Exploring the Influence of Cell Metabolism on Cell Fate through Protein Post-translational Modifications

Abstract

The connection between cell fate transitions and metabolic shifts is gaining momentum in the study of cell differentiation in embryonic development, adult stem cells, and cancer pathogenesis. Here, we explore how metabolic transitions influence post-translational modifications (PTMs), which play central roles in the activation of transcriptional programs. PTMs can control the function of transcription factors acting as master regulators of cell fate as well as activation/repression of cell identity genes by regulating chromatin state via histone tail modifications. It now becomes clear that cell metabolism is an integral part of the complex landscape of regulatory mechanisms underlying cell differentiation.

Introduction

The developmental regulation of cell fate depends on the exquisite execution and fine tuning of developmental gene expression programs. Correct specification of different embryonic cell lineages and differentiated cell types, from totipotent/multipotent stem cells across embryonic development, relies on the activation of specific transcriptional programs, which are triggered by a diverse array of inputs. While our understanding of cell fate specification has been dominated by the prevalent role of cell signaling pathways in the outside-in instruction of transcriptional programs during cell differentiation (Cantley et al., 2014; Tam et al., 2013), recent advances in the study of cellular metabolism have revealed that the metabolic state inside a cell has a major impact on the way cell signaling pathways are translated into the nucleo-cytosolic space. Consequently, the activation of transcriptional programs that control cell fate decisions can be modulated by the biochemical environment staged by the distinctive metabolism exhibited by a cell. Molecular mechanisms emerging from the study of the relationship between cellular metabolism and modulation of intracellular cell signaling pathways point to protein post-translational modifications (PTMs) as central inflection points in the control of cell fate specification. We are starting to learn that many of these PTMs depend on the relative concentration of intermediate metabolites from catabolic and anabolic metabolic networks. Regulation of cellular metabolism affects the relative abundance of some of these metabolites, as well as other biochemical parameters (i.e., reactive oxygen species, redox, and pH), which act as rate-limiting biochemical parameters in enzymatic and non-enzymatic reactions that produce these PTMs (Kaelin and McKnight, 2013; Li et al., 2018; Narita et al., 2019). Some of these PTMs operate at the end of cell signaling cascades and probably impart a broad influence on transcriptional activation by either modulating transcription factors directly or indirectly via chromatin regulation (Hoffmeyer et al., 2017; Kaelin and McKnight, 2013; Reid et al., 2017).

In this review, we explore how regulation of glucose and lipid metabolism in the cell can influence the activation of specific cell differentiation programs by creating a biochemical environment that facilitates PTM reactions, such as acylation, methylation, and O-GlcNAcylation, which could have a major impact on cell fate decisions during embryogenesis. We will also discuss the impact of cell metabolism on protein acetylation and its connection to the regulation of gene expression programs controlling cell fate transitions.

A Glimpse of Cell Metabolism through the Window of the Warburg Effect

Glycolysis and mitochondrial respiration are the cornerstones of cellular bioenergetics. It is generally believed that in normal physiological conditions cells engage principally in mitochondrial respiration, with glycolysis as a necessary intermediate series of steps to sustain the influx of pyruvate to the mitochondria, and, in doing so, feeding mitochondrial respiration through the tricarboxylic acid cycle (TCA). However, in stark contrast to most adult differentiated cells, many embryonic cells, as well as cancer cells, are metabolically programmed to show very active glycolysis even when exposed to normal levels of oxygen concentrations and steady levels of glucose. This intense glycolytic activity occurs despite the large disparity in ATP production per molecule of glucose when comparing glucose fermentation by glycolysis and complete glucose oxidation by mitochondrial respiration. A century ago, seminal studies by Otto Warburg revealed that even in aerobic conditions, energy metabolism in cancer cells is characterized by high levels of glucose uptake (Koppenol et al., 2011; Warburg, 1956; Weinhouse, 1956), and in contrast to differentiated adult cells, cancer cells engage in very high levels of glycolysis and massive generation of lactate (a byproduct of high glycolytic activity). Since then, aerobic glycolysis, also called “Warburg effect,” has been extensively studied in multiple cancer types, and it is now considered one of the hallmarks of cancer (Hanahan and Weinberg, 2011; Vander Heiden et al., 2009). Although glycolysis is an inefficient way to generate ATP per molecule of glucose when compared to mitochondrial respiration, the overall kinetics of glycolysis is orders of magnitude faster than that of the TCA cycle (Liberti and Locasale, 2016). Therefore, cells displaying aerobic glycolysis are not handicapped in terms of ATP synthesis. A large body of work on cancer metabolism has been deployed to understand the significance of Warburg metabolism in cell biology. This has led to the realization that the elevated levels of glucose uptake and high glycolytic activity in these cells reflect fundamental roles of glucose metabolism on multiple aspects of cell biology beyond the most immediate bioenergetic functions (Liberti and Locasale, 2016). Indeed, cancer metabolic reprogramming of malignant cells to Warburg metabolism seems to be of crucial importance for the pathogenesis of many cancer types (Hanahan and Weinberg, 2011; Hsu and Sabatini, 2008; Pavlova and Thompson, 2016).

