Dynamics and dispensability of variant-specific histone H1 Lys-26/Ser-27 and Thr-165 post-translational modifications

In mammals, the linker histone H1, involved in DNA packaging into chromatin, is represented by a family of variants. H1 tails undergo post-translational modifications (PTMs) that can be detected by mass spectrometry. We developed antibodies to analyze several of these as yet unexplored PTMs including the combination of H1.4 K26 acetylation or trimethylation and S27 phosphorylation. H1.2-T165 phosphorylation was detected at S and G2/M phases of the cell cycle and was dispensable for chromatin binding and cell proliferation; while the H1.4-K26 residue was essential for proper cell cycle progression. We conclude that histone H1 PTMs are dynamic over the cell cycle and that the recognition of modified lysines may be affected by phosphorylation of adjacent residues.     

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.     

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.     

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.     

Crosstalk between lysine methylation and phosphorylation of ATG16L1 dictates the apoptosis of hypoxia/reoxygenation-induced cardiomyocytes

Post-translational modifications of autophagy-related (ATG) genes are necessary to modulate their functions. However, ATG protein methylation and its physiological role have not yet been elucidated. The methylation of non-histone proteins by SETD7, a SET domain-containing lysine methyltransferase, is a novel regulatory mechanism to control cell protein function in response to various cellular stresses. Here we present evidence that the precise activity of ATG16L1 protein in hypoxia/reoxygenation (H/R)-treated cardiomyocytes is regulated by a balanced methylation and phosphorylation switch. We first show that H/R promotes autophagy and decreases SETD7 expression, whereas autophagy inhibition by 3-MA increases SETD7 level in cardiomyocytes, implying a tight correlation between autophagy and SETD7. Then we demonstrate that SETD7 methylates ATG16L1 at lysine 151 while KDM1A/LSD1 (lysine demethylase 1A) removes this methyl mark. Furthermore, we validate that this methylation at lysine 151 impairs the binding of ATG16L1 to the ATG12–ATG5 conjugate, leading to inhibition of autophagy and increased apoptosis in H/R-treated cardiomyocytes. However, the cardiomyocytes with shRNA-knocked down SETD7 or inhibition of SETD7 activity by a small molecule chemical, display increased autophagy and decreased apoptosis following H/R treatment. Additionally, methylation at lysine 151 inhibits phosphorylation of ATG16L1 at S139 by CSNK2 which was previously shown to be critical for autophagy maintenance, and vice versa. Together, our findings define a novel modification of ATG16L1 and highlight the importance of an ATG16L1 phosphorylation-methylation switch in determining the fate of H/R-treated cardiomyocytes.     

Middle‐Down MS is Ready to Answer Complex Questions in Chromatin Biology

Histones are the most abundant protein family in the cells of complex organisms such as mammals and, together with DNA, they define the backbone of chromatin. Histone PTMs are key players of chromatin biology, as they function as anchors for proteins that bind and modulate chromatin readout, including gene expression. Middle‐down mass spectrometry (MS) has been optimized for about 10 years to study histone N‐terminal tails, but it has been rarely applied to identify the role of coexisting histone marks in biology. In this work, Jiang et al. used middle‐down MS to study the dynamics of coexisting PTMs on histone H4 in two breast cancer cell lines. 1 They found that overall serine 1 phosphorylation (S1ph) is mildly regulated during the cell cycle, but S1ph coexistence frequency with acetylations and methylations on the lysine residues of the N‐terminal tail is remarkably tuned during S phase and G2/M phase. Together, the team placed another benchmark proving that MS analysis of combinatorial histone PTMs provides a more comprehensive view on chromatin state than studying individual marks. We should then constantly question ourselves regarding the limitations of analyzing single PTMs when we attempt to define their effect on protein functions.     

Sulfo‐NHS‐SS‐biotin derivatization: A versatile tool for MALDI mass analysis of PTMs in lysine‐rich proteins

The discovery of PTMs in proteins by MS requires nearly complete sequence coverage of the detected proteolytic peptides. Unfortunately, mass spectrometric analysis of the desired sequence fragments is often impeded due to low ionization efficiency and/or signal suppression in complex samples. When several lysine residues are in close proximity tryptic peptides may be too short for mass analysis. Moreover, modified peptides often appear in low stoichiometry and need to be enriched before analysis. We present here how the use of sulfo‐NHS‐SS‐biotin derivatization of lysine side chain can help to detect PTMs in lysine‐rich proteins. This label leads to a mass shift which can be adjusted by reduction of the SS bridge and alkylation with different reagents. Low intensity peptides can be enriched by use of streptavidin beads. Using this method, the functionally relevant protein kinase A phosphorylation site in 5‐lipoxygenase was detected for the first time by MS. Additionally, methylation and acetylation could be unambiguously determined in histones.     

