ABSTRACT
Super-enhancers are large clusters of enhancers that activate gene expression. Broad trimethyl histone H3 lysine 4 (H3K4me3) often defines active tumor suppressor genes. However, how these epigenomic signatures are regulated for tumor suppression is little understood. Here we show that brain-specific knockout of the H3K4 methyltransferase MLL4 (a COMPASS-like enzyme, also known as KMT2D) in mice spontaneously induces medulloblastoma. Mll4 loss upregulates oncogenic Ras and Notch pathways while downregulating neuronal gene expression programs. MLL4 enhances DNMT3A-catalyzed DNA methylation and SIRT1/BCL6-mediated H4K16 deacetylation, which antagonize expression of Ras activators and Notch pathway components, respectively. Notably, Mll4 loss downregulates tumor suppressor genes (e.g., Dnmt3a and Bcl6) by diminishing broad H3K4me3 and super-enhancers and also causes widespread impairment of these epigenomic signatures during medulloblastoma genesis. These findings suggest an anti-tumor role for super-enhancers and provide a unique tumor-suppressive mechanism in which MLL4 is necessary to maintain broad H3K4me3 and super-enhancers at tumor suppressor genes.
Subject(s)
Cerebellar Neoplasms/genetics , DNA Methylation , Genes, Tumor Suppressor , Histone-Lysine N-Methyltransferase/genetics , Medulloblastoma/genetics , Oncogenes , Protein Processing, Post-Translational , Acetylation , Animals , Cell Proliferation , Cerebellar Neoplasms/metabolism , Cerebellar Neoplasms/pathology , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methyltransferase 3A , Gene Expression Regulation, Neoplastic , Genes, ras , Histone-Lysine N-Methyltransferase/deficiency , Lysine , Medulloblastoma/metabolism , Medulloblastoma/pathology , Mice, Knockout , Proto-Oncogene Proteins c-bcl-6/genetics , Proto-Oncogene Proteins c-bcl-6/metabolism , Receptors, Notch/genetics , Receptors, Notch/metabolism , Signal Transduction , Sirtuin 1/genetics , Sirtuin 1/metabolismABSTRACT
Human epidermal growth factor receptor-2 (HER2), programmed death-ligand 1 (PD-L1), and microsatellite (MS) status are well-established biomarkers in gastroesophageal adenocarcinomas (GEAs). However, it is unclear how the combination of these biomarkers is associated with clinicopathological factors and prognosis. This retrospective study included baseline metastatic GEA patients who were tested for all three biomarkers (HER2, PD-L1, and MS status) at the MD Anderson Cancer Center between 2012 and 2022. Stratification was performed according to the combination of biomarker profiles: triple negative (TN), single positive (SP), and multiple positive (MP). Comparative analyses of clinicopathological factors and survival using combinations of biomarkers were performed. Among the 698 GEA patients analyzed, 251 (36.0%) were classified as TN, 334 (47.9%) as SP, and 113 (16.1%) as MP. The MP group showed a significant association with tumors located in the esophagus (p < .001), well to moderate differentiation (p < .001), and the absence of signet ring cells (p < .001). In the survival analysis, MP group had a significantly longer overall survival (OS) compared to the other groups (MP vs. TN, p < .001 and MP vs. SP, p < .001). Multivariate Cox regression analysis revealed that MP serves as an independent positive prognostic indicator for OS (hazard ratio = 0.63, p < .01). Our findings indicate that MP biomarkers are associated with a favorable prognosis in metastatic GEA. These results are reflective of clinical practice and offer valuable insights into how therapeutics and future biomarkers could influence therapy/prognosis.
ABSTRACT
Histone acetylation, including acetylated H3K14 (H3K14ac), is generally linked to gene activation. Monomethylated histone H3 lysine 4 (H3K4me1), together with other gene-activating marks, denotes active genes. In contrast to usual gene-activating functions of H3K14ac and H3K4me1, we here show that the dual histone modification mark H3K4me1-H3K14ac is recognized by ZMYND8 (also called RACK7) and can function to counteract gene expression. We identified ZMYND8 as a transcriptional corepressor of the H3K4 demethylase JARID1D. ZMYND8 antagonized the expression of metastasis-linked genes, and its knockdown increased the cellular invasiveness in vitro and in vivo. The plant homeodomain (PHD) and Bromodomain cassette in ZMYND8 mediated the combinatorial recognition of H3K4me1-H3K14ac and H3K4me0-H3K14ac by ZMYND8. These findings uncover an unexpected role for the signature H3K4me1-H3K14ac in attenuating gene expression and reveal a metastasis-suppressive epigenetic mechanism in which ZMYND8's PHD-Bromo cassette couples H3K4me1-H3K14ac with downregulation of metastasis-linked genes.
