Polo-like Kinase-1 Regulates Myc Stabilization and Activates a Feedforward Circuit Promoting Tumor Cell Survival.

MYCN amplification in human cancers predicts poor prognosis and resistance to therapy. However, pharmacological strategies that directly target N-Myc, the protein encoded by MYCN, remain elusive. Here, we identify a molecular mechanism responsible for reciprocal activation between Polo-like kinase-1 (PLK1) and N-Myc. PLK1 specifically binds to the SCFFbw7 ubiquitin ligase, phosphorylates it, and promotes its autopolyubiquitination and proteasomal degradation, counteracting Fbw7-mediated degradation of N-Myc and additional substrates, including cyclin E and Mcl1. Stabilized N-Myc in turn directly activates PLK1 transcription, constituting a positive feedforward regulatory loop that reinforces Myc-regulated oncogenic programs. Inhibitors of PLK1 preferentially induce potent apoptosis of MYCN-amplified tumor cells from neuroblastoma and small cell lung cancer and synergistically potentiate the therapeutic efficacies of Bcl2 antagonists. These findings reveal a PLK1-Fbw7-Myc signaling circuit that underlies tumorigenesis and validate PLK1 inhibitors, alone or with Bcl2 antagonists, as potential effective therapeutics for MYC-overexpressing cancers.

O-GlcNAcylation of TDP-43 suppresses proteinopathies and promotes TDP-43's mRNA splicing activity.

Pathological TDP-43 aggregation is characteristic of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP); however, how TDP-43 aggregation and function are regulated remain poorly understood. Here, we show that O-GlcNAc transferase OGT-mediated O-GlcNAcylation of TDP-43 suppresses ALS-associated proteinopathies and promotes TDP-43's splicing function. Biochemical and cell-based assays indicate that OGT's catalytic activity suppresses TDP-43 aggregation and hyperphosphorylation, whereas abolishment of TDP-43 O-GlcNAcylation impairs its RNA splicing activity. We further show that TDP-43 mutations in the O-GlcNAcylation sites improve locomotion defects of larvae and adult flies and extend adult life spans, following TDP-43 overexpression in Drosophila motor neurons. We finally demonstrate that O-GlcNAcylation of TDP-43 promotes proper splicing of many mRNAs, including STMN2, which is required for normal axonal outgrowth and regeneration. Our findings suggest that O-GlcNAcylation might be a target for the treatment of TDP-43-linked pathogenesis.

Malting barley carbon dots-mediated oxidative stress promotes insulin resistance in mice via NF-κB pathway and MAPK cascade.

BACKGROUND: Food-borne carbon dots (CDs) are widely generated during food processing and are inevitably ingested by humans causing toxicity. However, the toxic effects of food-borne CDs on the blood glucose metabolism are unknown. RESULTS: In this study, we brewed beer via a representative strategy and extracted the melting-barley CDs (MBCDs) to explore the toxic effects on blood glucose in mice. We found the accumulation of fluorescent labeled MBCDs in various organs and oral administration of MBCDs can cause visceral toxicity, manifested as liver damage. Mice were orally administered MBCDs (5 and 25 mg/kg) for 16 weeks, and increased levels of fasting blood glucose were observed in both MBCDs-treated groups. Transcriptomic analyses revealed that MBCDs activate oxidative stress, inflammatory responses, the MAPK cascade, and PI3K/Akt signaling in mice livers. Mechanistically, MBCDs exposure-induced reactive oxygen species (ROS) overproduction activates the nuclear factor-κB (NF-κB) signaling pathway and MAPK cascade, thereby promoting phosphorylated insulin receptor substrate (IRS)-1 at Ser307 and inducing insulin resistance (IR). Meanwhile, the IR promoted gluconeogenesis, which enhanced MBCDs-induced hyperglycemia of mice. Importantly, inhibition of the ROS significantly attenuated the MBCDs-induced inflammatory response and MAPK cascade, thereby alleviating IR and hyperglycemia in mice. CONCLUSION: In summary, this study revealed that MBCDs promote ROS overproduction and thus induced IR, resulting in imbalance of glucose homeostasis in mice. More importantly, this study was further assessed to reveal an imperative emphasis on the reevaluation of dietary and environmental CDs exposure, and has important implications for T2DM prevention research.

