Chemical and biochemical approaches in the study of histone methylation and demethylation

Abstract

Histone methylation represents one of the most critical epigenetic events in DNA function regulation in eukaryotic organisms. Classic molecular biology and genetics tools provide significant knowledge about mechanisms and physiological roles of histone methyltransferases and demethylases in various cellular processes. In addition to this stream line, development and application of chemistry and chemistry-related techniques are increasingly involved in biological study, and offer information otherwise difficult to obtain by standard molecular biology methods. Herein, we review recent achievements and progress in developing and applying chemical and biochemical approaches in the study of histone methylation, including chromatin immunoprecipitation, chemical ligation, mass spectrometry, biochemical methylation and demethylation assays, and inhibitor development. These technological advances allow histone methylation to be studied from genome-wide level to molecular and atomic levels. With ChIP technology, information can be obtained about precise mapping of histone methylation patterns at specific promoters, genes, or other genomic regions. MS is particularly useful in detecting and analyzing methylation marks in histone and nonhistone protein substrates. Chemical approaches that permit site-specific incorporation of methyl groups into histone proteins greatly facilitate the investigation of biological impacts of methylation at individual modification sites. Discovery and design of selective organic inhibitors of histone methyltransferases and demethylases provide chemical probes to interrogate methylation-mediated cellular pathways. Overall, these chemistry-related technological advances have greatly improved our understanding of the biological functions of histone methylation in normal physiology and diseased states, and also are of great potential to translate basic epigenetics research into diagnostic and therapeutic applications in the clinic.

Figure 1

Crystal structure of nucleosomes containing H3Kc79me2. The whole structure is shown on left and the zoomed area of H3K79 is shown on right. H2A is colored yellow, H2B colored pink, H3 without Kc79Me2 colored dark blue, H3 with Kc79Me2 colored light blue, and H4 colored green.

Figure 2

Biochemical mechanisms of histone methylation and demethylation. (A) Lysine is methylated by HKMTs. (B) Type I and type II PRMTs methylate arginines and generate MMA, ADMA and SDMA. (C) LSD demethylates lysine via an amine oxidation reaction using FAD as a cofactor. (D) JMJD enzymes use αKG and Fe(II) as cofactors to demethylate the methylated lysines.

Figure 3

High-resolution profiling of histone H3 methylation in a cancer genome. (permission from reference ).

Figure 4

Schematic of chromatin immunoprecipation (ChIP), ChIP-on-Chip and ChIP-seq.

Figure 5

Distribution of methyl marks (labeled with green squares)) on the core histones.

Figure 6

Determination of trimethylated N-terminal lysine residues by mass spectrometry. (A) Mass spectra of the P. falciparum H3 peptides: (upper) KacSTAGKacAPR and (lower) Kme3STAGKacAPR. The theoretical Δm is calculated as the mass difference between a trimethylation and an acetylation (42.04695 - 42.010565 = 0.03693). (B) MS/MS spectra of these two peptides. (permission from reference )

Figure 7

A chemical approach to generating histones that contain site-specific methyl-lysine analogs.

Figure 8

Native chemical ligation and its application in making site-specifically methylated histones.

Figure 9

A genetic codon approach to the site-selective incorporation of methyl lysines into recombinant histones.

Figure 10

Approaches in LBDD and SBDD.

Figure 11

The fragment-based drug design workflow.

Figure 12

General strategies for methyltransferase assays.

Figure 13

Coupled demethylation assays analyzing the production of hydrogen peroxide.

Figure 14

AdoMet analogs as methyltransferase inhibitors.

Figure 15

Inhibitors of HKMTs.

Figure 16

Inhibitors of PRMTs.

Figure 17

Inhibitors of LSD1.

Figure 18

Inhibitors of JMJD demethylases.