Subject(s)
Cell Movement , DNA Methylation , Gene Expression Regulation, Neoplastic , Histones/metabolism , Lung Neoplasms/metabolism , Prostatic Neoplasms/metabolism , Receptors, Cell Surface/metabolism , Acetylation , Animals , Binding Sites , Cell Line, Tumor , Cell Proliferation , Down-Regulation , Histone Demethylases/genetics , Histone Demethylases/metabolism , Histones/genetics , Humans , Lung Neoplasms/genetics , Lung Neoplasms/secondary , Male , Mice, Nude , Minor Histocompatibility Antigens/genetics , Minor Histocompatibility Antigens/metabolism , Models, Molecular , Neoplasm Invasiveness , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , RNA Interference , Receptors for Activated C Kinase , Receptors, Cell Surface/genetics , Time Factors , Transcription, Genetic , Transfection , Tumor Burden , Tumor Suppressor ProteinsABSTRACT
Mixed-lineage leukemia 4 (MLL4; also called MLL2 and ALR) enzymatically generates trimethylated histone H3 Lys 4 (H3K4me3), a hallmark of gene activation. However, how MLL4-deposited H3K4me3 interplays with other histone marks in epigenetic processes remains largely unknown. Here, we show that MLL4 plays an essential role in differentiating NT2/D1 stem cells by activating differentiation-specific genes. A tandem plant homeodomain (PHD(4-6)) of MLL4 recognizes unmethylated or asymmetrically dimethylated histone H4 Arg 3 (H4R3me0 or H4R3me2a) and is required for MLL4's nucleosomal methyltransferase activity and MLL4-mediated differentiation. Kabuki syndrome mutations in PHD(4-6) reduce PHD(4-6)'s binding ability and MLL4's catalytic activity. PHD(4-6)'s binding strength is inhibited by H4R3 symmetric dimethylation (H4R3me2s), a gene-repressive mark. The protein arginine methyltransferase 7 (PRMT7), but not PRMT5, represses MLL4 target genes by up-regulating H4R3me2s levels and antagonizes MLL4-mediated differentiation. Consistently, PRMT7 knockdown increases MLL4-catalyzed H3K4me3 levels. During differentiation, decreased H4R3me2s levels are associated with increased H3K4me3 levels at a cohort of genes, including many HOXA and HOXB genes. These findings indicate that the trans-tail inhibition of MLL4-generated H3K4me3 by PRMT7-regulated H4R3me2s may result from H4R3me2s's interference with PHD(4-6)'s binding activity and is a novel epigenetic mechanism that underlies opposing effects of MLL4 and PRMT7 on cellular differentiation.
Subject(s)
DNA-Binding Proteins/metabolism , Histones/metabolism , Cell Differentiation , Cell Line, Tumor , DNA-Binding Proteins/genetics , Epigenesis, Genetic , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , HEK293 Cells , Histone-Lysine N-Methyltransferase , Homeodomain Proteins/metabolism , Humans , Intermediate Filament Proteins/metabolism , Methylation , Nerve Tissue Proteins/metabolism , Nestin , Neurons/cytology , Protein Binding , Protein Structure, Tertiary , Protein-Arginine N-Methyltransferases/genetics , Stem Cells/cytologyABSTRACT
The stemness maintenance of embryonic stem cells (ESCs) requires pluripotency transcription factors, including Oct4, Nanog, and Sox2. We have previously reported that protein arginine methyltransferase 7 (PRMT7), an epigenetic modifier, is an essential pluripotency factor that maintains the stemness of mouse ESCs, at least in part, by down-regulating the expression of the anti-stemness microRNA (miRNA) miR-24-2. To gain greater insight into the molecular basis underlying PRMT7-mediated maintenance of mouse ESC stemness, we searched for new PRMT7-down-regulated anti-stemness miRNAs. Here, we show that miR-221 gene-encoded miR-221-3p and miR-221-5p are anti-stemness miRNAs whose expression levels in mouse ESCs are directly repressed by PRMT7. Notably, both miR-221-3p and miR-221-5p targeted the 3' untranslated regions of mRNA transcripts of the major pluripotency factors Oct4, Nanog, and Sox2 to antagonize mouse ESC stemness. Moreover, miR-221-5p silenced also the expression of its own transcriptional repressor PRMT7. Transfection of miR-221-3p and miR-221-5p mimics induced spontaneous differentiation of mouse ESCs. CRISPR-mediated deletion of the miR-221 gene, as well as specific antisense inhibitors of miR-221-3p and miR-221-5p, inhibited the spontaneous differentiation of PRMT7-depleted mouse ESCs. Taken together, these findings reveal that the PRMT7-mediated repression of miR-221-3p and miR-221-5p expression plays a critical role in maintaining mouse ESC stemness. Our results also establish miR-221-3p and miR-221-5p as anti-stemness miRNAs that target Oct4, Nanog, and Sox2 mRNAs in mouse ESCs.
