Lysine methylation: beyond histones

Posttranslational modifications (PTMs) of histone proteins, such as acetylation, methylation, phosphorylation, and ubiquitylation, play essential roles in regulating chromatin dynamics. Combinations of different modifications on the histone proteins, termed 'histone code' in many cases, extend the information potential of the genetic code by regulating DNA at the epigenetic level. Many PTMs occur on non-histone proteins as well as histones, regulating protein-protein interactions, stability, localization, and/or enzymatic activities of proteins involved in diverse cellular processes. Although protein phosphorylation, ubiquitylation, and acetylation have been extensively studied, only a few proteins other than histones have been reported that can be modified by lysine methylation. This review summarizes the current progress on lysine methylation of non-histone proteins, and we propose that lysine methylation, like phosphorylation and acetylation, is a common PTM that regulates proteins in diverse cellular processes.     

Influence of combinatorial histone modifications on antibody and effector protein recognition

Increasing evidence suggests that histone posttranslational modifications (PTMs) function in a combinatorial fashion to regulate the diverse activities associated with chromatin. Yet how these patterns of histone PTMs influence the adapter proteins known to bind them is poorly understood. In addition, how histone-specific antibodies are influenced by these same patterns of PTMs is largely unknown. Here we examine the binding properties of histone-specific antibodies and histone-interacting proteins using peptide arrays containing a library of combinatorially modified histone peptides. We find that modification-specific antibodies are more promiscuous in their PTM recognition than expected and are highly influenced by neighboring PTMs. Furthermore, we find that the binding of histone-interaction domains from BPTF, CHD1, and RAG2 to H3 lysine 4 trimethylation is also influenced by combinatorial PTMs. These results provide further support for the histone code hypothesis and raise specific concerns with the quality of the currently available modification-specific histone antibodies.     

Post-translational modifications of lysine-specific demethylase 1

Lysine-specific demethylase 1 (LSD1) is crucial for regulating gene expression by catalyzing the demethylation of mono- and di-methylated histone H3 lysine 4 (H3K4) and lysine 9 (H3K9) and non-histone proteins through the amine oxidase activity with FAD⁺ as a cofactor. It interacts with several protein partners, which potentially contributes to its diverse substrate specificity. Given its pivotal role in numerous physiological and pathological conditions, the function of LSD1 is closely regulated by diverse post-translational modifications (PTMs), including phosphorylation, ubiquitination, methylation, and acetylation. In this review, we aim to provide a comprehensive understanding of the regulation and function of LSD1 following various PTMs. Specifically, we will focus on the impact of PTMs on LSD1 function in physiological and pathological contexts and discuss the potential therapeutic implications of targeting these modifications for the treatment of human diseases.     

Global mapping of post-translational modifications on histone H3 variants in mouse testes

Mass spectrometry (MS)-based characterization is important in proteomic research for verification of structural features and functional understanding of gene expression. Post-translational modifications (PTMs) such as methylation and acetylation have been reported to be associated with chromatin remodeling during spermatogenesis. Although antibody- and MS-based approaches have been applied for characterization of PTMs on H3 variants during spermatogenesis, variant-specific PTMs are still underexplored. We identified several lysine modifications in H3 variants, including testis-specific histone H3 (H3t), through their successful separation with MS-based strategy, based on differences in masses, retention times, and presence of immonium ions. Besides methylation and acetylation, we detected formylation as a novel PTM on H3 variants in mouse testes. These patterns were also observed in H3t. Our data provide high-throughput structural information about PTMs on H3 variants in mouse testes and show possible applications of this strategy in future proteomic studies on histone PTMs.     

The functional diversity of protein lysine methylation

Large‐scale characterization of post‐translational modifications (PTMs), such as phosphorylation, acetylation and ubiquitination, has highlighted their importance in the regulation of a myriad of signaling events. While high‐throughput technologies have tremendously helped cataloguing the proteins modified by these PTMs, the identification of lysine‐methylated proteins, a PTM involving the transfer of one, two or three methyl groups to the ε‐amine of a lysine side chain, has lagged behind. While the initial findings were focused on the methylation of histone proteins, several studies have recently identified novel non‐histone lysine‐methylated proteins. This review provides a compilation of all lysine methylation sites reported to date. We also present key examples showing the impact of lysine methylation and discuss the circuitries wired by this important PTM.     