Rph1 coordinates transcription of ribosomal protein genes and ribosomal RNAs to control cell growth under nutrient stress conditions.

Coordinated regulation of ribosomal RNA (rRNA) synthesis and ribosomal protein gene (RPG) transcription by eukaryotic RNA polymerases (RNAP) is a key requirement for growth control. Although evidence for balance between RNPI-dependent 35S rRNA production and RNAPII-mediated RPG transcription have been described, the molecular basis is still obscure. Here, we found that Rph1 modulates the transcription status of both rRNAs and RPGs in yeast. We show that Rph1 widely associates with RNAPI and RNAPII-transcribed genes. Deletion of RPH1 remarkably alleviates cell slow growth caused by TORC1 inhibition via derepression of rRNA and RPG transcription under nutrient stress conditions. Mechanistically, Rim15 kinase phosphorylates Rph1 upon rapamycin treatment. Phosphorylation-mimetic mutant of Rph1 exhibited more resistance to rapamycin treatment, decreased association with ribosome-related genes, and faster cell growth compared to the wild-type, indicating that Rph1 dissociation from chromatin ensures cell survival upon nutrient stress. Our results uncover the role of Rph1 in coordination of RNA polymerases-mediated transcription to control cell growth under nutrient stress conditions.

Gcn5-mediated Rph1 acetylation regulates its autophagic degradation under DNA damage stress.

Histone modifiers regulate proper cellular activities in response to various environmental stress by modulating gene expression. In budding yeast, Rph1 transcriptionally represses many DNA damage or autophagy-related gene expression. However, little is known how Rph1 is regulated during these stress conditions. Here, we report that Rph1 is degraded upon DNA damage stress conditions. Notably, this degradation occurs via the autophagy pathway rather than through 26S proteasome proteolysis. Deletion of ATG genes or inhibition of vacuole protease activity compromises Rph1 turnover. We also determine that Rph1 and nuclear export protein Crm1 interact, which is required for Rph1 translocation from the nucleus to the cytoplasm. More importantly, Gcn5 directly acetylates Rph1 in vitro and in vivo, and Gcn5-containing complex, SAGA, is required for autophagic degradation of Rph1. Gcn5-mediated Rph1 acetylation is essential for the association of Rph1 with the nuclear pore protein Nup1. Finally, we show that sustaining high levels of Rph1 during DNA damage stress results in cell growth defects. Thus, we propose that Gcn5-mediated acetylation finely regulates Rph1 protein level and that autophagic degradation of Rph1 is important for cell homeostasis. Our findings may provide a general connection between DNA damage, protein acetylation and autophagy.

Cell cycle-dependent degradation of the methyltransferase SETD3 attenuates cell proliferation and liver tumorigenesis.

Histone modifications, including lysine methylation, are epigenetic marks that influence many biological pathways. Accordingly, many methyltransferases have critical roles in various biological processes, and their dysregulation is often associated with cancer. However, the biological functions and regulation of many methyltransferases are unclear. Here, we report that a human homolog of the methyltransferase SET (SU(var), enhancer of zeste, and trithorax) domain containing 3 (SETD3) is cell cycle-regulated; SETD3 protein levels peaked in S phase and were lowest in M phase. We found that the ß-isoform of the tumor suppressor F-box and WD repeat domain containing 7 (FBXW7ß) specifically mediates SETD3 degradation. Aligning the SETD3 sequence with those of well known FBXW7 substrates, we identified six potential non-canonical Cdc4 phosphodegrons (CPDs), and one of them, CPD1, is primarily phosphorylated by the kinase glycogen synthase kinase 3 (GSK3ß), which is required for FBXW7ß-mediated recognition and degradation. Moreover, depletion or inhibition of GSK3ß or FBXW7ß resulted in elevated SETD3 levels. Mutations of the phosphorylated residues in CPD1 of SETD3 abolished the interaction between FBXW7ß and SETD3 and prevented SETD3 degradation. Our data further indicated that SETD3 levels positively correlated with cell proliferation of liver cancer cells and liver tumorigenesis in a xenograft mouse model, and that overexpression of FBXW7ß counteracts the SETD3's tumorigenic role. We also show that SETD3 levels correlate with cancer malignancy, indicated by SETD3 levels that the 54 liver tumors are 2-fold higher than those in the relevant adjacent tissues. Collectively, these data elucidated that a GSK3ß-FBXW7ß-dependent mechanism controls SETD3 protein levels during the cell cycle and attenuates its oncogenic role in liver tumorigenesis.

