Salvia miltiorrhiza polysaccharide mitigates AFB1-induced liver injury in rabbits.

Aflatoxin B1 (AFB1) is one of the common dietary contaminants worldwide, which can harm the liver of humans and animals. Salvia miltiorrhiza polysaccharide (SMP) is a natural plant-derived polysaccharide with numerous pharmacological activities, including hepatoprotective properties. The purpose of this study is to explore the intervention effect of SMP on AFB1-induced liver injury and its underlying mechanisms in rabbits. The rabbits were administered AFB1 (25 µg/kg/feed) and or treatment with SMP (300, 600, 900 mg/kg/feed) for 42 days. The results showed that SMP effectively alleviated the negative impact of AFB1 on rabbits' productivity by increasing average daily weight gain (ADG) and feed conversion rate (FCR). SMP reduced aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels in serum, ameliorating AFB1-induced hepatic pathological changes. Additionally, SMP enhanced superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) activity, and inhibited reactive oxygen species (ROS), malondialdehyde (MDA), 4-Hydroxynonenal (4-HNE), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression, thus mitigating AFB1-induced oxidative stress and inflammatory responses. Moreover, SMP upregulated the expression of nuclear factor E2 related factor 2 (Nrf2), heme oxygenase 1 (HO-1), NADPH quinone oxidoreductase 1 (NQO1) and B-cell lymphoma 2 (Bcl2) while downregulating kelch like ECH associated protein 1 (Keap1), cytochrome c (cyt.c), caspase9, caspase3, and Bcl-2-associated X protein (Bax) expression, thereby inhibiting AFB1-induced hepatocyte apoptosis. Consequently, our findings conclude that SMP can mitigate AFB1-induced liver damage by activating the Nrf2/HO-1 pathway and inhibiting mitochondria-dependent apoptotic pathway in rabbits.

Potential of Tamarind Shell Extract against Oxidative Stress In Vivo and In Vitro.

Tamarind shell is rich in flavonoids and exhibits good biological activities. In this study, we aimed to analyze the chemical composition of tamarind shell extract (TSE), and to investigate antioxidant capacity of TSE in vitro and in vivo. The tamarind shells were extracted with 95% ethanol refluxing extraction, and chemical constituents were determined by ultra-performance chromatography-electrospray tandem mass spectrometry (UPLC-MS/MS). The free radical scavenging activity of TSE in vitro was evaluated using the oxygen radical absorbance capacity (ORAC) method. The antioxidative effects of TSE were further assessed in 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH)-stimulated ADTC5 cells and tert-butyl hydroperoxide (t-BHP)-exposed zebrafish. A total of eight flavonoids were detected in TSE, including (+)-catechin, taxifolin, myricetin, eriodictyol, luteolin, morin, apigenin, and naringenin, with the contents of 5.287, 8.419, 4.042, 6.583, 3.421, 4.651, 0.2027, and 0.6234 mg/g, respectively. The ORAC assay revealed TSE and these flavonoids had strong free radical scavenging activity in vitro. In addition, TSE significantly decreased the ROS and MDA levels but restored the SOD activity in AAPH-treated ATDC5 cells and t-BHP-exposed zebrafish. The flavonoids also showed excellent antioxidative activities against oxidative damage in ATDC5 cells and zebrafish. Overall, the study suggests the free radical scavenging capacity and antioxidant potential of TSE and its primary flavonoids in vitro and in vivo and will provide a theoretical basis for the development and utilization of tamarind shell.

New insights into the effects of caffeine on adult hippocampal neurogenesis in stressed mice: Inhibition of CORT-induced microglia activation.

Chronic stress-evoked depression has been implied to associate with the decline of adult hippocampal neurogenesis. Caffeine has been known to combat stress-evoked depression. Herein, we aim to investigate whether the protective effect of caffeine on depression is related with improving adult hippocampus neurogenesis and explore the mechanisms. Mouse chronic water immersion restraint stress (CWIRS) model, corticosterone (CORT)-established cell stress model, a coculture system containing CORT-treated BV-2 cells and hippocampal neural stem cells (NSCs) were utilized. Results showed that CWIRS caused obvious depressive-like disorders, abnormal 5-HT signaling, and elevated-plasma CORT levels. Notably, microglia activation-evoked brain inflammation and inhibited neurogenesis were also observed in the hippocampus of stressed mice. In comparison, intragastric administration of caffeine (10 and 20 mg/kg, 28 days) significantly reverted CWIRS-induced depressive behaviors, neurogenesis recession and microglia activation in the hippocampus. Further evidences from both in vivo and in vitro mechanistic experiments demonstrated that caffeine treatment significantly suppressed microglia activation via the A2AR/MEK/ERK/NF-κB signaling pathway. The results suggested that CORT-induced microglia activation contributes to stress-mediated neurogenesis recession. The antidepression effect of caffeine was associated with unlocking microglia activation-induced neurogenesis inhibition.

