H3K9 Demethylation Research Articles

The EZH2 inhibitor tazemetostat upregulates the expression of CCL17/TARC in B-cell lymphoma and enhances T-cell recruitment.

An inhibitor of the histone methyltransferase enhancer of zeste homologue 2 (EZH2), tazemetostat, has been developed for the treatment of B‐cell lymphoma, but its mechanisms of action are not fully elucidated. We screened for genes targeted by tazemetostat in eleven B‐cell lymphoma cell lines and found that tazemetostat significantly increased the expression of chemokine (C‐C motif) ligand 17 (CCL17)/thymus‐ and activation‐regulated chemokine (TARC) in all, which codes for a chemokine that is a hallmark of Hodgkin/Reed‐Sternberg (H/RS) cells in Hodgkin lymphoma. Notably, gene set enrichment analysis demonstrated a positive correlation between the genes upregulated by tazemetostat in five follicular lymphoma (FL) cell lines and those reported to be overexpressed in H/RS cells. The CCL17 promoter region was enriched in repressive histone modification H3K27me3, and tazemetostat induced H3K27 demethylation and activated gene transcription. CCL17 protein secretion was also induced by EZH2 inhibition, which was further enhanced by concurrent CpG stimulation. In vitro transwell migration assay demonstrated that CCL17 produced by tazemetostat‐treated B cells enhanced the recruitment of T cells, which had the potential to exert antilymphoma response. Analysis of publicly available human lymphoma databases showed that CCL17 gene expression was inversely correlated with the EZH2 activation signature and significantly paralleled the CD4+ and CD8+ T‐cell–rich signature in FL and germinal center B‐cell–like diffuse large B‐cell lymphoma. Our findings indicate that tazemetostat can potentially activate antilymphoma response by upregulating CCL17 expression in B‐cell lymphoma cells and promote T‐cell recruitment, which provides a rationale for its combination with immunotherapy.

Hairless regulates heterochromatin maintenance and muscle stem cell function as a histone demethylase antagonist

Skeletal muscle possesses remarkable regenerative ability because of the resident muscle stem cells (MuSCs). A prominent feature of quiescent MuSCs is a high content of heterochromatin. However, little is known about the mechanisms by which heterochromatin is maintained in MuSCs. By comparing gene-expression profiles from quiescent and activated MuSCs, we found that the mammalian Hairless (Hr) gene is expressed in quiescent MuSCs and rapidly down-regulated upon MuSC activation. Using a mouse model in which Hr can be specifically ablated in MuSCs, we demonstrate that Hr expression is critical for MuSC function and muscle regeneration. In MuSCs, loss of Hr results in reduced trimethylated Histone 3 Lysine 9 (H3K9me3) levels, reduced heterochromatin, increased susceptibility to genotoxic stress, and the accumulation of DNA damage. Deletion of Hr leads to an acceleration of the age-related decline in MuSC numbers. We have also demonstrated that despite the fact that Hr is homologous to a family of histone demethylases and binds to di- and trimethylated H3K9, the expression of Hr does not lead to H3K9 demethylation. In contrast, we show that the expression of Hr leads to the inhibition of the H3K9 demethylase Jmjd1a and an increase in H3K9 methylation. Taking these data together, our study has established that Hr is a H3K9 demethylase antagonist specifically expressed in quiescent MuSCs.

JMJ27-mediated histone H3K9 demethylation positively regulates drought-stress responses in Arabidopsis.

Dimethylation of histone H3 at lysine 9 (H3K9me2) is associated with heterochromatinization and transcriptional gene silencing in plants. The activation of sets of genes by drought stress is correlated with reduced H3K9me2 levels, but the role of H3K9 methylation in the regulation of drought-stress responses remains elusive. Here, we show that the Jumonji domain-containing H3K9 demethylase JMJ27 positively regulates drought-stress responses through its histone demethylase activity. RNA-seq analysis identified JMJ27-regulated genes, including positive regulators of drought stress GALACTINOL SYNTHASE 2 (GOLS2) and RESPONSE TO DESICCATION 20 (RD20). Genetic analysis demonstrated that JMJ27 positively regulates drought-stress responses at least partly through GOLS2 and RD20. JMJ27 directly associated with GOLS2 and RD20, and protected these loci from silencing by reducing H3K9me2 levels under normal conditions. REGULATORY PARTICLE NON-ATPASE 1a (RPN1a), a subunit of the 26S proteasome, interacted with JMJ27 and negatively regulated JMJ27 accumulation. Drought stress diminished RPN1a abundance, resulting in increased JMJ27 abundance. The drought stress-promoted occupancy of JMJ27 at GOLS2 and RD20 chromatin may reinforce their transcriptional induction by locally reducing the H3K9me2 levels. These results indicate that the RPN1a-JMJ27 module precisely regulates dynamic H3K9me2 deposition plasticity, ensuring proper adaptation to drought stress in Arabidopsis.

