EZH2
Histone-lysine N-methyltransferase EZH2 (EC 2.1.1.356) (ENX-1) (Enhancer of zeste homolog 2) (Lysine N-methyltransferase 6) [KMT6]
Publications[править]
Invariant natural killer T (iNKT) cells represent a subclass of T cells possessing a restricted repertoire of T cell receptors enabling them to recognize lipid derived ligands. iNKT cells are continuously generated in thymus and differentiate into three main subpopulations: iNKT1, iNKT2, and iNKT17 cells. We investigated the transcriptomes of these subsets comparing cells isolated from young adult (6-10 weeks old) and aged BALB/c mice (25-30 weeks of age) in order to identify genes subject to an age-related regulation of expression. These time points were selected to take into consideration the consequences of thymic involution that radically alter the existing micro-milieu. Significant differences were detected in the expression of histone genes affecting all iNKT subsets. Also the proliferative capacity of iNKT cells decreased substantially upon aging. Several genes were identified as possible candidates causing significant age-dependent changes in iNKT cell generation and/or function such as genes coding for granzyme A, ZO-1, EZH2, SOX4, IGF1 receptor, FLT4, and CD25. Moreover, we provide evidence that IL2 differentially affects homeostasis of iNKT subsets with iNKT17 cells engaging a unique mechanism to respond to IL2 by initiating a slow rate of proliferation.
Keywords
- IL2
- aging
- invariant natural killer T cells
- thymus
- transcriptome
Long non-coding RNAs (ncRNAs) are major regulators of gene expression and cell fate. The [i]INK4[/i] locus encodes the tumour suppressor proteins p15 , p16 and p14 required for cell cycle arrest and whose expression increases during senescence. [i]ANRIL[/i] is a ncRNA antisense to the [i]p15[/i] gene. In proliferative cells, [i]ANRIL[/i] prevents senescence by repressing [i]INK4[/i] genes through the recruitment of Polycomb-group proteins. In models of replicative and RASval12 oncogene-induced senescence (OIS), the expression of [i]ANRIL[/i] and Polycomb proteins decreases, thus allowing [i]INK4[/i] derepression. Here, we found in a model of RAF1 OIS that [i]ANRIL[/i] expression rather increases, due in particular to an increased stability. This led us to search for circular [i]ANRIL[/i] isoforms, as circular RNAs are rather stable species. We found that the expression of two circular [i]ANRIL[/i] increases in several OIS models (RAF1, MEK1 and BRAF). In proliferative cells, they repress [i]p15[/i] expression, while in RAF1 OIS, they promote full induction of [i]p15, p16[/i] and [i]p14 [/i] expression. Further analysis of one of these circular [i]ANRIL[/i] shows that it interacts with Polycomb proteins and decreases EZH2 Polycomb protein localization and H3K27me3 at the [i]p15[/i] and [i]p16[/i] promoters, respectively. We propose that changes in the ratio between Polycomb proteins and circular [i]ANRIL[/i] isoforms allow these isoforms to switch from repressors of [i]p15[/i] gene to activators of all [i]INK4[/i] genes in RAF1 OIS. Our data reveal that regulation of [i]ANRIL[/i] expression depends on the senescence inducer and underline the importance of circular [i]ANRIL[/i] in the regulation of [i]INK4[/i] gene expression and senescence.
Keywords
- INK4 locus
- Non-coding RNAs
- Polycomb proteins
- circular RNAs
- gene expression regulation
- oncogene-induced senescence
Bowhead whales are among the longest-lived mammals with an extreme lifespan of about 211 years. During the first 25 years of their lives, rib bones increase in mineral density and the medulla transitions from compact to trabecular bone. Molecular drivers associated with these phenotypic changes in bone remain unknown. This study assessed expression levels of osteogenic genes from samples of rib bones of bowheads. Samples were harvested from prenatal to 86-year-old whales, representing the first third of the bowhead lifespan. Fetal to 2-year-old bowheads showed expression levels consistent with the rapid deposition of the bone extracellular matrix. Sexually mature animals showed expression levels associated with low rates of osteogenesis and increased osteoclastogenesis. After the first 25 years of life, declines in osteogenesis corresponded with increased expression of EZH2, an epigenetic regulator of osteogenesis. These findings suggest EZH2 may be at least one epigenetic modifier that contributes to the age-related changes in the rib bone phenotype along with the transition from compact to trabecular bone. Ancient cetaceans and their fossil relatives also display these phenotypes, suggesting EZH2 may have shaped the skeleton of whales in evolutionary history.
