Dynamic protein methylation in chromatin biology

Abstract

Post-translational modification of chromatin is emerging as an increasingly important regulator of chromosomal processes. In particular, histone lysine and arginine methylation play important roles in regulating transcription, maintaining genomic integrity, and contributing to epigenetic memory. Recently, the use of new approaches to analyse histone methylation, the generation of genetic model systems, and the ability to interrogate genome wide histone modification profiles has aided in defining how histone methylation contributes to these processes. Here we focus on the recent advances in our understanding of the histone methylation system and examine how dynamic histone methylation contributes to normal cellular function in mammals.

Figure 1

Mass spectrometry based approaches for studying histone modifications. Histones are extracted from tissue or cultured cells and biochemically purified, usually by means of reverse phase high performance chromatography. As the histones elute from the column they are fractionated and individual histones are isolated (i.e. Histone H4 in the schematic). The purified histone is then analysed using bottom up or top down approaches. Bottom up analysis involves enzymatic digestion of the histone prior to ionization, whereas top down analysis relies on ionization the intact histone. Both approaches permit interrogation of histone modifications, but top down approaches have the advantage of retaining information about modifications that occur on the same histone. MS - mass spectrometry.

Figure 2

Effector proteins form an aromatic cage that recognizes methylated lysine residues (A–E). Cartoon representations corresponding to the three dimensional structure of effector protein methyl-lysine binding domains (top half of each section) with a close up view of the aromatic cage in association with a methylated ligand (bottom half of each section). (A) The KMT1D ankyrin repeat in complex with H3K9me2 (PDB 3b95), repeats 3, 4 and 5 shown only. (B) The chromodomain of heterochromatin protein 1 (HP1) in complex with H3K9me2 (PDB 1kna). (C) The MBT repeats of L3MBTL1 in complex with H4K20me2 (PDB 2pqw), repeat 2 shown only. (D) The PHD finger of bromodomain PHD transcription factor (BPTF) in complex with H3K4me3 (PDB 2f6j), PHD finger shown only. (E) The double tudor domain of KDM4A in complex with H4K20me3 (PDB 2qqs).

Figure 3

Unique substrate recognition properties of two methyl-lysine recognition domains. (A) Space filling representation of the three dimensional structure of the KDM4A tandem-tudor domain in complex with H3K4me3 (magenta) and H4K20me3 (yellow) histone substrates. The N- and C- terminus of the of histone peptides are labelled and the lysine side chain is depicted projecting into the aromatic methyl-lysine recognition cage (cyan). The two unique binding faces of KDM4a appear to permit the dual substrate recognition properties of this methyl lysine binding effector protein. (B) Space filling representation of the RAG2-PHD domain in association with an H3 peptide (green) containing H3K4me3 and H3R2me2s modifications. The methyl-lysine binding aromatic cage is coloured cyan and the Tyr445 residue that interacts with the symmetric dimethyl-arginine is coloured magenta.

Figure 4

Proposed lysine and arginine demethylase reaction mechanisms. (A) A schematic indicating potential lysine demethylation reaction mechanisms. (Top) Mono-methyl lysine demethylation catalyzed by JmjC domain-containing proteins using 2OG and Fe(II) as cofactors. The reaction produces succinate, CO₂, and formaldehyde as by-products. A ribbon structure of the catalytic domain of the KDM4A demethylase enzymes is depicted to the left (PDB 2oq6). (Bottom) The amine oxidase histone demethylase, KDM1, uses FAD as cofactor to demethylate mono-methyl lysine. The reaction produces H₂O₂, FADH₂, and formaldehyde as by products. The ribbon structure of the KDM1 catalytic domain is depicted to the left (PDB 2v1d). (B) A schematic indicating potential arginine deimination and demethylation reaction mechanisms. (Top) Deimination of mono-methyl arginine catalysed by the peptidylarginine PADI4 enzyme. The ribbon structure of the PADI4 enzyme is depicted to the right (PDB 2dex). This reaction antagonizes histone arginine methylation by converting it to citrulline. (Bottom) Demethylation of mono-methyl arginine by the JmjC domain-containing protein JMJD6. The atomic structure of JMJD6 remains to be solved.

Figure 5

Hydrogen peroxide produced by LSD1 during demethylation contributes to transcriptional activation. (A) During transcriptional activation of estrogen receptor target genes, KDM1 removes repressive H3K9me2 marks. (B) A by-product of this demethylation reaction is hydrogen peroxide (H₂O₂) which is reactive and can cause 8-oxo-guanine (8-OG) lesions on DNA. (C) LSD1 mediated 8-OG is targeted for removal by the 8-oxo-guanine DNA glycosylase-1 (OGG1) and perhaps other components of the base excision repair system. This repair process leads to single stranded DNA nicks that are a substrate for topoisomersase IIb (TOPO). Recruitment of topoisomerase IIb can lead to alterations in DNA architecture. (D) Changes in DNA architecture may aid in RNApolII loading onto target genes by promoting chromatin accessibility or DNA bending and therefore contribute to transcriptional activation. ER — estrogen receptor.

Figure 6

p53 is regulated by protein methylation and demethylation. The p53 protein is made up of an N-terminal transcriptional activation domain (TAD), a central DNA binding domain (DBD), and a C-terminal domain (CTD). Methylation of the CTD on Lys372 by SET7/9 occurs following DNA damage and stabilizes p53 leading to transactivation of p53 target genes. Conversely, mono-methylation of Lys370 by KMT3C/Smyd2 antagonizes binding of p53 to target genes and inhibits p53 mediated transactivation. Surprisingly, di-methylation of the same Lys370 residue creates a binding site for 53BP1 leading to transactivation of p53 target genes. Di-methylation of p53 is enzymatically reversed by the LSD1 histone demethylase indicating that this post-translational modification and its effects on p53 function are regulated. Recent evidence also indicates the KMT5/Set8 enzyme methylates Lys382 in the CTD, indicating that p53 is even more broadly regulated by lysine methylation than previously realized.