Ribosome biogenesis is a crucial process requiring enormous transcriptional output. In budding yeast, the expression of 138 ribosomal protein (RP) genes and over 200 ribosome biogenesis (RiBi) genes is regulated by an intricate network of factors, including the nutrient-sensitive transcription activator Sfp1 and the NuA4 coactivator/acetyltransferase complex. Nutrient starvation or inhibition of target of rapamycin complex 1 by rapamycin leads to repression of RP and RiBi genes, in part through blocking Sfp1 nuclear localization and NuA4-dependent chromatin acetylation. Here, we demonstrate that Sfp1 physically interacts with NuA4 in a target of rapamycin complex 1-dependent manner. Our results indicate that Sfp1, along with NuA4, regulate the transcription of RiBi and RP genes via distinct mechanisms depending on promoter architectures. Sfp1 promotes histone acetylation at the promoters without affecting NuA4 recruitment. In contrast, NuA4 does impact Sfp1 binding but specifically at two classes of RP genes. Importantly, NuA4 acetylates Sfp1 at lysines 655 and 657, regulating its function. Cells expressing Sfp1 with acetyl-mimicking mutations exhibit increased expression of RiBi genes while RP genes remain stable. However, the same mutants lead to the loss of Sfp1 binding/activity at RiBi genes when cells are under non-optimal growth conditions. Mimicking constitutive acetylation of Sfp1 also limits the transcriptional burst of RP genes upon addition of glucose. Altogether, these results draw an intricate functional relationship between Sfp1 and NuA4 to control ribosome biogenesis, fine-tuning transcription output in different growth conditions.

Polyphosphates (polyP) are linear chains of inorganic phosphate (Pi) that are highly conserved among various organisms. In Escherichia coli, PolyP is synthesized primarily by polyphosphate kinase 1 (PPK1, encoded by ppk) in response to environmental stressors. PolyP contributes to bacterial survival by regulating biofilm formation, stress responses, and antibiotic resistance. The mechanisms controlling PolyP accumulation and its effects on bacterial physiology and resistance remain incompletely understood. Notably, PPK-mediated PolyP accumulation modulates lipopolysaccharide (LPS) structure, contributing to polymyxin resistance during starvation stress. Compared to the wild-type (WT) strain, Δppk mutants exhibit significant growth defects when transferred to MOPS minimal medium, whereas WT cells show minimal growth defects under this nutrient-starved condition. This differential phenotype serves as a useful readout for evaluating PPK inhibitors. Deletion of the low-affinity phosphate transporter PitA rescues the growth defect of Δppk mutants; this interaction is still under investigation. Different repurposed drugs act as PPK inhibitors, including mesalamine (widely used to treat inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis) and gallein (originally a histological dye and Gβγ inhibitor). Both compounds inhibit bacterial PPK enzymes. In this study, we evaluated the effects of mesalamine and gallein on the growth phenotypes of four E. coli strains (WT, Δppk, ΔpitA, and the double mutant ΔppkΔpitA) under nutrient-starved conditions (MOPS minimal medium). Both inhibitors produced only mild, non-specific growth impairment across strains, and the result was not improved by higher concentrations of drug use. Gallein treatment showed no clear effect on PolyP levels, and polyP levels following mesalamine treatment has not been evaluated. These preliminary results highlighted the need for optimized drug delivery or alternative inhibition strategies to fully recapitulate the phenotypes of genetic PolyP deficiency.

Many prokaryotic and eukaryotic cells store inorganic phosphate in the form of polymers called polyphosphate (polyP). There has been an explosion of interest in polyP over the past decade, in part due to newly suggested roles related to diverse aspects of human health. The physical interaction of polyP chains with specific proteins has been proposed to regulate cellular homeostasis and modulate signaling pathways in response to environmental changes. Recently, several studies have challenged existing models for how polyP interacts with its protein targets, while identifying new motifs that are capable of binding to polyP. In this review, we summarize these findings, delineate the functional implications for polyP-protein interactions at the molecular level, and define open questions that should be addressed to propel the field forward.

Lysine acetylation is a post-translational modification (PTM) that is traditionally studied as a modifier of histones. In recent years, nonhistone protein acetylation has also emerged as a ubiquitous modification in eukaryotes. Recent advances in mass spectrometry (MS) workflows suggest that a majority of proteins are acetylated at some point during their life cycle. However, only a few of these acetylations have been studied for their functional significance. Here, we review the function of acetylations on key nonhistone proteins involved in chromatin remodeling and DNA damage repair, protein homeostasis, and metabolic coordination of the cell cycle in Saccharomyces cerevisiae. We discuss the diverse roles of acetylation in regulating these pathways, while highlighting emerging themes and open questions in the field.