Warburg-type metabolism is not an exclusive metabolic feature of cancer cell biology. In fact, there is increasing evidence that aerobic glycolysis also plays a very important role in normal cell differentiation in multiple developmental contexts (Odegaard and Chawla, 2011; Oginuma et al., 2017; Peng et al., 2016; Zhou et al., 2019), such as embryonic stem cell differentiation (Cliff and Dalton, 2017; Moussaieff et al., 2015). The significance of this type of metabolism remains an open question; however, significant research efforts in the last few decades have revealed important cellular functions of aerobic glycolysis that are not necessarily related to bioenergetic roles (Liberti and Locasale, 2016). Since the aerobic glycolysis phenotype is in many cases correlated with high levels of cell proliferation, one hypothesis seeking to explain the benefits of Warburg metabolism, as a mechanism to bolster cell proliferation, derives from the realization that ATP synthesis is not the limiting factor for cell division. Rather, the production of the necessary lipids, proteins, carbohydrates, and nucleotides might represent a more significant barrier for cell proliferation (Vander Heiden et al., 2009; Liberti and Locasale, 2016). Given that glycolysis serves as a hub connecting many anabolic pathways, this type of metabolism might be instrumental in keeping many of the intermediate glycolytic metabolite pools full in order to steer the flux of anabolic reactions toward the generation of cell biomass.

An important caveat of this hypothesis resides in the fact that during aerobic glycolysis, most of the carbon coming from glucose ends up converted into lactate, which is then excreted to the extracellular space, making the net biomass production per molecule of glucose substantially lower than originally appreciated (DeBerardinis and Chandel, 2020; Liberti and Locasale, 2016). Therefore, the idea of a beneficial role of aerobic glycolysis on providing carbons to primarily boost anabolic pathways needs to be revisited. Specifically, better quantitative assessments in different developmental or pathological contexts are necessary to quantify the relative flux from glucose to lactate or toward the biosynthetic pathways that ramify from glycolysis. Interestingly, in some highly proliferative embryonic tissues, the expression of the aerobic glycolysis phenotype does not necessarily map to increased levels of cell proliferation (Bulusu et al., 2017; Oginuma et al., 2017). Thus, the significance of this type of metabolism in embryonic development and cancer might be neither primarily biosynthetic nor limited only to sustain high levels of cell proliferation.

The Interplay between Metabolism, Cell Signaling, and Transcriptional Regulation

The recognition that aerobic glycolysis plays a role in cell differentiation and cancer pathogenesis, which is parallel to its roles to ATP generation and biomass production, makes imperative that we contemplate alternative functions (non-bioenergetic/ non-biosynthetic) for this metabolic phenotype. We need to consider in a broader sense the plausibility of alternative accessory functions of other cell metabolic networks, which could significantly alter the extracellular and intracellular biochemical state of the cell when dynamically regulated. The study of how aerobic glycolysis is modulated during cell differentiation and carcinogenesis has shed light on the possible molecular mechanisms that link cell metabolic state and cell identity.

Cells exhibiting elevated levels of glucose uptake, high glycolytic flux, and massive production of lactate, as seen in cancer cells under Warburg metabolism, alter the biochemical state of the extracellular and intracellular space by changing the relative concentrations of metabolites, as well as physico-chemical parameters, such as pH, NADH/NAD+ ratio, and generation of reactive oxygen species (ROS) (Liberti and Locasale, 2016; Reid et al., 2017; Tatapudy et al., 2017). Intermediate glycolytic or TCA metabolites can act as rate-limiting factors in some enzymatic and non-enzymatic PTMs (Kaelin and McKnight, 2013; Reid et al., 2017), which in turn can trigger major functional effects by switching protein activity (Figure 1A). The rates of some of these PTM reactions are also affected by physico-chemical factors, such as pH or NADH/ NAD+ ratio, which can be altered during sustained high rates of aerobic glycolysis (Liberti and Locasale, 2016). The influence of cell metabolism in modulating protein PTMs has also been reported in response to modulation of other metabolic pathways besides glycolysis, such as increased lysine acetylation during high levels of fatty acid oxidation (Xiong et al., 2018), protein methylation driven by alterations in the relative abundance of intermediate metabolites of the TCA cycle and the homocysteine-methionine cycle (Kaelin and McKnight, 2013; Lu and Thompson, 2012), or changes in levels of protein O-GlcNAcylation due to high flux of the hexosamine biosynthetic pathway (Chi et al., 2020; Li et al., 2018). Although the cellular roles of PTMs can be quite diverse, there is evidence of a connection between metabolically regulated PTMs and the execution of cell differentiation programs through epigenetic regulation of chromatin state, activation/repression of gene transcription, and modulation of cell signaling outputs. In parallel to PTMs, epigenetic regulation of gene transcription can be also influenced by cell metabolism at the level of DNA methylation, since the activity of DNA methyl transferases is affected by the cellular concentration of the homocysteine-methionine cycle metabolite S-adenosylmethionine (SAM). This metabolite also serves as the methyl donor for protein methylation reactions by methyltransferases (Figure 1A) (Kaelin and McKnight, 2013; Li et al., 2018).

Figure 1. Connections of Metabolic Pathways with PTM Reactions through Intermediate Metabolites and Impact of Growth Factor Signaling on Modulation of Glucose Metabolism.

(A) Diagram depicting the interplay between some of the cell metabolic pathways with some of the fundamental protein post-translational modification reactions known to modulate protein functions. DNA methylation is also included, since DNA methylation and protein methylation share the same methyl donor substrates and also the metabolites needed for demethylation. Intermediate metabolites that affect the rate of these reactions are in red. This diagram is not meant to be comprehensive, as many other metabolites can affect the rate of these reactions; however, the metabolites highlighted here are some of the best understood, and a comprehensive listing of all other metabolites is beyond the scope of this review. Stoichiometry of ATP and NADH production per molecule of glucose is omitted for simplicity. Uptake of acetate from the extracellular space is omitted for simplicity as well, but it is noteworthy that an endogenous pyruvate by keto acid dehydrogenases (such as PDH, pyruvate dehydrogenase). B-Ox, β-oxidation pathway; HBP, hexosamine biosynthesis pathway; PPP, pentode phosphate pathway; ROS, reactive oxygen species; TCA, tricarboxylic acid cycle; SAM, S-adenosyl methionine; UDP-GLcNAc, uridine diphosphate N-acetylglucosamine.

