In 1809 Lamarck published Philosophie zoologique describing an intriguing theory by which the environment would exert a driving force on animals whose body plans, physiology, and behavior would have to adapt.1 This hypothesis, refuted by modern geneticists, was consigned to oblivion in favor of Darwin’s theory of evolution.
We now believe, however, that both theories can be reconciled to establish the principles for modern epigenetics: inheritance can play an important role in defining the phenotype of organisms by leaving the DNA sequence unaltered while modifying the expression of the genes.
Epigenetics may be defined in terms of the mechanisms that initiate and maintain heritable patterns of gene function and regulation in an inheritable manner without affecting the sequence of the genome. This explains how two identical genotypes can result in different phenotypes in responding to environmental stimuli.
The different mechanisms underlying epigenetics are post-translational modification in histone proteins, DNA methylation, chromatin remodeling, histone variants, and noncoding RNAs. During the last two decades the best-studied epigenetic processes have been histone modification and DNA methylation, although the others should lead to auspicious results in the near future.
DNA methylation was the first factor to be defined as an epigenetic marker based on the X-chromosome inactivation taking place in somatic cells.2,3 A major step forward in the field was the introduction of methylation-sensitive restriction enzymes as a way to detect the methylation state of CpG sites in higher eukaryotic genomes. This clarified the genomic distribution of methylated CpGs and their role in controlling the activity of the genes.4
Then in 1983, Bestor and Ingram purified the first DNA methyl transferase—dnmt15. In the early 90s, knock-out models led to the discovery of methyl binding domain (MBD) proteins. The first genetic ablation was DNA methyltransferase 1 (DNMT1), resulting in a profound deregulation of epigenetically silenced loci in the mouse genome.6
However, the transmission of heritable changes that impact gene activity in organisms with extremely low levels of DNA methylation, such as Drosophila melanogaster, suggested the existence of additional epigenetic mechanisms yet to be discovered.
Before the 70s, the activity of histone-mediated gene regulation had been misunderstood, considered to act as proteins that only helped in coiling DNA to optimize its packaging.7 However, in 1964, Alfrey proposed an active role for histone acetylation in gene expression.8 By using biochemical techniques with radioactive precursors, new histone modifications were discovered during the 1960s, although their functional significance remained unclear. Since then, research around these new players has experienced an awakening.
A major advance came with the determination of the crystal structure of the nucleosome at different resolutions, which provided evidence that histone tails could be modified.9,10 Later on, a critical connection between histone acetylation and gene activation was reported with the isolation of the Tetrahymena histone acetyltransferase gcn5 through an “in-gel” assay approach.11
At the same time, the first histone deacetylase was purified.12 Afterward, many other epigenetic-modifying complexes were discovered via biochemical studies using purified factors and DNA templates (e.g., the ATP-dependent nucleosome remodeling complex SWI/SNF).13
Different investigations finally demonstrated the connection between all epigenetic players that had been reported so far, by describing physical interactions among MBDs, histone deacetylases (HDACs), and chromatin remodelers.14,15 All this work gave rise to an exciting research field that has yielded important contributions to our understanding of human development and disease.
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