In many bacteria, polyphosphate kinase (PPK) enzymes use ATP to synthesize polyphosphate (polyP) in response to cellular stress. These chains of inorganic phosphates are joined by high-energy bonds and can reach hundreds of residues in length. PolyP plays diverse functions in helping bacteria adjust to changing environmental conditions. However, the molecular mechanisms underlying these functions are poorly understood. In eukaryotic cells, polyacidic serine- and lysine-rich (PASK) motifs of proteins can mediate binding to polyP chains. Whereas PASK motifs are relatively common in yeast and human cells, we report that these sequences are rare in bacteria commonly used for polyP research. Thus, to identify novel polyP-binding proteins in Escherichia coli, we carried out a screen and identified seven novel targets with links to translation control and ribosome biogenesis. For two targets, the GTPase activating protein YihI and the ribonuclease Rnr, we mapped the regions of polyP interaction to non-PASK sequences and identified lysine residues critical for binding. We found that deletion of rnr suppressed the slow-growth phenotype of Δppk mutants grown on minimal media. Conversely, ppk deletion resulted in decreased Rnr protein expression. These phenotypes were dependent on the polyP-binding region of Rnr but independent of polyP binding itself, suggesting a complex interplay between PPK and Rnr function in E. coli. Overall, our work provides new insights into the scope of polyP-binding proteins and extends the connections between polyP and the regulation of protein translation in E. coli. IMPORTANCE In bacteria, polyphosphate (polyP) molecules are important regulators of cellular stress responses. Accordingly, cells that cannot make polyP display defects in processes that are important for bacterial survival, infection, and antibiotic resistance. The molecular mechanisms by which polyP exerts its functions are poorly understood. In eukaryotic cells, there has been much interest in the identification and characterization of polyP-binding proteins that act as effectors of polyP in vivo. By comparison, much less is known about polyP-binding proteins in bacteria. In this study, we take advantage of large-scale collections of Escherichia coli strains expressing epitope-tagged proteins to carry out the first systematic search for bacterial polyP-binding proteins. We describe seven novel polyP-binding proteins with links to ribosome biogenesis or translation. We further identify a complex genetic and molecular interplay between polyphosphate kinase, the enzyme that makes polyP, and the polyP-binding protein RNase R. Given the importance of translational control for bacteria survival, investigation of these pathways is expected to reveal new targets that can be leveraged for therapeutic exploration.

Here, we report a novel role for the yeast lysine acetyltransferase NuA4 in regulating phospholipid availability for organelle morphology. Disruption of the NuA4 complex results in 70% of cells displaying nuclear deformations and nearly 50% of cells exhibiting vacuolar fragmentation. Cells deficient in NuA4 also show severe defects in the formation of nuclear-vacuole junctions (NJV), as well as a decrease in piecemeal microautophagy of the nucleus (PMN). To determine the cause of these defects we focused on Pah1, an enzyme that converts phosphatidic acid into diacylglycerol, favoring accumulation of lipid droplets over phospholipids that are used for membrane expansion. NuA4 subunit Eaf1 was required for Pah1 localization to the inner nuclear membrane and artificially tethering of Pah1 to the nuclear membrane rescued nuclear deformation and vacuole fragmentation defects, but not defects related to the formation of NVJs. Mutation of a NuA4-dependent acetylation site on Pah1 also resulted in aberrant Pah1 localization and defects in nuclear morphology and NVJ. Our work suggests a critical role for NuA4 in organelle morphology that is partially mediated through the regulation of Pah1 subcellular localization.

