Protein lysine four-carbon acylations in health and disease

Abstract

Lysine acylation, a type of posttranslational protein modification sensitive to cellular metabolic states, influences the functions of target proteins involved in diverse cellular processes. Particularly, lysine butyrylation, crotonylation, β-hydroxybutyrylation, and 2-hydroxyisobutyrylation, four types of four-carbon acylations, are modulated by intracellular concentrations of their respective acyl-CoAs and sensitive to alterations of nutrient metabolism induced by cellular and/or environmental signals. In this review, we discussed the metabolic pathways producing these four-carbon acyl-CoAs, the regulation of lysine acylation and deacylation, and the functions of individual lysine acylation.

1 |. INTRODUCTION

Cellular metabolism plays an essential role in mediating environmental influences on cellular functions. In addition to providing energy and biosynthetic building blocks, metabolism also produces intermediate metabolites that modulate protein activities or stabilities by allosteric regulation or through covalent posttranslational modifications (PTMs). Acetylation and methylation are two classically characterized PTMs. In recent years, the advance of high-resolution liquid chromatography with tandem mass spectrometry (LC-MS/MS) has led to the identification of a variety of new PTMs from acylation, including lysine butyrylation (Kbu), crotonylation (Kcr), β-hydroxybutyrylation (Kbhb), 2-hydroxyisobutyrylation (Khib), propionylation (Kpr), malonylation (Kmal), glutarylation (Kglu), benzoylation (Kbz), succinylation (Ksucc), and lactylation (Kla) (Figlia et al., 2020; Sabari et al., 2017). In many cases, the levels of these lysine acylations are subjected to the alterations of the intracellular concentration of their corresponding acyl-CoAs, thus providing a direct link between cellular metabolic states and functions of target proteins. These modifications, therefore, function as metabolic sensors to regulate cellular processes.

Great advance has been achieved in understanding the functions of these acylations in recent years. In this review, we will focus on a group of acylation with four-carbon in length: Kbu, Kcr, Kbhb, and Khib, as they are metabolically or structurally related (Table 1, Figure 1). Kbu and Kcr are hydrophobic acyl group, while Kcr contains a carbon-carbon (C-C) π-bond which leads to a rigid planar conformation. This rigid conformation of histone Kcr hinders the formation of water-mediated hydrogen bond with DNA backbone and promote histone–DNA dissociation and chromatin opening for gene transcription (Suzuki et al., 2016). Kbhb and Khib are polar acyl groups. Histone Kbu, Kcr, Kbhb, and Khib have all been shown to be associated with active gene expression (Bhattacharya et al., 2022; L. Dai et al., 2014; Fang et al., 2021; H. Huang et al., 2021; Tan et al., 2011). Here, we will review the metabolic pathways producing the four acyl-CoAs, the writers and erasers modulating these acylations, and the functions and physiological roles of individual acylation in a variety of cellular processes and pathophysiological conditions. Finally, we will discuss opening questions in the field.

TABLE 1.

Structures, writers, and erasers of Kbu, Kcr, Kbhb, and Khib.

Name	Structure	Writer	Eraser	References
Kbu		CBP		Chen et al. (2007); Simithy et al. (2017)
		GCN5		Ringel and Wolberger (2016); Simithy et al. (2017)
		p300		Bhattacharya et al. (2022); Chen et al. (2007); Goudarzi et al. (2016); Simithy et al. (2017)
		PCAF		Simithy et al. (2017)
		NatA		Simithy et al. (2017)
		Tip60		Simithy et al. (2017)
		MOF		Simithy et al. (2017)
			SIRT1, 2, 3	Feldman et al. (2013); Smith and Denu (2007)
			HDAC	G. Xu et al. (2014)
Kcr		CBP		X. Liu, Wei, et al. (2017); Simithy et al. (2017)
		GCN5, Gcn5		W. Liao et al. (2022); Simithy et al. (2017)
		P300		X. Liu, Wei, et al. (2017); Simithy et al. (2017)
		PCAF		Simithy et al. (2017)
		NatA		Simithy et al. (2017)
		Tip60		Simithy et al. (2017); Song et al. (2021)
		MOF, Esa1		X. Liu, Wei, et al. (2017); Simithy et al. (2017)
		KAT7		Yan et al. (2022)
			SIRT1, 2	Feldman et al. (2013); Wei, Liu, et al. (2017)
			HDAC1,2	Fellows et al. (2018); Kelly et al. (2018); Wei, Liu et al. (2017)
			HDAC3	Fellows et al. (2018); Madsen and Olsen (2012); Wei, Liu, et al. (2017)
Kbhb		CBP		K. Liu, Li, et al. (2019); Simithy et al. (2017)
		GCN5		Simithy et al. (2017)
		P300		H. Huang et al. (2021); Simithy et al. (2017)
		PCAF		Simithy et al. (2017)
		NatA		Simithy et al. (2017)
		Tip60		Simithy et al. (2017)
		MOF		Simithy et al. (2017)
			HDAC1,2	H. Huang et al. (2021)
			SIRT1,2,3,5	X. Zhang et al. (2019)
Khib		Tip60, Esa1p		H. Huang, Luo, et al. (2018)
		P300		H. Huang, Tang, et al., (2018)
			HDAC2,3	H. Huang, Luo, et al. (2018)
			SIRT3,5	X. Zhang et al. (2019)

Abbreviations: HDAC, histone deacetylase; KAT7, acetyltransferase 7; SIRT, sirtuin.

FIGURE 1.

Metabolic pathways producing the 4-carbon acyl-CoAs. During fatty acid β-oxidation, butyryl-CoA is produced from the degradation of fatty acyl-CoA, converted to crotonyl-CoA by ACADS in mitochondria and by ACOX1,3 in peroxisomes. Crotonyl-CoA is then converted to β-hydroxybutyryl-CoA by ECHS1, then to acetoacetyl-CoA by HADH, and finally to 2 molecules of acetyl-CoA by ACAA2. During fatty acid synthesis, acetyl-CoA produced from glucose glycolysis is converted to malonyl-CoA by ACC. Malonyl-CoA then is used to synthesize butyryl-CoA by FASN. In the amino acid degradation pathway, lysine and tryptophan are degraded to produce glutaryl-CoA, which is converted to crotonyl-CoA by GCDH. In the ketogenesis pathway, acetyl-CoA produced by fatty acid β-oxidation is utilized to synthesize acetoacetyl-CoA, which is further converted to HMG-CoA by HMGCS and to acetoacetate by HMGCL. Acetoacetate is degraded to generate acetate and β-hydroxybutyrate, which is converted to β-hydroxybutyryl-CoA by ACSS2. Likewise, crotonate and butyrate can be converted to crotonyl-CoA and butyryl-CoA by ACSS2, respectively. ACAA2, acetyl-coenzyme acyltransferase 2; ACADS, acyl-CoA dehydrogenase short chain; ACAT, acetyl-CoA acetyltransferase; ACC, acetyl-CoA carboxylase; ACOX1, 3, acyl-CoA oxidase 1, 3; ACSS2, acyl-CoA synthetase short-chain family member 2; BDH, β-hydroxybutyrate dehydrogenase; ECHS1, enoyl-CoA hydratase, short chain 1; FASN, fatty acid synthase; GCDH, glutaryl-CoA dehydrogenase; HADH, hydroxyacyl-CoA dehydrogenase; HMGCS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGCL, 3-hydroxy-3-methylglutaryl-CoA lyase; MCD, malonyl-CoA decarboxylase.