Keywords
- Cetacea
- aging
- bone
- hyperostosis
- osteoblasts
- whales
Microglial activation and pro-inflammatory cytokines expression is closely related to pathogenesis of depression. Aging is a known risk factor for neuroinflammation in the central nervous system and subsequent behavioral impairment. Enhancer of zeste homolog 2 (EZH2), a methyltransferase of histone H3 lysine 27 which regulates microglial activation, plays a crucial role in proinflammatory cytokines expression. However, whether the EZH2 is involved in susceptibility to depression in different ages remains elusive. Young and aged C57BL/6 mice were exposed to chronic unpredictable mild stress for three weeks. Depression- and anxiety-like behaviors, spatial memory impairment, and the expression of pro-inflammatory cytokines, P-p65, EZH2, H3K27me3 and SOCS3 in the prefrontal cortex and hippocampus were measured using an established behavioral battery, ELISA, immunohistochemistry and western blotting techniques. Moreover, EPZ-6438, an inhibitor of EZH2, was utilized to detect the role of EZH2 in neuroinflammation and behavioral abnormalities. CUMS induced depression-like behaviors and spatial memory impairment, elevated levels of proinflammatory cytokines and P-p65, enhanced M1 microglia activation, and increased levels of EZH2, H3K27me3 and SOCS3 in the prefrontal cortex and hippocampus in young and aged mice. Both unstressed and stressed aged mice displayed attention-deficit behavioral outcomes, alteration of protein levels compared with young mice. However, inhibition of EZH2 could relieve most of behavioral and molecular alterations. A relative small sample size is a limitation. EZH2 might be involved in susceptibility to neuroinflammation and depression-like behaviors in different aged mice.
Keywords
- Aging
- CUMS
- Cytokines
- Depresion
- EZH2
- Microglia
The senescence of nucleus pulposus (NP) cells plays a vital role in the pathogenesis of intervertebral disc (IVD) degeneration (IDD). NADPH oxidase 4 (NOX4)-associated oxidative stress has been shown to induce premature NP cell senescence. Enhancer of zeste homolog 2 (EZH2) is a crucial gene regulating cell senescence. The aim of this study was to investigate the roles of EZH2 in NOX4-induced NP cell senescence and a feedback loop between EZH2 and NOX4. The down-regulation of EZH2 and the up-regulation of NOX4 and p16 were observed in the degenerative discs of aging rats. EZH2 regulated NP cell senescence via the H3K27me3-p16 pathway. Also, EZH2 regulated the expression of NOX4 in NP cells through the histone H3 lysine 27 trimethylation (H3K27me3) in the promoter of NOX4 gene. Furthermore, NOX4 down-regulated EZH2 expression in NP cells via the canonical Wnt/β-catenin pathway. A positive feedback loop between EZH2 and NOX4 is involved in regulating NP cell senescence, which provides a novel insight into the mechanism of IDD and a potential therapeutic target for IDD.
Keywords
- Epigenetic histone modification
- Intervertebral disc degeneration
- Nucleus pulposus cell senescence
- Wnt/β-catenin signaling pathway
The endocrine disruptive effects caused by bisphenol A (BPA) are well known. Despite this, to date, evaluation of its long term effects is limited, meaning that there is still much to be unveiled in terms of alterations caused by perinatal exposure to BPA. Our aim was to determine if perinatal exposure to two different doses of BPA causes long term morphological and molecular alteration effects in the mammary gland (MG). We evaluated MG from Mongolian gerbil offspring exposed perinatally (during gestation and lactation) to 50 or 5000 μg/kg/day BPA. At 90 days of age the animals were subjected to a single dose of N-nitroso-N-methylurea in order to mimic a carcinogenic environment. At 6 months of age, animals in estrous were euthanized for morphological evaluation of the MGs. The MG architecture presented considerable changes in terms of detached epithelial cells, inflammation, glandular hyperplasia, and collagen fiber deposition. Furthermore, a higher index of epithelial cell proliferation was detected in comparison to the intact control group. In addition, we verified a higher molecular expression of EZH2 in the vehicle treated group, indicating that corn oil applied alone can alter the expression of this epigenetic biomarker. In conclusion, BPA perinatal exposure promotes significant changes in glandular cytoarchitecture and increases glandular epithelium proliferation rate, leading to the retention of stem-like properties. This event could compromise the fate and differentiation potential of mammary epithelium.
