During the late 1980s and into the 1990s, research on DNA methylation intensified. At that time, there were two different strategies to assess the DNA-methylation patterns in organisms: a candidate-gene approach and restriction-enzyme-based methods. The advent of bisulphite conversion was a crucial step in epigenetic research. Through this chemical reaction, unmethylated cytosine residues are transformed into uracil while leaving 5-methylcytosine unaffected.
The implementation of this technique with genomic sequencing or PCR amplification (methylation specific PCR—MSP) allowed a sensitive and fast interrogation for DNA methylation at any target sequence and proved suitable for identification of DNA-methylation alterations in selected candidate-genes.
Restriction-enzyme-based methods conversely utilized different techniques to analyze DNA methylation in a genome-wide manner: restriction landmark genomic scanning16; differential methylation hybridization17 and amplification of intermethylated sites18. All of these tools took advantage of methylation-sensitive restriction enzymes to analyze a limited number of genomic sites.
However, use of these techniques does come with some drawbacks, such as the limited number of sequences that can be interrogated and the varied sensitivity exhibited by the restriction enzymes depending on the CG density, etc.
The advent of the chromatin immunoprecipitation technique (ChIP) was a fundamental contribution to the study of other epigenetic factors, mainly in histone modifications. ChIP is a powerful technique for analyzing targeted proteins that bind to particular sequences of DNA. From that moment on, many ChIP-grade antibodies that recognized most of the histone modifications and chromatin-modifying players were produced, increasing exponentially the knowledge of the relationship between epigenetic players and control of gene expression.
In 2001, Strahl and Allis compiled all this information about the interplay of different epigenetic marks and players, and formulated the histone code hypothesis.19