2 |. METABOLIC PATHWAYS PRODUCING BUTYRYL-COA, CROTONYL-COA, β-HYDROXYBUTYRYL-COA, AND 2-HYDROXYISOBUTYRYL-COA

Metabolic pathways producing acyl-CoA, the precursor to histone/protein acylation, can heavily impact the levels of histone/protein acylation, indicating that histone/protein acylation reflects cellular metabolism and could function as a bridge to link metabolism to cellular functions.

Production of butyryl-CoA, crotonyl-CoA, and β-hydroxybutyryl-CoA involves an interactive metabolic network containing fatty acid β-oxidation, ketogenesis, fatty acid synthesis, and amino acid degradation (Figure 1). Butyryl-CoA is an important intermediate short-chain fatty acyl-CoA from both fatty acid synthesis from carbohydrates and fatty acid breakdown process. It then enters the last cycle of fatty acid β oxidation to be broken into two molecules of acetyl-CoA. In mitochondria, butyryl-CoA is first converted to crotonyl-CoA by acyl-CoA dehydrogenase short chain (ACADS). Crotonyl-CoA is then converted to β-hydroxybutyryl-CoA by Enoyl-CoA hydratase, short chain 1 (ECHS1). In peroxisomes, butyryl-CoA is converted to crotonyl-CoA by ACOX1,3, two peroxisomal acyl-CoA oxidases. β-Oxidation is a key cellular source of butyryl-CoA for histone short-chain fatty acylations. Reduction of the overall β-oxidation rate by acetyl-coenzyme A acyltransferase 2 (ACAA2) deletion-resulted accumulation of acetoacetyl-CoA and subsequent inhibition of the enoyl-CoA hydratase long-chain enzyme leads to a decrease in H3K9bu and H3K9cr (Yang et al., 2021). On the other hand, for cells grown in the glucose-rich but not fatty acid-rich medium, butyryl-CoA synthesized from glucose metabolism appears to be an important short-chain fatty acid (SCFA) source, as glucose deprivation or deletion of fatty acid synthetase (FASN) dramatically reduces the levels of histone acylation including Kbu, Kcr, and Kac. Yet treatment of BSA-conjugated oleic acid (OA) rescues the indicated histone acylation levels (Jo et al., 2020; Yang et al., 2021). The importance of SCFA β-oxidation in Kbu and Kcr is further validated by genetic manipulation of enzymes involved in different steps. For instance, increased expression of ACADS results in a decrease of H3K9bu level in the hearts of high-fat fed mice and enhances histone crotonylation level during endoderm development (Fang et al., 2021; Yang et al., 2021). Deficiency of ACADS or ACOX3, on the other hand, enhances lysine butyrylation level and reduces crotonylation level (Fang et al., 2021; Pougovkina et al., 2014). High expression of ECHS1 in hearts reduces histone crotonylation level (Tang et al., 2021). Besides glucose and fatty acid metabolism, crotonyl-CoA can also be generated from glutaryl-CoA by glutaryl-CoA dehydrogenase (GCDH) during lysine and tryptophan degradation (Figure 1).

Short-chain fatty acyl-CoA can also be produced from their corresponding SCFAs by acyl-CoA synthetase short-chain family member 2 (ACSS2) (Sabari et al., 2015) (Figure 1). For example, β-hydroxybutyrate produced during starvation-induced ketogenesis can be converted to β-hydroxybutyryl-CoA to enhance histone β-hydorxybutyrylation (Kbhb) levels (Xie et al., 2016). In CD133⁺ hepatocellular carcinoma (HCC) cells, the expression of BDH1, a rate-limiting enzyme during ketogenesis to catalyze the conversion between β-hydroxybutyrate and acetoacetate, is transcriptionally inhibited by MTA2-triggered R-loop, leading to an increase in Kbhb, including H3K9hbh (K. Liu, Li, et al., 2019; H. Zhang et al., 2021). One key source of SCFAs is bacterial fermentation in the gastrointestinal tract, which provides abundant butyrate, crotonate, and acetate for protein short-chain acylation through ACSS2. 2-hydroxyisobutyrate is highly abundant in several bio-fluids in humans, including blood, urine, saliva, and feces (Bouatra et al., 2013; Dame et al., 2015; Guneral & Bachmann, 1994; Hoffmann et al., 1993; Hušek et al., 2016; Psychogios et al., 2011), though the physiological pathways generating it remain unclear. The observation that it is a key intermediate metabolite during microbial degradation of methyl tert-butyl ether (MTBE), a wide used gasoline additive and the second most common contaminant of urban aquifers in the United States, indicates that it could be an environmental metabolite impacting human health (Dekant et al., 2001; Ferreira et al., 2006; François et al., 2002; Steffan et al., 1997). Moreover, increased urinary 2-hydroxyisobutyrate level has been observed in alcohol consumers, pregnant women, as well as obese subjects, including obese and type 2 diabetic mice, suggesting that 2-hydroxyisobutyrate metabolism might be correlated with dysregulation in glucose and lipid metabolism (Calvani et al., 2010; Diaz et al., 2011; Elliott et al., 2015; Gil et al., 2018; Irwin et al., 2018; Y. Li et al., 2017; Schifano et al., 2022).