Subject(s)
Gene Expression Regulation , MicroRNAs/genetics , Mouse Embryonic Stem Cells/cytology , Nanog Homeobox Protein/metabolism , Octamer Transcription Factor-3/metabolism , Protein-Arginine N-Methyltransferases/metabolism , SOXB1 Transcription Factors/metabolism , Animals , Cell Differentiation , Cells, Cultured , Mice , Mouse Embryonic Stem Cells/metabolism , Nanog Homeobox Protein/genetics , Octamer Transcription Factor-3/genetics , Protein-Arginine N-Methyltransferases/genetics , SOXB1 Transcription Factors/geneticsABSTRACT
Self-renewal and pluripotency are two fundamental characteristics of embryonic stem cells (ESCs) and are controlled by diverse regulatory factors, including pluripotent factors, epigenetic regulators and microRNAs (miRNAs). Although histone methyltransferases are key epigenetic regulators, whether and how a histone methyltransferase forms a network with miRNAs and the core pluripotent factor system to regulate ESC stemness is little known. Here, we show that the protein arginine methyltransferase 7 (PRMT7) is a pluripotent factor essential for the stemness of mouse ESCs. PRMT7 repressed the miR-24-2 gene encoding miR-24-3p and miR-24-2-5p by upregulating the levels of symmetrically dimethylated H4R3. Notably, miR-24-3p targeted the 3' untranslated regions (UTRs) of the major pluripotent factors Oct4, Nanog, Klf4 and c-Myc, whereas miR-24-2-5p silenced Klf4 and c-Myc expression. miR-24-3p and miR-24-2-5p also targeted the 3'UTR of their repressor gene Prmt7 miR-24-3p and miR-24-2-5p induced mouse ESC differentiation, and their anti-sense inhibitors substantially reversed spontaneous differentiation of PRMT7-depleted mouse ESCs. Oct4, Nanog, Klf4 and c-Myc positively regulated Prmt7 expression. These findings define miR-24-3p and miR-24-2-5p as new anti-pluripotent miRNAs and also reveal a novel epigenetic stemness-regulatory mechanism in which a double-negative feedback loop consisting of PRMT7 and miR-24-3p/miR24-2-5p interplays with Oct4, Nanog, Klf4 and c-Myc to control ESC stemness.
Subject(s)
MicroRNAs/physiology , Mouse Embryonic Stem Cells/physiology , Protein-Arginine N-Methyltransferases/metabolism , 3' Untranslated Regions , Animals , Base Sequence , Cell Differentiation , Cell Self Renewal , Cells, Cultured , Down-Regulation , Gene Expression , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Mice , Nanog Homeobox Protein/genetics , Nanog Homeobox Protein/metabolism , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Promoter Regions, Genetic , Protein-Arginine N-Methyltransferases/genetics , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , RNA InterferenceABSTRACT
Trimethylated histone H3 lysine 27 (H3K27me3) is linked to gene silencing, whereas H3K4me3 is associated with gene activation. These two marks frequently co-occupy gene promoters, forming bivalent domains. Bivalency signifies repressed but activatable states of gene expression and can be resolved to active, H3K4me3-prevalent states during multiple cellular processes, including differentiation, development and epithelial mesenchymal transition. However, the molecular mechanism underlying bivalency resolution remains largely unknown. Here, we show that the H3K27 demethylase UTX (also called KDM6A) is required for the resolution and activation of numerous retinoic acid (RA)-inducible bivalent genes during the RA-driven differentiation of mouse embryonic stem cells (ESCs). Notably, UTX loss in mouse ESCs inhibited the RA-driven bivalency resolution and activation of most developmentally critical homeobox (Hox) a-d genes. The UTX-mediated resolution and activation of many bivalent Hox genes during mouse ESC differentiation were recapitulated during RA-driven differentiation of human NT2/D1 embryonal carcinoma cells. In support of the importance of UTX in bivalency resolution, Utx-null mouse ESCs and UTX-depleted NT2/D1 cells displayed defects in RA-driven cellular differentiation. Our results define UTX as a bivalency-resolving histone modifier necessary for stem cell differentiation.