MeSH Terms
- Actins
- Aging
- Animals
- Benzhydryl Compounds
- Cell Proliferation
- Collagen
- Enhancer of Zeste Homolog 2 Protein
- Female
- Gerbillinae
- Histones
- Mammary Glands, Animal
- Phenols
- Pregnancy
- Prenatal Exposure Delayed Effects
Keywords
- BPA
- EZH2
- Environment pollutant
- Estrogen
- Morphologic alterations
- Phospho-histone-h3
Gallbladder cancer (GBC) is the most malignant cancer occurring in the biliary tract cancer featured with undesirable prognosis, in which most patients die within a year of cholecystectomy. Long noncoding RNAs (lncRNAs) function as critical regulators of multiple stages of cancers. Herein, the mechanism of lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) in GBC is investigated. Microarray-based analysis initially provided data suggesting that the expression of MALAT1 was up-regulated while that of the ABI family member 3 binding protein (ABI3BP) was down-regulated in GBC tissues and cell lines. Kaplan-Meier method was then adopted to analyze the relationship between the MALAT1 expression and overall survival and disease-free survival of patients with GBC. A set of in vitro and in vivo experiments were conducted by transducing ABI3BP-vector or sh-MALAT1 into GBC cells. The results confirmed that the cancer prevention effects triggered by restored ABI3BP and depleted MALAT1 as evidenced by suppressed cell growth and enhanced cell senescence. MALAT1 was observed to down-regulate ABI3BP expression through recruitment of the enhancer of zeste homolog 2 (EZH2) to the ABI3BP promoter region while the silencing of MALAT1 or suppression of H3K27 methylation was observed to promote the expression of ABI3BP. Furthermore, GBC patients with high expression of MALAT1 indicated poor prognosis. The current study clarifies that MALAT1 silencing and ABI3BP elevation impede the GBC development through the H3K27 methylation suppression induced by EZH2, highlighting a promising competitive paradigm for therapeutic approaches of GBC.
MeSH Terms
- Adult
- Animals
- Carrier Proteins
- Cell Line, Tumor
- Cell Movement
- Cell Proliferation
- Cell Transformation, Neoplastic
- Cellular Senescence
- Disease Models, Animal
- Female
- Gallbladder Neoplasms
- Gene Expression Regulation, Neoplastic
- Heterografts
- Histones
- Humans
- Male
- Methylation
- Mice
- Middle Aged
- Models, Biological
- Prognosis
- RNA Interference
- RNA, Long Noncoding
- Young Adult
Keywords
- ABI family member 3 binding protein
- Enhancer of zeste homolog 2
- Gallbladder cancer
- Growth
- Histone
- Metastasis associated lung adenocarcinoma transcript 1
- Methylation
- Senescence
Triple-negative breast cancer (TNBC) cells lack the expression of ER, PR and HER2. Thus, TNBC patients cannot benefit from hormone receptor-targeted therapy as non-TNBC patients, but can only receive chemotherapy as the systemic treatment and have a worse overall outcome. More effective therapeutic targets and combination therapy strategies are urgently needed to improve the treatment effectiveness. We analyzed the expression levels of EZH2 and TET1 in TCGA and our own breast cancer patient cohort, and tested their correlation with patient survival. We used TNBC and non-TNBC cell lines and mouse xenograft tumor model to unveil novel EZH2 targets and investigated the effect of EZH2 inhibition or TET1 overexpression in cell proliferation and viability of TNBC cells. In TNBC cells, EZH2 decreases TET1 expression by H3K27me3 epigenetic regulation and subsequently suppresses anti-tumor p53 signaling pathway. Patients with high EZH2 and low TET1 presented the poorest survival outcome. Experimentally, targeting EZH2 in TNBC cells with specific inhibitor GSK343 or shRNA genetic approach could induce cell cycle arrest and senescence by elevating TET1 expression and p53 pathway activation. Using mouse xenograft model, we have tested a novel therapy strategy to combine GSK343 and chemotherapy drug Adriamycin and could show drastic and robust inhibition of TNBC tumor growth by synergistic induction of senescence and apoptosis. We postulate that the well-controlled dynamic pathway EZH2-H3K27me3-TET1 is a novel epigenetic co-regulator module and provide evidence regarding how to exploit it as a novel therapeutic target via its pivotal role in senescence and apoptosis control. Of clinical and therapeutic significance, the present study opens a new avenue for TNBC treatment by targeting the EZH2-H3K27me3-TET1 pathway that can modulate the epigenetic landscape.