3 |. WRITERS AND ERASERS OF LYSINE ACYLATION (TABLE 1)

Similar to acetyl-CoA, the indicated 4-carbon acyl-CoA are catalyzed to modify histones as well as nonhistone proteins by the canonically characterized histone acetyltransferases (HATs). Acyl-specific transfer-ases have yet to be identified. For instance, p300/CBP has been reported to catalyze Kcr, Kbu, Kbhb, and Khib (Bhattacharya et al., 2022; Chen et al., 2007; H. Huang, Luo, et al., 2018; H. Huang et al., 2021; Sabari et al., 2015; Simithy et al., 2017). p300 is the major acyltransferase for crotonylation and 2-hydroxyisobutyrylation in mammalian cells (H. Huang, Luo, et al., 2018; X. Liu, Wei, et al., 2017) and displays different activities and/or substrate preferences toward different acylations. For instance, p300 catalyzes acetylation primarily on proteins involved in RNA biology but 2-hydroxyisobutyrylation mostly on proteins in the metabolic pathways, including carbon metabolism, amino acid biosynthesis, and glycolysis/gluconeogenesis (H. Huang, Luo, et al., 2018). p300 exhibits a reduced transferase activity toward acyl-CoAs with long acyl-chains compared to those with short chains and has an enhanced activity toward butyryl-coA compared to crotonyl-CoA and β-hydroxybutyryl-CoA (Kaczmarska et al., 2017; Simithy et al., 2017). Not surprisingly, comparison of the acyltransferase activities of other HATs, including CBP, GCN5, PCAF, NatA, Tip60, and MOF toward different short-chain fatty acyl-CoAs in an in vitro assay using H3 or H4 as protein substrates, also revealed a much higher activity of these enzymes toward acetyl-CoA and butyryl-CoA than crotonyl-CoA and β-hydroxybutyryl-CoA, probably due to their specific structures (Simithy et al., 2017). However, nBRPF2-HBO1 complex displays comparable affinities to these acyl-CoAs, with 2.05 μM for acetyl-CoA, 2.22 μM for butyryl-CoA, and 6.56 μM for crotonyl-CoA, indicating other factors may facilitate the binding of different acyl-CoAs to HATs (Xiao et al., 2021). MOF also catalyzes histone crotonylation in mammalian cells, and Esa1, the yeast homolog of MOF, is the major histone crotonyltransferase in yeast (X. Liu, Wei, et al., 2017). Despite the promiscuous nature of these tested HATs, the acyltransferase activities of some enzymes could be separated by genetic mutations or chemicals. For example, the HAT and crotonyltransferase (HCT) activities of p300 and CBP can be separated by p300 I1395G and CBP I1432G mutation. Both mutants maintain the HCT but impair HAT activity, as the mutations may enlarge the size of the pocket for substrate binding (X. Liu, Wei, et al., 2017). LTK-14A, a semi-synthetic derivative of garcinol, has been shown to specifically inhibit p300-mediated histone butyrylation but not acetylation activity (Bhattacharya et al., 2022). It, therefore, could be helpful in studying histone butyrylation, though the effects of this compound on other acylation activity remain to be characterized.

The histone deacetylases (HDACs), including the NAD⁺-dependent deacetylases sirtuins (SIRTs), also have a broad deacylase activity toward a variety of acylations. HDAC1, 2, 3, 8 and SIRT1,2,3 have been shown to remove lysine crotonylation (Bao et al., 2014; Feldman et al., 2013; Fellows et al., 2018; Kelly et al., 2018; Madsen & Olsen, 2012; Wei, Liu, et al., 2017; W. Xu et al., 2017). Consistently, suberoylanilide hydroxamic acid (SAHA), a pan-HDAC inhibitor, could also dramatically increase the levels of histone butyrylation in addition to histone acetylation, indicating that HDACs could be involved in removing lysine butyrylation (G. Xu, 2014). Again, the deacylase activities of some enzymes toward different acylations could be separated by genetic mutations. For instance, the HDAC1-VRPP and HDAC3-VRPP mutants which replace the original AGG by VRPP, display impaired deacetylation activity but retain their decrotonylation activity. Consequently, overexpression of the mutants decreases histone Kcr but not Kac and leads to global gene transcription repression, supporting that histone Kcr promotes gene expression (Wei, Liu, et al., 2017).

4 |. FUNCTION OF PROTEIN CROTONYLATION (FIGURE 2)

FIGURE 2.

Functions of protein Kcr in health and disease. Histone Kcr, particularly H4K77cr and H4K91cr, promotes meso/endoderm differentiation from human embryonic stem cells; Kcr promotes self-renewal of mouse embryonic stem cells; histone Kcr is positively correlated with the expression of genes involved in cardiac hypertrophy; histone Kcr inhibits depression and promotes neuron development; decrotonylation of H2AK119cr attenuates replication stress induced transcription-replication conflicts; histone Kcr enhances PGC-1α and Sirt3 expression to ameliorate acute kidney injury; the dynamic crotonylation at EB1 K66 is critical to the correct positioning of the spindle structures during mitosis and cell cycle progression; H3K27cr inhibits GLUT4 expression, promoting type 2 diabetes; CANX K525cr, induced by leucine deprivation, inhibits CANX to activate GEF and leads to MTORC1 inactivation; histone Kcr promotes HIV latency reversal; histone Kcr promotes spermatogenesis by activating the postmeiotic expression of X-linked genes and histone replacement. CANX, calnexin; EB1, end binding 1; HIV, human immunodeficiency virus; Kcr, crotonylation; mESC, mouse embryonic stem cell; NSPC, neural stem/progenitor cell.

4.1 |. Pluripotency maintenance and differentiation of pluripotent stem cells (PSCs)

Protein Kcr plays a crucial role in maintaining pluripotency and regulating differentiation of PSCs. During meso/endoderm differentiation of human embryonic stem cells (hESCs), a primed state of PSCs, histone Kcr is enhanced to promote endoderm and mesoderm differentiation (Fang et al., 2021). In line with a metabolic switch from glycolysis to oxidative phosphorylation during endoderm/mesoderm differentiation from hESCs, two crotonyl-CoA producing enzymes ACADS and ACOX3, which are both involved in fatty acid β-oxidation, are upregulated to enhance histone Kcr levels on the regulatory elements of meso/endodermal genes. ACSS2, which produces crotonyl-CoA by transferring CoA to crotonate, is also upregulated to promote histone Kcr deposition during endoderm differentiation. Reducing histone Kcr by knocking-out any of the enzymes or mutating H4K77 or H4K91 to R to abolish crotonylation at the sites, impairs endoderm differentiation, while increasing histone Kcr by addition of exogeneous crotonate to the cells rescues the defect, demonstrating histone Kcr is required for endodermal gene expression. H4K77 and H4K91 are located on the histone globular cores on the nucleosome lateral surface and on the histone–histone interface, respectively. High-level crotonylation at these two sites suggests a role of Kcr in regulating DNA/nucleosome interaction and nucleosome dynamics and stability when promoting endoderm differentiation (Fang et al., 2021).