Pathological concentration of zinc dramatically accelerates abnormal aggregation of full-length human Tau and thereby significantly increases Tau toxicity in neuronal cells.

A pathological hallmark of Alzheimer disease and other tauopathies is the formation of neurofibrillary tangles mainly composed of bundles of fibrils formed by microtubule-associated protein Tau. Here we study the effects of Zn2+ on abnormal aggregation and cytotoxicity of a pathological mutant ΔK280 of full-length human Tau. As revealed by Congo red binding assays, transmission electron microscopy, immunofluorescence, Western blot, and immunogold electron microscopy, pathological concentration of Zn2+ dramatically accelerates the fibrillization of ΔK280 both in vitro and in SH-SY5Y neuroblastoma cells. As evidenced by annexin V-FITC apoptosis detection assay and MTT reduction assay, pathological concentration of Zn2+ remarkably enhances ΔK280 fibrillization-induced apoptosis and toxicity in SH-SY5Y cells. Substitution of Cys-291 and Cys-322 with Ala, however, essentially eliminates such enhancing effects of Zn2+ on the fibrillization and the consequent cytotoxicity of ΔK280. Furthermore, Zn2+ is co-localized with and highly enriched in amyloid fibrils formed by ΔK280 in SH-SY5Y cells. The results from isothermal titration calorimetry show that Zn2+ binds to full-length human Tau by interacting with Cys-291 and Cys-322, forming a 1:1 Zn2+-Tau complex. Our data demonstrate that zinc dramatically accelerates abnormal aggregation of human Tau and significantly increases Tau toxicity in neuronal cells mainly via bridging Cys-291 and Cys-322. Our findings could explain how pathological zinc regulates Tau aggregation and toxicity associated with Alzheimer disease.

Polyubiquitination of the demethylase Jhd2 controls histone methylation and gene expression.

The identification of histone methyltransferases and demethylases has uncovered a dynamic methylation system needed to modulate appropriate levels of gene expression. Gene expression levels of various histone demethylases, such as the JARID1 family, show distinct patterns of embryonic and adult expression and respond to different environmental cues, suggesting that histone demethylase protein levels must be tightly regulated for proper development. In our study, we show that the protein level of the yeast histone H3 Lys 4 (H3 K4) demethylase Jhd2/Kdm5 is modulated through polyubiquitination by the E3 ubiquitin ligase Not4 and turnover by the proteasome. We determine that polyubiquitin-mediated degradation of Jhd2 controls in vivo H3 K4 trimethylation and gene expression levels. Finally, we show that human NOT4 can polyubiquitinate human JARID1C/SMCX, a homolog of Jhd2, suggesting that this is likely a conserved mechanism. We propose that Not4 is an E3 ubiquitin ligase that monitors and controls a precise amount of Jhd2 protein so that the proper balance between histone demethylase and histone methyltransferase activities occur in the cell, ensuring appropriate levels of H3 K4 trimethylation and gene expression.

Construction of a series of pCS2+ backbone-based Gateway vectors for overexpressing various tagged proteins in vertebrates.

Gateway vectors have been extensively developed to facilitate gene cloning in numerous species; however, a universal system that is compatible for multiple organisms was lacking. As a multipurpose expression vector, pCS2+ backbone-based expression plasmids are widely used for high-level expression of messenger RNAs (mRNAs) or proteins in mammalian/avian culture cells or Xenopus/zebrafish embryos. To date, a suite of vectors with pCS2+ backbone applicable for Gateway cloning system were unavailable yet. Here, we generated a set of Gateway destination vectors, named as pGCS (plasmids of Gateway in pCS2+) vectors, which can be fused to a choice of frequently used amino- or carboxyl-terminal tags, including MYC, HA, FLAG, His, GST, as well as eGFP fluorescent epitope. The systematic generation of this set of pCS2+ backbone-based Gateway destination vectors allows for in vitro recombination of DNA with high speed, accuracy, and reliability compared with the traditional 'digestion-ligation' cloning approach. Thus, our system accelerates the production of functional proteins, which could be widely expressed in a large variety of vertebrate organisms without tediously transferring genes into different expression vectors. Moreover, we make this series of Gateway vectors available to the research community via the non-profit Addgene Plasmid Repository.