(B) A simplified generalized schematic of glycolysis upregulation by growth factor signaling, focusing on the intracellular connections between some of the major players controlling cellular metabolism, such as AKT, AMPK, ERK, mTOR, PI3K, and RAS kinases and FOXO1/3a, MYC, and HIF1α transcription factors. Glucose metabolism is profoundly affected by AKT, mTORC1, and AMPK signaling cascades, which involve the phosphorylation activation/inhibition of many enzymes and transcription factors, which then shape the cell metabolic landscape of a cell; here only major regulatory proteins were included for simplicity.

Tight regulation of cell signaling pathways and precise activation of gene transcriptional programs are critical for cell differentiation, and in this context, PTMs act as key players of cell differentiation programs, since they can act as switches controlling the activation or repression of protein functions. Protein PTMs provide a more energy-efficient and faster flexible mechanism than protein turnover to regulate protein function in a reversible manner. In the case of kinases, the normal physiological range of ATP concentration does not limit the rate of kinase activity. In contrast to protein phosphorylation reactions, the catalysis of protein acylations, methylations, and glycosylations is sensitive to the concentration of their metabolite substrates. The concentration of intermediate metabolites, which function as substrates, co-factors, and allosteric regulators, exhibit physiological dynamic ranges that can limit the rate of catalysis of these PTM reactions (Reid et al., 2017). This is the case for protein acetylation/deacetylation and methylation/demethylation reactions, as well as with DNA methyl-ation/ demethylation. For those biochem- ical modifications, the relationship between the physiological concentration range of the metabolic substrates (i.e., acetyl-CoA, NAD+, SAM, α-ketoglutarate) with the catalytic kinetic constant of the corresponding enzymes (Km) carrying out each of these reactions makes the rate of catalysis responsive to changes in the intracellular abundance of metabolic substrates (Reid et al., 2017; Sivanand et al., 2018). Therefore, PTM reactions are controlled by the dynamics of cell metabolism.

Metabolic reprogramming during cell differentiation has the potential to create permissive biochemical conditions allowing PTM reactions to occur at higher rates, changing the stoichiometric balance of these modifications in response to the cell metabolic state. When these PTMs occur in proteins like transcription factors or epigenetic modulators of gene expression, then the metabolic state of the cell can directly impact gene transcription and have a major effect on the activation of gene expression programs controlling cell fate. The activity of many transcription factors can be significantly changed by PTMs. Depending on the location of the modified residues, these PTMs can promote/inhibit protein-protein interactions, increase protein stability by blocking ubiquitylation, promote kinase phosphorylation, enhance nuclear/cytoplasmic localization, or in-crease/reduce DNA binding affinity. Similarly, PTMs of histone tails (mainly on histone 3 and 4) affect the chromatin state of promoters, gene bodies, and non-coding cis-regulatory elements, which then affect chromatin structure and function. Many histone PTMs can promote the specific interaction of tagged histone tails with “reader” proteins that also carry chromatin modifying proteins of diverse functions. This eventually leads to the establishment and maintenance of chromatin states and the expression or silencing of genes (Grossniklaus and Paro, 2014; Kingston and Tamkun, 2014). Moreover, histone methylation and DNA methylation can display reciprocal stimulation; in the case of histone methylation, trimethylated histone 3 at lysine 9 (H3K9me3) can recruit DNA methyltransferase enzymes (DNMT1) through the adaptor proteins carrying “reader” domains for methylated lysine (Rothbart et al., 2012). DNA methylation and histone methylation (H3K9 and H3K27) are interconnected and can be potentially altered by cell metabolism regulation, with important implications in the metabolic control of gene silencing during cell differentiation (Kaelin and McKnight, 2013; Li et al., 2018).

Metabolic Shifts and Cell Fate Transitions

Metabolic shifts during cell differentiation can often be correlated with the acquisition of specific cell identities. A shift between aerobic glycolysis and oxidative phosphorylation, for instance, is observed in different lineages during cell differentiation toward specific cell fates. The importance of this type of metabolic reprogramming has been uncovered in a broad range of developmental contexts, such as in the control of the differentiation of embryonic stem cells (ESCs) (Cliff and Dalton, 2017; Moussaieff et al., 2015), induced pluripotent stem cells (Panopoulos et al., 2012; Wu et al., 2016), adult stem cells (i.e., muscle satellite, hematopoietic, hair follicles, intestinal crypts) (Flores et al., 2017; Ito and Suda, 2014; Takubo et al., 2013), macrophage polarization (Odegaard and Chawla, 2011), neural stem cells (Zhou et al., 2019), neuromesodermal progenitors (Oginuma et al., 2017, 2020), and T cell activation (Peng et al., 2016). In some cases, glycolysis is enhanced, whereas in others, increase in oxidative phosphorylation is needed. The permissive or instructive nature of these metabolic shifts during cell fate transitions remains an open question. A common pattern seems to be the enhancement of glycolysis in order to maintain the stem cell/progenitor state, and the transition to a more differentiated state is usually associated with an increase in oxidative phosphorylation. However, in some developmental cell fate transitions, high levels of glucose uptake does not necessarily translate into merely boosting the glycolytic flux, but in some instances high glucose uptake serves to increase the flux toward parallel pathways that branch out from glycolysis at early points of the glycolysis pathway, such as the pentose phosphate pathway (PPP) and the hexosamine biosynthesis pathway (HBP) (Chi et al., 2020). For instance, one of the earliest cell fate decisions in mammalian embryogenesis is the segregation between trophectoderm and inner cell mass cell fates. Despite high levels of glucose uptake, trophectoderm cell fate is accomplished by diverting glucose metabolism early in the glycolysis pathway to feed the PPP and the HBP. Crosstalk between the two pathways increases the intracellular concentration of UDP-GlcNAc, a critical metabolite for protein O-glycosylation. Then, glycosylation-mediated nuclear translocation of the transcription factor YAP allows the activation of gene transcription of trophectoderm identity genes (Chi et al., 2020). Regulation of lipid metabolism also plays significant roles in the control of cell identity. Regulation of fatty acid oxidation is a critical component of the cell identity network controlling some cell states. For instance, suppression of fatty acid oxidation is needed in endothelial to mesenchymal transition (discussed below) and also during chondrogenesis from skeletal progenitor cells toward the chondrocyte fate (van Gastel et al., 2020).