Narciclasine attenuates diet-induced obesity by promoting oxidative metabolism in skeletal muscle.

Obesity develops when caloric intake exceeds metabolic needs. Promoting energy expenditure represents an attractive approach in the prevention of this fast-spreading epidemic. Here, we report a novel pharmacological strategy in which a natural compound, narciclasine (ncls), attenuates diet-induced obesity (DIO) in mice by promoting energy expenditure. Moreover, ncls promotes fat clearance from peripheral metabolic tissues, improves blood metabolic parameters in DIO mice, and protects these mice from the loss of voluntary physical activity. Further investigation suggested that ncls achieves these beneficial effects by promoting a shift from glycolytic to oxidative muscle fibers in the DIO mice thereby enhancing mitochondrial respiration and fatty acid oxidation (FAO) in the skeletal muscle. Moreover, ncls strongly activates AMPK signaling specifically in the skeletal muscle. The beneficial effects of ncls treatment in fat clearance and AMPK activation were faithfully reproduced in vitro in cultured murine and human primary myotubes. Mechanistically, ncls increases cellular cAMP concentration and ADP/ATP ratio, which further lead to the activation of AMPK signaling. Blocking AMPK signaling through a specific inhibitor significantly reduces FAO in myotubes. Finally, ncls also enhances mitochondrial membrane potential and reduces the formation of reactive oxygen species in cultured myotubes.

Histone H3 lysine 56 methylation regulates DNA replication through its interaction with PCNA.

Histone modifications play important roles in regulating DNA-based biological processes. Of the modified sites, histone H3 lysine 56 (H3K56) is unique in that it lies within the globular core domain near the entry-exit sites of the nucleosomal DNA superhelix and its acetylation state in yeast is a marker for newly synthesized histones in transcription, DNA repair, and DNA replication. We now report the presence of H3K56 monomethylation (H3K56me1) in mammalian cells and find that the histone lysine methytransferase G9a/KMT1C is required for H3K56me1 both in vivo and in vitro. We also find that disruption of G9a or H3K56 impairs DNA replication. Furthermore, H3K56me1 associates with the replication processivity factor PCNA primarily in G1 phase of the cell cycle and, directly, in vitro. These results find H3K56me1 in mammals and indicate a role for H3K56me1 as a chromatin docking site for PCNA prior to its function in DNA replication.

Comparative Transcriptomic and Epigenomic Analyses Reveal New Regulators of Murine Brown Adipogenesis.

Increasing energy expenditure through brown adipocyte recruitment is a promising approach to combat obesity. We report here the comprehensive profiling of the epigenome and transcriptome throughout the lineage commitment and differentiation of C3H10T1/2 mesenchymal stem cell line into brown adipocytes. Through direct comparison to datasets from differentiating white adipocytes, we systematically identify stage- and lineage-specific coding genes, lncRNAs and microRNAs. Utilizing chromatin state maps, we also define stage- and lineage-specific enhancers, including super-enhancers, and their associated transcription factor binding motifs and genes. Through these analyses, we found that in brown adipocytes, brown lineage-specific genes are pre-marked by both H3K4me1 and H3K27me3, and the removal of H3K27me3 at the late stage is necessary but not sufficient to promote brown gene expression, while the pre-deposition of H3K4me1 plays an essential role in poising the brown genes for expression in mature brown cells. Moreover, we identify SOX13 as part of a p38 MAPK dependent transcriptional response mediating early brown cell lineage commitment. We also identify and subsequently validate PIM1, SIX1 and RREB1 as novel regulators promoting brown adipogenesis. Finally, we show that SIX1 binds to adipogenic and brown marker genes and interacts with C/EBPα, C/EBPß and EBF2, suggesting their functional cooperation during adipogenesis.

Epigenomic Control of Thermogenic Adipocyte Differentiation and Function.