Di- and tri-methylation of histone H3K36 play distinct roles in DNA double-strand break repair.

Histone H3 Lys36 (H3K36) methylation and its associated modifiers are crucial for DNA double-strand break (DSB) repair, but the mechanism governing whether and how different H3K36 methylation forms impact repair pathways is unclear. Here, we unveil the distinct roles of H3K36 dimethylation (H3K36me2) and H3K36 trimethylation (H3K36me3) in DSB repair via non-homologous end joining (NHEJ) or homologous recombination (HR). Yeast cells lacking H3K36me2 or H3K36me3 exhibit reduced NHEJ or HR efficiency. yKu70 and Rfa1 bind H3K36me2- or H3K36me3-modified peptides and chromatin, respectively. Disrupting these interactions impairs yKu70 and Rfa1 recruitment to damaged H3K36me2- or H3K36me3-rich loci, increasing DNA damage sensitivity and decreasing repair efficiency. Conversely, H3K36me2-enriched intergenic regions and H3K36me3-enriched gene bodies independently recruit yKu70 or Rfa1 under DSB stress. Importantly, human KU70 and RPA1, the homologs of yKu70 and Rfa1, exclusively associate with H3K36me2 and H3K36me3 in a conserved manner. These findings provide valuable insights into how H3K36me2 and H3K36me3 regulate distinct DSB repair pathways, highlighting H3K36 methylation as a critical element in the choice of DSB repair pathway.

Polystyrene Nanoplastics Induce Lipid Metabolism Disorder by Activating the PERK-ATF4 Signaling Pathway in Mice.

In recent years, the study of microplastics (MPs) and nanoplastics (NPs) and their effects on human health has gained significant attention. The impacts of NPs on lipid metabolism and the specific mechanisms involved remain poorly understood. To address this, we utilized high-throughput sequencing and molecular biology techniques to investigate how endoplasmic reticulum (ER) stress might affect hepatic lipid metabolism in the presence of polystyrene nanoplastics (PS-NPs). Our findings suggest that PS-NPs activate the PERK-ATF4 signaling pathway, which in turn upregulates the expression of genes related to lipid synthesis via the ATF4-PPARγ/SREBP-1 pathway. This activation leads to an abnormal accumulation of lipid droplets in the liver. 4-PBA, a known ER stress inhibitor, was found to mitigate the PS-NPs-induced lipid metabolism disorder. These results demonstrate the hepatotoxic effects of PS-NPs and clarify the mechanisms of abnormal lipid metabolism induced by PS-NPs.

Methylparaben induces hepatic glycolipid metabolism disorder by activating the IRE1α-XBP1 signaling pathway in male mice.

Methylparaben (MP), a preservative widely used in daily supplies, exists in both the environment and the human body. However, the potential health risks posed by MP remain unclear. This study aimed to unravel the mechanisms by which MP disrupts glucose and lipid homeostasis. For this, we administered MP to mice and observed changes in glucose and lipid metabolism. MP exposure led to hyperglycemia, hyperlipidemia, visceral organ injury, and hepatic lipid accumulation. RNA sequencing results from mice livers indicated a close association between MP exposure and endoplasmic reticulum (ER) stress, inflammatory response, and glucose and lipid homeostasis. Western blotting and quantitative reverse transcription-polymerase chain reaction revealed that MP activated ER stress, particularly the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) pathway, which further promoted the activation of the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. The activation of these pathways phosphorylated insulin receptor substrate-1 (IRS1) (ser 307), resulting in decreased phosphorylation of protein kinase B (Akt) (ser 473), leading to insulin resistance. Additionally, MP exposure promoted lipogenesis through ER stress. To explore potential remedies, we administered the ER stress inhibitor 4-phenylbutyric acid (4-PBA) and the IRE1α-XBP1 pathway inhibitor toyocamycin to mice, both of which protected against metabolic disorders and organ injury caused by MP. These findings suggest that MP induces disruptions in glucose and lipid metabolism through ER stress, primarily through the IRE1α-XBP1 pathway.

