DNA methylation analysis of steroid hormone receptor genes.
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.
Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México, Av. Universidad 3000, Ciudad Universitaria, Coyoacán, México, DF, 04510, México, camachoarroyo@gmail.com.
Ignacio
I
Camacho-Arroyo
Valeria
V
Hansberg-Pastor
Mauricio
M
Rodríguez-Dorantes
D003001:Cloning, Molecular; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000187:drug effects; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000494:pharmacology; D014170:Transformation, Genetic
D017931:DNA Primers; D018160:Receptors, Cytoplasmic and Nuclear; D013447:Sulfites; C009279:sodium bisulfite