Obesity and its associated metabolic disorders are spreading at a fast pace throughout the world; thus, effective therapeutic approaches are necessary to combat this epidemic. Obesity develops when there is a greater caloric intake than energy expenditure. Promoting energy expenditure has recently attracted much attention as a promising approach for the management of body weight. Thermogenic adipocytes are capable of burning fat to dissipate chemical energy into heat, thereby enhancing energy expenditure. After the recent re-discovery of thermogenic adipocytes in adult humans, much effort has focused on understanding the molecular mechanisms, especially the epigenetic mechanisms, which regulate thermogenic adipocyte development and function. A number of chromatin signatures, such as histone modifications, DNA methylation, chromatin accessibilities, and interactions, have been profiled at the genome level and analyzed in various murine and human thermogenic fat cell systems. Moreover, writers and erasers, as well as readers of the epigenome are also investigated using genomic tools in thermogenic adipocytes. In this review, we summarize and discuss the recent advance in these studies and highlight the insights gained into the epigenomic regulation of thermogenic program as well as the pathogenesis of human metabolic diseases.

Quantitative proteomics reveals a role for epigenetic reprogramming during human monocyte differentiation.

The differentiation of monocytes into macrophages and dendritic cells involves mechanisms for activation of the innate immune system in response to inflammatory stimuli, such as pathogen infection and environmental cues. Epigenetic reprogramming is thought to play an important role during monocyte differentiation. Complementary to cell surface markers, the characterization of monocytic cell lineages by mass spectrometry based protein/histone expression profiling opens a new avenue for studying immune cell differentiation. Here, we report the application of mass spectrometry and bioinformatics to identify changes in human monocytes during their differentiation into macrophages and dendritic cells. Our data show that linker histone H1 proteins are significantly down-regulated during monocyte differentiation. Although highly enriched H3K9-methyl/S10-phos/K14-acetyl tri-modification forms of histone H3 were identified in monocytes and macrophages, they were dramatically reduced in dendritic cells. In contrast, histone H4 K16 acetylation was found to be markedly higher in dendritic cells than in monocytes and macrophages. We also found that global hyperacetylation generated by the nonspecific histone deacetylase HDAC inhibitor Apicidin induces monocyte differentiation. Together, our data suggest that specific regulation of inter- and intra-histone modifications including H3 K9 methylation, H3 S10 phosphorylation, H3 K14 acetylation, and H4 K16 acetylation must occur in concert with chromatin remodeling by linker histones for cell cycle progression and differentiation of human myeloid cells into macrophages and dendritic cells.

TELP, a sensitive and versatile library construction method for next-generation sequencing.

Next-generation sequencing has been widely used for the genome-wide profiling of histone modifications, transcription factor binding and gene expression through chromatin immunoprecipitated DNA sequencing (ChIP-seq) and cDNA sequencing (RNA-seq). Here, we describe a versatile library construction method that can be applied to both ChIP-seq and RNA-seq on the widely used Illumina platforms. Standard methods for ChIP-seq library construction require nanograms of starting DNA, substantially limiting its application to rare cell types or limited clinical samples. By minimizing the DNA purification steps that cause major sample loss, our method achieved a high sensitivity in ChIP-seq library preparation. Using this method, we achieved the following: (i) generated high-quality epigenomic and transcription factor-binding maps using ChIP-seq for murine adipocytes; (ii) successfully prepared a ChIP-seq library from as little as 25 pg of starting DNA; (iii) achieved paired-end sequencing of the ChIP-seq libraries; (iv) systematically profiled gene expression dynamics during murine adipogenesis using RNA-seq and (v) preserved the strand specificity of the transcripts in RNA-seq. Given its sensitivity and versatility in both double-stranded and single-stranded DNA library construction, this method has wide applications in genomic, epigenomic, transcriptomic and interactomic studies.

Antialcohol and Hepatoprotective Effects of Tamarind Shell Extract on Ethanol-Induced Damage to HepG2 Cells and Animal Models.

