Histidine Nτ-methylation identified as a new posttranslational modification in histone H2A at His-82 and H3 at His-39.

Histone posttranslational modifications play critical roles in a variety of eukaryotic cellular processes. In particular, methylation at lysine and arginine residues is an epigenetic mark that determines the chromatin state. In addition, histone "histidine" methylation was initially reported over 50 years ago; however, further studies in this area were not conducted, leaving a gap in our understanding. Here, we aimed to investigate the occurrence of histidine methylation in histone proteins using highly sensitive mass spectrometry. We found that acid hydrolysates of whole histone fraction from calf thymus contained Nτ-methylhistidine, but not Nπ-methylhistidine. Both core and linker histones carried a Nτ-methylhistidine modification, and methylation levels were relatively high in histone H3. Furthermore, through MALDI-TOF MS, we identified two histidine methylation sites at His-82 in the structured globular domain of histone H2A and His-39 in the N-terminal tail of histones H3. Importantly, these histidine methylation signals were also detected in histones purified from a human cell line HEK293T. Moreover, we revealed the overall methylation status of histone H3, suggesting that methylation is enriched primarily at lysine residues and to a lesser extent at arginine and histidine residues. Thus, our findings established histidine Nτ-methylation as a new histone modification, which may serve as a chemical flag that mediates the epigenetic mark of adjacent residues of the N-terminal tail and the conformational properties of the globular domain.

SOX4 reversibly induces phenotypic changes by suppressing the epithelial marker genes in human keratinocytes.

BACKGROUND: SOX4 is a transcription factor belonging to the SOX (Sry-related High Mobility Group [HMG] box) family and plays a pivotal role in various biological processes at various stages of life. SOX4 is also expressed in the skin in adults and has been reported to be involved in wound healing, tumor formation, and metastasis. METHODS AND RESULTS: In this study, we investigated the role of SOX4 in keratinocyte phenotypic changes. We generated a SOX4-overexpressing keratinocyte cell line that expresses SOX4 in a doxycycline (DOX)-inducible manner. DOX treatment induced a change from a paving stone-like morphology to a spindle-like morphology under microscopic observation. Comprehensive gene analysis by RNA sequencing revealed increased expression of genes related to anatomical morphogenesis and cell differentiation as well as decreased expression of genes related to epithelial formation and keratinization, suggesting that SOX4 induced EMT-like phenotype in keratinocytes. Differentially expressed genes (DEGs) obtained by RNA-seq were confirmed using qRT-PCR. DOX-treated TY-1 SOX4 showed a decrease in the epithelial markers (KRT15, KRT13, KRT5, and CLDN1) and an increase in the mesenchymal marker FN1. Protein expression changes by Western blotting also showed a decrease in the epithelial marker proteins keratin 15, keratin 13, and claudin 1, and an increase in the mesenchymal marker fibronectin. Removal of DOX from DOX-treated cells also restored the epithelial and mesenchymal markers altered by SOX4. CONCLUSION: Our results indicate that SOX4 reversibly induces an EMT-like phenotype in human keratinocytes via suppression of epithelial marker genes.

Epigenetic control of rDNA loci in response to intracellular energy status.

Intracellular energy balance is important for cell survival. In eukaryotic cells, the most energy-consuming process is ribosome biosynthesis, which adapts to changes in intracellular energy status. However, the mechanism that links energy status and ribosome biosynthesis is largely unknown. Here, we describe eNoSC, a protein complex that senses energy status and controls rRNA transcription. eNoSC contains Nucleomethylin, which binds histone H3 dimethylated Lys9 in the rDNA locus, in a complex with SIRT1 and SUV39H1. Both SIRT1 and SUV39H1 are required for energy-dependent transcriptional repression, suggesting that a change in the NAD(+)/NADH ratio induced by reduction of energy status could activate SIRT1, leading to deacetylation of histone H3 and dimethylation at Lys9 by SUV39H1, thus establishing silent chromatin in the rDNA locus. Furthermore, eNoSC promotes restoration of energy balance by limiting rRNA transcription, thus protecting cells from energy deprivation-dependent apoptosis. These findings provide key insight into the mechanisms of energy homeostasis in cells.

siRNA screening identifies METTL9 as a histidine Nπ-methyltransferase that targets the proinflammatory protein S100A9.

