Functions and mechanisms of lysine crotonylation

Abstract Lysine crotonylation is a newly discovered post‐translational modification, which is structurally and functionally different from the widely studied lysine acetylation. Recent advances in the identification and quantification of lysine crotonylation by mass spectrometry have revealed that non‐histone proteins are frequently crotonylated, implicating it in many biological processes through the regulation of chromatin remodelling, metabolism, cell cycle and cellular organization. In this review, we summarize the writers, erasers and readers of lysine crotonylation, and their physiological functions, including gene transcription, acute kidney injury, spermatogenesis, depression, telomere maintenance, HIV latency and cancer process. These findings not only point to the new functions for lysine crotonylation, but also highlight the mechanisms by which crotonylation regulates various cellular processes.

Lysine crotonylation is a newly discovered histone PTM, which is specifically enriched at active gene promoters and potential enhancers in mammalian cell genomes. 11 Crotonylation can be catalysed reversibly by protein crotonyltransferases and decrotonylases. The crotonylation of lysine was first identified on histones. 11 Afterwards, more eukaryotic non-histone proteins were identified as being crotonylated, and they were involved in cellular metabolism, cell cycle and cellular organization process. [13][14][15][16][17][18][19][20][21][22] In this review, we concentrate mainly on recent studies about lysine crotonylation and discuss its implications.

| THE D ISCOVERY OF LYS INE CROTONYL ATION
Tan et al 11 first reported protein lysine crotonylation in 2011.
They used an integrated, mass spectrometry-based proteomics approach, which takes advantage of in vitro propionylation, efficient peptide separation using isoelectric focusing (OFFGEL) and the high sensitivity of the LTQ Orbitrap Velos mass spectrometer to carry out a comprehensive analysis of histone PTMs. With this approach, they identified lysine crotonylation as a novel histone mark type. A total of 28 human histone peptides were found to have lysine crotonylation in this study ( Figure 1). In addition, they also generated a specific anti-crotonyl-lysine antibody and applied it in corroborating Western blot and immunostaining experiments.
Interestingly, they confirmed the existence of 19 crotonylation marks in HeLa cells using isotopic labelling with D4-crotonate. The crotonyl group contains a unique C-C π-bond, which results in a rigid and planar configuration. Following the initial discovery of lysine crotonylation, the landscape of these modifications is rapidly expanding.

| PROTEIN CROTONYLOME
Although initially identified on histones, lysine crotonylation has expanded to large number of non-histone proteins. To characterize the global crotonylation proteome, the proteomic method based on sensitive immune-affinity purification and high-resolution liquid chromatography-tandem (LC-MS/MS) was applied to identify new crotonylated proteins and modification sites. The utilization of antibodies with high specificity to the crotonylated peptides involved in immunoprecipitation significantly improved the ability to enrich and identify crotonylated lysine residues. In recent years, several landmark studies have revealed dramatically improved number of crotonylated lysine residues and crotonylated proteins (Table 1), and these proteins play roles in nearly all cellular processes including chromatin remodelling, metabolism, cell cycle and cellular organization. [13][14][15][16][17][18][19][20][21][22] Crotonylome mapping provides an important resource for discovering novel properties and regulatory functions of crotonylation.

| WRITER S OF LYS INE CROTONYL ATI ON
Lysine crotonylation is enzymatically regulated by the dynamic balance between crotonyltransferases and decrotonylases. 23 The crotonyltransferases were colloquially termed writers, which catalyse the covalent modification of lysine crotonylation. The histone acetyltransferases (HATs) were also shown to have histone crotonyltransferase (HCT) activities ( Figure 2; Table 2). There are three major HATs families, which can be categorized into three major families by their sequence and structural features: p300/ CBP (CREB-binding protein), GNAT (Gcn5-related N-acetyltransferases) and MYST (MOZ, Ybf2/Sas3, Sas2 and Tip60) families. 24 The p300/CBP and MYST families are identified only in eukaryotic cells, while the GNAT family is present and conserved in all domains of life.
Sabari et al 25 first reported that p300 has both HAT and HCT activities. In the cell-free assays, they demonstrated that p300-catalysed histone crotonylation directly stimulated transcription to a greater degree than p300-catalysed histone acetylation. Besides, the level of histone crotonylation was sensitive to changes in the cellular concentration of crotonyl-CoA, due to p300's dual enzymatic activities. Afterwards, MOF was also identified to catalyse histone crotonylation, including histone H3 at lysine residues 4, 9, 18 and 23, and histone H4 at lysine 8 and 12. 26  In their study, they found non-histone protein, NPM1, is strongly crotonylated by CBP and MOF, and moderately crotonylated by PCAF.
However, the crotonylation of DEAD-box RNA helicase, DDX5, can only be catalysed by CBP.