Butylparaben induces glycolipid metabolic disorders in mice via disruption of gut microbiota and FXR signaling.

Butylparaben, a common preservative, is widely used in food, pharmaceuticals and personal care products. Epidemiological studies have revealed the close relationship between butylparaben and diabetes; however the mechanisms of action remain unclear. In this study, we administered butylparaben orally to mice and observed that exposure to butylparaben induced glucose intolerance and hyperlipidemia. RNA sequencing results demonstrated that the enrichment of differentially expressed genes was associated with lipid metabolism, bile acid metabolism, and inflammatory response. Western blot results further validated that butylparaben promoted hepatic lipogenesis, inflammation, gluconeogenesis, and insulin resistance through the inhibition of the farnesoid X receptor (FXR) pathway. The FXR agonists alleviated the butylparaben-induced metabolic disorders. Moreover, 16 S rRNA sequencing showed that butylparaben reduced the abundance of Bacteroidetes, S24-7, Lactobacillus, and Streptococcus, and elevated the Firmicutes/Bacteroidetes ratio. The gut microbiota dysbiosis caused by butylparaben led to decreased bile acids (BAs) production and increased inflammatory response, which further induced hepatic glycolipid metabolic disorders. Our results also demonstrated that probiotics attenuated butylparaben-induced disturbances of the gut microbiota and hepatic metabolism. Taken collectively, the findings reveal that butylparaben induced gut microbiota dysbiosis and decreased BAs production, which further inhibited FXR signaling, ultimately contributing to glycolipid metabolic disorders in the liver.

G9a Inhibition Promotes Neuroprotection through GMFB Regulation in Alzheimer's Disease.

Epigenetic alterations are a fundamental pathological hallmark of Alzheimer's disease (AD). Herein, we show the upregulation of G9a and H3K9me2 in the brains of AD patients. Interestingly, treatment with a G9a inhibitor (G9ai) in SAMP8 mice reversed the high levels of H3K9me2 and rescued cognitive decline. A transcriptional profile analysis after G9ai treatment revealed increased gene expression of glia maturation factor ß (GMFB) in SAMP8 mice. Besides, a H3K9me2 ChIP-seq analysis after G9a inhibition treatment showed the enrichment of gene promoters associated with neural functions. We observed the induction of neuronal plasticity and a reduction of neuroinflammation after G9ai treatment, and more strikingly, these neuroprotective effects were reverted by the pharmacological inhibition of GMFB in mice and cell cultures; this was also validated by the RNAi approach generating the knockdown of GMFB/Y507A.10 in Caenorhabditis elegans. Importantly, we present evidence that GMFB activity is controlled by G9a-mediated lysine methylation as well as we identified that G9a directly bound GMFB and catalyzed the methylation at lysine (K) 20 and K25 in vitro. Furthermore, we found that the neurodegenerative role of G9a as a GMFB suppressor would mainly rely on methylation of the K25 position of GMFB, and thus G9a pharmacological inhibition removes this methylation promoting neuroprotective effects. Then, our findings confirm an undescribed mechanism by which G9a inhibition acts at two levels, increasing GMFB and regulating its function to promote neuroprotective effects in age-related cognitive decline.

Charge-based interaction conserved within histone H3 lysine 4 (H3K4) methyltransferase complexes is needed for protein stability, histone methylation, and gene expression.