Protein Kcr is also important for the self-renewal of mouse ESCs (mESCs). Histone Kcr deposition has been reported to be more abundant in CGR8 mESCs than in differentiated embryoid bodies (EB) (Wei, Liu, et al., 2017). When HDAC1-VRPP, a HDAC1 mutant removing only crotonylation but not acetylation, was overexpressed in mESCs, the expression of pluripotency core factors including Sox2, Oct4, and Nanog was decreased, the colony number of mESCs was reduced, and differentiation was promoted, indicating that protein Kcr is important for mESC pluripotency maintenance (Wei, Liu, et al., 2017). Additionally, nonhistone proteins are widely crotonylated in mESCs. Lv et al. (2021) systematically profiled protein crotonylation in mESCs of ground, metastable, and primed states as well as in mESCs undergoing early pluripotency exit and identified 3628 high-confidence crotonylated sites in 1426 proteins (Lv et al., 2021). Cellular processes like RNA biogenesis, central carbon metabolism, and proteasome function are enriched in the crotonylated proteins. Crotonylation enhances proteasome activity in metastable mESCs to maintain pluripotency and delay differentiation (Lv et al., 2021).

Interestingly, crotonate has been reported to facilitate somatic cell reprogramming. For instance, crotonate induces the expression of 2 cell stage genes, including Zscan4, lengthening telomeres and promoting the generation of chemical-induced PSCs (CiPSCs) from mouse embryonic fibroblasts (MEF) (Fu et al., 2018). As telomere length is critical to unlimited self-renewal and genomic stability, ciPSCs with longer telomeres possess higher pluripotency. With addition of crotonate, the Lin group generated germline-competent PSCs (gPSCs) from adult somatic cells through chemical reprogramming. These gPSCs can be induced to differentiate into primodrdial-germ-cell like cells and form functional oocytes that produce fertile mice (Tian et al., 2019).

4.2 |. Spermatogenesis

Histone crotonylation is essential for spermatogenesis as reducing histone crotonylation leads to a defect in male fertility associated with a lower epididymal sperm count and motility/velocity as well as a marked increase in cell apoptosis in testis (S. Liu, Yu, et al., 2017).

During spermatogenesis, the sex chromosomes (X and Y) are originally transcriptionally active during spermatogonial divisions and early meiotic stages but undergo a quick silencing in the rest stages of meiosis. This inactivation is followed by reactivation of a subset of sex-chromosome-linked genes (especially those on X chromosomes) in postmeiotic round spermatids (Y. Liu, Li, et al., 2019; Mueller et al., 2008; Turner, 2007). Histone Kcr is abundant on the promoters of the subset of X-liked genes which escape X chromosome inactivation after meiosis (Crespo et al., 2020; S. Liu, Yu, et al., 2017; Tan et al., 2011). When Cdyl, a crotonyl-CoA hydratase hydrating crotonyl-CoA to β-hydroxybutyral-CoA, was expressed in testis to reduce crotonyl-CoA concentration, both the histone Kcr deposition on the promoters and the expression of these X-linked genes were greatly reduced, demonstrating that histone Kcr is essential for expressing these genes after meiosis (S. Liu, Yu, et al., 2017). Additionally, histone Kcr is highly enriched in elongating spermatids, where transcription is essentially ceased and genome-wide histones are replaced by transition proteins (Tnps) and protamines (Prms) (Meistrich et al., 2003; Montellier et al., 2013; Oliva, 2006). Over-expressing Cdyl in mouse testis leads to a reduction in chromatin-associated Tnp1 and Prm2, suggesting a potential role of histone crotonylation in regulating histone replacement (S. Liu, Yu, et al., 2017).

4.3 |. Nervous system development and diseases

Changes of histone Kcr have been observed during neuronal differentiation in vitro and neural development in mouse embryos. During mouse embryo development, H3K9cr and H3K18cr are enriched in the cortical plate (CP) instead of the intermediate zone (LZ) or the ventricular/subventricular zone (VZ/SVZ), and their levels are increased from E12.5 to E16.5. Specifically, H3K9cr is enriched at active promoters co-marked by H3K4me3 and H3K27ac or H3K9ac with low DNA methylation levels, and primarily regulates genes involved in nuclei acid metabolism, protein quality control, and cell proliferation (S. K. Dai et al., 2021; S. K. Dai et al., 2022). During in vitro neuronal differentiation of neural stem/progenitor cells (NSPCs), treatment with crotonate or MS-275, a pan-inhibitor of HDAC1–3, increases histone Kcr mainly on bivalent promoters and stimulates expression of genes involved in neuronal differentiation and cell proliferation (S. K. Dai et al., 2021).

Consistently, alterations of histone/protein Kcr was observed in many disorders and pathogenesis of nervous systems. For instances, total protein Kcr and succinylation levels were elevated in the cortices of the BTBR T⁺ Itpr3^tf/J (BTBR) mice, which display defects in social communication and functioning and are often used as a model for human autism spectrum disorder (ASD) (McFarlane et al., 2008). In the early stage of Alzhemier’s disease (AD), the expression of Nuclear Paraspeckle Assembly Transcript 1 (NEAT1), a P300/CBP complex-associated long noncoding RNA is repressed, and this repression reduces the clearance of intracellular β-amyloid peptide (Aβ) via inhibiting the expression of endocytosis-related genes like CAV2, TGFB2, and TGFBR1 (Wang et al., 2019). Interestingly, NEAT1 knockdown resulted reduction of above genes is associated with enhanced H3K27cr deposition on their promoters (Wang et al., 2019). Histone Kcr may also play a role in suppression of stress-induced vulnerability to depression. In a subset of mice which develop depressive-like behaviors upon chronic social defeat stress, overall histone Kcr level is reduced specifically in the medial prefrontal cortex (mPFC), a brain region heavily implicated in major depressive disorders (Chan & Maze, 2019; Y. Liu, Li, et al., 2019). This reduction is associated with an increased expression of a crotonyl-CoA hydratase Cdyl. Manipulating the expression level of Cdyl by RNAi or overexpression changes histone Kcr levels, the expression of Cdyl target genes, and depressive-behaviors, supporting a causal role of Cdyl and histone Kcr in stress-induced depression (Y. Liu, Li, et al., 2019). Taken together, histone Kcr is an important epigenetic regulator during normal neural development and pathogenesis.

4.4 |. Kidney diseases

Histone crotonylation has also been reported to have a beneficial effect on acute kidney injury (AKI), a potentially lethal condition with high morbidity and mortality rates (Ruiz-Andres, Sanchez-Niño, et al., 2016). Histone Kcr is detectable in tubular cells from healthy mouse and human kidney tissues. After AKI in vivo or in cultured proximal tubule epithelial cells after incubation of TWEAK, a proinflammatory cytokine driving kidney injury, histone Kcr is increased at genes of PGC-1α, a driver of mitochondrial biogenesis and a major regulator of gluconeogenesis, and Sirt3, a member of the SIRT family of NAD⁺-dependent deacetylase localized in mitochondria (Ruiz-Andres, Sanchez-Niño, et al., 2016). This increase in histone Kcr stimulates their expression and is important to maintain mitochondrial function and prevent decrease in renal function in AKI (Ruiz-Andres, Suarez-Alvarez, et al., 2016).