Protein methylation is one of the most common post-translational modifications observed in basic amino acid residues, including lysine, arginine, and histidine. Histidine methylation occurs on the distal or proximal nitrogen atom of its imidazole ring, producing two isomers: Nτ-methylhistidine or Nπ-methylhistidine. However, the biological significance of protein histidine methylation remains largely unclear owing in part to the very limited knowledge about its contributing enzymes. Here, we identified mammalian seven-ß-strand methyltransferase METTL9 as a histidine Nπ-methyltransferase by siRNA screening coupled with methylhistidine analysis using LC-tandem MS. We demonstrated that METTL9 catalyzes Nπ-methylhistidine formation in the proinflammatory protein S100A9, but not that of myosin light chain kinase MYLK2, in vivo and in vitro. METTL9 does not affect the heterodimer formation of S100A9 and S100A8, although Nπ-methylation of S100A9 at His-107 overlaps with a zinc-binding site, attenuating its affinity for zinc. Given that S100A9 exerts an antimicrobial activity, probably by chelation of zinc essential for the growth of bacteria and fungi, METTL9-mediated S100A9 methylation might be involved in the innate immune response to bacterial and fungal infection. Thus, our findings suggest a functional consequence for protein histidine Nπ-methylation and may add a new layer of complexity to the regulatory mechanisms of post-translational methylation.

Identification of a novel nucleolar protein complex required for mitotic chromosome segregation through centromeric accumulation of Aurora B.

The nucleolus is a membrane-less nuclear structure that disassembles when cells undergo mitosis. During mitosis, nucleolar factors are thus released from the nucleolus and dynamically change their subcellular localization; however, their functions remain largely uncharacterised. Here, we found that a nucleolar factor called nucleolar protein 11 (NOL11) forms a protein complex with two tryptophan-aspartic acid (WD) repeat proteins named WD-repeat protein 43 (WDR43) and Cirhin in mitotic cells. This complex, referred to here as the NWC (NOL11-WDR43-Cirhin) complex, exists in nucleoli during interphase and translocates to the periphery of mitotic chromosomes, i.e., perichromosomal regions. During mitotic progression, both the congression of chromosomes to the metaphase plate and sister chromatid cohesion are impaired in the absence of the NWC complex, as it is required for the centromeric enrichment of Aurora B and the associating phosphorylation of histone H3 at threonine 3. These results reveal the characteristics of a novel protein complex consisting of nucleolar proteins, which is required for regulating kinetochores and centromeres to ensure faithful chromosome segregation.

Tricarboxylic acid cycle activity suppresses acetylation of mitochondrial proteins during early embryonic development in Caenorhabditis elegans.

The tricarboxylic acid (TCA) cycle (or citric acid cycle) is responsible for the complete oxidation of acetyl-CoA and formation of intermediates required for ATP production and other anabolic pathways, such as amino acid synthesis. Here, we uncovered an additional mechanism that may help explain the essential role of the TCA cycle in the early embryogenesis of Caenorhabditis elegans. We found that knockdown of citrate synthase (cts-1), the initial and rate-limiting enzyme of the TCA cycle, results in early embryonic arrest, but that this phenotype is not because of ATP and amino acid depletions. As a possible alternative mechanism explaining this developmental deficiency, we observed that cts-1 RNAi embryos had elevated levels of intracellular acetyl-CoA, the starting metabolite of the TCA cycle. Of note, we further discovered that these embryos exhibit hyperacetylation of mitochondrial proteins. We found that supplementation with acetylase-inhibiting polyamines, including spermidine and putrescine, counteracted the protein hyperacetylation and developmental arrest in the cts-1 RNAi embryos. Contrary to the hypothesis that spermidine acts as an acetyl sink for elevated acetyl-CoA, the levels of three forms of acetylspermidine, N1-acetylspermidine, N8-acetylspermidine, and N1,N8-diacetylspermidine, were not significantly increased in embryos treated with exogenous spermidine. Instead, we demonstrated that the mitochondrial deacetylase sirtuin 4 (encoded by the sir-2.2 gene) is required for spermidine's suppression of protein hyperacetylation and developmental arrest in the cts-1 RNAi embryos. Taken together, these results suggest the possibility that during early embryogenesis, acetyl-CoA consumption by the TCA cycle in C. elegans prevents protein hyperacetylation and thereby protects mitochondrial function.

Arginine methylation of FOXO transcription factors inhibits their phosphorylation by Akt.