Alcohol liver disease (ALD) is one of the leading outcomes of acute and chronic liver injury. Accumulative evidence has confirmed that oxidative stress is involved in the development of ALD. In this study, we used chick embryos to establish ALD model to study the hepatoprotective effects of tamarind shell exttract (TSE). Chick embryos received 25% ethanol (75 µL) and TSE (250, 500, 750 µg/egg/75 µL) from embryonic development day (EDD) 5.5. Both ethanol and TSE were administrated every two days until EDD15. Ethanol-exposed zebrafish and HepG2 cell model were also employed. The results suggested that TSE effectively reversed the pathological changes, liver dysfunction and ethanol-metabolic enzyme disorder in ethanol-treated chick embryo liver, zebrafish and HepG2 cells. TSE suppressed the excessive reactive oxygen species (ROS) in zebrafish and HepG2 cells, as well as rebuilt the irrupted mitochondrial membrane potential. Meanwhile, the declined antioxidative activity of glutathione peroxidase (GPx) and superoxide dismutase (SOD), together with the content of total glutathione (T-GSH) were recovered by TSE. Moreover, TSE upregulated nuclear factor erythroid 2-related factor 2 (NRF2) and heme oxyense-1 (HO-1) expression in protein and mRNA level. All the phenomena suggested that TSE attenuated ALD through activating NRF2 to repress the oxidative stress induced by ethanol.

Cyclo(-Phe-Phe) alleviates chick embryo liver injury via activating the Nrf2 pathway.

Excessive reactive oxygen species (ROS) accumulation is involved in the pathogenesis of liver fibrosis and damage, specifically in the developing embryo that is extremely sensitive to oxidative stress. Herein, a liver injury model in chick embryo was established by using 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH), which was used to investigate the effect of cyclo(-Phe-Phe) (CPP), a natural dipeptide found in foods and beverages. The results showed that CPP significantly alleviated AAPH-induced liver pathological damage, hepatic dysfunction and inhibited the excessive production of ROS in both chick embryo liver and HepG2 cells. Additionally, CPP increased the antioxidative activity of glutathione peroxidase (GPx) and superoxide dismutase (SOD), as well as elevated the level of glutathione (GSH), suggesting that CPP combating liver injury probably depends on its antioxidant capability. Mechanistically, CPP upregulated the mRNA and protein expression of heme oxyense-1 (HO-1) and NADPH quinone oxidoreductase 1 (NQO1) in vivo and in vitro, along with promoting the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) while inhibiting its degradation through binding with Kelch-like ECH-associated protein 1 (Keap1). In conclusion, this study proposes a potential peptide drug for the treatment of hepatic damage induced by oxidative stress and also unravels its mechanism of action.

Tripeptide Leu-Pro-Phe from Corn Protein Hydrolysates Attenuates Hyperglycemia-Induced Neural Tube Defect in Chicken Embryos.

Neural tube defect (NTD) is the most common and severe embryopathy causing embryonic malformation and even death associated with gestational diabetes mellitus (GDM). Leu-Pro-Phe (LPF) is an antioxidative tripeptide isolated from hydrolysates of corn protein. However, the biological activity of LPF in vivo and in vitro remains unclear. This study is aimed at investigating the protective effects of tripeptide LPF against NTD in the high glucose exposure condition and delineate the underlying biological mechanism. We found that LPF alleviated NTD in the high glucose-exposed chicken embryo model. In addition, DF-1 chicken embryo fibroblast was loaded with high glucose for induction of oxidative stress and abnormal O-GlcNAcylation in vitro. LPF significantly decreased accumulation of reactive oxygen species and content of malondialdehyde in DF-1 cells but increased the ratio of reduced glutathione and oxidized glutathione in chick embryo. Oxygen radical absorbance capacity results showed that LPF itself had good free radical scavenging capacity and could enhance antioxidant activity of the cell content. Mechanistic studies suggested that the resistance of LPF to oxidative damage may be related to promotion of NRF2 expression and nuclear translocation. LPF alleviated the overall O-GlcNAcylation level of cellular proteins under high glucose conditions and restored the level of Pax3 protein. Collectively, our findings indicate that LPF peptide could act as a nutritional supplement for the protection of development of embryonic neural tube affected by GDM.

Phospholipid peroxidation inhibits autophagy via stimulating the delipidation of oxidized LC3-PE.