4.5 |. Cardiovascular function and pathogenesis

Histone crotonylation regulated by ECHS1, a hydratase hydrolyzing crotonyl-CoA to β-hydroxybutyryl-CoA, has recently been associated with cardiac hypertrophy (Tang et al., 2021). In human hearts with hypertrophic cardiomyopathy, ECHS1 is reduced and the levels of H3K18cr and H2BKcr are increased (Tang et al., 2021). Consistently, mutations of ECHS1 gene are associated with cardiomyopathies such as hypertrophic cardiomyopathy in human newborns or children and deletion of one copy of Echs1 in mice promotes Angiotensin II-induced hypertrophy, whereas overexpression of Echs1 in mice reduces the level of H3K18cr and H2BK12cr and suppresses Angiotensin II induced hypertrophy (Ganetzky & Stojinski, 2019; Tang et al., 2021; Yamada et al., 2015). At the molecular level, hypertrophic cardiomyopathy genes are enriched in Echs1 deficient or crotonate-treated cardiomyocytes, indicating a potential role of histone crotonylation in promoting the expression of hypertrophic genes (Tang et al., 2021).

4.6 |. Cell cycle

Protein Kcr also regulates cell cycle at both posttranslational and transcriptional levels. Kcr is crucial to the correct spindle positioning during mitosis and cell cycle progression. End binding 1 (EB1) is a microtubule plus-end-tracking protein modulating microtubule elongation and shrinkage, thereby stabilizing astral microtubules for proper orientation of the mitotic spindle (Akhmanova & Steinmetz, 2008; K. Jiang et al., 2009). In metaphase, TIP60 relocates from centromeres to the spindle poles during chromosome alignment, where it crotonylates EB1 at K66, leading to a reduction of EB1 tracking efficiency on microtubule plus ends (Song et al., 2021). Reducing EB1 K66cr via a TIP60 inhibitor (NU9056) results in spindle misorientation, anaphase delay, and chromosome segregation defects, while constitutively expressing genetically encoded and chemically engineered EB1 K66cr leads to aberrant spindle orientation by uncoupling the NuMA-EB1 interaction with astral microtubules (Song et al., 2021). Therefore, TIP60-mediated crotonylation of EB1 forms a dynamic link between astral microtubules and the lateral cell cortex for fine tuning of spindle positioning during mitosis (Song et al., 2021). Additionally, crotonylome analysis in Hela cells has identified crotonylation in many proteins involved in chromatin organization and cell cycle, including CDK7 and MCM3, and crotonate treatment leads to a reduction of cells in S phase but an increase in G2/M phase and H3 phosphorylation at Ser10 in a dose-dependent manner, indicating that protein crotonylation modulates cell cycle (Wei, Mao, et al., 2017).

4.7 |. Carcinogenesis

Protein crotonylation has been shown to be critically involved in carcinogenesis in multiple tissues. First, the overall protein crotonylation levels has been reported to be altered in different types of human tumors (Wan et al., 2019; X. Xu et al., 2021). Specifically, in cervical cancer cells, p300-mediated histone H3 Kcr increases the expression of heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), an oncogene associated with cancer development, and thus promotes cancer development (Han et al., 2020). In human colorectal cancer (CRC), the Kcr level of α enolase (ENO1) is significantly elevated compared to the paratumoral tissues, and ENO1 K420cr, which enhances ENO1 activity, promotes the growth, migration, and invasion of CRC cells in vitro (Hou et al., 2021). In mouse intestinal epithelial cells, H3K18cr is enriched in the transcription start sites (TSSs) of genes involved in cancer and cell cycle progression (Fellows et al., 2018). In mouse colon, microbiota-derivedSCFAs enhance histone Kcr, while antibiotic administration reduces bacterial loads and global histone crotonylation. These observations suggest that gut microbiota could be critical in cancer development by regulating histone Kcr levels on oncogenic genes (Fellows et al., 2018). Protein crotonylation can also promote tumor cell proliferation via posttranslational modulation of tumor suppressor p53. It has recently been shown that crotonate treatment induces p53 crotonylation at serine 46, which suppresses its protein level without change of mRNA. This modification leads to increased glycolysis and mitochondrial activity, and thus augments p53-dependent tumor cell proliferation in response to glucose starvation (P. Liao et al., 2020).

4.8 |. HIV latency reversal

Histone Kcr has recently shown to play an important role in HIV latency reversal. In primary CD4⁺ cells, treatment with crotonate induces ACSS2 expression, increases the levels of H3K4cr, H3K4ac and H3K18ac, and reactivates HIV from latency, while suppression of ACSS2 dampens HIV reactivation (G. Jiang et al., 2018). The combination of ACSS2 induction with either a PKC agonist (PEP005) or an HDAC inhibitor (vorinostat) further leads to a dramatic synergistic effect in reactivating latent HIV (G. Jiang, 2018). In addition, Kcr enhances AZD5582-induced noncanonical NF-κB (ncNF-κB) signaling which also reactivates HIV, by promoting TRIM27, a ubiquitin E3 ligase, to cleave p100 to the active p52 (D. Li et al., 2022). Taken together, Kcr is a critical regulator for HIV reactivation.

4.9 |. DNA damage response

Kcr also plays a role in regulation of DNA repair upon DNA damage. It has been recently reported that upon CPT-induced DNA damage, the Kcr levels of RPA1 on K88, K379, and K595 are upregulated (Yu et al., 2020). Mutation of the Kcr sites in RAP1 impairs its interaction with single-stranded DNA (ssDNA) and/or with components of resection machinery, supporting an important role of RAP1 Kcr in promoting HR and cell survival during DNA damage (Yu et al., 2020). On the other hand, following DNA damage by exposure to ionizing radiation, UV light, laser-microirradiation or etoposide damaging agents, recruitment of CDYL1 to the DNA damage sites induces a rapid and transient reduction of histone Kcr, including H3K9cr levels (Abu-Zhayia et al., 2018, 2019, 2022; Miller et al., 2010). This action of CDYL1 facilitates DSB-induced transcriptional silencing without affecting the homologous recombination (HR) efficiency (Abu-Zhayia et al., 2022).

Dynamic regulation of Kcr is also critical to the replication stress response. Because DNA replication and RNA transcription share the same template, the uncoordinated transcription-replication conflicts (TRC) due to collision of replication and transcription machineries could induce stalling of DNA replication forks and lead to DNA breaks (Hamperl et al., 2017; Merrikh et al., 2011; T. S. Sankar et al., 2016). It has recently been shown that SIRT1-mediated decrotonylation of H2AK119cr allows its subsequent ubiquitination and accumulation at reversed replication forks, which releases RNA polymerase II and thereby repressing transcription in the vicinity of stalled replication forks and attenuating TRC (Hao et al., 2022). Therefore, the coordinated decrotonylation and ubiquitination at H2Ak119 play critical roles in resolving replication stress-induced TRC and maintaining genome integrity (Hao et al., 2022).