Subject(s)
Cell Differentiation/genetics , Histone Demethylases/physiology , Nuclear Proteins/physiology , Promoter Regions, Genetic , Transcriptional Activation , Animals , Cell Differentiation/drug effects , Cell Line, Tumor , Cells, Cultured , Embryonic Stem Cells/cytology , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Genes, Homeobox , Histone Demethylases/metabolism , Humans , Mice , Nuclear Proteins/metabolism , Tretinoin/pharmacologyABSTRACT
Dysregulated expression of histone methyltransferases and demethylases is an emerging epigenetic mechanism underlying cancer development and metastasis. We recently showed that the histone H3 lysine 36 (H3K36) demethylase KDM2A (also called FBXL11 and JHDM1A) is necessary for tumorigenic and metastatic capabilities of KDM2A-overexpressing non-small cell lung cancer (NSCLC) cells. Here, we report that KDM2A transcriptionally represses the histone deacetylase 3 (HDAC3) gene by removing methyl groups from dimethylated H3K36 at the HDAC3 promoter in KDM2A-overexpressing NSCLC cells. KDM2A depletion reduced expression levels of cell cycle-associated genes (e.g. CDK6) and cell invasion-related genes (e.g. NANOS1); these levels were rescued by ectopic expression of KDM2A but not its catalytic mutant. These genes were occupied and down-regulated by HDAC3. HDAC3 knockdown significantly recovered the proliferation and invasiveness of KDM2A-depleted NSCLC cells as well as the levels of CDK6 and NANOS1 expression in these cells. Similar to their previously reported functions in other cell types, CDK6 and NANOS1 were required for the proliferation and invasion, respectively, of KDM2A-overexpressing NSCLC cells. In a mouse xenograft model, HDAC3 depletion substantially restored the tumorigenic ability of KDM2A knockdown cells. These findings reveal a novel cancer-epigenetic pathway in which the antagonistic effect of KDM2A on HDAC3 expression releases cell cycle-associated genes and cell invasion-related genes from HDAC3 repression and indicate the importance of this pathway for tumorigenicity and invasiveness of KDM2A-overexpressing NSCLC cells.
Subject(s)
Carcinoma, Non-Small-Cell Lung/metabolism , Epigenesis, Genetic , F-Box Proteins/metabolism , Gene Expression Regulation, Neoplastic , Histone Deacetylases/metabolism , Jumonji Domain-Containing Histone Demethylases/metabolism , Lung Neoplasms/metabolism , Transcription, Genetic , Animals , Carcinoma, Non-Small-Cell Lung/genetics , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Cyclin-Dependent Kinase 6/metabolism , F-Box Proteins/genetics , Gene Knockdown Techniques , Gene Silencing , Histone Deacetylases/genetics , Humans , Jumonji Domain-Containing Histone Demethylases/genetics , Lung Neoplasms/genetics , Male , Mice , Mice, Nude , Neoplasm Transplantation , Promoter Regions, Genetic , RNA-Binding Proteins/metabolismABSTRACT
The histone H3 lysine 4 (H3K4) methyltransferase KMT2D (also called MLL4) is one of the most frequently mutated epigenetic modifiers in medulloblastoma (MB) and other types of cancer. Notably, heterozygous loss of KMT2D is prevalent in MB and other cancer types. However, what role heterozygous KMT2D loss plays in tumorigenesis has not been well characterized. Here, we show that heterozygous Kmt2d loss highly promotes MB driven by heterozygous loss of the MB suppressor gene Ptch in mice. Heterozygous Kmt2d loss upregulated tumor-promoting programs, including oxidative phosphorylation and G-protein-coupled receptor signaling, in Ptch-mutant-driven MB genesis. Mechanistically, both downregulation of the transcription-repressive tumor suppressor gene NCOR2 by heterozygous Kmt2d loss and upregulation of the oncogene MycN by heterozygous Ptch loss increased the expression of tumor-promoting genes. Moreover, heterozygous Kmt2d loss extensively diminished enhancer signals (e.g., H3K27ac) and H3K4me3 signature, including those for tumor suppressor genes (e.g., Ncor2). Combinatory pharmacological inhibition of oxidative phosphorylation and the H3K4 demethylase LSD1 drastically reduced tumorigenicity of MB cells bearing heterozygous Kmt2d loss. These findings reveal the mechanistic basis underlying the MB-promoting effect of heterozygous KMT2D loss, provide a rationale for a therapeutic strategy for treatment of KMT2D-deficient MB, and have mechanistic implications for the molecular pathogenesis of other types of cancer bearing heterozygous KMT2D loss.