MeSH Terms
- Animals
- Antibiotics, Antineoplastic
- Apoptosis
- Cancer Survivors
- Cell Line, Tumor
- Cellular Senescence
- DNA Methylation
- DNA, Neoplasm
- Doxorubicin
- Drug Delivery Systems
- Enhancer of Zeste Homolog 2 Protein
- Epigenesis, Genetic
- Female
- Gene Expression Regulation, Neoplastic
- Humans
- Indazoles
- Mice
- Mice, Nude
- Mixed Function Oxygenases
- Proto-Oncogene Proteins
- Pyridones
- Triple Negative Breast Neoplasms
- Xenograft Model Antitumor Assays
Keywords
- EZH2
- TET1
- TNBC
- cellular senescence
- epigenetic co-deregulation
Aging is the most potent of carcinogens, especially for the bone marrow stem cell clonal disorders called myelodysplastic syndromes (MDS). Age-associated changes in the microenvironment or the soil of the bone marrow (BM) as well as in the cell or the seed provide a growth advantage for clonal myeloid cells. Slowly accumulating senescent cells which can no longer divide because they have reached the end of their proliferative life cycle, but which continue to produce metabolic debris, overwhelm the natural autophagy mechanisms resulting in pro-inflammatory changes in the BM soil. In addition, the seed or stem cells acquire passenger mutations with each round of proliferation resulting from DNA copying errors. Some mutations commonly associated with MDS can be found in older, otherwise healthy individuals; however, when combined with other passenger mutations or in the setting of a noxious soil, the result could be a proliferative advantage for one stem cell over others, leading to its clonal expansion and development of the clinical syndrome. When considering therapeutic options for MDS patients, the important considerations are related to both the common co-morbidities of an elderly population along with the heterogeneous passenger mutations and the inflammatory changes in the soil. At present, allogeneic stem cell transplant is the only potentially curative option in MDS. Palliative strategies are directed at improving the quality of life and prolonging survival. Only three drugs are FDA approved, two being the hypomethylating agents azacytidine and decitabine while the third is lenalidomide which is restricted to lower risk MDS patients with deletion 5q. Promising future therapies are directed at reversing the pro-inflammatory changes in the microenvironment (luspatercept) or targeting specific mutations isocitrate dehydrogenase (IDH)1, IDH2, p53, EZH2. More durable responses are to be expected when the seed and soil are targeted simultaneously through a combination of drugs.
MeSH Terms
- Aging
- Angiogenesis Inhibitors
- Antimetabolites, Antineoplastic
- Azacitidine
- Decitabine
- Enzyme Inhibitors
- Humans
- Lenalidomide
- Myelodysplastic Syndromes
- Stem Cell Transplantation
- Transplantation, Homologous
Keywords
- Anemia
- Bone marrow
- Cytopenia
- Myelodysplasia
- Treatment
Physicians have observed that surgical wounds in the elderly heal with thinner scars than wounds in young patients. Understanding this phenomenon may reveal strategies for promoting scarless wound repair. We show that full-thickness skin wounds in aged but not young mice fully regenerate. Exposure of aged animals to blood from young mice by parabiosis counteracts this regenerative capacity. The secreted factor, stromal-derived factor 1 (SDF1), is expressed at higher levels in wounded skin of young mice. Genetic deletion of SDF1 in young skin enhanced tissue regeneration. In aged mice, enhancer of zeste homolog 2 (EZH2) and histone H3 lysine 27 trimethylation are recruited to the SDF1 promoter at higher levels, and pharmacologic inhibition of EZH2 restores SDF1 induction and prevents tissue regeneration. Similar age-dependent EZH2-mediated SDF1 suppression occurs in human skin. Our findings counter the current dogma that tissue function invariably declines with age and suggest new therapeutic strategies in regenerative medicine.