4.10 |. Metabolic responses

Protein crotonylation plays an important role in regulation of cellular response to amino acid limitation. Amino acids trigger the translocation of MTORC1 to lysosome surface, where it binds to several regulatory proteins for its activation (Sancak et al., 2010). Yan et al. (2022) recently reported that leucine deprivation-induced protein crotonylation plays a key role in inhibition of lysosomal translocation and, thereby, activation of MTORC1 (Yan et al., 2022). In mouse primary hepatocyte cell line AML12, leucine deprivation induces a global increase of protein lysine crotonylation, but not acetylation, ubiquitination, or succinylation (Yan et al., 2022). Specifically, leucine deprivation triggers lysine acetyltransferase 7 (KAT7) to crotonylate calnexin (CANX) at K525. Crotonylated CANX then translocates to lysosomes, where it binds to lysosomal associated membrane protein 2 (LAMP2) for its interaction with v-ATPase/Ragulator complex. The resulting complex regulates the interaction of Ragulator to RRAG GTPases, inhibits Ragulator GEF activity toward RRAGA, and eventually impairs the lysosomal translocation and activation of MTORC1 (Yan et al., 2022). Mutating CANX K525 to R prevents its translocation to lysosome and interaction with LAMP2, confirming that CANX K525 crotonylation is critical for leucine deprivation-induced inactivation of MTORC1 (Yan et al., 2022).

H3K27cr has recently been reported to negatively regulate GLUT4 expression in cells undergoing persistent hyperglycemia (W. Liao et al., 2022). EPB41L4A-AS1, a long noncoding RNA, is abnormally increased in the liver of T2DM patients and in the muscle cells of patients with insulin resistance and in T2DM cell models, where it inhibits GLUT4 transcription and reduces glucose uptake (W. Liao et al., 2022). Mechanistically, EPB41L4A-AS1 binds to GCN5, a HAT, to enhance H3K27cr level in the promoter region of GLUT4, which in turn inhibits GLUT4 expression. Incubation of cells with crotonate decreases GLUT4 expression in the absence or presence of high glucose, while knockdown EPB41L4A-AS1 reduces H3K27cr level and rescues the expression of GLUT4 in the presence of high glucose (W. Liao et al., 2022). It is worth noting that rather than activating gene expression, H3K27cr seems to inhibit gene expression, indicating that histone crotonylation may have both positive and negative effects on gene expression (W. Liao et al., 2022; Wang et al., 2019). Another example showing histone crotonylation repressing gene expression is H3K9cr which inhibits pro-growth gene expression in the periods of low oxygen consumption (LOC) in the yeast metabolic cycle (YMC) (Gowans et al., 2019).

5 |. FUNCTION OF PROTEIN β-HYDROXYBUTYRYLATION (FIGURE 3)

FIGURE 3.

Function of protein Kbhb in health and disease. Kbhb at H3K9 and nonhistone proteins mediates starvation responses; enhanced H3K9bhb level promotes FoxO4 and PGC-1α expression, which upregulates the expression of cytosolic Pck1, an enzyme controlling the formation and maintenance of CD8⁺ T_mem cells by regulating redox homeostasis; Kbhb at K389 and K405 of AHCY inhibits AHCY activity, impairing the conversion from SAH to Hcy in methionine metabolism; enhanced H3K9bhb promotes HCC stemness and progression; Kbhb at K120, K319, and K370 reduces p53 acetylation level and attenuates p53 activity, leading to reduced cell growth arrest and apoptosis; H3K9bhb enhances the expression of ferroptosis-suppressor genes and thereby prevents pancreatic damage during acute liver failure. AHCY, S-adenosyl-l-homocysteine hydrolase; HCC, hepatocellular carcinoma; Kbhb, b β-hydroxybutyrylation; Pck1, phosphoenolpyruvate carboxykinase; SAH, S-adenosylhomocycteine.

Similar to histone crotonylation, histone lysine β-hydroxybutyrylation (Kbhb) is widely distributed across the genomes and is sensitive to metabolism. For instance, Kbhb is increased in response to elevated β-hydroxybutyrate levels in cultured cells, and is elevated in livers from mice subjected to prolonged fasting or streptozotocin-induced diabetic ketoacidosis, a condition to greatly enhance physiological β-hydroxybutyrate levels (K. Liu, Li, et al., 2019; Xie et al., 2016). In vitro assay shows that p300 catalyzes histone Kbhb and promotes gene transcription (H. Huang et al., 2021). ChIP-seq and RNA-analysis found that histone Kbhb is enriched in the promoters of actively expressed genes (Xie et al., 2016). Starvation-enhanced H3K9bhb levels on the promoters of genes that are upregulated in starvation-responsive metabolic pathways including amino acid catabolism, circadian rhythm, redox balance (selenoamino acid metabolism, cysteine and methionine metabolism), PPAR signaling pathway and oxidative phosphorylation, showing that histone Kbhb represents a new epigenetic regulatory mark linking metabolism to gene expression (Xie et al., 2016).

Histone Kbhb also regulates the development of CD8⁺ T-cell memory (CD8⁺T_mem). In CD8⁺T_mem cells, H3K9bhb is enriched in Foxo1 and Ppargc1a (encoding PGC-1α) to upregulate the expression of these genes (H. Zhang, Tang, et al., 2020). FoxO1 and PGC-1α then cooperatively upregulates the expression of cytosolic phosphoenolpyruvate carboxykinase (Pck1), which controls the formation and maintenance of CD8⁺T_mem cells by regulating redox homeostasis (H. Zhang, Tang, et al., 2020).

Histone Kbhb has recently been reported to promote tumor progression. The expression of BDH1 is inhibited in CD133⁺ HCC, which results in accumulation of intracellular β-hydroxybutyrate and enhancement of H3K9bhb levels. This enhancement is associated with increased expression of JMJD6, GREB3, GTPBP4, NPM1 and TIMM23, leading to poor prognosis (H. Zhang et al., 2021). Kbhb also occurs on a variety of nonhistone proteins. In starved liver, proteins involved in energy and detoxification pathways like fatty acid metabolism, TCA cycle, glycolysis/gluconeogenesis, ketone body, amino acid and ATP metabolism, glutathione metabolism and redox homeostasis, are enriched in the Kbhb-modified proteins (Koronowski et al., 2021). Kbhb inhibits S-adenosyl-l-homocysteine hydrolase (AHCY), a rate-limiting enzyme that hydrolyzes S-adenosylhomocycteine (SAH) to homocysteine and adenosine in the methionine cycle, and mutation of K389 and K405 to R abolishes β-hydroxybutyrate-induced inhibition of AHCY activity, supporting an important role of Kbhb in regulating AHCY activity (Koronowski et al., 2021). Additionally, Kbhb at K120, K319 and K370 of p53 by CBP results in a reduction in p53 acetylation, thereby attenuating p53 activity in both β-hydroxybutyrate cultured cells and in the thymus of fasted mice and leading to decreased cell growth arrest and apoptosis (K. Liu, Li, et al., 2019).

