Modern approaches for investigating epigenetic signaling pathways

Abstract

Epigenetics is increasingly being recognized as a central component of physiological processes as diverse as obesity and circadian rhythms. Primarily acting through DNA methylation and histone posttranslational modifications, epigenetic pathways enable both short- and long-term transcriptional activation and silencing, independently of the underlying genetic sequence. To more quantitatively study the molecular basis of epigenetic regulation in physiological processes, the present review informs the latest techniques to identify and compare novel DNA methylation marks and combinatorial histone modifications across different experimental conditions, and to localize both DNA methylation and histone modifications over specific genomic regions.

Fig. 1.

Major branches of epigenetic regulation. Independent of genetic sequence, CpG island methylation and histone (white, gray, and black circles) posttranslational modifications (PTMs) are capable of positively and negatively regulating transcription. me, methylation (mono-, di-, and tri-methylated are possibilities for lysine residues, while mono- and di- are possibilities for arginine); p, phosphorylation; ac, acetylation.

Fig. 2.

Local and global approaches to identifying cytosine methylation sites are shown. Local detection involves denaturing and bisulfite treatment of DNA before amplification of a particular locus. The unmethylated cytosines are shown in bold and are converted to thymines during the process. Unmethylated cytosines are indicated and can be identified during sequencing as remaining as cytosines. A method for global methylation detection is shown where the DNA is sonicated before bisulfite treatment. Adapters are used to ensure only converted DNA is used for sequencing. The second adapters are specific for next-generation sequencing chips.

Fig. 3.

“Bottom-up” and “middle-down” mass spectrometry (MS). A: the principles of analyzing a hypothetical histone with K4 and K14 acetylation using “bottom up” or “middle down” MS. B: derivatization of the protein with propionic anhydride effectively caps unmodified and monomethylated lysine residues with a propionyl group (black circle), preventing trypsin digestion at those sites. Lysines containing other PTMs, like acetylation (white triangle), usually are not digested efficiently by trypsin. C: trypsin digestion produces various histone peptides. D: MS/MS spectra are collected for a particular peptide precursor (gray dashed circle from C) to reveal peptide sequence information (MS spectra of all the precursors are not shown). Note that the connectivity between K4 and K14 acetylation is lost as both modifications are on different peptides. Thus one does not know if original histone protein contains the 2 marks together or individually. E: GluC digest results in a larger histone peptide for “middle-down” MS analysis and preserves the connectivity of K4 and K14 acetylation.