Polyphosphates (polyP) are chains of inorganic phosphates that can reach over 1,000 residues in length. InEscherichia coli, polyP is produced by the polyP kinase (PPK) and is thought to play a protective role during the response to cellular stress. However, the molecular pathways impacted by PPK activity and polyP accumulation remain poorly characterized. In this work, we used label-free mass spectrometry to study the response of bacteria that cannot produce polyP (Δppk) during starvation to identify novel pathways regulated by PPK. In response to starvation, we found 92 proteins significantly differentially expressed between wild-type and Δppkmutant cells. Wild-type cells were enriched for proteins related to amino acid biosynthesis and transport, while Δppkmutants were enriched for proteins related to translation and ribosome biogenesis, suggesting that without PPK, cells remain inappropriately primed for growth even in the absence of the required building blocks. From our data set, we were particularly interested in Arn and EptA proteins, which were down-regulated in Δppkmutants compared to wild-type controls, because they play a role in lipid A modifications linked to polymyxin resistance. Using western blotting, we confirm differential expression of these and related proteins in K-12 strains and a uropathogenic isolate, and provide evidence that this mis-regulation in Δppkcells stems from a failure to induce the BasRS two-component system during starvation. We also show that Δppkmutants unable to up-regulate Arn and EptA expression lack the respective L-Ara4N and pEtN modifications on lipid A. In line with this observation, loss ofppkrestores polymyxin sensitivity in resistant strains carrying a constitutively activebasRallele. Overall, we show a new role for PPK in lipid A modification during starvation and provide a rationale for targeting PPK to sensitize bacteria towards polymyxin treatment. We further anticipate that our proteomics work will provide an important resource for researchers interested in the diverse pathways impacted by PPK.

Ribosome biogenesis is a crucial process requiring enormous transcriptional output. In budding yeast, the expression of 138 ribosomal protein (RP) genes and over 200 ribosome biogenesis (RiBi) genes is regulated by and intricate network of factors, including the nutrient-sensitive transcription activator Sfp1 and the NuA4 coactivator/acetyltransferase complex. Nutrient starvation or inhibition of TORC1 by rapamycin leads to repression of RP and RiBi genes, in part through blocking Sfp1 nuclear localization and NuA4-dependent chromatin acetylation. Here, we demonstrate that Sfp1 physically interacts with NuA4 in a TORC1-independent manner. Our results indicate that Sfp1, along with NuA4, regulate transcription of RiBi and RP genes via distinct mechanisms depending on promoter architectures. Sfp1 promotes NuA4-dependent histone acetylation at the promoter of RiBi and RP genes without affecting NuA4 recruitment. In contrast, NuA4 does impact Sfp1 binding but specifically at two classes of RP genes. Importantly, we found that NuA4 acetylates Sfp1 at lysines 655 and lysine 657, regulating its function. Cells expressing Sfp1 with acetyl-mimicking mutations exhibit increased expression of RiBi genes while RP genes remain stable. However, the same mutants lead to the loss of Sfp1 binding/activity at RiBi genes when cells are under non-optimal growth conditions. Mimicking constitutive acetylation of Sfp1 also limits the transcriptional burst of RP genes upon addition of glucose. Altogether, these results draw an intricate functional relationship between Sfp1 and NuA4 to control ribosome biogenesis, fine-tuning transcription output in different growth conditions. ### Competing Interest Statement The authors have declared no competing interest.

Polyphosphate (polyP) is a chain of inorganic phosphate that is present in all domains of life and affects diverse cellular phenomena, ranging from blood clotting to cancer. A study by Azevedo et al. described a protein modification whereby polyP is attached to lysine residues within polyacidic serine and lysine (PASK) motifs via what the authors claimed to be covalent phosphoramidate bonding. This was based largely on the remarkable ability of the modification to survive extreme denaturing conditions. Our study demonstrates that lysine polyphosphorylation is non-covalent, based on its sensitivity to ionic strength and lysine protonation and absence of phosphoramidate bond formation, as analyzed via 31P NMR. Ionic interaction with lysine residues alone is sufficient for polyP modification, and we present a new list of non-PASK lysine repeat proteins that undergo polyP modification. This work clarifies the biochemistry of polyP-lysine modification, with important implications for both studying and modulating this phenomenon. This Matters Arising paper is in response to Azevedo et al. (2015), published in Molecular Cell. See also the Matters Arising Response by Azevedo et al. (2024), published in this issue.

Organs are composed of diverse cell types that change across space and time during development. To interrogate this diversity, we micro-dissected developing human lungs along the proximal-distal axis during the late pseudoglandular stage and generated an integrated analysis of single-nucleus sequencing and spatial transcriptomics, creating a cellularly-resolved atlas of the lung. These rich datasets revealed positional niches and cellular heterogeneity along the proximal-distal axis, including the identification of a unique population of TP63+ basal cells, the primary stem cell of the airway, marked by expression of LGR5 and LGR6 . Analysis of the LGR5+ basal cell niche and functional experiments with primary organoid models suggest a tonic level of WNT pathway activity in LGR5+ basal cells that is potentiated by mesenchyme-derived R-SPONDIN. We found that basal cell self-renewal is enhanced by WNT activity, suggesting that the WNT pathway plays a previously unappreciated but critical role in airway stem cell maintenance during human development. These results enhance our fundamental understanding of the positional and cellular heterogeneity in the developing human lungs and begin to reveal unique niches that maintain homeostasis throughout the lung. ### Competing Interest Statement The authors have declared no competing interest.