Histone Kbhb is also important in preventing ferroptosis, an intracellular iron-dependent form of programmed cell death, which in turn leads to pancreatic damage during acute liver failure (ALF) (Zheng et al., 2022). During ALF, the β-hydroxybutyrate level is reduced. Supplementation of β-hydroxybutyrate increases H3K9bhb and chromatin accessibility in the promoter regions of ferroptosis-suppressor genes, enhancing their expression and alleviating pancreatic injury in ALF mice (Zheng et al., 2022). These observations suggest that histone Kbhb is a key epigenetic modification controlling the expression of ferroptosis-suppressor genes.

6 |. FUNCTION OF PROTEIN 2-HYDROXYISOBUTYRYLATION (FIGURE 4)

FIGURE 4.

Function of protein Khib in health and disease. H4K8hib is associated with active gene transcription in meiotic and postmeiotic cells, indicating a potential role in spermatogenesis; Khib at proteins located in sperm tails inhibits motility of sperm; Khib at ENO1 increases its activity, thereby promoting glycolysis; H4K8hib promotes yeast CLS. CLS, chronological life span; ENO1, an enolase; Khib, 2-hydroxyisobutyrylation.

Consistent with the observation that 2-hydroxyisobutyrate, the precursor of 2-hydroxyisobutyrylation, is an abundant environmental metabolite, widespread histone/protein lysine 2-hydroxyisobutyrylation (Khib) has been identified in testis and dermal tissues. During male germ cell differentiation, histone Khib shows distinct genomic distribution from histone Kac or Kcr. Particularly, H4K8hib is associated with active gene transcription in meiotic and postmeiotic cells, suggesting histone Khib could play a role in regulating gene expression (L. Dai et al., 2014). Khib is also present in several proteins mainly located in the tail of human sperm to regulate sperm motility. Higher level of Khib due to increased 2-hydroxyisobutyrate reduces total and progressive motility, penetration ability, and ATP level of human sperms (Cheng et al., 2020). Protein Khib was also found to be abundantly present in dermal tissues and is differentially distributed between normal and psoriasis patients’ skin (Ge et al., 2019). Protein Khib was upregulated at 94 sites in 72 proteins and downregulated at 51 sites in 44 proteins in lesional skin. Particularly, the PI3K-Akt signaling pathway is more enriched with Khib in lesional psoriasis skin than normal skin (Ge, 2019). Future work needs to examine whether Khib regulates the PI3K-Akt signaling pathway and plays a role in psoriasis pathogenesis.

Protein Khib also regulates metabolism. Different from p300-mediated Kac, which targets spliceosome and ribosome, p300-mediated Khib is specifically enriched in multiple metabolism-related pathways including carbon metabolism, biosynthesis of amino acids and glycolysis/gluconeogenesis, in human cells (H. Huang, Luo, et al., 2018). Particularly, 5 out of the 10 glycolytic enzymes are 2-hydroxyisobutylated by p300, including glucose-6-phophate isomerase (GPI), ATP-dependent-6-phosphofrucokinase muscle type (PFKM), fructose-bisphosphate aldolase A (ALDOA), phosphoglycerate kinase I (PGK1), and α/γ-enolase (ENO1/2). Deletion of p300 reduces Khib levels on these key glycolytic enzymes including ENO1, leading to decreased catalytic activities and impaired glycolysis as well as hypersensitivity to glucose depletion-induced cell death (H. Huang, Luo, et al., 2018). Protein Khib also impacts glucose homeostasis in Saccharomyces cerevisiae (J. Huang et al., 2017). Removal of 2-hydroxyisobutyrylation at H4K8 changes transcription of carbon transport/metabolism genes and reduces chronological life span (CLS). Moreover, 2-hydroxyisobutyrylation is enriched in the ribosome, glycolysis/glycogenesis pathways, and some amino acid pathways, suggesting a potential role of Khib in carbon and nitrogen metabolism (J. Huang et al., 2017).

7 |. FUNCTION OF PROTEIN BUTYRYLATION (FIGURE 5)

FIGURE 5.

Function of protein Kbu in health and disease. Kbu is enriched in the promoters of Pparg, Cebpd, and Lep to promote their expression and thereby adipogenesis; H4K5bu influences spermatogenesis via inhibiting H4K5ac, which is bound by Brdt to promote the expression of certain spermatogenic-specific genes; Butyrylated H4 shows a delayed replacement than acetylated H4 during spermatogenesis, indicating that the sequential histone replacement might be important for spermatogenesis; H4K5bu reduces H4K5ac level, releasing more free BRD4 from interacting with H4K5ac and promoting BRD4 to enhance active gene expression in ALL and B-ALL. ALL, acute lymphoblastic leukemia; B-ALL, B-precursor ALL; BRD4, bromodomain-containing protein 4.

Histone butyrylation, which is evolutionally conserved and widely distributed across genomes, plays important roles in spermatogenesis and adipogenesis. ChIP-seq analysis shows that acetylation and butyrylation of H4 are both particularly enriched at the TSSs of the most active genes during mouse spermatogenesis (Goudarzi et al., 2016). Brdt, a testis-specific bromodomain-containing proteins, binds to H4K5ac and H4K8ac to promote the expression of certain spermatogenic-specific genes (Gaucher et al., 2012). Butyrylation of H4K5 inhibits Brdt binding, which implies that a differential ratio of acetylation and butyrylation could impact stage-specific gene expression and genome reorganization during spermatogenesis (Goudarzi et al., 2016). Moreover, H4K5/8ac facilitates the replacement of histones by nonhistone sperm-specific transition proteins (Tnps) and Prms, which leads to the removal and degradation of acetylated histones in late elongating spermatids (Gaucher et al., 2012). But butyrylated H4 escapes this wave of histone removal and finally disappears in condensing spermatids (Goudarzi et al., 2016). In fact, the histone acetylation/other acylations ratio could be a common mechanism regulating the bromodomain factors’ genomic distribution (Gao et al., 2021). It was found that an increased ratio of butyrylation or crotonylation over acetylation on H4K5 reduces the chromatin interaction of bromodomain-containing protein 4 (BRD4), which enhances BRD4 nuclear mobility and availability for binding to TSS regions of active genes in acute lymphoblastic leukemia (ALL) cells and blasts from patients with B-precursor ALL (B-ALL) (Gao et al., 2021).