The rice histone methylation regulates hub species of the root microbiota

Plants have a close relationship with their root microbiota, which comprises a complex microbial network. Histone methylation is an important epigenetic modification influencing multiple plant traits; however, little is known about the role of plant histone methylation in the assembly and network structure of the root microbiota. In this study, we established that the rice (Oryza sativa) histone methylation regulates the structure and composition of the root microbiota, especially the hub species in the microbial network. DJ-jmj703 (defective in histone H3K4 demethylation) and ZH11-sdg714 (defective in H3K9 methylation) showed significant different root microbiota compared with the corresponding wild types at the phylum and family levels, with a consistent increase in the abundance of Betaproteobacteria and a decrease in the Firmicutes. In the root microbial network, 35 of 44 hub species in the top 10 modules in the tested field were regulated by at least one histone methylation-related gene. These observations establish that the rice histone methylation plays a pivotal role in regulating the assembly of the root microbiota, providing insights into the links between plant epigenetic regulation and root microbiota.

Removal of H3K27me3 by JMJ Proteins Controls Plant Development and Environmental Responses in Arabidopsis.

Trimethylation of histone H3 lysine 27 (H3K27me3) is a highly conserved repressive histone modification that signifies transcriptional repression in plants and animals. In Arabidopsis thaliana, the demethylation of H3K27 is regulated by a group of JUMONJI DOMAIN-CONTANING PROTEIN (JMJ) genes. Transcription of JMJ genes is spatiotemporally regulated during plant development and in response to the environment. Once JMJ genes are transcribed, recruitment of JMJs to target genes, followed by demethylation of H3K27, is critically important for the precise control of gene expression. JMJs function synergistically and antagonistically with transcription factors and/or other epigenetic regulators on chromatin. This review summarizes the latest advances in our understanding of Arabidopsis H3K27me3 demethylases that provide robust and flexible epigenetic regulation of gene expression to direct appropriate development and environmental responses in plants.

The H3K9 histone methyltransferase G9a modulates renal ischemia reperfusion injury by targeting Sirt1

Ischemia reperfusion (IR) injury dampens renal function and usually confers a great risk of renal failure. Aberrant expression of G9a, a H3K9 methyltransferase of mammalian histone, has been implicated as a driving event in various kidney diseases. However, the role of G9a plays in renal IR injury is required to be clarified. Herein, our results showed that renal IR injury resulted in a rapid elevation of G9a, accompanying the down-regulation of Sirt1, a deacetylase that has been reported to afford renoprotection. Genetic overexpression or therapeutic activation of Sirt1 efficiently ameliorated renal IR injury by elevating anti-oxidative genes expression and reducing the accumulation of reactive oxygen species, including O2·- and ·OH. In addition, inhibition of G9a activity by BIX01294 (BIX) alleviated IR injury through abolishing O2·- and ·OH levels in a Sirt1-dependent manner. Mechanistically, we observed that demethylated H3K9 was accumulated on the Sirt1 promoter in renal IR injury. Silencing or suppression of G9a activity erased H3K9me2 from Sirt1 promoter and normalized Sirt1 expression. Further exploration revealed that G9a interacted with chromobox homolog 1 (CBX1) to catalyze H3K9 de-methylation and formed a transcription repressor complex on the Sirt1 promoter, ultimately repressing Sirt1 transcription. In this study, we provided strong evidence that G9a modulated renal IR injury through cooperation with CBX1 to form a transcription repressor complex on the Sirt1 promoter and regulate O2·- and ·OH generation, indicating that G9a-Sirt1 axis might be a promising therapeutic target in an epigenetic manner.