Influence of Growth Factor Signaling in Cell Metabolism Regulation

Growth factor signaling is central in triggering the activation of the cytoplasmic responses and eventually for the activation of the necessary gene expression programs to specify cell fate. Metabolic reprograming to an aerobic glycolysis phenotype is initiated in numerous cases by growth factor stimulation, where intracellular activation of signaling pathways generally results in a large increase in glucose uptake and a ramp up of glycolytic metabolism at the expense of mitochondrial respiration (Figure 1B). This usually occurs by controlling the activity of multiple proteins at different levels of regulation, such as rates of transcription and translation, as well as allosterically via PTMs. A glycolytic phenotype can be induced by activation of different types of receptor tyrosine kinases (RTKs) after ligand stimulation resulting in downstream activation of phosphatidylinositol 3-kinase (PI3K). Subsequent activation of the serine/threonine kinase AKT (also known as protein kinase B) generates a phosphorylation cascade that enhances the glycolytic flux either by cranking up the activity of glycolysis enzymes via phosphorylation, by increasing their transcription through inhibition of FOXO transcription factors, or by increasing the stability of the MYC transcription factor via glycogen synthase kinase-3 (GSK-3) inhibition (Manning and Toker, 2017). Activation of the PI3K/AKT pathway also increases the activity of the serine/threonine kinase mechanistic target of rapamycin complex 1 (mTORC1) through the phosphorylation and inhibition of the mTORC1 inhibitor TSC; mTORC1 then serves as a major stimu- lant of anabolic metabolism (Liu and Sabatini, 2020; Manning and Toker, 2017; Mossmann et al., 2018). In the context of glucose metabolism, mTORC1 can act in synergy with AKT stimulation in boosting a glycolytic phenotype through enhanced transcription of glycolytic enzymes as well as hypoxia-inducible factor 1a (HIF-1α) and glucose transporters (Liu and Sabatini, 2020; Manning and Toker, 2017; Mossmann et al., 2018). Growth factor activation of the MAPK/ERK pathway can also stimulate mTORC1 activity by repression of TSC proteins (Liu and Sabatini, 2020); thus, there is an important intracellular crosstalk of different cell signaling pathways at the level of the integration of specific intracellular responses to coordinate the transitions in metabolic shifts.

While AKT and mTOR kinases can be regulated on different levels, a major regulator of the cell metabolic state and an inhibitor of mTORC1 is the AMP-activated protein kinase (AMPK). AMPK can counterbalance the stimulation of anabolic metabolism by mTORC1 by activating TSC2 and by inhibition of the mTORC1 component RAPTOR (Herzig and Shaw, 2018; Lin and Hardie, 2018). Repression of mTORC1 together with regulation of metabolic enzymes through phosphorylation causes AMPK to promote the enhancement of catabolic processes, increasing oxidative phosphorylation and upregulating autophagy (Garcia and Shaw, 2017; Lin and Hardie, 2018). AMPK is an important inhibitor of the aerobic glycolysis phenotype seen in cancer cells under Warburg metabolism (Faubert et al., 2013). However, its role in the regulation of metabolic shifts in normal cell differentiation is poorly understood. We are starting to gain a better understanding of the interplay between growth factor stimulation and the intracellular activation of master regulators of energy metabolism, like PI3K/AKT, mTOR, and AMPK, as well as the degree of intracellular crosstalk between these metabolic regulatory pathways. However, most of this work comes from the study of cancer metabolism, and it is unclear how these higher-level regulators of cell metabolism regulate metabolic shifts during embryonic development and how they impact cell fate transitions.

