the regulation of progesterone receptor isoforms expression in human astrocytoma cell lines. Steroids 78:500–507
Sasaki M, Kaneuchi M, Fujimoto S et al (2003) Hypermethylation can selectively silence multiple promoters of steroid receptors in cancers. Mol Cell Endocrinol 202:201–207
Turner JD, Pelascini LP, Macedo JA et al (2008) Highly individual methylation patterns of alternative glucocorticoid receptor promoters suggest individualized epigenetic regulatory mechanisms. Nucleic Acids Res 36:7207–7218
Breslin MB, Geng CD, Vedeckis WV (2001) Multiple promoters exist in the human GR gene, one of which is activated by glucocorticoids. Mol Endocrinol 15:1381–1395
Takai D, Jones PA (2003) The CpG island searcher: a new WWW resource. In Silico Biol 3:235–240
Li LC, Dahiya R (2002) MethPrimer: designing primers for methylation PCRs. Bioinformatics 18:1427–1431
Lefever S, Hoebeeck J, Pattyn F et al (2010) methGraph: a genome visualization tool for PCR-based methylation assays. Epigenetics 5:159–163
Bonin S, Hlubek F, Benhattar J et al (2010) Multicentre validation study of nucleic acids extraction from FFPE tissues. Virchows Arch 457:309–317
Bonin S, Stanta G (2013) Nucleic acid extraction methods from fixed and paraffin-embedded tissues in cancer diagnostics. Expert Rev Mol Diagn 13:271–282
Darst RP, Pardo CE, Ai L et al (2010) Bisulfite sequencing of DNA. Curr Protoc Mol Biol Chapter 7, Unit 7 9:1–17.
Bock C, Reither S, Mikeska T et al (2005) BiQ analyzer: visualization and quality control for DNA methylation data from bisulfite sequencing. Bioinformatics 21:4067–4068
Rohde C, Zhang Y, Reinhardt R et al (2010) BISMA-fast and accurate bisulfite sequencing data analysis of individual clones from unique and repetitive sequences. BMC Bioinformatics 11:230. doi:10.1186/1471-2105-11-230
Boyd VL, Zon G (2004) Bisulfite conversion of genomic DNA for methylation analysis: protocol simplification with higher recovery applicable to limited samples and increased throughput. Anal Biochem 326:278–280
Dallol A, Al-Ali W, Al-Shaibani A et al (2011) Analysis of DNA methylation in FFPE tissues using the MethyLight technology. Methods Mol Biol 724:191–204
Pedersen IS, Krarup HB, Thorlacius-Ussing O et al (2012) High recovery of cell-free methylated DNA based on a rapid bisulfite-treatment protocol. BMC Mol Biol 13:12. doi:10.1186/1471-2199-13-12
Ralser M, Querfurth R, Warnatz HJ et al (2006) An efficient and economic enhancer mix for PCR. Biochem Biophys Res Commun 347:747–751
Alba FJ, Bermudez A, Daban JR (2001) Green-light transilluminator for the detection without photodamage of proteins and DNA labeled with different fluorescent dyes. Electrophoresis 22:399–403
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Camacho-Arroyo, I., Hansberg-Pastor, V., Rodríguez-Dorantes, M. (2014). DNA Methylation Analysis of Steroid Hormone Receptor Genes. In: Robles-Flores, M. (eds) Cancer Cell Signaling. Methods in Molecular Biology, vol 1165. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0856-1_8
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DOI: https://doi.org/10.1007/978-1-4939-0856-1_8
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