H3K27 demethylases are dispensable for activation of Polycomb-regulated injury response genes in peripheral nerve

The induction of nerve injury response genes in Schwann cells depends on both transcriptional and epigenomic reprogramming. The nerve injury response program is regulated by the repressive histone mark H3K27 trimethylation (H3K27me3), deposited by Polycomb repressive complex 2 (PRC2). Loss of PRC2 function leads to early and augmented induction of the injury response gene network in peripheral nerves, suggesting H3K27 demethylases are required for derepression of Polycomb-regulated nerve injury genes. To determine the function of H3K27 demethylases in nerve injury, we generated Schwann cell–specific knockouts of H3K27 demethylase Kdm6b and double knockouts of Kdm6b/Kdm6a (encoding JMJD3 and UTX). We found that H3K27 demethylases are largely dispensable for Schwann cell development and myelination. In testing the function of H3K27 demethylases after injury, we found early induction of some nerve injury genes was diminished compared with control, but most injury genes were largely unaffected at 1 and 7 days post injury. Although it was proposed that H3K27 demethylases are required to activate expression of the cyclin-dependent kinase inhibitor Cdkn2a in response to injury, Schwann cell–specific deletion of H3K27 demethylases affected neither the expression of this gene nor Schwann cell proliferation after nerve injury. To further characterize the regulation of nerve injury response genes, we found that injury genes are associated with repressive histone H2AK119 ubiquitination catalyzed by PRC1, which declines after injury. Overall, our results indicate H3K27 demethylation is not required for induction of injury response genes and that other mechanisms likely are involved in activating Polycomb-repressed injury genes in peripheral nerve.

Polycomb-dependent differential chromatin compartmentalization determines gene coregulation in Arabidopsis.

In animals, distant H3K27me3-marked Polycomb targets can establish physical interactions forming repressive chromatin hubs. In plants, growing evidence suggests that H3K27me3 acts directly or indirectly to regulate chromatin interactions, although how this histone modification modulates 3D chromatin architecture remains elusive. To decipher the impact of the dynamic deposition of H3K27me3 on the Arabidopsis thaliana nuclear interactome, we combined genetics, transcriptomics, and several 3D epigenomic approaches. By analyzing mutants defective for histone H3K27 methylation or demethylation, we uncovered the crucial role of this chromatin mark in short- and previously unnoticed long-range chromatin loop formation. We found that a reduction in H3K27me3 levels led to a decrease in the interactions within Polycomb-associated repressive domains. Regions with lower H3K27me3 levels in the H3K27 methyltransferase clf mutant established new interactions with regions marked with H3K9ac, a histone modification associated with active transcription, indicating that a reduction in H3K27me3 levels induces a global reconfiguration of chromatin architecture. Altogether, our results reveal that the 3D genome organization is tightly linked to reversible histone modifications that govern chromatin interactions. Consequently, nuclear organization dynamics shapes the transcriptional reprogramming during plant development and places H3K27me3 as a key feature in the coregulation of distant genes.

NFAT5 Amplifies Antipathogen Responses by Enhancing Chromatin Accessibility, H3K27 Demethylation, and Transcription Factor Recruitment.

The ability of innate immune cells to respond to pathogen-associated molecular patterns across a wide range of intensities is fundamental to limit the spreading of infections. Studies on transcription responses to pathogen-activated TLRs have often used relatively high TLR ligand concentrations, and less is known about their regulation under mild stimulatory conditions. We had shown that the transcription factor NFAT5 facilitates expression of antipathogen genes under TLR stimulation conditions corresponding to low pathogen loads. In this study, we analyze how NFAT5 optimizes TLR-activated responses in mouse macrophages. We show that NFAT5 was required for effective recruitment of central effectors p65/NF-κB and c-Fos to specific proinflammatory target genes, such as Nos2, Il6, and Tnf in primary macrophages responding to low doses of the TLR4 ligand LPS. By contrast, NFAT5 was not required for p65/NF-κB recruitment in response to high LPS doses. Using the transposase-accessible chromatin with high-throughput sequencing assay, we show that NFAT5 facilitated chromatin accessibility mainly at promoter regions of multiple TLR4-responsive genes. Analysis of various histone marks that regulate gene expression in response to pathogens identified H3K27me3 demethylation as an early NFAT5-dependent mechanism that facilitates p65 recruitment to promoters of various TLR4-induced genes. Altogether, these results advance our understanding about specific mechanisms that optimize antipathogen responses to limit infections.

Histone demethylase complexes KDM3A and KDM3B cooperate with OCT4/SOX2 to define a pluripotency gene regulatory network.