ABSTRACT
The peritoneal cavity is a common site of gastric adenocarcinoma (GAC) metastasis. Peritoneal carcinomatosis (PC) is resistant to current therapies and confers poor prognosis, highlighting the need to identify new therapeutic targets. CD47 conveys a "don't eat me" signal to myeloid cells upon binding its receptor signal regulatory protein alpha (SIRPα), which helps tumor cells circumvent macrophage phagocytosis and evade innate immune responses. Previous studies demonstrated that the blockade of CD47 alone results in limited clinical benefits, suggesting that other target(s) might need to be inhibited simultaneously with CD47 to elicit a strong antitumor response. Here, we found that CD47 was highly expressed on malignant PC cells, and elevated CD47 was associated with poor prognosis. Galectin-3 (Gal3) expression correlated with CD47 expression, and coexpression of Gal3 and CD47 was significantly associated with diffuse type, poor differentiation, and tumor relapse. Depletion of Gal3 reduced expression of CD47 through inhibition of c-Myc binding to the CD47 promoter. Furthermore, injection of Gal3-deficient tumor cells into either wild-type and Lgals3-/- mice led to a reduction in M2 macrophages and increased T-cell responses compared with Gal3 wild-type tumor cells, indicating that tumor cell-derived Gal3 plays a more important role in GAC progression and phagocytosis than host-derived Gal3. Dual blockade of Gal3 and CD47 collaboratively suppressed tumor growth, increased phagocytosis, repolarized macrophages, and boosted T-cell immune responses. These data uncovered that Gal3 functions together with CD47 to suppress phagocytosis and orchestrate immunosuppression in GAC with PC, which supports exploring a novel combination therapy targeting Gal3 and CD47. SIGNIFICANCE: Dual inhibition of CD47 and Gal3 enhances tumor cell phagocytosis and reprograms macrophages to overcome the immunosuppressive microenvironment and suppress tumor growth in peritoneal metastasis of gastric adenocarcinoma.
Subject(s)
Adenocarcinoma , Neoplasms , Peritoneal Neoplasms , Stomach Neoplasms , Animals , Mice , Antigens, Differentiation/metabolism , CD47 Antigen/genetics , Galectin 3/genetics , Neoplasms/drug therapy , Phagocytosis , T-Lymphocytes/metabolism , Tumor MicroenvironmentABSTRACT
The kinesin superfamily of motor proteins is known to be ATP-dependent transporters of various types of cargoes. In neurons, KIF17 is found to transport vesicles containing the N-methyl-D-aspartate receptor NR2B subunit from the cell body specifically to the dendrites. These subunits are intimately associated with glutamatergic neurotransmission as well as with learning and memory. Glutamatergic synapses are highly energy-dependent, and recently we found that the same transcription factor, nuclear respiratory factor 1 (NRF-1), co-regulates energy metabolism (via its regulation of cytochrome c oxidase and other mitochondrial enzymes) and neurochemicals of glutamatergic transmission (NR1, NR2B, GluR2, and nNOS). The present study tested our hypothesis that NRF-1 also transcriptionally regulates KIF17. By means of in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation assays, promoter mutations, and real-time quantitative PCR, we found that NRF-1 (but not NRF-2) functionally regulates Kif17, but not Kif1a, gene. NRF-1 binding sites on Kif17 gene are highly conserved among mice, rats, and humans. Silencing of NRF-1 with small interference RNA blocked the up-regulation of Kif17 mRNA and proteins (and of Grin1 and Grin2b) induced by KCl-mediated depolarization, whereas over-expressing NRF-1 rescued these transcripts and proteins from being suppressed by TTX. Thus, NRF-1 co-regulates oxidative enzymes that generate energy and neurochemicals that consume energy related to glutamatergic neurotransmission, such as KIF17, NR1, and NR2B, thereby ensuring that energy production matches energy utilization at the molecular and cellular levels.