Forkhead box O (FOXO) transcription factors, the key regulators of cell survival, are negatively controlled through the PI3K-Akt signaling pathway. Phosphorylation of FOXO by Akt leads to cytoplasmic localization and subsequent degradation via the ubiquitin-proteasome system. Here we show a paradigm of FOXO1 regulation by the protein arginine methyltransferase PRMT1. PRMT1 methylated FOXO1 at conserved Arg248 and Arg250 within a consensus motif for Akt phosphorylation; this methylation directly blocked Akt-mediated phosphorylation of FOXO1 at Ser253 in vitro and in vivo. Silencing of PRMT1 by small interfering RNA enhanced nuclear exclusion, polyubiquitination, and proteasomal degradation of FOXO1. PRMT1 knockdown led to a decrease in oxidative-stress-induced apoptosis depending on the PI3K-Akt signaling pathway. Furthermore, stable expression of enzymatic inactive PRMT1 mutant increased resistance to apoptosis, whereas this effect was reversed by expression of phosphorylation-deficient FOXO1. Our findings predict a role for arginine methylation as an inhibitory modification against Akt-mediated phosphorylation.

Detection of choline and phosphatidic acid (PA) catalyzed by phospholipase D (PLD) using MALDI-QIT-TOF/MS with 9-aminoacridine matrix.

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), the most abundant phospholipids of plasma membrane, resulting in the production of choline and phosphatidic acid (PA). Choline is a precursor of the neurotransmitter acetylcholine, whereas PA functions as an intracellular lipid mediator of diverse biological functions. For assessing PLD activity in vitro, PLD-derived choline has been often analyzed with radioactive or non-radioactive methods. In this study, we have developed a new method for detecting choline and PA with MALDI-QIT-TOF/MS by using 9-aminoacridine as a matrix. The standard calibration curves showed that choline and PA could be detected with linearity over the range from 0.05 and 1 pmol, respectively. Importantly, this method enables the concomitant detection of choline and PA as a reaction product of PC hydrolysis by PLD2 proteins. Thus, our simple and direct method would be useful to characterize the enzymatic properties of PLD, thereby providing insight into mechanisms of PLD activation.

Arginine methylation of BCL-2 antagonist of cell death (BAD) counteracts its phosphorylation and inactivation by Akt.

Protein arginine methylation is a common posttranslational modification catalyzed by a family of the protein arginine methyltransferases (PRMTs). We have previously reported that PRMT1 methylates Forkhead box O transcription factors at two arginine residues within an Akt consensus phosphorylation motif (RxRxxS/T), and that this methylation blocks Akt-mediated phosphorylation of the transcription factors. These findings led us to hypothesize that the functional crosstalk between arginine methylation and phosphorylation could be extended to other Akt target proteins as well as Forkhead box O proteins. Here we identify BCL-2 antagonist of cell death (BAD) as an additional substrate for PRMT1 among several Akt target proteins. We show that PRMT1 specifically binds and methylates BAD at Arg-94 and Arg-96, both of which comprise the Akt consensus phosphorylation motif. Consistent with the hypothesis, PRMT1-mediated methylation of these two arginine residues inhibits Akt-mediated phosphorylation of BAD at Ser-99 in vitro and in vivo. We also demonstrate that the complex formation of BAD with 14-3-3 proteins, which occurs subsequent to Akt-mediated phosphorylation, is negatively regulated by PRMT1. Furthermore, PRMT1 knockdown prevents mitochondrial localization of BAD and its binding to the antiapoptotic BCL-X(L) protein. BAD overexpression causes an increase in apoptosis with concomitant activation of caspase-3, whereas PRMT1 knockdown significantly suppresses these apoptotic processes. Taken together, our results add a new dimension to the complexity of posttranslational BAD regulation and provide evidence that arginine methylation within an Akt consensus phosphorylation motif functions as an inhibitory modification against Akt-dependent survival signaling.

Diverting the food-freezing technology improves the cryopreservation efficiency of induced pluripotent stem cells and derived neurospheres.