Cell Metabolism Is an Important Regulator of Protein Acetylation

The activity of numerous proteins can be dynamically modulated by lysine acetylation (Narita et al., 2019), and regulation of the relative abundance of intermediate metabolites of glycolysis, oxidative phosphorylation, and fatty acid oxidation can impact the level of acetylation at the level of the entire proteome (Kaelin and McKnight, 2013; Li et al., 2018; Sivanand et al., 2018). Lysine acetylation is generated by the transfer of an acetylation group from acetyl-CoA to the ε-amino side chain of lysine by a diverse group of lysine acetyl transferases (KATs), whereas removal of lysine acetylation is catalyzed by lysine deacetylases (KDACs) (Marmorstein and Zhou, 2014; Seto and Yoshida, 2014). In contrast to protein kinases, the specificity of protein acetylation substrate motifs of KAT is poorly understood, and many KATs have overlapping protein substrates and display functional redundancy (Choudhary et al., 2014; Li et al., 2018; Narita et al., 2019). Metabolic regulation of acetyl-CoA levels is an important parameter to modulate protein acetylation levels by KAT (and non-enzymatically; see below). CoA also binds with high affinity to KATs in competition with acetyl-CoA, suggesting that acetyl-CoA:CoA ratio could be a more relevant parameter dictating KAT activity than concentrations of acetyl-CoA alone (Choudhary et al., 2014; Pietrocola et al., 2015). Since the mitochondria is not permeable to acetyl-CoA, compartment-specific acetyl-CoA production can locally drive acetylation. Mitochondrial and nucleocytoplasmic pools of acetyl-CoA are independently synthesized. Mitochondrial acetyl-CoA can be produced by pyruvate dehydrogenase (PDH), β-oxidation of fatty acids, and amino acid metabolism. Nuclear and cytoplasmic acetyl-CoA is generated by nuclear PDH, but also by ATP-citrate lyase (ACLY) using citrate as a substrate, by acetyl-CoA synthetase short-chain family member 2 (ACSS2) using acetate as a substrate, and by carnitine acetyltransferase (CrAT) using acetylcarnitine as a substrate (Madiraju et al., 2009; Pietrocola et al., 2015; Sivanand et al., 2018). Therefore, nucleocytoplasmic levels of the intermediate metabolites, which are mainly derived from glycolysis, mitochondrial respiration, and fatty acid oxidation and serve as substrates for these enzymes, play a fundamental role in the rate of nucleocytoplasmic acetyl-CoA generation (Kaelin and McKnight, 2013; Li et al., 2018; Narita et al., 2019). High levels of glucose consumption and aerobic glycolysis is known to increase the nucleocytoplasmic levels of acetyl -CoA level and to lead to increased histone acetylation in an acetyl-CoA-dose-dependent manner (Cluntun et al., 2015; Lee et al., 2014). Therefore, by changing the acetylation levels of the active histone marks H3k9ac and H3K27ac, glycolysis-dependent histone acetylation can affect the epigenetic state of genes controlling cell identity during early differentiation of embryonic stem cells (Figure 2A) (Moussaieff et al., 2015) and T cell activation (Peng et al., 2016).

Figure 2. Schematic Representation of Three Examples of Metabolic Transitions Influencing the Activation of Transcriptional Programs That Have an Instrumental Effect on Cell Fate Transitions.

(A) Human embryonic stem cells and induced pluripotent stem cells display a highly glycolytic phenotype that is critical for maintenance of the stem cellphenotype. Differentiation to some cell lineages involves the downregulation of glycolysis and upregulation of oxidative phosphorylation. Activation of stem cell genes is maintained at the level of epigenetic regulation, exerted by chromatin acetylation. Histone acetylation is dependent on glycolysis-mediated accumulation of acetyl-CoA (Ac-CoA). While active chromatin is here represented by nucleosomes decorated by acetyl groups (i.e., H3K9ac), repressed chromatin is decorated by methyl groups (H3K9me3, H3K27me3; although other methylated histone marks label active chromatin, H3K4me1, H3K4me3).

(B) Neuromesodermal cells give rise to neural progenitor and paraxial mesodermal cells. Mesodermal cell fate is dependent on upregulation of glycolysis, which drives intracellular alkalization (via monocarboxylate acid extruders, MCT) and promotes β-CATENIN acetylation. Activation of WNT/β-CATENIN signaling pathway in conjunction with glycolysis-mediated acetylation of β-CATENIN at lysine 49 (green circle labeled Ac) is necessary for the expression of mesodermal β-CATENIN target genes and the acquisition of paraxial mesodermal identity.

(C) Maintenance of endothelial identity is controlled by active fatty acid oxidation, whereas downregulation of fatty acid oxidation is necessary for endothelial tomesenchymal transition (EdMT). High levels of fatty acid oxidation are critical to keep high levels of acetyl-CoA and promote the acetylation of the TGFβ signaling pathway inhibitor SMAD7 (green circles labeled Ac). Acetylation of SMAD7 stabilize the protein by blocking its poly-ubiquitination (gray circles labeled Ub) and its eventual proteasomal degradation. TGFb signaling pathway (via activated SMAD2), including bringing SMAD7 levels down, is necessary for the activation of EdMT genes and the acquisition of a mesenchymal identity.

On the other hand, neural differentiation from neural stem cells also depends on increasing the nucleocytosolic levels of acetyl-CoA to elevate the levels of H3K9 acetylation in neuron identity genes. In this case, metabolic reprogramming is triggered in part by the expression of TP53 inducible glycolysis and apoptosis regulator (TIGAR) (Zhou et al., 2019). TIGAR inhibits the glycolytic flux (at the level of fructose-6-phosphate) by diverting most glucose metabolism to the PPP. But TIGAR also enhances mitochondrial respiration by increasing pyruvate levels in the mitochondria through the increase in lactate dehydrogenase B (LDHB) expression. LDHB isoform preferentially converts lactate to pyruvate, whereas LDHA promotes the reverse reaction. Thus, upregulation of LDHB expression during neuron differentiation, concomitant to the loss of LDHA expression (Zheng et al., 2016), boosts pyruvate catabolism through the mitochondrial TCA cycle. This elevates the efflux of mitochondrial derived citrate, which is then used for the generation of acetyl-CoA in the nucleocytosolic space by ACLY (Zhou et al., 2019). In a different developmental context, early cleavage stages of mouse and human embryos (preimplantation stages) engage in active pyruvate metabolism in the nucleus, which has an important influence in the complex balance of histone acetylation and methylation in these cells (Nagaraj et al., 2017). Mammalian preimplantation embryos depend on exogenous pyruvate for the zygotic genome activation (ZGA); however, pyruvate is not metabolized in the mitochondria at significant levels in preimplantation embryos (due to low levels of mitochondrial respiration through PDH inhibition). Instead, some of the TCA cycle enzymes are redirected to the nucleus in order to metabolize pyruvate into a subset series of TCA-related enzymatic reactions. This leads to the nuclear synthesis of acetyl-CoA and α-ketoglutarate; these two metabolites are critical for proper epigenetic regulation of ZGA through the regulation of histone acetylation and methylation levels (Nagaraj et al., 2017).