Using scRNA-seq and microscopy, we describe a cell that is enriched in the lower airways of the developing human lung and identified by the unique coexpression of SCGB3A2/SFTPB/CFTR. To functionally interrogate these cells, we apply a single-cell barcode-based lineage tracing method, called CellTagging, to track the fate of SCGB3A2/SFTPB/CFTR cells during airway organoid differentiation in vitro. Lineage tracing reveals that these cells have a distinct differentiation potential from basal cells, giving rise predominantly to pulmonary neuroendocrine cells and a subset of multiciliated cells distinguished by high C6 and low MUC16 expression. Lineage tracing results are supported by studies using organoids and isolated cells from the lower noncartilaginous airway. We conclude that SCGB3A2/SFTPB/CFTR cells are enriched in the lower airways of the developing human lung and contribute to the epithelial diversity and heterogeneity in this region.

ABSTRACTPolyphosphates (polyP) are chains of inorganic phosphates that can reach over 1000 residues in length. InEscherichia coli, polyP is produced by the polyP kinase (PPK) and is thought to play a protective role during the response to cellular stress. However, the molecular pathways impacted by PPK activity and polyP accumulation remain poorly characterized. In this work we used label-free mass spectrometry to study the response of bacteria that cannot produce polyP (Δppk) during starvation to identify novel pathways regulated by PPK.In response to starvation, we found 92 proteins significantly differentially expressed between wild-type and Δppkmutant cells. Wild-type cells were enriched for proteins related to amino acid biosynthesis and transport, whileΔppkmutants were enriched for proteins related to translation and ribosome biogenesis, suggesting that without PPK, cells remain inappropriately primed for growth even in the absence of required building blocks.From our dataset, we were particularly interested in Arn and EptA proteins, which were downregulated in Δppkmutants compared to wild-type controls, because they play a role in lipid A modifications linked to polymyxin resistance. Using western blotting, we confirm differential expression of these and related proteins, and provide evidence that this mis-regulation in Δppkcells stems from a failure to induce the BasS/BasR two-component system during starvation. We also show that Δppkmutants unable to upregulate Arn and EptA expression lack the respective L-Ara4N and pEtN modifications on lipid A. In line with this observation, loss ofppkrestores polymyxin sensitivity in resistant strains carrying a constitutively activebasRallele.Overall, we show a new role for PPK in lipid A modification during starvation and provide a rationale for targeting PPK to sensitize bacteria towards polymyxin treatment. We further anticipate that our proteomics work will provide an important resource for researchers interested in the diverse pathways impacted by PPK.

Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate that is present in nearly all organisms studied to date. A remarkable function of polyP involves its attachment to lysine residues via non-enzymatic post-translational modification (PTM), which is presumed to be covalent. Here, we show that proteins containing tracts of consecutive histidine residues exhibit a similar modification by polyP, which confers an electrophoretic mobility shift on NuPAGE gels. Our screen uncovers 30 human and yeast histidine repeat proteins that undergo histidine polyphosphate modification (HPM). This polyP modification is histidine dependent and non-covalent in nature, although remarkably it withstands harsh denaturing conditions-a hallmark of covalent PTMs. Importantly, we show that HPM disrupts phase separation and the phosphorylation activity of the human protein kinase DYRK1A, and inhibits the activity of the transcription factor MafB, highlighting HPM as a potential protein regulatory mechanism.

Polyphosphate (polyP) is a conserved polymer of inorganic phosphate residues that can reach thousands of moieties in length. PolyP has been implicated in cellular functions ranging from energy and phosphate homeostasis to cell signalling in eukaryotes from yeast to humans. Despite the interest in the role of polyP as a signalling molecule, the spatiotemporal regulation of polyP itself remains poorly understood. This knowledge gap limits our ability to understand how polyP impacts the physiology of normal and diseased cells and how this might be exploited in a therapeutic context. Polyphosphatases, enzymes that degrade polyP to generate shorter chains and free inorganic phosphate are ideally positioned to mediate polyP dynamics. However, little is known about how the activities of these enzymes are linked to specific cellular functions and how they might be regulated. Here, we provide an in‐depth overview of polyphosphatase enzymes in budding yeast, which has served as a workhorse for polyP research, and in mammalian cells where the enzymes that make and degrade polyP have remained elusive. We identify critical open questions in both systems and propose strategies to guide future work.