The pluripotency gene regulatory network of porcine induced pluripotent stem cells(piPSCs), especially in epigenetics, remains elusive. To determine the biological function of epigenetics, we cultured piPSCs in different culture conditions. We found that activation of pluripotent gene- and pluripotency-related pathways requires the erasure of H3K9 methylation modification which was further influenced by mouse embryonic fibroblast (MEF) served feeder. By dissecting the dynamic change of H3K9 methylation during loss of pluripotency, we demonstrated that the H3K9 demethylases KDM3A and KDM3B regulated global H3K9me2/me3 level and that their co-depletion led to the collapse of the pluripotency gene regulatory network. Immunoprecipitation-mass spectrometry (IP-MS) provided evidence that KDM3A and KDM3B formed a complex to perform H3K9 demethylation. The genome-wide regulation analysis revealed that OCT4 (O) and SOX2 (S), the core pluripotency transcriptional activators, maintained the pluripotent state of piPSCs depending on the H3K9 hypomethylation. Further investigation revealed that O/S cooperating with histone demethylase complex containing KDM3A and KDM3B promoted pluripotency genes expression to maintain the pluripotent state of piPSCs. Together, these data offer a unique insight into the epigenetic pluripotency network of piPSCs.

The KDM4B\u2013CCAR1\u2013MED1 axis is a critical regulator of osteoclast differentiation and bone homeostasis

Bone undergoes a constant and continuous remodeling process that is tightly regulated by the coordinated and sequential actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Recent studies have shown that histone demethylases are implicated in osteoblastogenesis; however, little is known about the role of histone demethylases in osteoclast formation. Here, we identified KDM4B as an epigenetic regulator of osteoclast differentiation. Knockdown of KDM4B significantly blocked the formation of tartrate-resistant acid phosphatase-positive multinucleated cells. Mice with myeloid-specific conditional knockout of KDM4B showed an osteopetrotic phenotype due to osteoclast deficiency. Biochemical analysis revealed that KDM4B physically and functionally associates with CCAR1 and MED1 in a complex. Using genome-wide chromatin immunoprecipitation (ChIP)-sequencing, we revealed that the KDM4B–CCAR1–MED1 complex is localized to the promoters of several osteoclast-related genes upon receptor activator of NF-κB ligand stimulation. We demonstrated that the KDM4B–CCAR1–MED1 signaling axis induces changes in chromatin structure (euchromatinization) near the promoters of osteoclast-related genes through H3K9 demethylation, leading to NF-κB p65 recruitment via a direct interaction between KDM4B and p65. Finally, small molecule inhibition of KDM4B activity impeded bone loss in an ovariectomized mouse model. Taken together, our findings establish KDM4B as a critical regulator of osteoclastogenesis, providing a potential therapeutic target for osteoporosis.

Kdm2a deficiency in macrophages enhances thermogenesis to protect mice against HFD-induced obesity by enhancing H3K36me2 at the Pparg locus

Kdm2a catalyzes H3K36me2 demethylation to play an intriguing epigenetic regulatory role in cell proliferation, differentiation, and apoptosis. Herein we found that myeloid-specific knockout of Kdm2a (LysM-Cre-Kdm2af/f, Kdm2a−/−) promoted macrophage M2 program by reprograming metabolic homeostasis through enhancing fatty acid uptake and lipolysis. Kdm2a−/− increased H3K36me2 levels at the Pparg locus along with augmented chromatin accessibility and Stat6 recruitment, which rendered macrophages with preferential M2 polarization. Therefore, the Kdm2a−/− mice were highly protected from high-fat diet (HFD)-induced obesity, insulin resistance, and hepatic steatosis, and featured by the reduced accumulation of adipose tissue macrophages and repressed chronic inflammation following HFD challenge. Particularly, Kdm2a−/− macrophages provided a microenvironment in favor of thermogenesis. Upon HFD or cold challenge, the Kdm2a−/− mice manifested higher capacity for inducing adipose browning and beiging to promote energy expenditure. Collectively, our findings demonstrate the importance of Kdm2a-mediated H3K36 demethylation in orchestrating macrophage polarization, providing novel insight that targeting Kdm2a in macrophages could be a viable therapeutic approach against obesity and insulin resistance.

IOX1 Suppresses Wnt Target Gene Transcription and Colorectal Cancer Tumorigenesis through Inhibition of KDM3 Histone Demethylases.