Histone butyrylation is important for adipogenesis. During adipogenesis, histone butyrylation is increased and specifically enriched in the promoters of Pparg, Lep, and Cebpd to promote their expression and adipogenesis (Bhattacharya et al., 2022). Treatment of LTK-14, a small molecule that selectively inhibits p300-catalyzed histone butyrylation, reduces histone butyrylation level and significantly inhibits adipogenesis in vitro as well as weight gain of high-fat diet-fed or genetically obese db/db mice in vivo (Bhattacharya et al., 2022).

8 |. CONCLUSION

It has been increasingly recognized that metabolism, via producing specific metabolites, actively modifies histones and other proteins to regulate cellular processes and functions. Butyryl-CoA, crotonyl-CoA, and β-hydroxybutyryl-CoA are intermediates of glucose and fatty acid metabolisms capable of modifying epigenetics and nonhistone proteins posttranslationally. Recent research has demonstrated that these 4-C acylations play important roles in a variety of cell processes including stem cell biology, cancer development, spermatogenesis, and stress response.

The findings from the current 4-C acylation research have several important clinical implications. On the one hand, histone Kcr-associated reduction of inflammation and mitochondrial stress during AKI and crotonate pretreatment-induced amelioration of kidney injury and dysfunction suggest that administration of crotonate might be beneficial for patients undergoing AKI (Ruiz-Andres, Sanchez-Niño, et al., 2016). The observation that increased Kcr could inhibit depression and promote HIV latency reversal further suggests that administration of crotonate could also potentially alleviate depression and activate HIV from latency for eradication (Chan & Maze, 2019; G. Jiang et al., 2018; D. Li et al., 2022; Y. Liu, Li, et al., 2019). Furthermore, crotonate-promoted endoderm differentiation in vitro could facilitate potential cell therapy using endoderm-derived cells (Fang & Li, 2021; Fang et al., 2021). On the other hand, considering the impact of histone Kcr on promotion of cardiac hypertrophy, inhibitors targeting histone crotonylation could be potentially beneficial for treatment of cardiac hypertrophy (Tang et al., 2021). Particularly, LTK-14A, a nonmutagenic and nontoxic butyrylation-specific inhibitor that inhibits adipogenesis and reduces the body weight of db/db mice, could be a potential antiobesity drug (Bhattacharya et al., 2022). Finally, the observations that increased H3K9bhb promotes the expression of ferroptosis-suppressor genes and supplementation of β-hydroxybutyrate alleviates pancreatic damage during ALF make it interesting to test whether administration of β-hydroxybutyrate could prevent pancreatic injury in human (Zheng et al., 2022). In conclusion, the research on protein acylation will provide molecular basis for potential new therapeutic strategies against a number of human diseases.

Although great advance has been achieved to understand the functions of lysine acylation in past years, a lot remain unknown. First, the intracellular crotonyl-CoA concentration is about 600–1000-folds less than that of acetyl-CoA, and most of the HATs have a higher activity toward acetyl-CoA in vitro (Fang et al., 2021; Sabari et al., 2015; Simithy et al., 2017). How could crotonyl-CoA compete acetyl-CoA to be transferred to histones/nonhistone proteins? How is individual acylation specifically deposited at target genes during different cellular processes? A possibility is that other factors may exist to facilitate the processes. Human MOF, yeast Esa1, or Gcn5 has little HCT activity in vitro, but shows HCT activity in vivo (Andrews et al., 2016; X. Liu, Wei, et al., 2017; Sabari et al., 2015). Actually, Gcn5 can form ADA complex with Ada2 and Ada3 to crotonylate H3 in yeast (Kollenstart et al., 2019). Therefore, it will be valuable to identify these potential factors for understanding the specificity of lysine acylation.

Second, as most acyl-CoAs are generated in mitochondria, it is not clear whether they need to be transported into the nucleus for acylation of histones and other nuclear proteins, or there is a nuclear pool of acyl-CoAs given that some acyl-CoA-producing metabolic enzymes have been reported to be in the nucleus (Fang et al., 2021; Yang et al., 2021). Additional studies are required to address these questions.

Third, differential histone and protein acylation have been detected between many health and disease conditions. However, the in-depth roles of lysine acylations in these processes are not clear. It is unclear whether the change of lysine acylations, particularly at histones, is causal or simply as an association. For instance, H3K27ac, a well-known marker for active enhancers, has been recently shown to be dispensable for activation of gene transcription (A. Sankar et al., 2022; T. Zhang, Zhang, et al., 2020). In contrast, histone Kbu, Kcr, Kbhb, and Khib have all been shown to be associated with active gene expression currently (Bhattacharya et al., 2022; L. Dai et al., 2014; Fang et al., 2021; H. Huang et al., 2021; Tan et al., 2011) and are able to activate gene expression in cell-free systems (Goudarzi et al., 2016; H. Huang, Luo et al., 2018; H. Huang et al., 2021; Sabari et al., 2015). Moreover, crotonylation of H4K77 and H4K91 during endoderm differentiation of hESCs appears to be critical for induction of endodermal gene expression as mutation of either of these two K to R impairs endodermal gene expression (Fang et al., 2021). Future mutagenesis studies are needed to dissect the causal relationship between a specific histone acylation, changes of the gene expression pattern, and subsequent biological outcomes. Given the fact that 4-C actylations are bulkier than acetylation, it will be also interesting to determine whether these modifications are more impactful in opening chromatin and activating gene expression than acetylation.

Fourth, as individual lysine can be modified by various epigenetic modifications, such as different acylations, methylation, ubiquitination, and other modifications, how different modifications interplay to regulate a particular cellular process is another area of intense research. For example, it has been recently reported that the subsequent ubiquitination at H2AK119 after decrotonylation of H2AK119cr helps to release transcriptional machineries from TRC (Hao et al., 2022). Butrylylation of H4K5 reduces its acetylation, which decreases the interaction between chromatin and Brdt or BRD4 and modulates gene transcription (Gao et al., 2021; Goudarzi et al., 2016). In addition to the interplay at the same lysine sites, there are extensive cross-talks between different acylations at different sites. For example, K168cr and K400cr of AKT1 reduce its phosphorylation at several key sites, including T308 and S473, and thereby inhibiting myogenic differentiation (Qian et al., 2022). p53 acetylation can be inhibited by Kbhb at K120, K319, and K370, leading to an attenuated p53 activity (K. Liu, Li, et al., 2019). The interplay of different posttranslational modifications confers another layer of modulation on gene expression, protein activity, and biological function in response to different stimuli. Future studies deciphering these mysteries would shed new lights on metabolisms regulating specific cellular processes/functions, which could open door for development of novel therapy strategies against human diseases.