Polyphosphates (polyP) are chains of inorganic phosphates that can reach over 1000 residues in length. In Escherichia coli, polyP is produced by the polyP kinase (PPK) and is thought to play a protective role during the response to cellular stress. However, the molecular pathways impacted by PPK activity and polyP accumulation remain poorly characterized. In this work we used label-free mass spectrometry to study the response of bacteria that cannot produce polyP (∆ppk) during starvation to identify novel pathways regulated by PPK. In response to starvation, we found 92 proteins significantly differentially expressed between wild-type and ∆ppk mutant cells. Wild-type cells were enriched for proteins related to amino acid biosynthesis and transport, while Δppk mutants were enriched for proteins related to translation and ribosome biogenesis, suggesting that without PPK, cells remain inappropriately primed for growth even in the absence of required building blocks. From our dataset, we were particularly interested in Arn and EptA proteins, which were downregulated in ∆ppk mutants compared to wild-type controls, because they play a role in lipid A modifications linked to polymyxin resistance. Using western blotting, we confirm differential expression of these and related proteins, and provide evidence that this mis-regulation in ∆ppk cells stems from a failure to induce the BasS/BasR two-component system during starvation. We also show that ∆ppk mutants unable to upregulate Arn and EptA expression lack the respective L-Ara4N and pEtN modifications on lipid A. In line with this observation, loss of ppk restores polymyxin sensitivity in resistant strains carrying a constitutively active basR allele. Overall, we show a new role for PPK in lipid A modification during starvation and provide a rationale for targeting PPK to sensitize bacteria towards polymyxin treatment. We further anticipate that our proteomics work will provide an important resource for researchers interested in the diverse pathways impacted by PPK.

In diverse cells from bacterial to mammalian species, inorganic phosphate is stored in long chains called polyphosphate (polyP). These nearly universal polymers, ranging from three to thousands of phosphate moieties in length, are associated with molecular functions, including energy homeostasis, protein folding, and cell signaling. In many cell types, polyphosphate is concentrated in subcellular compartments or organelles. In the budding yeast Saccharomyces cerevisiae, polyP synthesis by the membrane-bound vacuolar transporter chaperone (VTC) complex is coupled to its translocation into the lumen of the vacuole, a lysosome-like organelle, where it is stored at high concentrations. In contrast, the ectopic expression of the bacterial polyphosphate kinase (PPK) results in the toxic accumulation of polyP outside the vacuole. In this study, we used label-free mass spectrometry to investigate the mechanisms underlying this toxicity. We find that PPK expression results in the activation of a stress response mediated in part by the Hog1 and Yak1 kinases and the Msn2/Msn4 transcription factors as well as by changes in protein kinase A (PKA) activity. This response is countered by the combined action of the Ddp1 and Ppx1 polyphosphatases that function together to counter polyP accumulation and downstream toxicity. In contrast, the ectopic expression of previously proposed mammalian polyphosphatases did not impact PPK-mediated toxicity in this model, suggesting either that these enzymes do not function directly as polyphosphatases in vivo or that they require cofactors unique to higher eukaryotes. Our work provides insight into why polyP accumulation outside lysosome-like organelles is toxic. Furthermore, it serves as a resource for exploring how polyP may impact conserved biological processes at a molecular level. IMPORTANCE Cells from bacteria to humans have a molecule called polyphosphate (polyP) that functions in diverse processes. In many microbes, polyP is sequestered in granules or lysosome-related organelles such as vacuoles. In this study, we use an ectopic expression system to force budding yeast to accumulate polyP outside the vacuole. We use proteomics to demonstrate that this nonvacuolar polyP initiates a stress response mediated by a signaling cascade involving the Yak1 and Hog1 kinases and the Msn2 and Msn4 transcription factors. This response is countered by a pair of polyphosphatases with different enzymatic activities that function in concert to degrade polyP. Our results provide new insights into why polyP is confined to specific cell locations in many microbial cells.