MeSH Terms
- Aging
- Animals
- Cells, Cultured
- Chemokine CXCL12
- Enhancer of Zeste Homolog 2 Protein
- Female
- Humans
- Mice
- Mice, Inbred C57BL
- Skin
- Wound Healing
Keywords
- CXCL12
- SDF1
- aging
- epigenetics
- organ regeneration
- scar
- skin
- tissue regeneration
Myeloid leukemia treatment is quite successful nowadays; nevertheless the development of new therapies is still necessary. In the present study, we investigated the potential of epigenetic modulators EGCG (epigallocatechin-3-gallate) and BIX-01294 (N-(1-benzylpiperidin-4-yl)-6,7-dimethoxy-2-(4-methyl-1,4-diazepan-1-yl)quinazolin-4-amine) to alter epigenetic state and cause cellular senescence in acute and chronic myeloid leukemia NB4 and K562 cells. We have shown that after leukemia cell treatment with EGCG and BIX-01294 the proliferation and survival were inhibited of both cell lines; however, only NB4 cells underwent apoptosis. Both epigenetic modulators caused cell cycle arrest in G0/G1 phase as assessed by RT-qPCR (p53, p21, Rb) and flow cytometry analysis. Increased levels of ATM, HMGA2, phosphorylated ATM, and SA-β-galactosidase staining indicated that EGCG caused cellular senescence, whereas BIX-01294 did not. Immunoblot analysis of epigenetic players DNMT1, HP1α, H3K9me3, EZH2, and SUZ12 demonstrated beneficial epigenetic modulation by both agents with exception of mainly no epigenetic changes caused in K562 cells by EGCG. Therefore, we suggest EGCG as a promising epigenetic modulator for acute promyelocytic leukemia therapy and as a potential cellular senescence inducer in both acute and chronic myeloid leukemia treatment, whereas BIX-01294 could be beneficial as an epigenetic modifier for both myeloid leukemias treatment.
MeSH Terms
- Antineoplastic Agents
- Azepines
- Catechin
- Cellular Senescence
- Drug Screening Assays, Antitumor
- Epigenesis, Genetic
- G1 Phase Cell Cycle Checkpoints
- Gene Expression Regulation, Neoplastic
- Humans
- K562 Cells
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive
- Leukemia, Promyelocytic, Acute
- Quinazolines
Keywords
- BIX-01294
- EGCG
- Epigenetic regulation
- Myeloid leukemia
- Senescence
To date, several cell proliferation markers, apoptosis, vascular markers, oncogenes, tumor suppressor genes, cell cycle mediators, microRNA (miRNAs), and long noncoding RNAs (lncRNAs) have been identified to be involved in the tumorigenesis, migration, proliferation and invasiveness of pituitary adenomas. There are still no reliable morphologic markers predictive of pituitary adenoma recurrence. Recent scientific research introduced new techniques to enable us to attain new information on the genesis and biologic behavior of pituitary adenomas. Areas covered: This review covers selected, compelling and cumulative information in regards to TACSTD family (EpCAM, TROP2), neuropilin (NRP-1), oncogene-induced senescence (OIS), fascins (FSCN1), invasion-associated genes (CLDN7, CNTNAP2, ITGA6, JAM3, PTPRC and CTNNA1) EZH2, and ENC1 genes and endocan. Expert commentary: Ongoing research provides clinicians, surgeons and researchers with new information not only on diverse pathways in tumorigenesis but also on the clinical aggressive behavior of pituitary adenomas. Newly developed molecular techniques, bioinformatics and new pharmaceutical drug options are helpful tools to widen the perspectives in our understanding of the complex nature of pituitary tumorigenesis. The discovery of new molecular biomarkers can only be accomplished by continuing to investigate pituitary embryogenesis, histogenesis and tumorigenesis.