Phospholipid peroxidation of polyunsaturated fatty acids at the bis-allylic position drives ferroptosis. Here we identify a novel role for phospholipid peroxidation in the inhibition of autophagy. Using in vitro and in vivo models, we report that phospholipid peroxidation induced by glutathione peroxidase-4 inhibition and arachidonate 15-lipoxygenase overexpression leads to overload of peroxidized phospholipids and culminate in inhibition of autophagy. Functional and lipidomics analysis further demonstrated that inhibition of autophagy was associated with an increase of peroxidized phosphatidylethanolamine (PE) conjugated LC3. We further demonstrate that autophagy inhibition occurred due to preferential cleavage of peroxidized LC3-PE by ATG4B to yield delipidated LC3. Mouse models of phospholipid peroxidation and autophagy additionally supported a role for peroxidized PE in autophagy inhibition. Our results agree with the recognized role of endoplasmic reticulum as the primary source for autophagosomal membranes. In summary, our studies demonstrated that phospholipid peroxidation inhibited autophagy via stimulating the ATG4B-mediated delipidation of peroxidized LC3-PE.

GASC1 promotes hepatocellular carcinoma progression by inhibiting the degradation of ROCK2.

Hepatocellular carcinoma (HCC) is a devastating malignancy without targeted therapeutic options. Our results indicated that the histone demethylase GASC1 signature is associated with later tumor stage and poorer survival in HCC patients. GASC1 depletion led to diminished HCC proliferation and tumor growth. A distinct heterogeneity in GASC1 levels was observed among HCC cell populations, predicting their inherent high or low tumor-initiating capacity. Mechanistically, GASC1 is involved in the regulation of several components of the Rho-GTPase signaling pathway including its downstream target ROCK2. GASC1 demethylase activity ensured the transcriptional repression of FBXO42, a ROCK2 protein-ubiquitin ligase, thereby inhibiting ROCK2 degradation via K63-linked poly-ubiquitination. Treatment with the GASC1 inhibitor SD70 impaired the growth of both HCC cell lines and xenografts in mice, sensitizing them to standard-of-care chemotherapy. This work identifies GASC1 as a malignant-cell-selective target in HCC, and GASC1-specific therapeutics represent promising candidates for new treatment options to control this malignancy.

Regulation of Thermogenic Adipocyte Differentiation and Adaptive Thermogenesis Through Histone Acetylation.

Over the past decade, the increasing prevalence of obesity and its associated metabolic disorders constitutes one of the most concerning healthcare issues for countries worldwide. In an effort to curb the increased mortality and morbidity derived from the obesity epidemic, various therapeutic strategies have been developed by researchers. In the recent years, advances in the field of adipocyte biology have revealed that the thermogenic adipose tissue holds great potential in ameliorating metabolic disorders. Additionally, epigenetic research has shed light on the effects of histone acetylation on adipogenesis and thermogenesis, thereby establishing the essential roles which histone acetyltransferases (HATs) and histone deacetylases (HDACs) play in metabolism and systemic energy homeostasis. In regard to the therapeutic potential of thermogenic adipocytes for the treatment of metabolic diseases, herein, we describe the current state of knowledge of the regulation of thermogenic adipocyte differentiation and adaptive thermogenesis through histone acetylation. Furthermore, we highlight how different HATs and HDACs maintain the epigenetic transcriptional network to mediate the pathogenesis of various metabolic comorbidities. Finally, we provide insights into recent advances of the potential therapeutic applications and development of HAT and HDAC inhibitors to alleviate these pathological conditions.

Regulation of white and brown adipocyte differentiation by RhoGAP DLC1.

Adipose tissues constitute an important component of metabolism, the dysfunction of which can cause obesity and type II diabetes. Here we show that differentiation of white and brown adipocytes requires Deleted in Liver Cancer 1 (DLC1), a Rho GTPase Activating Protein (RhoGAP) previously studied for its function in liver cancer. We identified Dlc1 as a super-enhancer associated gene in both white and brown adipocytes through analyzing the genome-wide binding profiles of PPARγ, the master regulator of adipogenesis. We further observed that Dlc1 expression increases during differentiation, and knockdown of Dlc1 by siRNA in white adipocytes reduces the formation of lipid droplets and the expression of fat marker genes. Moreover, knockdown of Dlc1 in brown adipocytes reduces expression of brown fat-specific genes and diminishes mitochondrial respiration. Dlc1-/- knockout mouse embryonic fibroblasts show a complete inability to differentiate into adipocytes, but this phenotype can be rescued by inhibitors of Rho-associated kinase (ROCK) and filamentous actin (F-actin), suggesting the involvement of Rho pathway in DLC1-regulated adipocyte differentiation. Furthermore, PPARγ binds to the promoter of Dlc1 gene to regulate its expression during both white and brown adipocyte differentiation. These results identify DLC1 as an activator of white and brown adipocyte differentiation, and provide a molecular link between PPARγ and Rho pathways.

miRNA-32 Drives Brown Fat Thermogenesis and Trans-activates Subcutaneous White Fat Browning in Mice.