DNA damage response mechanisms have meiotic roles that ensure successful gamete formation. While completion of meiotic double-strand break (DSB) repair requires the canonical RAD9A-RAD1-HUS1 (9A-1-1) complex, mammalian meiocytes also express RAD9A and HUS1 paralogs, RAD9B and HUS1B, predicted to form alternative 9-1-1 complexes. The RAD1 subunit is shared by all predicted 9-1-1 complexes and localizes to meiotic chromosomes even in the absence of HUS1 and RAD9A. Here, we report that testis-specific disruption of RAD1 in mice resulted in impaired DSB repair, germ cell depletion, and infertility. Unlike Hus1 or Rad9a disruption, Rad1 loss in meiocytes also caused severe defects in homolog synapsis, impaired phosphorylation of ATR targets such as H2AX, CHK1, and HORMAD2, and compromised meiotic sex chromosome inactivation. Together, these results establish critical roles for both canonical and alternative 9-1-1 complexes in meiotic ATR activation and successful prophase I completion.

After lung injury, damage-associated transient progenitors (DATPs) emerge, representing a transitional state between injured epithelial cells and newly regenerated alveoli. DATPs express profibrotic genes, suggesting that they might promote idiopathic pulmonary fibrosis (IPF). However, the molecular pathways that induce and/or maintain DATPs are incompletely understood. Here we show that the bifunctional kinase/RNase—IRE1α—a central mediator of the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress is a critical promoter of DATP abundance and function. Administration of a nanomolar-potent, monoselective kinase inhibitor of IRE1α (KIRA8)—or conditional epithelial IRE1α gene knockout—both reduce DATP cell number and fibrosis in the bleomycin model, indicating that IRE1α cell-autonomously promotes transition into the DATP state. IRE1α enhances the profibrotic phenotype of DATPs since KIRA8 decreases expression of integrin αvβ6, a key activator of transforming growth factor β (TGF-β) in pulmonary fibrosis, corresponding to decreased TGF-β-induced gene expression in the epithelium and decreased collagen accumulation around DATPs. Furthermore, IRE1α regulates DNA damage response (DDR) signaling, previously shown to promote the DATP phenotype, as IRE1α loss-of-function decreases H2AX phosphorylation, Cdkn1a (p21) expression, and DDR-associated secretory gene expression. Finally, KIRA8 treatment increases the differentiation of Krt19 CreERT2 -lineage-traced DATPs into type 1 alveolar epithelial cells after bleomycin injury, indicating that relief from IRE1α signaling enables DATPs to exit the transitional state. Thus, IRE1α coordinates a network of stress pathways that conspire to entrap injured cells in the DATP state. Pharmacological blockade of IRE1α signaling helps resolve the DATP state, thereby ameliorating fibrosis and promoting salutary lung regeneration.

General control nonderepressible 5 protein (Gcn5) and its homologs, including p300/CBP-associated factor (PCAF), are lysine acetyltransferases that modify both histone and non-histone proteins using acetyl coenzyme A as a donor substrate. While decades of studies have uncovered a vast network of cellular processes impacted by these acetyltransferases, including gene transcription and metabolism, far less is known about how these enzymes are themselves regulated. In this review, we summarize the type and functions of posttranslational modifications proposed to control Gcn5 in both yeast and human cells. We further outline common themes, open questions, and strategies to guide future work.

Objective: Aberrant ketogenesis is correlated with the degree of steatosis in non-alcoholic fatty liver disease (NAFLD) patients, and an inborn error of ketogenesis (mitochondrial HMG-CoA synthase deficiency) is commonly associated with the development of the fatty liver. Here we aimed to determine the impact of Hmgcs2-mediated ketogenesis and its modulations on the development and treatment of fatty liver disease. Methods: Loss- and gain-of-ketogenic function models, achieved by Hmgcs2 knockout and overexpression, respectively, were utilized to investigate the role of ketogenesis in the hepatic lipid accumulation during postnatal development and in a high-fat diet-induced NAFLD mouse model. Results: Ketogenic function was decreased in NAFLD mice with a reduction in Hmgcs2 expression. Mice lacking Hmgcs2 developed spontaneous fatty liver phenotype during postnatal development, which was rescued by a shift to a low-fat dietary composition via early weaning. Hmgcs2 heterozygous adult mice, which exhibited lower ketogenic activity, were more susceptible to diet-induced NAFLD development, whereas HMGCS2 overexpression in NAFLD mice improved hepatosteatosis and glucose homeostasis. Conclusions: Our study adds new knowledge to the field of ketone body metabolism and shows that Hmgcs2-mediated ketogenesis modulates hepatic lipid regulation under a fat-enriched nutritional environment. The regulation of hepatic ketogenesis may be a viable therapeutic strategy in the prevention and treatment of hepatosteatosis.