Keywords
- ENC1 gene
- EZH2
- FSCN1
- TACSTD family (EpCAM
- TROP2)
- endocan
- invasion-associated genes
- molecular biomarkers
- neuropilin (NRP-1)
- oncogene-induced senescence
- pituitary adenoma
Polycomb group (PcG) factors maintain facultative heterochromatin and mediate many important developmental and differentiation processes. EZH2, a PcG histone H3 lysine-27 methyltransferase, is repressed in senescent cells. We show here that downregulation of EZH2 promotes senescence through two distinct mechanisms. First, depletion of EZH2 in proliferating cells rapidly initiates a DNA damage response prior to a reduction in the levels of H3K27me3 marks. Second, the eventual loss of H3K27me3 induces p16 (CDKN2A) gene expression independent of DNA damage and potently activates genes of the senescence-associated secretory phenotype (SASP). The progressive depletion of H3K27me3 marks can be viewed as a molecular "timer" to provide a window during which cells can repair DNA damage. EZH2 is regulated transcriptionally by WNT and MYC signaling and posttranslationally by DNA damage-triggered protein turnover. These mechanisms provide insights into the processes that generate senescent cells during aging.
MeSH Terms
- Cell Differentiation
- Cellular Senescence
- Chromatin
- Cyclin-Dependent Kinase Inhibitor p16
- DNA Damage
- DNA Replication
- Down-Regulation
- Enhancer of Zeste Homolog 2 Protein
- HEK293 Cells
- Histones
- Humans
- Methylation
- Polycomb-Group Proteins
- Up-Regulation
Keywords
- CDKN1A
- CDKN2A
- DNA damage response
- Polycomb group
- WNT pathway
- cell cycle checkpoints
- cellular senescence
- chromatin
- proinflammatory cytokines
- senescence-associated secretory phenotype
Histone methyltransferases (HMTs) are present in heterogeneous cell populations within the adult brain including neurogenic niches. Yet the question remains whether loss of HMTs and the resulting changes in histone methylation alter cell fate in a region-specific manner. We utilized stereotaxic injection of Cre recombinant protein into the adult neurogenic niches, the subventricular zone (SVZ) adjacent to the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus. We confirmed that Cre protein was enzymatically active in vivo and recombination events were restricted to the vicinity of injection areas. In this study, we focus on using Cre mediated recombination in mice harboring floxed HMT: enhancer of zeste homolog 2 (EZH2) or suppressor of variegation homolog (Suv4-20h). Injectable Cre protein successfully knocked out either EZH2 or Suv4-20h, allowing assessment of long-term effects in a region-specific fashion. We performed meso-scale imaging and flow cytometry for phenotype analysis and unbiased quantification. We demonstrated that regional loss of EZH2 affects the differentiation paradigm of neural stem progenitor cells as well as the maintenance of stem cell population. We further demonstrated that regional loss of Suv4-20h influences the cell cycle but does not affect stem cell differentiation patterns. Therefore, Cre protein mediated knock-out a given HMT unravel their distinguishable and important roles in adult neurogenic niches. This Cre protein-based approach offers tightly-controlled knockouts in multiple cell types simultaneously for studying diverse regulatory mechanisms and is optimal for region-specific manipulation within complex, heterogeneous brain architectures.
MeSH Terms
- Aging
- Animals
- Cell Differentiation
- Cell Movement
- Chromatin
- Enhancer of Zeste Homolog 2 Protein
- Gene Knockout Techniques
- Histone-Lysine N-Methyltransferase
- Histones
- Homologous Recombination
- Integrases
- Lysine
- Methylation
- Mice
- Neurogenesis
- Neurons
- Organ Specificity
- Recombinant Proteins
- S Phase
Keywords
- enhancer of zeste homolog 2 (EZH2)
- subgranular zone
- subventricular zone
- suppressor of variegation homolog (Suv4-20h)
In humans, DNA methylation at specific CpG sites can be used to estimate the 'epigenetic clock', a biomarker of aging and health. The mechanisms that regulate the aging epigenome and level of conservation are not entirely clear. We performed affinity-based enrichment with methyl-CpG binding domain protein followed by high-throughput sequencing (MBD-seq) to assay DNA methylation in mouse samples. Consistent with previous reports, aging is associated with increase in methylation at CpG islands that likely overlap regulatory regions of genes that have been implicated in cancers (e.g., C1ql3, Srd5a2 and Ptk7). The differentially methylated regions in mice have high sequence conservation in humans and the pattern of methylation is also largely conserved between the two species. Based on human ENCODE data, these sites are targeted by polycomb proteins, including EZH2. Chromatin immunoprecipitation confirmed that these regions interact with EZH2 in mice as well, and there may be reduction in EZH2 occupancy with age at C1ql3. This adds to the growing evidence that EZH2 is part of the protein machinery that shapes the aging epigenome. The conservation in both sequence and methylation patterns of the age-dependent CpGs indicate that the epigenetic clock is a fundamental feature of aging in mammals.