In addition to glycolysis and TCA cycle metabolic pathways, stable isotope tracing in mouse liver cells (McDonnell et al., 2016) has demonstrated that increased mitochondrial fatty acid oxidation can augment the nucleocytoplasmic acetyl-CoA synthesis as well. This probably occurs by augmenting the efflux of mitochondrial-derived citrate and acetyl-carnitine toward the cytoplasm, after which high acetyl-CoA levels promote the increase in histone acetylation and the transcriptional activation of specific gene programs (McDonnell et al., 2016; Pietrocola et al., 2015; Shi and Tu, 2015).

Glycolysis-mediated lysine acetylation in histone tails increases the negative charge of chromatin and has the net effect of decompacting the chromatin fiber (Liu et al., 2015). Importantly, acetylated histone-labeled chromatin is also permissive for transcription, whereas deacetylated chromatin, on the other hand, is associated with compacted chromatin and silencing transcription (Grossniklaus and Paro, 2014; Kingston and Tamkun, 2014; Seto and Yoshida, 2014). Acetylated lysines in histone and non-histone proteins are recognized by acetyl-lysine reader domains, such as bromodomain (BRD), double plant homeodomain (DPH), and YEATS domain. All these protein domains are present in multiple proteins and are used to interact with acetyl-lysine docking sites (Gong et al., 2016). This allows the assemblage of protein complex at specific loci, such as promoters and non-coding cis-regulatory elements, to regulate gene transcription (Allis and Jenuwein, 2016; Schoenfelder and Fraser, 2019). Future studies will be needed to explore the mechanism controlling metabolically driven changes in histone acetylation causing loci-specific epigenetic effects in contrast to epigenome-wide impacts on chromatin acetylation. It is possible that similar metabolic reprogramming in different cell types can generate divergent epigenetic outcomes. This would probably depend on the epigenomic, transcriptomic, and proteomic profiles of the cell undergoing a metabolic shift, especially when factoring cell-type-specific profiles of transcription factors, transcriptional co-activators, chromatin remodelers, and chromatin architectural proteins.

Metabolically regulated acetylation of non-histone proteins also represents an important inflection point in the control of cell fate. Acetylation of non-histone proteins can switch the activity of a protein on or off and in that way regulate the outputs of cell signaling pathways with a major impact on cell fate. The connection between cell metabolism, cell fate, and non-histone protein acetylation can be illustrated by the specification of paraxial mesodermal cells (precursors of the axial musculoskeletal system) from neuromesodermal cell precursors during embryonic axial elongation in vertebrates (Figure 2B). In mice and chicken embryos, specification of paraxial mesoderm at the posterior end of the embryo (in the tail bud) occurs within a population of cells that, in normoxic conditions, displays high levels of glucose uptake, glycolytic activity, and lactate production (Bulusu et al., 2017; Oginuma et al., 2017). Disruption of glycolysis in neuromesodermal cells biases these cells to generate neural precursors at the expense of paraxial mesodermal cells, indicating that regulation of glycolysis influences neuromesodermal cell fate during differentiation (Oginuma et al., 2017, 2020). FGF signaling activation at the posterior end of the embryo causes ERK and AKT activation (Delfini et al., 2005; Dubrulle and Pourquié, 2004), and pharmacological inhibition of ERK signaling demonstrates that ERK activation is a key driver of glycolytic enzymes and glucose transporters transcriptional upregulation (Oginuma et al., 2017).

Specification of paraxial mesoderm cell fate also requires Wnt/ β-Catenin signaling in neuromesodermal cells (Henrique et al., 2015; Nowotschin et al., 2012). Although posterior expression of Wnt ligands activates the pathway, proper intracellular Wnt/ β-Catenin activation necessary for paraxial mesoderm specification requires high glycolytic activity in these cells (Oginuma et al., 2017). During mesodermal cell specification, β-Catenin transcriptional regulatory functions are in large degree controlled through the opposite effects elicited by acetylation or trimethylation of lysine 49 (K49) (Hoffmeyer et al., 2017). Thus, in mouse ESCs, Wnt/β-Catenin-dependent mesodermal differentiation requires K49 acetylation. This PTM abrogates the association of β-Catenin with the polycomb repressive complex 2 (PRC2). This complex carries the methyltransferases Ezh1 and Ezh2, responsible for the generation of the H3K27me3 repressive chromatin mark, as well as K49 β-Catenin trimethylation (Hoffmeyer et al., 2017). Conversely, interaction of β-Catenin and the acetyl transferase p300 occurs at the level of active chromatin loci (Hoffmeyer et al., 2017), which further drives local increase of histone acetylation. Whether K49 acetylation is needed for p300 interaction via p300 bromodomain needs further investigation. Recent studies demonstrate that high glycolysis leads to an increase in transcription of mesodermal Wnt/β-Catenin target genes during embryonic paraxial mesoderm differentiation in vivo in the tail bud of chicken embryos as well as in vitro during paraxial mesoderm differentiation from hiPS cells. The high glycolytic activity enhances β-Catenin K49 acetylation, thus regulating neuromesodermal cell fate by enhancing WNT/ β-Catenin signaling (Oginuma et al., 2020).