Epigenetic activation of Wnt/β-catenin signaling plays a critical role in Wnt-induced tumorigenesis, notably in colorectal cancers. KDM3 and KDM4 histone demethylases have been reported to promote oncogenic Wnt signaling through demethylation of H3K9 on Wnt target gene promoters and are suggested to be potential therapeutic targets. However, potent inhibitors for these regulators are still not available. In addition, which family is most responsible for activation of Wnt target genes and Wnt-induced oncogenesis is not well documented, specifically in colorectal cancer. In this study, we characterized the functional redundancy and differences between KDM3 and KDM4 in regard to regulating Wnt signaling. Our data suggest that KDM3 may play a more essential role than KDM4 in regulating oncogenic Wnt signaling in human colorectal cancer. We also identified that IOX1, a known histone demethylase inhibitor, significantly suppresses Wnt target gene transcription and colorectal cancer tumorigenesis. Mechanistically, IOX1 inhibits the enzymatic activity of KDM3 by binding to the Jumonji C domain and thereby preventing the demethylation of H3K9 on Wnt target gene promoters. Taken together, our data not only identified the critical mechanisms by which IOX1 suppressed Wnt/β-catenin signaling and colorectal cancer tumorigenesis through inhibition of KDM3, but also suggested that IOX1 may represent an attractive small molecule lead for future drug design and discovery.

Vitamin C sensitizes triple negative breast cancer to PI3K inhibition therapy.

Rationale: The clinical use of PI3K inhibitors, such as buparlisib, has been plagued with toxicity at effective doses. The aim of this study is to determine if vitamin C, a potent epigenetic regulator, can improve the therapeutic outcome and reduce the dose of buparlisib in treating PIK3CA-mutated triple negative breast cancer (TNBC).Methods: The response of TNBC cells to buparlisib was assessed by EC50 measurements, apoptosis assay, clonogenic assay, and xenograft assay in mice. Molecular approaches including Western blot, immunofluorescence, RNA sequencing, and gene silencing were utilized as experimental tools.Results: Treatment with buparlisib at lower doses, along with vitamin C, induced apoptosis and inhibited the growth of TNBC cells in vitro. Vitamin C via oral delivery rendered a sub-therapeutic dose of buparlisib able to inhibit TNBC xenograft growth and to markedly block metastasis in mice. We discovered that buparlisib and vitamin C coordinately reduced histone H3K4 methylation by enhancing the nuclear translocation of demethylase, KDM5, and by serving as a cofactor to promote KDM5-mediated H3K4 demethylation. The expression of genes in the PI3K pathway, such as AKT2 and mTOR, was suppressed by vitamin C in a KDM5-dependent manner. Vitamin C and buparlisib cooperatively blocked AKT phosphorylation. Inhibition of KDM5 largely abolished the effect of vitamin C on the response of TNBC cells to buparlisib. Additionally, vitamin C and buparlisib co-treatment changed the expression of genes, including PCNA and FILIP1L, which are critical to cancer growth and metastasis.Conclusion: Vitamin C can be used to reduce the dosage of buparlisib needed to produce a therapeutic effect, which could potentially ease the dose-dependent side effects in patients.

NAD+ metabolism regulates preadipocyte differentiation by enhancing α-ketoglutarate-mediated histone H3K9 demethylation at the PPARγ promoter.

NAD⁺ metabolism regulates preadipocyte differentiation by enhancing α-ketoglutarate-mediated histone H3K9 demethylation at the PPARγ promoter.

Lipopolysaccharide-Induced H3K27 Demethylation Inhibits Activated Macrophage Apoptosis by Targeting Adora2a and C/EBPβ Expression in Acute Lung Injury

Lipopolysaccharide-Induced H3K27 Demethylation Inhibits Activated Macrophage Apoptosis by Targeting Adora2a and C/EBPβ Expression in Acute Lung Injury

Melatonin protects against oxybenzone-induced deterioration of mouse oocytes during maturation.

Oxybenzone (OBZ), an ultraviolet light filter that is widely used in sunscreens and cosmetics, is an emerging contaminant found in humans and the environment. Recent studies have shown that OBZ has been detected in women’s plasma, urine, and breast milk. However, the effects of OBZ exposure on oocyte meiosis have not been addressed. In this study, we investigated the detrimental effects of OBZ on oocyte maturation and the protective roles of melatonin (MT) in OBZ-exposed mouse models. Our in vitro and in vivo results showed that OBZ suppressed oocyte maturation, while MT attenuated the meiotic defects induced by OBZ. In addition, OBZ facilitated H3K4 demethylation by increasing the expression of the Kdm5 family of genes, elevating ROS levels, decreasing GSH, impairing mitochondrial quality, and disrupting spindle configuration in oocytes. However, MT treatment resulted in significant protection against OBZ-induced damage during oocyte maturation and improved oocyte quality. The mechanisms underlying the beneficial roles of MT involved reduction of oxidative stress, inhibition of apoptosis, restoration of abnormal spindle assembly and up-regulation of H3K4me3. Collectively, our results suggest that MT protects against defects induced by OBZ during mouse oocyte maturation in vitro and in vivo.