MeSH Terms
- 3-Oxo-5-alpha-Steroid 4-Dehydrogenase
- Aging
- Animals
- Cell Adhesion Molecules
- CpG Islands
- DNA Methylation
- Enhancer of Zeste Homolog 2 Protein
- Humans
- Membrane Proteins
- Mice
- Receptor Protein-Tyrosine Kinases
Keywords
- DNA methylation
- EZH2
- Epigenetics
- MBD sequencing
- Polycomb repressive complex
As senescence develops, cells sequentially acquire diverse senescent phenotypes along with simultaneous multistage gene reprogramming. It remains unclear what acts as the key regulator of the collective changes in gene expression at initiation of senescent reprogramming. Here we analyzed time series gene expression profiles obtained in two different senescence models in human diploid fibroblasts: replicative senescence and H O -induced senescence. Our results demonstrate that suppression of DNA methyltransferase 1 (DNMT1)-mediated DNA methylation activity was an initial event prior to the display of senescent phenotypes. We identified seven DNMT1-interacting proteins, ubiquitin-like with PHD and ring finger domains 1 (UHRF1), EZH2, CHEK1, SUV39H1, CBX5, PARP1, and HELLS (also known as LSH (lymphoid-specific helicase) 1), as being commonly down-regulated at the same time point as DNMT1 in both senescence models. Knockdown experiments revealed that, among the DNMT1-interacting proteins, only UHRF1 knockdown suppressed DNMT1 transcription. However, UHRF1 overexpression alone did not induce DNMT1 expression, indicating that UHRF1 was essential but not sufficient for DNMT1 transcription. Although UHRF1 knockdown effectively induced senescence, this was significantly attenuated by DNMT1 overexpression, clearly implicating the UHRF1/DNMT1 axis in senescence. Bioinformatics analysis further identified WNT5A as a downstream effector of UHRF1/DNMT1-mediated senescence. Senescence-associated hypomethylation was found at base pairs -1569 to -1363 from the transcription start site of the WNT5A gene in senescent human diploid fibroblasts. As expected, WNT5A overexpression induced senescent phenotypes. Overall, our results indicate that decreased UHRF1 expression is a key initial event in the suppression of DNMT1-mediated DNA methylation and in the consequent induction of senescence via increasing WNT5A expression.
MeSH Terms
- CCAAT-Enhancer-Binding Proteins
- Cellular Senescence
- DNA (Cytosine-5-)-Methyltransferase 1
- DNA (Cytosine-5-)-Methyltransferases
- DNA Methylation
- Fibroblasts
- Gene Expression Profiling
- Gene Expression Regulation
- HEK293 Cells
- Histones
- Humans
- Hydrogen Peroxide
- Male
- Oligonucleotide Array Sequence Analysis
- Phenotype
- Promoter Regions, Genetic
- Protein Binding
- Protein Domains
- RNA, Small Interfering
- Ubiquitin-Protein Ligases
- Wnt-5a Protein
- beta-Galactosidase
Keywords
- DNA methylation
- cellular senescence
- gene expression
- gene regulation
- microarray
Mesenchymal stem cells (MSCs) are multipotent precursors that can undergo multilineage differentiation, including osteogenesis and adipogenesis, which are two mutually exclusive events. Previously, we demonstrated that enhancer of zeste homolog 2 (EZH2), the catalytic component of the Polycomb-repressive complex 2, mediates epigenetic silencing of histone deacetylase 9c (HDAC9c) in adipocytes but not in osteoblasts and that HDAC9c accelerates osteogenesis while attenuating adipogenesis of MSCs through inactivation of peroxisome proliferator-activated receptor gamma 2 activity. Importantly, disrupting the balance between adipogenesis and osteogenesis can lead to age-associated bone loss (osteoporosis) and obesity. Here, we investigated the relationship between age, and osteogenic and adipogenic differentiation potential of MSCs by comparing EZH2 and HDAC9c expression in osteoblasts and adipocytes of both human and mice origins to determine whether the EZH2-HDAC9c axis regulates age-associated osteoporosis and obesity. Our findings indicated that a decline in HDAC9c expression over time was accompanied by increased EZH2 expression and suggested that a therapeutic intervention for age-associated osteoporosis and obesity may be feasible by targeting the EZH2-HDAC9c axis. Stem Cells 2016;34:2183-2193.