High mobility group proteins and their post-translational modifications

The high mobility group (HMG) proteins, including HMGA, HMGB and HMGN, are abundant and ubiquitous nuclear proteins that bind to DNA, nucleosome and other multi-protein complexes in a dynamic and reversible fashion to regulate DNA processing in the context of chromatin. All HMG proteins, like histone proteins, are subjected to extensive post-translational modifications (PTMs), such as lysine acetylation, arginine/lysine methylation and serine/threonine phosphorylation, to modulate their interactions with DNA and other proteins. There is a growing appreciation for the complex relationship between the PTMs of HMG proteins and their diverse biological activities. Here, we reviewed the identified covalent modifications of HMG proteins, and highlighted how these PTMs affect the functions of HMG proteins in a variety of cellular processes.     

Examining post‐translational modification‐mediated protein–protein interactions using a chemical proteomics approach

Post‐translational modifications (PTM) of proteins can control complex and dynamic cellular processes via regulating interactions between key proteins. To understand these regulatory mechanisms, it is critical that we can profile the PTM‐dependent protein–protein interactions. However, identifying these interactions can be very difficult using available approaches, as PTMs can be dynamic and often mediate relatively weak protein–protein interactions. We have recently developed CLASPI (cross‐linking‐assisted and stable isotope labeling in cell culture‐based protein identification), a chemical proteomics approach to examine protein–protein interactions mediated by methylation in human cell lysates. Here, we report three extensions of the CLASPI approach. First, we show that CLASPI can be used to analyze methylation‐dependent protein–protein interactions in lysates of fission yeast, a genetically tractable model organism. For these studies, we examined trimethylated histone H3 lysine‐9 (H3K9Me₃)‐dependent protein–protein interactions. Second, we demonstrate that CLASPI can be used to examine phosphorylation‐dependent protein–protein interactions. In particular, we profile proteins recognizing phosphorylated histone H3 threonine‐3 (H3T3‐Phos), a mitotic histone “mark” appearing exclusively during cell division. Our approach identified survivin, the only known H3T3‐Phos‐binding protein, as well as other proteins, such as MCAK and KIF2A, that are likely to be involved in weak but selective interactions with this histone phosphorylation “mark”. Finally, we demonstrate that the CLASPI approach can be used to study the interplay between histone H3T3‐Phos and trimethylation on the adjacent residue lysine 4 (H3K4Me₃). Together, our findings indicate the CLASPI approach can be broadly applied to profile protein–protein interactions mediated by PTMs.     

Methylation of H3-lysine 79 is mediated by a new family of HMTases without a SET domain

The N-terminal tails of core histones are subjected to multiple covalent modifications, including acetylation, methylation, and phosphorylation. Similar to acetylation, histone methylation has emerged as an important player in regulating chromatin dynamics and gene activity. Histone methylation occurs on arginine and lysine residues and is catalyzed by two families of proteins, the protein arginine methyltransferase family and the SET-domain-containing methyltransferase family. Here, we report that lysine 79 (K79) of H3, located in the globular domain, can be methylated. K79 methylation occurs in a variety of organisms ranging from yeast to human. In budding yeast, K79 methylation is mediated by the silencing protein DOT1. Consistent with conservation of K79 methylation, DOT1 homologs can be found in a variety of eukaryotic organisms. We identified a human DOT1-like (DOT1L) protein and demonstrated that this protein possesses intrinsic H3-K79-specific histone methyltransferase (HMTase) activity in vitro and in vivo. Furthermore, we found that K79 methylation level is regulated throughout the cell cycle. Thus, our studies reveal a new methylation site and define a novel family of histone lysine methyltransferase.