| ER A S ER S OF LYS INE CROTONYL ATI ON
Histone deacetylases (HDACs) were also reported to have histone decrotonylase (HDCR) activity ( Figure 2; Table 2). The decrotonylases were colloquially termed erasers, which could remove the covalent modification of lysine crotonylation. Currently, two major families of KDACs have been divided into four classes: the NADdependent sirtuin family (class III Sirt1-7) and the zinc-dependent HDAC3 was firstly reported to exhibit HDCR activity in vitro.
By utilizing a collection of fluorogenic substrates, HDAC3-NCoR1 was exhibited decrotonylase activity with a catalytic efficiency that is comparable to the deacetylase activity of other KDAC isoforms. 28 After analysing the deacylase activity of Sirt1 through SIRT7 using catalytic amounts of enzyme and different expansive panel of acylated H3K9 peptides, Sirt1 and Sirt2 were also indicated act as efficient decrotonylases. 29 With the application of an optimized CLASPI approach to comprehensively profile 'eraser' enzymes, Bao et al 30 identified human Sirt1, Sirt2 and Sirt3 as decrotonylases in vitro, and using X-ray crystallography, they examined the molecular basis of how the enzymes recognize crotonylated group. Afterwards, class I HDACs and Sirt1 were proved as active HDCR enzymes, 31 by screening ectopically expressed HDAC and Sirt family proteins in HeLa cells, with immunofluorescence staining using an anti-crotonyl-lysine specific antibody. Kelly et al 32 found that knock-down HDAC1/2 in embryonic stem (ES) cells increased global histone crotonylation levels and caused a great reduction in total decrotonylase activity. Xu et al 13

| RE ADER S OF LYS INE CROTONYL ATI ON
Double PHD finger (DPF), bromodomain and YEATS are three major classes of acetylation and non-acetyl acylation readers. 6 Histone crotonylations can act as docking marks to recruit downstream readers ( Figure 2; Table 2). Using ChIP-qPCR and immunofluorescence assays, Xiong et al 33 suggested the colocalization of MOZ and H3K14 crotonylation, and they also indicated that DPF domains of MOZ and DPF2 maintained high affinity for crotonylation at H3K14. A recent study reported that the binding affinity between AF9 YEATS domain and crotonyl-lysine was higher than that to acetyl-lysine. 34 Furthermore, using heteronuclear NMR spectroscopy, they determined a 3D solution structure of the AF9 YEATS domain bound to an H3 crotonylated at Lys 18 (H3K18cr) peptide. Afterwards, Taf14 YEATS domain was identified as a reader of crotonyl-lysine binding to histone H3 crotonylated at Lys 9 (H3K9cr) via a unique π-π-π stacking mechanism. 35 Using a modified histone peptide array, Zhao et al 36 found that YEATS2 bound to acylated histone peptides with the best preference for histone H3 crotonylated at Lys 27 (H3K27cr) peptide.

| Regulation of spermatogenesis
Genome-wide removal of histones from chromatin and their replacement is a unique epigenetic event during spermatogenesis. 40 Liu et al 41

| Regulation of depression
Histone crotonylation was found decreased in the medial prefrontal cortex of susceptible rodents exposed to chronic social defeat stress. 42 In addition, when knocking down CDYL in prelimbic cortex, the histone crotonylation was increased. Using ChIP-quantitative

| Regulation of telomere maintenance
Telomere elongation with increasing passage depends on the mechanisms of both telomerase and recombination-based alternative lengthening of telomeres. 43 When crotonylation induced by crotonic acid, Zscan4 was found activated, and T-SCE level increased, which maintained telomeres and reduced telomere damage during chemical induction. 44 In addition, chemically induced pluripotent stem cells' (CiPSC) clone formation at stage II during middle induction was improved by crotonylation, indicating that extraembryonic endoderm state is primed for action by crotonic acid.

| Regulation of HIV latency
Latent HIV reservoirs in the host are established early before viral infection. 45,46 ACSS2, the crotonyl-CoA-producing enzyme acyl-CoA synthetase short-chain family member 2, was identified to influence HIV replication and viral latency by regulating histone crotonylation at HIV long-terminal repeat. 47 After histone crotonylation was induced by ACSS2 in vitro and ex vivo, HIV latency was disrupted.
Furthermore, when inhibiting histone crotonylation with suppressing ACSS2, latent HIV reactivation was dampened, suggesting a potential role of histone decrotonylation in HIV latency establishment.
For the first time, this finding linked maintenance of viral latency to histone crotonylation by ACSS2 at the HIV long-terminal repeat.

| Regulation of cancer
Cancer is a life-threatening malignancy that has become a global healthcare problem. [48][49][50] Using immunohistochemical methods, we recently reported that levels of crotonylation in eight types of cancer. 51 We found the expression level of crotonylation was down-regulated in liver, stomach and kidney carcinomas, and up-regulated in thyroid, oesophagus, colon, pancreas and lung carcinomas, suggesting that crotonylation may modulate different cancer progression.
Besides, lysine crotonylation was identified involving in hepatoma cell motility and proliferation. For the first time, we indicated that status of lysine crotonylation may represent an important type of post-translational modifications accounting for cancer progression.

| CON CLUS IONS
Lysine crotonylation is recently identified as a novel evolutionarily conserved histone PTM. 11 The recent discovery of a large panel of new histone PTMs, including lysine crotonylation, may change our vision of epigenome. 6,52 Lysine crotonylation is involved in many pathways that regulate diverse cellular functions ranging from gene expression to telomere maintenance (Table 3)  to provide a wealth of regulatory potential.
Over the past years, we have witnessed the tremendous ad- In the further research, we should focus on how human physiological processes and related diseases mechanistically regulated by crotonylation. Beside, more studies should search for understanding the role of crotonylation in non-histones. We can also discovery more sites about lysine crotonylation, including the mechanisms by which they occur. Therefore, future studies are needed to uncover the effects of crotonylation on regulating protein functions and to interpret the underlying mechanisms behind protein crotonylation's ability to modulate diverse physiological and pathological processes.

CO N FLI C T O F I NTE R E S T
The authors declared no conflict of interest.

AUTH O R CO NTR I B UTI O N S
JHW and HQZ provided direction and guidance throughout the preparation of this manuscript. JHW draw the graph. HYL and JC collected and prepared the related literature. All authors have read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data will be available upon reasonable request.