Suppression of canonical TGF-β signaling enables GATA4 to interact with H3K27me3 demethylase JMJD3 to promote cardiomyogenesis

Direct reprogramming of fibroblasts into cardiomyocytes (CMs) represents a promising strategy to regenerate CMs lost after ischemic heart injury. Overexpression of GATA4, HAND2, MEF2C, TBX5, miR-1, and miR-133 (GHMT2m) along with transforming growth factor beta (TGF-β) inhibition efficiently promote reprogramming. However, the mechanisms by which TGF-β blockade promotes cardiac reprogramming remain unknown. Here, we identify interactions between the histone H3 lysine 27 trimethylation (H3K27me3) demethylase JMJD3, the SWI/SNF remodeling complex subunit BRG1, and cardiac transcription factors. Furthermore, canonical TGF-β signaling regulates the interaction between GATA4 and JMJD3. TGF-β activation impairs the ability of GATA4 to bind target genes and prevents demethylation of H3K27 at cardiac gene promoters during cardiac reprogramming. Finally, a mutation in GATA4 (V267M) that is associated with congenital heart disease exhibits reduced binding to JMJD3 and impairs cardiomyogenesis. Thus, we have identified an epigenetic mechanism wherein canonical TGF-β pathway activation impairs cardiac gene programming, in part by interfering with GATA4-JMJD3 interactions.

RNA interference-independent reprogramming of DNA methylation in Arabidopsis.

DNA methylation is important for silencing transposable elements (TEs) in diverse eukaryotes, including plants. In plant genomes, TEs are silenced by methylation of histone H3 lysine 9 (H3K9) and cytosines in both CG and non-CG contexts. The role of RNA interference (RNAi) in establishing TE-specific silent marks has been extensively studied, but the importance of RNAi-independent pathways remains largely unexplored. Here, we directly investigated transgenerational de novo DNA methylation of TEs after the loss of silent marks. Our analyses uncovered potent and precise RNAi-independent pathways for recovering non-CG methylation and H3K9 methylation in most TE genes (that is, coding regions within TEs). Characterization of a subset of TE genes without the recovery revealed the effects of H3K9 demethylation, replacement of histone H2A variants and their interaction with CG methylation, together with feedback from transcription. These chromatin components are conserved among eukaryotes and may contribute to chromatin reprogramming in a conserved manner.

NAD+ Metabolism Regulates Preadipocyte Differentiation by Enhancing α-Ketoglutarate-Mediated Histone H3K9 Demethylation at the PPARγ Promoter.

Obesity has become a serious problem in public health worldwide, causing numerous metabolic diseases. Once the differentiation to mature adipocytes is disrupted, adipocyte hypertrophy and ectopic lipid accumulation leads to the inflammation in adipose tissue and systemic metabolic disorders. Intracellular metabolic state is known to change during cell differentiation and it affects the cell fate or the differentiation through epigenetic mechanism. Although the mechanism of preadipocyte differentiation has been well established, it is unknown how metabolic state changes and how it affects the differentiation in predipocyte differentiation. Nicotinamide adenine dinucleotide (NAD+) plays crucial roles in energy metabolism as a coenzyme in multiple redox reactions in major catabolic pathways and as a substrate of sirtuins or poly(ADP-ribose)polymerases. NAD+ is mainly synthesized from salvage pathway mediated by two enzymes, Nampt and Nmnat. The manipulation to NAD+ metabolism causes metabolic change in each tissue and changes in systemic metabolism. However, the role of NAD+ and Nampt in adipocyte differentiation remains unknown. In this study, we employed liquid chromatography-mass spectrometry (LC-MS)- and gas chromatography-mass spectrometry (GC-MS)-based targeted metabolomics to elucidate the metabolic reprogramming events that occur during 3T3-L1 preadipocyte differentiation. We found that the tricarboxylic acid (TCA) cycle was enhanced, which correlated with upregulated NAD+ synthesis. Additionally, increased alpha-ketoglutarate (αKG) contributed to histone H3K9 demethylation in the promoter region of PPARγ, leading to its transcriptional activation. Thus, we concluded that NAD+-centered metabolic reprogramming is necessary for the differentiation of 3T3-L1 preadipocytes.