Histone H3 lysine 4 (H3K4) methyltransferases are conserved from yeast to humans, assemble in multisubunit complexes, and are needed to regulate gene expression. The yeast H3K4 methyltransferase complex, Set1 complex or complex of proteins associated with Set1 (COMPASS), consists of Set1 and conserved Set1-associated proteins: Swd1, Swd2, Swd3, Spp1, Bre2, Sdc1, and Shg1. The removal of the WD40 domain-containing subunits Swd1 and Swd3 leads to a loss of Set1 protein and consequently a complete loss of H3K4 methylation. However, until now, how these WD40 domain-containing proteins interact with Set1 and contribute to the stability of Set1 and H3K4 methylation has not been determined. In this study, we identified small basic and acidic patches that mediate protein interactions between the C terminus of Swd1 and the nSET domain of Set1. Absence of either the basic or acidic patches of Set1 and Swd1, respectively, disrupts the interaction between Set1 and Swd1, diminishes Set1 protein levels, and abolishes H3K4 methylation. Moreover, these basic and acidic patches are also important for cell growth, telomere silencing, and gene expression. We also show that the basic and acidic patches of Set1 and Swd1 are conserved in their human counterparts SET1A/B and RBBP5, respectively, and are needed for the protein interaction between SET1A and RBBP5. Therefore, this charge-based interaction is likely important for maintaining the protein stability of the human SET1A/B methyltransferase complexes so that proper H3K4 methylation, cell growth, and gene expression can also occur in mammals.

Lambda-cyhalothrin induces lipid accumulation in mouse liver is associated with AMPK inactivation.

Lambda-cyhalothrin (LCT) is a critical synthetic Type II pyrethroid insecticide widely applied. Several studies suggest pyrethroids could induce fat accumulation, promote adipogenesis, and impair liver function. Now, the influences of LCT on the hepatic lipid metabolism and the cellular mechanism is still unknown. AMPK has important function in regulating cellular energy balance. To indicate the potential pathogenesis of liver injury caused by LCT exposure, ICR mice were orally administrated with LCT at a dose of 0.4 mg/kg and 2 mg/kg. The results suggest that LCT induced obesity, dyslipidemia and hepatic steatosis. In addition, LCT also induced oxidative stress, liver function injury, and disorganized structure of the liver. Furthermore, upregulation of PPARγ, FASN, and SREBP1c expression, as well as reduction of PPARα and FGF21 expression, bringing with decreases of phosphorylated ratios of AMPK and ACC were found in LCT-L group. These results indicate that LCT at 0.4 mg/kg could result in dyslipidemia and hepatic steatosis in mice. In addition, activation of AMPK in hepatocytes effectively attenuated the effects of LCT. The detailed mechanism of LCT-induced hepatic steatosis is associated with AMPK and its downsteam genes. Activation of AMPK might be a novel protection against the progression of hepatic steatosis induced by LCT.

High-fat diet and palmitate inhibits FNDC5 expression via AMPK-Zfp57 pathway in mouse muscle cells.

Irisin, a muscle-secreted cytokine involved in maintaining glucose homeostasis and improving insulin resistance, is generated from the precursor fibronectin type Ⅲ domain-containing protein 5 (FNDC5) by specific proteases. Zinc-finger protein Zfp57, a transcription factor that maintains the methylation during early embryonic development, is also reported to be associated with diabetes mellitus. However, the association between Zfp57 and FNDC5 is still unclear. In our study, we explored the detailed regulatory effect of Zfp57 on FNDC5 expression. In this study, we found that high-fat diet or saturated fatty acid palmitate increased the Zfp57 expression and decreased FNDC5 expression in muscle tissue or C2C12 myotubes. RNA sequencing analysis disclosed effects of the high-fat diet on genes associated with insulin resistance and the AMP-activated protein kinase (AMPK) signaling pathway in muscle tissue of mice. Chromatin immunoprecipitation experiments revealed that Zfp57 binds the FNDC5 gene promoter at positions -308 to -188. Moreover, Zfp57 overexpression inhibited FNDC5 expression, and Zfp57 knockdown alleviated the inhibitory effect of palmitate on FNDC5 expression in C2C12 myotubes. In addition, in vivo and in vitro studies demonstrated that activation of the AMPK pathway by 5-Aminoimidazole-4-carboxamide riboside (AICAR) or metformin mitigated the inhibitory effect of Zfp57 on FNDC5 expression and improved insulin resistance. These findings collectively suggest that high-fat diet and palmitate inhibit the AMPK pathway to increase Zfp57 expression, which in turn induces FNDC5 inhibition, to further aggravate insulin resistance.