Introduction: Recent advances in induced pluripotent stem (iPS) technology and regenerative medicine require effective cryopreservation of iPSC-derived differentiated cells and three-dimensional cell aggregates (eg. Spheroids and organoids). Moreover, innovative freezing technologies for keeping food fresh over the long-term rapidly developed in the food industry. Therefore, we examined whether one of such freezing technologies, called "Dynamic Effect Powerful Antioxidation Keeping (DEPAK)," could be effective for the cryopreservation of biological materials. Methods: We evaluated the efficiency of cryopreservation using DEPAK and Proton freezers, both of which are used in the food industry, compared with conventional slow-freezing methods using a programmable freezer and a cell-freezing vessel. As they are highly susceptible cells to freeze-thaw damage, we selected two suspension cell lines (KHYG-1 derived from human natural killer cell leukemia and THP-1 derived from human acute monocyte leukemia) and two adherent cell lines (OVMANA derived from human ovarian tumors and HuH-7 derived from human hepatocarcinoma). We used two human iPS cell lines, 201B7-Ff and 1231A3, which were either undifferentiated or differentiated into neurospheres. After freezing using the above methods, the frozen cells and neurospheres were immediately transferred to liquid nitrogen. After thawing, we assessed the cryopreservation efficiency of cell viability, proliferation, neurosphere formation, and neurite outgrowth after thawing. Results: Among the four cryopreservation methods, DEPAK freezing resulted in the highest cell proliferation in suspension and adherent cell lines. Similar results were obtained for the cryopreservation of undifferentiated human iPS cells. In addition, we demonstrated that the DEPAK freezing method sustained the neurosphere formation capacity of differentiated iPS cells to the same extent as unfrozen controls. In addition, we observed that DEPAK-frozen neurospheres exhibited higher viability after thawing and underwent neural differentiation more efficiently than slow-freezing methods. Conclusions: Our results suggest that diversifying food-freezing technologies can overcome the difficulties associated with the cryopreservation of various biological materials, including three-dimensional cell aggregates.

Conserved SAMS function in regulating egg-laying in C. elegans.

S-adenosyl-L-methionine (SAM) is an intermediate metabolite of methionine and serves as the methyl donor for many biological methylation reactions. The synthesis of SAM is catalyzed by SAM synthetase (SAMS), which transfers the adenosyl moiety of adenosine-5'-triphosphate to methionine. In the nematode Caenorhabditis elegans, four sams family genes, sams-1, -3, -4 and -5, are predicted to encode SAMS proteins. However, their physiological roles remain unclear. Here we show that the four predicted SAMS proteins in fact have the ability to catalyze the formation of SAM in vitro, and revealed that only sams-1 mutant animals among the family genes exhibited a significant reduction in egg-laying. Using transgenic animals carrying a transcriptional reporter for each sams gene promoter, we observed that each sams promoter confers a distinct expression pattern with respect to tissue, time of expression and expression level (i.e. promoter specificity). Promoter-swap experiments revealed that the ectopic expression of SAMS-3, -4 or -5 driven by the sams-1 promoter completely rescued egg-laying in sams-1 mutants. These data indicate that SAMS protein function is conserved throughout the entire family.

γ-enolase (ENO2) is methylated at the Nτ position of His-190 among enolase isozymes.

Protein methylation is mainly observed in lysine, arginine and histidine residues. Histidine methylation occurs at one of two different nitrogen atoms of the imidazole ring, producing Nτ-methylhistidine and Nπ-methylhistidine, and it has recently attracted attention with the identification of SETD3, METTL18 and METTL9 as catalytic enzymes in mammals. Although accumulating evidence had suggested the presence of more than 100 proteins containing methylated histidine residues in cells, much less information has been known regarding histidine-methylated proteins than lysine- and arginine-methylated ones, because no method has been developed to identify substrates for histidine methylation. Here, we established a method to screen novel target proteins for histidine methylation, using biochemical protein fractionation combined with the quantification of methylhistidine by LC-MS/MS. Interestingly, the differential distribution pattern of Nτ-methylated proteins was found between the brain and skeletal muscle, and identified γ-enolase where the His-190 at the Nτ position is methylated in mouse brain. Finally, in silico structural prediction and biochemical analysis showed that the His-190 in γ-enolase is involved in the intermolecular homodimeric formation and enzymatic activity. In the present study, we provide a new methodology to find histidine-methylated proteins in vivo and suggest an insight into the importance of histidine methylation.

Rhomboid protease RHBDL4/RHBDD1 cleaves SREBP-1c at endoplasmic reticulum monitoring and regulating fatty acids.