Subject(s)
Kinesins/metabolism , Neurons/metabolism , Nuclear Respiratory Factor 1/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Animals , Base Sequence , Cell Line, Tumor , Cells, Cultured , Humans , Kinesins/genetics , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , NF-E2-Related Factor 2/metabolism , Nuclear Respiratory Factor 1/genetics , Promoter Regions, Genetic , Protein Binding , RNA Interference , Rats , Receptors, N-Methyl-D-Aspartate/genetics , Up-RegulationABSTRACT
Epigenetic mechanisms are central to understanding the molecular basis underlying tumorigenesis. Aberrations in epigenetic modifiers alter epigenomic landscapes and play a critical role in tumorigenesis. Notably, the histone lysine methyltransferase KMT2D (a COMPASS/ Set1 family member; also known as MLL4, ALR, and MLL2) is among the most frequently mutated genes in many different types of cancer. Recent studies have demonstrated how KMT2D loss induces abnormal epigenomic reprograming and rewires molecular pathways during tumorigenesis. These findings also have clinical and therapeutic implications for cancer treatment. In this review, we summarize recent advances in understanding the role of KMT2D in regulating tumorigenesis and discuss therapeutic opportunities for the treatment of KMT2D-deficient tumors.
ABSTRACT
Neuronal activity and energy metabolism are tightly coupled processes. Regions high in neuronal activity, especially of the glutamatergic type, have high levels of cytochrome c oxidase (COX). Perturbations in neuronal activity affect the expressions of COX and glutamatergic NMDA receptor subunit 1 (NR1). The present study sought to test our hypothesis that the coupling extends to the transcriptional level, whereby NR1 and possibly other NR subunits and COX are coregulated by the same transcription factor, nuclear respiratory factor 1 (NRF-1), which regulates all COX subunit genes. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation, promoter mutations, and real-time quantitative PCR, NRF-1 was found to functionally bind to the promoters of Grin 1 (NR1), Grin 2b (NR2b) and COX subunit genes, but not of Grin2a and Grin3a genes. These transcripts were upregulated by KCl and downregulated by tetrodotoxin (TTX) in cultured primary neurons. However, silencing of NRF-1 with small interference RNA blocked the upregulation of Grin1, Grin2b, and COX induced by KCl, and overexpression of NRF-1 rescued these transcripts that were suppressed by TTX. NRF-1 binding sites on Grin1 and Grin2b genes are also highly conserved among mice, rats, and humans. Thus, NRF-1 is an essential transcription factor critical in the coregulation of NR1, NR2b, and COX, and coupling exists at the transcriptional level to ensure coordinated expressions of proteins important for synaptic transmission and energy metabolism.
Subject(s)
Electron Transport Complex IV/genetics , Energy Metabolism/physiology , Nuclear Respiratory Factor 1/metabolism , Receptors, N-Methyl-D-Aspartate/genetics , Synaptic Transmission/physiology , Animals , Animals, Newborn , Cells, Cultured , Cerebral Cortex/cytology , Chromatin Immunoprecipitation/methods , Electrophoretic Mobility Shift Assay/methods , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Humans , Mice , Mutation/physiology , Neuroblastoma , Neurons/cytology , Neurons/drug effects , Neurons/physiology , Potassium Chloride/pharmacology , Promoter Regions, Genetic/genetics , Protein Binding , Protein Subunits/genetics , Protein Subunits/metabolism , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacology , Rats , Sodium Channel Blockers/pharmacology , Synaptic Transmission/drug effects , Tetrodotoxin/pharmacology , Transfection/methodsABSTRACT
Neuronal activity is highly dependent on energy metabolism; yet, the two processes have traditionally been regarded as independently regulated at the transcriptional level. Recently, we found that the same transcription factor, nuclear respiratory factor 1 (NRF-1) co-regulates an important energy-generating enzyme, cytochrome c oxidase, as well as critical subunits of glutamatergic receptors. The present study tests our hypothesis that the co-regulation extends to the next level of glutamatergic synapses, namely, neuronal nitric oxide synthase, which generates nitric oxide as a downstream signaling molecule. Using in silico analysis, electrophoretic mobility shift assay, chromatin immunoprecipitation, promoter mutations, and NRF-1 silencing, we documented that NRF-1 functionally bound to Nos1, but not Nos2 (inducible) and Nos3 (endothelial) gene promoters. Both COX and Nos1 transcripts were up-regulated by depolarizing KCl treatment and down-regulated by TTX-mediated impulse blockade in neurons. However, NRF-1 silencing blocked the up-regulation of both Nos1 and COX induced by KCl depolarization, and over-expression of NRF-1 rescued both Nos1 and COX transcripts down-regulated by TTX. These findings are consistent with our hypothesis that synaptic neuronal transmission and energy metabolism are tightly coupled at the molecular level.