Phosphorylation of H3S10 blocks the access of H3K9 by specific antibodies and histone methyltransferase. Implication in regulating chromatin dynamics and epigenetic inheritance during mitosis

Post-translational modifications of histones play a critical role in regulating genome structures and integrity. We have focused on the regulatory relationship between covalent modifications of histone H3 lysine 9 (H3K9) and H3S10 during the cell cycle. Immunofluorescence microscopy revealed that H3S10 phosphorylation in HeLa, A549, and HCT116 cells was high during prophase, prometaphase, and metaphase, whereas H3K9 monomethylation (H3K9me1) and dimethylation (H3K9me2), but not H3K9 trimethylation (H3K9me3), were significantly suppressed. When H3S10 phosphorylation started to diminish during anaphase, H3K9me1 and H3K9me2 signals reemerged. Western blot analyses confirmed that mitotic histones, extracted in an SDS-containing buffer, had little H3K9me1 and H3K9me2 signals but abundant H3K9me3 signals. However, when mitotic histones were extracted in the same buffer without SDS, the difference in H3K9me1 and H3K9me2 signals between interphase and mitotic cells disappeared. Removal of H3S10 phosphorylation by pretreatment with lambda-phosphatase unmasked mitotic H3K9me1 and H3K9me2 signals detected by both fluorescence microscopy and Western blotting. Further, H3S10 phosphorylation completely blocked methylation of H3K9 but not demethylation of the same residue in vitro. Given that several conserved motifs consisting of a Lys residue immediately followed by a Ser residue are present in histone tails, our studies reveal a potential new mechanism by which phosphorylation not only regulates selective access of methylated lysines by cellular factors but also serves to preserve methylation patterns and epigenetic programs during cell division.     

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.     

Characterization of Protein Methyltransferases Rkm1, Rkm4, Efm4, Efm7, Set5 and Hmt1 Reveals Extensive Post-Translational Modification

Protein methylation is one of the major post-translational modifications (PTMs) in the cell. In Saccharomyces cerevisiae, over 20 protein methyltransferases (MTases) and their respective substrates have been identified. However, the way in which these MTases are modified and potentially subject to regulation remains poorly understood. Here, we investigated six overexpressed S. cerevisiae protein MTases (Rkm1, Rkm4, Efm4, Efm7, Set5 and Hmt1) to identify PTMs of potential functional relevance. We identified 48 PTM sites across the six MTases, including phosphorylation, acetylation and methylation. Forty-two sites are novel. We contextualized the PTM sites in structural models of the MTases and revealed that many fell in catalytic pockets or enzyme–substrate interfaces. These may regulate MTase activity. Finally, we compared PTMs on Hmt1 with those on its human homologs PRMT1, PRMT3, CARM1, PRMT6 and PRMT8. This revealed that several PTMs are conserved from yeast to human, whereas others are only found in Hmt1. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD006767.     

Lysine methylation modulates the protein–protein interactions of yeast cytochrome C Cyc1p

In recent years, protein methylation has been established as a major intracellular PTM. It has also been proposed to modulate protein‐protein interactions (PPIs) in the interactome. To investigate the effect of PTMs on PPIs, we recently developed the conditional two‐hybrid (C2H) system. With this, we demonstrated that arginine methylation can modulate PPIs in the yeast interactome. Here, we used the C2H system to investigate the effect of lysine methylation. Specifically, we asked whether Ctm1p‐mediated trimethylation of yeast cytochrome c Cyc1p, on lysine 78, modulates its interactions with Erv1p, Ccp1p, Cyc2p and Cyc3p. We show that the interactions between Cyc1p and Erv1p, and between Cyc1p and Cyc3p, are significantly increased upon trimethylation of lysine 78. This increase of interaction helps explain the reported facilitation of Cyc1p import into the mitochondrial intermembrane space upon methylation. This first application of the C2H system to the study of methyllysine‐modulated interactions further confirms its robustness and flexibility.     

DOT1—— 一类新的组蛋白赖氨酸甲基转移酶

组蛋白甲基化是一种重要的组蛋白共价修饰, 在染色质结构和基因表达的调控过程中起着重要的、多样化的作用。DOT1催化核心球体部位的组蛋白H3第79位赖氨酸(H3K79)使其发生甲基化, 是首个被发现的无SET结构域的组蛋白赖氨酸甲基转移酶, 代表了一类新的组蛋白赖氨酸甲基转移酶。DOT1及H3K79甲基化的特点决定了其可能具有重要的、特殊的生物学功能。文章重点综述了DOT1蛋白的结构及特点, DOT1及H3K79甲基化的生物学功能以及组蛋白泛素化修饰对H3K79甲基化的反式调控。     

Methylation of a histone mimic within the histone methyltransferase G9a regulates protein complex assembly

Epigenetic gene silencing in eukaryotes is regulated in part by lysine methylation of the core histone proteins. While histone lysine methylation is known to control gene expression through the recruitment of modification-specific effector proteins, it remains unknown whether nonhistone chromatin proteins are targets for similar modification-recognition systems. Here we show that the histone H3 methyltransferase G9a contains a conserved methylation motif with marked sequence similarity to H3 itself. As with methylation of H3 lysine 9, autocatalytic G9a methylation is necessary and sufficient to mediate in vivo interaction with the epigenetic regulator heterochromatin protein 1 (HP1), and this methyl-dependent interaction can be reversed by adjacent G9a phosphorylation. NMR analysis indicates that the HP1 chromodomain recognizes methyl-G9a through a binding mode similar to that used in recognition of methyl-H3K9, demonstrating that the chromodomain functions as a generalized methyl-lysine binding module. These data reveal histone-like modification cassettes - or "histone mimics" - as a distinct class of nonhistone methylation targets and directly extend the principles of the histone code to the regulation of nonhistone proteins.