The endoplasmic reticulum (ER)-embedded transcription factors, sterol regulatory element-binding proteins (SREBPs), master regulators of lipid biosynthesis, are transported to the Golgi for proteolytic activation to tune cellular cholesterol levels and regulate lipogenesis. However, mechanisms by which the cell responds to the levels of saturated or unsaturated fatty acids remain underexplored. Here, we show that RHBDL4/RHBDD1, a rhomboid family protease, directly cleaves SREBP-1c at the ER. The p97/VCP, AAA-ATPase complex then acts as an auxiliary segregase to extract the remaining ER-embedded fragment of SREBP-1c. Importantly, the enzymatic activity of RHBDL4 is enhanced by saturated fatty acids (SFAs) but inhibited by polyunsaturated fatty acids (PUFAs). Genetic deletion of RHBDL4 in mice fed on a Western diet enriched in SFAs and cholesterol prevented SREBP-1c from inducing genes for lipogenesis, particularly for synthesis and incorporation of PUFAs, and secretion of lipoproteins. The RHBDL4-SREBP-1c pathway reveals a regulatory system for monitoring fatty acid composition and maintaining cellular lipid homeostasis.

Regulation of FoxO transcription factors by acetylation and protein-protein interactions.

The forkhead box O transcription factors convert a variety of external stimuli, including growth factors, nutrients, and oxidative stress, into diverse biological responses through modulation of specific gene expression. Forkhead box O regulation is principally achieved by two distinct mechanisms: post-translational modifications and protein-protein interactions. Among several modifications of forkhead box O factors, we focus on reversible acetylation, describing past research and current advances. In the latter part of this review, we also provide an overview of forkhead box O-binding partners that control the transcriptional activity of forkhead box O factors. These two layers of regulation mostly overlap and thereby enable a more precise fine-tuning of forkhead box O functions involved in metabolism, longevity, and tumor suppression. This article is part of a Special Issue entitled: PI3K-AKT-FoxO axis in cancer and aging.

GSK3ß regulates gluconeogenic gene expression through HNF4α and FOXO1.

Hepatic gluconeogenesis is important for the maintenance of blood glucose homeostasis under fasting condition. Hepatocyte nuclear factor 4α (HNF4α) and FOXO1 transcription factors have implicated in this process through transcriptional regulation of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK), which are rate-limiting enzymes in gluconeogenesis. In this study, we demonstrate that glycogen synthase kinase 3ß (GSK3ß) regulates the expression of gluconeogenic genes through HNF4α and FOXO1. Silencing of GSK3ß leads to reduction in the expression of gluconeogenic genes, including G6Pase, PEPCK, and peroxisome proliferator-activated receptor γ coactivator-1α. We show that GSK3ß directly binds to both HNF4α and FOXO1. Inhibition of GSK3 by SB-216763 abolishes HNF4α-mediated activation of G6Pase promoter. We also found that overexpression of GSK3ß potentiates G6Pase promoter activation by FOXO1 in a manner dependent on its kinase activity. Treatment of SB-216763 diminishes FOXO1-mediated activation of G6Pase promoter. Taken together, these results reveal a previously unrecognized mechanism for the regulation of gluconeogenic gene expression.

The C. elegans PRMT-3 possesses a type III protein arginine methyltransferase activity.

Protein arginine methylation is a common post-translational modification in eukaryotes that is catalyzed by a family of the protein arginine methyltransferases (PRMTs). PRMTs are classified into three types: type I and type II add asymmetrically and symmetrically dimethyl groups to arginine, respectively, while type III adds solely monomethyl group to arginine. However, although the enzymatic activity of type I and type II PRMTs have been reported, the substrate specificity and the methylation activity of type III PRMTs still remains unknown. Here, we report the characterization of Caenorhabditis elegans PRMT-2 and PRMT-3, both of which are highly homologous to human PRMT7. We find that these two PRMTs can bind to S-adenosyl methionine (SAM), but only PRMT-3 has methyltransferase activity for histone H2A depending on its SAM-binding domain. Importantly, thin-layer chromatographic analysis demonstrates that PRMT-3 catalyzes the formation of monomethylated, but not dimethylated arginine. Our study thus identifies the first type III PRMT in C. elegans and provides a means to elucidate the physiological significance of arginine monomethylation in multicellular organisms.

PCAF represses transactivation function of FOXO1 in an acetyltransferase-independent manner.

The FOXO transcription factors play a key role in cell cycle control, apoptosis, DNA repair, oxidative stress resistance, and longevity. In this study, we demonstrated that the acetyltransferase p300/CBP associated factor (PCAF) functions as a negative regulator of FOXO1. We showed that PCAF bound to the forkhead domain of FOXO1 and acetylated FOXO1 at the K242 and K245 residues. However, PCAF repressed FOXO1-induced transcription in an enzymatic activity-independent manner. In contrast, the transcriptional activity of FOXO1 S253A mutant, in which an Akt phosphorylation site is replaced by alanine, was not repressed by PCAF. Akt-induced phosphorylation of FOXO1 is required for its binding to PCAF, whereas the binding between FOXO1 and CBP is independent on FOXO1 S253 phosphorylation. Furthermore, overexpression of PCAF increased nuclear accumulation of FOXO1 even in the presence of serum. These results suggest that PCAF binds to phosphorylated FOXO1 by Akt and acts as a transcriptional corepressor in the nucleus.