Fatty acid oxidation can affect nucleocytoplasmic levels of acetyl-CoA as well, and it has been linked to the control of acetylation of non-histone proteins to regulate the output of cell signaling pathways and influence cell fate (Figure 2C). Mitochondrial-dependent fatty acid oxidation involves the translocation of fatty acids to the mitochondrial matrix via the carnitine shuttle, after which mitochondrial β-oxidation of fatty acly-CoA yields acetyl-CoA to the TCA cycle. The high flux into the TCA cycle then allows the outflow of citrate to the cytoplasm (Pietrocola et al., 2015). Conversion of cytoplasmic citrate into acetyl-CoA by ACLY increases the abundance of acetyl-CoA in the cytoplasm (Pietrocola et al., 2015). Endothelial to mesenchymal transition (EdMT), a morphogenetic process underlying the development of cardiac valves and involved in the pathogenesis of vascular diseases such as atherosclerosis (Piera-Velazquez and Jimenez, 2019), is controlled by the activation of TGF-β signaling pathway in endothelial cells, which involves the intracellular phosphorylation and activation of SMAD2 transcription factor (Xiong et al., 2018). Homeostasis of the endothelial cell identity state is regulated by sustaining a fatty acid oxidation metabolic phenotype, which maintains high levels of cytosolic acetyl-CoA and enhances the acetylation-dependent stabilization of the TGF-β signaling pathway inhibitor protein SMAD7 (Xiong et al., 2018). Acetylation of SMAD7 at two lysine residues (K64 and K70) by p300 acetyl-transferase protects it from polyubiquitylation by the Smurf family of E3 ubiquitin ligases that otherwise would target SMAD7 for proteasomal degradation (Grö nroos et al., 2002; Xiong et al., 2018). This strategy is similar to that used by the transcription factor p53, which exhibits acetylation-dependent stabilization, blocking MDM2-catalyzed ubiquitylation of the same lysine residues (Nihira et al., 2017). In contrast to acetylated SMAD7, deacetylation of SMAD7 by KDAC1 (also known as HDAC1) allows ubiquitylation and decreases its stability (Grö nroos et al., 2002; Simonsson et al., 2005; Xiong et al., 2018). Control of SMAD7 stability by acetylation-mediated blockage of lysine ubiquitylation in endothelial cells allows the intracellular inhibition of the TGF-β signaling pathway and in that way is firmly engrained in maintenance of endothelial cell identity. During mouse heart development, EdMT is responsible for endocardial cell migration to form the cardiac atrioventricular valves mesenchyme, and disruption of fatty acid oxidation results in thickening of the valves due to unrestrained EdMT (Xiong et al., 2018). This underscores the influence of fatty acid oxidation in the control of cell fate decisions during embryogenesis.

Other Lysine-Acylations Are Metabolically Regulated

Besides acetylation, lysines are also modified by other types of acylations of variable length in their carbon chain, such as formylation, propionylation, crotonylation, or succinylation, to name a few (Choudhary et al., 2014). Some of these lysine-acylations are generated by KAT, as well as by non-enzymatic acylation, and some lysine-acylations can be removed by Sirtuins (NAD+ dependent KDACs) (Choudhary et al., 2014). However, we know much less about these types of modifications, and it is not clear whether incorporation and/or removal of these acylations are carried out by proteins specific for a given acylation type or the promiscuous activity of acetyl-lysine writers and erasers. Similarly, little is known about the biosynthetic pathways regulating the formation of different acyl-CoA compounds, which function as acyl donors for acyl-lysine reactions. The incorporation of lactate to lysine residues in histone tails is a recently discovered lysine acylation. This PTM occurs in cells showing high levels of aerobic glycolysis, and it is enriched in the glycolytic M1 macrophages during macrophage polarization (acquisition of a distinct macrophage functional phenotype). Elevated levels of intracellular lactate generated by aerobic glycolysis enhance histone lysine-lactylation, whereas inhibition of glycolysis or mitochondrial respiration, both affecting lactate generation in opposite directions, affect lactylation positively or negatively depending on nucleo-cytosolic lactate concentrations (Zhang et al., 2019). Genome-wide analysis of H3K18 lactylation (H3K18la) during acquisition of a M1 glycolytic phenotype during macrophage polarization indicates that H3K18la predominantly decorates chromatin at active gene promoters and shows significant enrichment in response to the increased availability of lactate due to the glycolytic metabolic shift (Zhang et al., 2019). Furthermore, the metabolic effect on chromatin lactylation in M1 macrophages also correlates with the activation of a transcriptomic program controlling M1 macrophage cell identity. This, together with in vitro biochemical experiments, demonstrates that histone lactylation marks can promote gene transcription (Zhang et al., 2019). Further studies on lysine-lactylation will be needed to determine how widespread lactylation is proteome-wide and whether this new PTM could also act as a switch for the activation of non-histone protein functions downstream of signaling pathways. This could broaden the scope of lysine-lactylation to metabolically modulate specific transcriptional outcomes for cell fate specification in different developmental contexts where aerobic glycolysis plays a central role in cell differentiation.