Foodborne Carbon Dot Exposure Induces Insulin Resistance through Gut Microbiota Dysbiosis and Damaged Intestinal Mucus Layer.

Foodborne carbon dots (CDs), an emerging food nanocontaminant, are an increasing risk factor for metabolic toxicity in mammals. Here, we report that chronic CD exposure induced glucose metabolism disorders via disruption of the gut-liver axis in mice. 16s rRNA analysis demonstrated that CD exposure decreased the abundance of beneficial bacteria (Bacteroides, Coprococcus, and S24-7) and increased the abundance of harmful bacteria (Proteobacteria, Oscillospira, Desulfovibrionaceae, and Ruminococcaceae), as well as increased the Firmicutes/Bacteroidetes ratio. Mechanistically, the increased pro-inflammatory bacteria release the endotoxin lipopolysaccharide, which induces an intestinal inflammation and disruption of the intestinal mucus layer, activating systemic inflammation and inducing hepatic insulin resistance in mice via the TLR4/NFκB/MAPK signaling pathway. Furthermore, these changes were almost completely reversed by probiotics. Fecal microbiota transplantation from CD-exposed mice induced glucose intolerance, damaged liver function, intestinal mucus layer injury, hepatic inflammation, and insulin resistance in the recipient mice. However, microbiota-depleted mice exposed to CDs had normal levels of these biomarkers consistent with microbiota-depleted control mice, which revealed that gut microbiota dysbiosis contributes to CD-induced inflammation-mediated insulin resistance. Together, our findings revealed that gut microbiota dysbiosis contributes to CD-induced inflammation-mediated insulin resistance and attempted to elucidate the specific underlying mechanism. Furthermore, we emphasized the importance of assessing the hazards associated with foodborne CDs.

Gcn5- and Bre1-mediated Set2 degradation promotes chronological aging of Saccharomyces cerevisiae.

Loss of transcription-coupled histone H3 lysine 36 trimethylation (H3K36me3) contributes to shorter lifespans in eukaryotes. However, the molecular mechanism of the decline of H3K36me3 during aging remains poorly understood. Here, we report that the degradation of the methyltransferase Set2 is the cause of decreased H3K36me3 levels during chronological aging in budding yeast. We show that Set2 protein degradation during cellular senescence and chronological aging is mainly mediated by the ubiquitin-conjugating E2 enzyme Ubc3 and the E3 ligase Bre1. Lack of Bre1 or abolishment of the ubiquitination stabilizes Set2 protein, sustains H3K36me3 levels at the aging-related gene loci, and upregulates their gene expression, thus leading to extended chronological lifespan. We further illustrate that Gcn5-mediated Set2 acetylation is a prerequisite for Bre1-catalyzed Set2 polyubiquitination and proteolysis during aging. We propose that two sequential post-translational modifications regulate Set2 homeostasis, suggesting a potential strategy to target the Gcn5-Bre1-Set2 axis for intervention of longevity.

Dynamic Phosphorylation of G9a Regulates its Repressive Activity on Chromatin Accessibility and Mitotic Progression.

Phosphorylation of Ser10 of histone H3 (H3S10p), together with the adjacent methylation of Lys9 (H3K9me), has been proposed to function as a 'phospho-methyl switch' to regulate mitotic chromatin architecture. Despite of immense understanding of the roles of H3S10 phosphorylation, how H3K9me2 are dynamically regulated during mitosis is poorly understood. Here, it is identified that Plk1 kinase phosphorylates the H3K9me1/2 methyltransferase G9a/EHMT2 at Thr1045 (pT1045) during early mitosis, which attenuates its catalytic activity toward H3K9me2. Cells bearing Thr1045 phosphomimic mutant of G9a (T1045E) show decreased H3K9me2 levels, increased chromatin accessibility, and delayed mitotic progression. By contrast, dephosphorylation of pT1045 during late mitosis by the protein phosphatase PPP2CB reactivates G9a activity and upregulates H3K9me2 levels, correlated with decreased levels of H3S10p. Therefore, the results provide a mechanistic explanation of the essential of a 'phospho-methyl switch' and highlight the importance of Plk1 and PPP2CB-mediated dynamic regulation of G9a activity in chromatin organization and mitotic progression.