Brown adipose tissue (BAT) activation and subcutaneous white fat browning are essential components of the thermogenic response to cold stimulus in mammals. microRNAs have been shown to regulate both processes in cis. Here, we identify miR-32 as a BAT-specific super-enhancer-associated miRNA in mice that is selectively expressed in BAT and further upregulated during cold exposure. Inhibiting miR-32 in vivo led to impaired cold tolerance, decreased BAT thermogenesis, and compromised white fat browning as a result of reduced serum FGF21 levels. Further examination showed that miR-32 directly represses its target gene Tob1, thereby activating p38 MAP kinase signaling to drive FGF21 expression and secretion from BAT. BAT-specific miR-32 overexpression led to increased BAT thermogenesis and serum FGF21 levels, which further promotes white fat browning in trans. Our results suggested miR-32 and Tob1 as modulators of FGF21 signaling that can be manipulated for therapeutic benefit against obesity and metabolic syndrome.

An Alternative Strategy for Pan-acetyl-lysine Antibody Generation.

Lysine acetylation is an important post-translational modification in cell signaling. In acetylome studies, a high-quality pan-acetyl-lysine antibody is key to successful enrichment of acetylated peptides for subsequent mass spectrometry analysis. Here we show an alternative method to generate polyclonal pan-acetyl-lysine antibodies using a synthesized random library of acetylated peptides as the antigen. Our antibodies are tested to be specific for acetyl-lysine peptides/proteins via ELISA and dot blot. When pooled, five of our antibodies show broad reactivity to acetyl-lysine peptides, complementing a commercial antibody in terms of peptide coverage. The consensus sequence of peptides bound by our antibody cocktail differs slightly from that of the commercial antibody. Lastly, our antibodies are tested in a proof-of-concept to analyze the acetylome of HEK293 cells. In total we identified 1557 acetylated peptides from 416 proteins. We thus demonstrated that our antibodies are well-qualified for acetylome studies and can complement existing commercial antibodies.

SIRT1 Interacts with and Deacetylates ATP6V1B2 in Mature Adipocytes.

SIRT1 plays a key role in maintaining metabolic homeostasis in mammals by directly modulating the activities of various transcription factors and metabolic enzymes through lysine deacetylation. White adipose tissue plays a key role in lipid storage and metabolism. To identify novel molecular targets of SIRT1 in fat cells, we used a non-biased proteomic approach. We identified a number of proteins whose acetylation status was significantly affected by SIRT1 modulator treatment in 3T3-L1 adipocytes. Among them, ATP6V1B2, a subunit of the vacuolar (H+)-ATPase, was further shown to be associated with SIRT1 by co-immunoprecipitation assay. Moreover, SIRT1 deacetylates ATP6V1B2 in vitro and in vivo. Taken together, our study demonstrates that ATP6V1B2 is a molecular target of SIRT1 in fat cells and the role of SIRT1 and ATP6V1B2 acetylation in the vacuolar (H+)-ATPase function warrants further investigation.

Histone H3 K27 acetylation marks a potent enhancer element on the adipogenic master regulator gene Pparg2.

PPARγ2 is expressed almost exclusively in adipose tissue and plays a central role in adipogenesis. Despite intensive studies over the last 2 decades, the mechanism regulating the expression of the Pparg2 gene, especially the role of cis-regulatory elements, is still not completely understood. Here, we report a comprehensive investigation of the enhancer elements within the murine Pparg2 gene. Utilizing the combined techniques of sequence conservation analysis and chromatin marker examination, we identified a potent enhancer element that augmented the expression of a reporter gene under the control of the Pparg2 promoter by 20-fold. This enhancer element was first identified as highly conserved non-coding sequence 10 (CNS10) and was later shown to be enriched with the enhancer marker H3 K27 acetylation. Further studies identified a binding site for p300 as the essential enhancer element in CNS10. Moreover, p300 physically binds to CNS10 and is required for the enhancer activity of CNS10. The depletion of p300 by siRNA resulted in significantly impaired activation of Pparg2 at the early stages of 3T3-L1 adipogenesis. In summary, our study identified a novel enhancer element on the murine Pparg2 gene and suggested a novel mechanism for the regulation of Pparg2 expression by p300 in 3T3-L1 adipogenesis.