The LXXLL motif of murine forkhead transcription factor FoxO1 mediates Sirt1-dependent transcriptional activity.

The forkhead transcription factor FoxO1 has been identified as a negative regulator of insulin/IGF-1 signaling. Its function is inhibited by phosphorylation and nuclear exclusion through a PI3K-dependent pathway. However, the structure/function relationship of FoxO1 has not been elucidated completely. In this study, we carried out mutation analysis of the FoxO1 coactivator-interacting LXXLL motif (amino acids 459-463). Expression of a 3A/LXXAA mutant, in which 3 Akt phosphorylation sites (T24, S253, and S316) and 2 leucine residues in the LXXLL motif (L462 and L463) were replaced by alanine, decreased both Igfbp-1 and G6Pase promoter activity and endogenous Igfbp-1 and G6Pase gene expression in simian virus 40-transformed (SV40-transformed) hepatocytes. Importantly, mutagenesis of the LXXLL motif eliminated FoxO1 interaction with the nicotinamide adenine dinucleotide-dependent (NAD-dependent) deacetylase sirtuin 1 (Sirt1), sustained the acetylated state of FoxO1, and made FoxO1 nicotinamide and resveratrol insensitive, supporting a role for this motif in Sirt1 binding. Furthermore, intravenous administration of adenovirus encoding 3A/LXXAA FoxO1 into Lepr db/db mice decreased fasting blood glucose levels and improved glucose tolerance and was accompanied by reduced G6Pase and Igfbp-1 gene expression and increased hepatic glycogen content. In conclusion, the LXXLL motif of FoxO1 may have an important role for its transcriptional activity and Sirt1 binding and should be a target site for regulation of gene expression of FoxO1 target genes and glucose metabolism in vivo.

Foxo1 increases pro-inflammatory gene expression by inducing C/EBPbeta in TNF-alpha-treated adipocytes.

Obesity is associated with a low-grade inflammation in adipose tissue resulting from increased production of pro-inflammatory cytokines and which can subsequently contribute to the development of insulin resistance. However, the mechanisms underlying the transcriptional regulation of pro-inflammatory genes are still unclear. Here we show that tumor necrosis factor (TNF)-alpha treatment attenuated Akt-dependent phosphorylation of Foxo1 and enhanced transcriptional activity of Foxo1. We found that Foxo1 increased the expression of CCAAT/enhancer binding protein (C/EBPbeta, a positive regulator of monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-6 genes, through directly binding to its promoter. Furthermore, knockdown of Foxo1 as well as C/EBPbeta inhibits TNF-alpha-induced expression of MCP-1 and IL-6 in 3T3-L1 adipocytes. These findings suggest that activation of Foxo1 triggered by TNF-alpha up-regulates the expression of C/EBPbeta in 3T3-L1 adipocytes, thereby leading to an increased production of pro-inflammatory cytokines, MCP-1 and IL-6.

Regulation of FOXO1-mediated transcription and cell proliferation by PARP-1.

Forkhead box O (FOXO) transcription factors play an important role in a wide range of biological processes, including cell cycle control, apoptosis, detoxification of reactive oxygen species, and gluconeogenesis through regulation of gene expression. In this study, we demonstrated that PARP-1 functions as a negative regulator of FOXO1. We showed that PARP-1 directly binds to and poly(ADP-ribosyl)ates FOXO1 protein. PARP-1 represses FOXO1-mediated expression of cell cycle inhibitor p27(Kip1) gene. Notably, poly(ADP-ribosyl)ation activity was not required for the repressive effect of PARP-1 on FOXO1 function. Furthermore, knockdown of PARP-1 led to a decrease in cell proliferation in a manner dependent on FOXO1 function. Chromatin immunoprecipitation experiments confirmed that PARP-1 is recruited to the p27(Kip1) gene promoter through a binding to FOXO1. These results suggest that PARP-1 acts as a corepressor for FOXO1, which could play an important role in proper cell proliferation by regulating p27(Kip1) gene expression.