Subject(s)
Electron Transport Complex IV/genetics , Energy Metabolism/genetics , Energy Metabolism/physiology , Neurons/metabolism , Nitric Oxide Synthase Type I/genetics , Nitric Oxide Synthase/genetics , Nuclear Respiratory Factor 1/metabolism , Synaptic Transmission/genetics , Synaptic Transmission/physiology , Animals , Base Sequence , Binding Sites/genetics , Cell Line , Cells, Cultured , DNA Primers/genetics , In Vitro Techniques , Mice , Models, Neurological , Mutagenesis, Site-Directed , Neurons/drug effects , Nuclear Respiratory Factor 1/antagonists & inhibitors , Nuclear Respiratory Factor 1/genetics , Promoter Regions, Genetic , Protein Interaction Domains and Motifs , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Tetrodotoxin/pharmacology , Transcription, Genetic/drug effectsABSTRACT
Neuronal activity and energy metabolism are tightly coupled processes. Recently, we found that nuclear respiratory factor 1 co-regulates all subunits of cytochrome c oxidase (COX, representing oxidative energy metabolism) and glutamatergic neurochemicals, including NR1 (Grin1) and NR2B (Grin2b) of NMDA receptors, GluR2 (Gria2) of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and neuronal nitric oxide synthase (Nos1). Moreover, all 10 nuclear-encoded COX subunit genes and three transcription factor genes for the three mitochondrial-encoded COX subunits are transcribed in the same transcription factory. The goal of the present study was to test our hypothesis that genomic loci for Grin1, Grin2b, Gria2, and Nos1 interact with those for COX at the transcriptional level. By means of chromosome conformation capture, interactions were found among all of these genes in neurons, but not in C2C12 muscle cells. COX subunit genes also did not interact with neurochemical genes not regulated by nuclear respiratory factor 1, nor with genes for calreticulin, a non-mitochondrial protein. Depolarizing stimulation up-regulated interaction frequencies between COX and neurochemical genes, whereas impulse blockade with tetrodotoxin or inhibition of COX with KCN down-regulated them in neurons. Thus, an efficient mechanism is in place for coordinating the transcriptional coupling of energy metabolism and glutamatergic neurotransmission at the molecular level in neurons.
Subject(s)
Chromosomes/genetics , Electron Transport Complex IV/genetics , Gene Expression Regulation, Enzymologic/physiology , Glutamates/physiology , Neurons/physiology , Synaptic Transmission/physiology , Animals , Cell Line, Tumor , Cells, Cultured , Computer Simulation , Electron Transport Complex IV/metabolism , Energy Metabolism/physiology , Gene Expression Regulation, Enzymologic/drug effects , Mice , Neurons/drug effects , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type I , Nuclear Respiratory Factor 1/genetics , Nuclear Respiratory Factor 1/physiology , Potassium Chloride/pharmacology , Potassium Cyanide/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, N-Methyl-D-Aspartate/drug effects , Receptors, N-Methyl-D-Aspartate/genetics , Reverse Transcriptase Polymerase Chain Reaction , Synaptic Transmission/drug effects , Tetrodotoxin/pharmacologyABSTRACT
Peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) coactivates a number of transcription factors critical for mitochondrial biogenesis. Previously, we found that the expression of PGC-1alpha is governed by neuronal activity, but the signaling mechanism is poorly understood. The present study aimed at testing our hypothesis that depolarizing activation of PGC-1alpha in neurons is mediated by p38 mitogen-activated protein kinase (MAPK) and calcium channels. Cultured primary neurons and N2a cells were depolarized with 20 mM KCl for varying times, and increases in PGC-1alpha mRNA and protein levels were found after 0.5 and 1 hr of stimulation, respectively. These levels returned to those of controls after the withdrawal of KCl. Significantly, 15 min of KCl stimulation induced an up-regulation of both p38 MAPK and phosphorylated p38 MAPK that were suppressed by 30 min of pretreatment with SB203580, a blocker of p38 MAPK that also blocked the up-regulation of PGC-1alpha by KCl. Likewise, 30 min of pretreatment with nifedipine, a calcium channel blocker, also prevented the up-regulation of PGC-1alpha mRNA and proteins by KCl. Furthermore, a knockdown of p38 MAPK with small interference hairpin RNA significantly suppressed PGC-1alpha mRNA and protein levels. Our results indicate that both p38 MAPK and calcium play important roles in mediating signaling in depolarization-induced activation of PGC-1alpha at the protein and message levels in neurons.