Glycolysis Affects Intracellular pH and Protein Acetylation

Despite large amounts of lactic acid production as a result of glycolysis, cells under Warburg metabolism increase their intracellular cellular pH (pHi) (Webb et al., 2011). However, changes in expression and activity of membrane transporters that increase the efflux of protons to the extracellular space, lowering the extracellular pH (pHe) but raising the pHi, are also associated to high glycolytic activity in cancer cells (Martinez-Zaguilan et al., 1993; McLean et al., 2000; Webb et al., 2011). Monocarboxylate transporters (MCTs) such as MCT-1 and MCT-4 are symporters, which can excrete lactic acid from the cell together with protons. This results in an H⁺-linked efflux into to the extracellular space, thus decreasing the pHe while promoting intracellular alkalization in cancer cells undergoing aerobic glycolysis (Halestrap, 2013). Cancer cells exhibit an inverted pHe/pHi gradient relative to normal differentiated adult cells in which the pHi is lower that the pHe. Increasing the pHi can promote cell survival by limiting apoptosis, antagonizing pH-sensitive activation of apoptosis pathways that are mediated by cytosolic acidification (Webb et al., 2011). It also promotes cell proliferation by allowing cells to bypass cell-cycle checkpoints, thus boosting the rate of S phase entry (Webb et al., 2011). Extracellular acidification, on the other hand, is associated with promoting metastatic behaviors by affecting cell adhesion and remodeling of the extracellular matrix via activation of pH-dependent metallo-proteases (Stock and Schwab, 2009). Some of these promigratory behaviors are also enhanced by increased pHi, which can lead to cytoskeletal remodeling (Stock and Schwab, 2009; Webb et al., 2011).

Intracellular regulation of pH can also influence histone and non-histone protein acetylation. In cancer cells under Warburg metabolism, acidification of the nucleo-cytoplasm causes global histone deacetylation, whereas alkalization causes the opposite effect (McBrian et al., 2013). Lysine residues at neutral pH must be first deprotonated at the ε amino group by KAT enzymes for acetylation to occur. At a more alkaline pH, the lysine ε amino group is naturally deprotonated, thus favoring non-enzymatic modification by acetyl-CoA and other acyl-CoA metabolites (Choudhary et al., 2014; Narita et al., 2019). In vitro experiments demonstrated that histone lysine acetylation can occur non-enzymatically in the presence of acetyl-CoA in a reaction which is pH-dependent (Paik et al., 1970). Thus, a more alkaline pHi should create a more permissive environment promoting non-enzymatic lysine acetylation. High pHi and elevated concentrations of acetyl-CoA are common in the mitochondrial matrix and are sufficient for the generation of acetyl-lysine in mitochondrial proteins in an enzyme-independent manner (Wagner and Payne, 2013). Other types of acylations might also be subject to non-enzymatic catalysis, such as lysine succinylation (Wagner and Payne, 2013). Non-enzymatic acetylation is favored in lysine residues flanked by positively charged amino acids or proximal to cysteine residues, resulting in a relatively higher stoichiometry when compared to other lysine residues (Baeza et al., 2015; James et al., 2017, 2018). Non-histone proteins might be also subject to non-enzymatic lysine acetylation in special metabolic states, such as during high levels of aerobic glycolysis.

Specification of paraxial mesoderm during embryonic development depends on high rates of aerobic glycolysis at the posterior end of the elongating embryo, which leads to the glycolysis-dependent acetylation of β-Catenin and the transcription of mesodermal β-Catenin target genes (Oginuma et al., 2017, 2020). Interestingly, inversion of the pHe/pHi gradient seen during the Warburg effect is also recapitulated during aerobic glycolysis in mesodermal differentiation. Experimental manipulations causing cytosolic acidification reduce the levels of β-Catenin acetylation and the transcriptional activation of mesodermal genes. This consequently interferes with pH-dependent β-Catenin acetylation (Figure 2B) and negatively impacts the specification of mesodermal cell fate from neuromesodermal progenitors, which in turn opt for a neural fate (Oginuma et al., 2020). Furthermore, acetylation of β-Catenin can also be catalyzed non-enzymatically when incubated with acetyl-CoA, and its acetylation levels positively respond to increasing pH (Oginuma et al., 2020). Therefore, metabolically driven pHi regulation has the potential to dramatically affect cell fate trajectories by controlling the activation of cell identity programs via lysine acetylation of proteins that function as master regulators of cell differentiation.

Future Directions

Metabolic reprogramming can cause changes in PTM levels at a global proteome scale, yet it is not clear how cell/lineage-specific PTM profiles are regulated, especially in different developmental or pathological contexts. For instance, how are divergent transcriptional responses achieved when similar kinds of metabolic shifts (i.e., aerobic glycolysis to oxidative phosphorylation) are executed? Taking protein acetylation as a case study, it is reasonable to predict that the acquisition of specific protein acetylation profiles probably depends on the initial proteome expression of cells before engaging in a metabolic shift, which would affect the possible acetylation substrates as well as the balance of enzyme-mediated acetylation/deacetylation given the specific combination of KAT and KDAC expression. This could explain, for instance, whether one specific type of protein acetylation modification shows different stoichiometry when comparing cell type “A” to cell type “B” undergoing a similar metabolic shift. Additionally, different cell types will likely express a different combination of proteins containing acetyl-lysine binding domains (BRD, DPH, and YEATS) and/or non-acetylated lysine-rich binding domains (acidic domains) (Gong et al., 2016). This could potentially bring together specific protein complexes in one cell type, but not in others, in a context-dependent manner. In general, the unique starting conditions of different cells before undergoing a metabolic reprogramming could explain why similar kinds of metabolic shifts might affect the stoichiometry of an acetylated residue in one developmental context but not in others. Finally, although the state of protein acetylation is in large part controlled by the balance of KAT and KDAC activities, as well as by the intracellular pH of the cell, future studies should explore how acetylation is also controlled by other parameters, such as the crosstalk with other PTMs, which depend not only on the cell metabolic state but also the intracellular activity of specific signaling pathways, for instance, phosphorylation-dependent acetylation and vice versa (Matsuzaki et al., 2005; Narita et al., 2019). Thus, further studies of PTM crosstalk could shed some light on possible mechanisms that bring context dependence of protein acetylation in a cell-type-specific manner, including the crosstalk with multiple types of acylations, methylation, and ubiquitylation.