Subject(s)
Calcium Channels/metabolism , Membrane Potentials/physiology , Neurons/physiology , RNA-Binding Proteins/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Cell Line, Tumor , Cells, Cultured , Membrane Potentials/drug effects , Mice , Neurons/drug effects , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Phosphorylation/drug effects , Potassium Chloride/metabolism , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Time Factors , Up-Regulation/drug effects , Visual Cortex/drug effects , Visual Cortex/physiology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
Epigenetic modifiers frequently harbor loss-of-function mutations in lung cancer, but their tumor-suppressive roles are poorly characterized. Histone methyltransferase KMT2D (a COMPASS-like enzyme, also called MLL4) is among the most highly inactivated epigenetic modifiers in lung cancer. Here, we show that lung-specific loss of Kmt2d promotes lung tumorigenesis in mice and upregulates pro-tumorigenic programs, including glycolysis. Pharmacological inhibition of glycolysis preferentially impedes tumorigenicity of human lung cancer cells bearing KMT2D-inactivating mutations. Mechanistically, Kmt2d loss widely impairs epigenomic signals for super-enhancers/enhancers, including the super-enhancer for the circadian rhythm repressor Per2. Loss of Kmt2d decreases expression of PER2, which regulates multiple glycolytic genes. These findings indicate that KMT2D is a lung tumor suppressor and that KMT2D deficiency confers a therapeutic vulnerability to glycolytic inhibitors.
Subject(s)
Adenocarcinoma of Lung/pathology , DNA-Binding Proteins/antagonists & inhibitors , Deoxyglucose/pharmacology , Enhancer Elements, Genetic , Gene Expression Regulation, Neoplastic , Glycolysis , Histone-Lysine N-Methyltransferase/physiology , Myeloid-Lymphoid Leukemia Protein/physiology , Neoplasm Proteins/antagonists & inhibitors , Adenocarcinoma of Lung/drug therapy , Adenocarcinoma of Lung/metabolism , Animals , Antimetabolites/pharmacology , Apoptosis , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cell Proliferation , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Histones/genetics , Histones/metabolism , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice , Mice, Knockout , Mice, Nude , Mutation , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Period Circadian Proteins/genetics , Period Circadian Proteins/metabolism , Prognosis , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Neuronal activity, especially of the excitatory glutamatergic type, is highly dependent on energy from the oxidative pathway. We hypothesized that the coupling existed at the transcriptional level by having the same transcription factor to regulate a marker of energy metabolism, cytochrome c oxidase (COX) and an important subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors, GluR2 (Gria2). Nuclear respiratory factor 1 (NRF-1) was a viable candidate because it regulates all COX subunits and potentially activates Gria2. By means of in silico analysis, electrophoretic mobility shift and supershift, chromatin immunoprecipitation, and promoter mutational assays, we found that NRF-1 functionally bound to Gria2 promoter. Silencing of NRF-1 with small interference RNA prevented the depolarization-stimulated up-regulation of Gria2 and COX, and over-expression of NRF-1 rescued neurons from tetrodotoxin-induced down-regulation of Gria2 and COX transcripts. Thus, neuronal activity and energy metabolism are tightly coupled at the molecular level, and NRF-1 is a critical agent in this process.
Subject(s)
Electron Transport Complex IV/metabolism , Energy Metabolism/physiology , Neurons/metabolism , Nuclear Respiratory Factor 1/metabolism , Receptors, AMPA/metabolism , Animals , Animals, Newborn , Cells, Cultured , Chromatin Immunoprecipitation/methods , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Electrophoretic Mobility Shift Assay/methods , Isoquinolines/metabolism , Mice , Mutagenesis/physiology , Neuroblastoma , Potassium Chloride/pharmacology , Promoter Regions, Genetic/physiology , RNA, Small Interfering/metabolism , Rats , Rats, Sprague-Dawley , Transfection , Visual Cortex/cytologyABSTRACT
MLL3 and MLL4 are two closely related members of the SET1/MLL family of histone H3K4 methyltransferases and are responsible for monomethylating histone H3K4 on enhancers, which are essential in regulating cell-type-specific gene expression. Mutations of MLL3 or MLL4 have been reported in different types of cancer. Recently, the PHD domains of MLL3/4 have been reported to recruit the MLL3/4 complexes to their target genes by binding to histone H4 during the NT2/D1 stem cell differentiation. Here we show that an extended PHD domain (ePHD6) involving the sixth PHD domain and its preceding zinc finger in MLL3 and MLL4 specifically recognizes an H4H18-containing histone H4 fragment and that modifications of residues surrounding H4H18 modulate H4 binding to MLL3/4. Our in vitro methyltransferase assays and cellular experiments further reveal that the interaction between ePHD6 of MLL3/4 and histone H4 is required for their nucleosomal methylation activity and MLL4-mediated neuronal differentiation of NT2/D1 cells.