MeSH Terms
- Adipocytes
- Adipogenesis
- Adolescent
- Adult
- Aged
- Aged, 80 and over
- Aging
- Animals
- Cell Differentiation
- Child
- Enhancer of Zeste Homolog 2 Protein
- Gene Knockdown Techniques
- Histone Deacetylases
- Humans
- Mesenchymal Stem Cells
- Mice
- Middle Aged
- Models, Biological
- Osteoblasts
- Osteogenesis
- Repressor Proteins
- Young Adult
Keywords
- Adipogenesis
- Aging
- EZH2
- HDAC9c
- Mesenchymal stem cells
- Osteogenesis
Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by abnormal cellular differentiation and maturation with variable progression to acute leukemia. Over the last decade, scientific discoveries have unraveled specific pathways involved in the complex pathophysiology of MDS. Prominent examples include aberrations in cytokines and their signaling pathways (such as tumor necrosis factor-alpha, interferon-gamma, SMAD proteins), mutations in genes encoding the RNA splicing machinery (SF3B1, SRSF2, ZRSR2, and U2AF1 genes), mutations in genes disrupting the epigenetic machinery (TET2, DNMT3A, DNMT3B, EZH2, ASXL1). In addition, abnormalities in regulatory T-cell dynamics and atypical interactions between the bone marrow microenvironment, stroma and progenitor cells, and abnormal maintenance of telomeres are also notable contributors to the complex pathogenesis of MDS. These pathways represent potential targets for novel therapies. Specific therapies include drugs targeting aberrant DNA methylation and chromatin remodeling, modulating/activating the immune system to enhance tumor-specific cellular immune responses and reduce anomalous cytokine signaling, and blocking abnormal interaction between hematopoietic progenitors and stromal cells.
MeSH Terms
- Animals
- Bone Marrow
- Cellular Microenvironment
- Cellular Senescence
- Cytokines
- DNA Methylation
- Epigenesis, Genetic
- Gene Expression Regulation
- Gene Silencing
- Genetic Variation
- Humans
- Immune System Diseases
- Molecular Targeted Therapy
- Myelodysplastic Syndromes
- RNA Splicing
- Signal Transduction
- Stromal Cells
- Telomere
Keywords
- bone marrow microenvironment
- cellular senescence
- cytokines
- epigenetic regulation
- immune dysregulation
- myelodysplastic syndromes
- pathogenesis
- targeted therapies
- telomeric erosion
The activity of the CDK inhibitor p21 is associated with diverse biological activities, including cell proliferation, senescence, and tumorigenesis. However, the mechanisms governing transcription of p21 need to be extensively studied. In this study, we demonstrate that the high-mobility group box-containing protein 1 (HBP1) transcription factor is a novel activator of p21 that works as part of a complex mechanism during senescence and tumorigenesis. We found that HBP1 activates the p21 gene through enhancing p53 stability by inhibiting Mdm2-mediated ubiquitination of p53, a well known positive regulator of p21. HBP1 was also found to enhance p21 transcription by inhibiting Wnt/β-catenin signaling. We identified histone methyltransferase EZH2, the catalytic subunit of polycomb repressive complex 2, as a target of Wnt/β-catenin signaling. HBP1-mediated repression of EZH2 through Wnt/β-catenin signaling decreased the level of trimethylation of histone H3 at lysine 27 of overall and specific histone on the p21 promoter, resulting in p21 transactivation. Although intricate, the reciprocal partnership of HBP1 and p21 has exceptional importance. HBP1-mediated elevation of p21 through the Mdm2/p53 and TCF4/EZH2 pathways contributes to both cellular senescence and tumor inhibition. Together, our results suggest that the HBP1 transcription factor orchestrates a complex regulation of key genes during cellular senescence and tumorigenesis with an impact on protein ubiquitination and overall histone methylation state.
MeSH Terms
- A549 Cells
- Animals
- Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
- Blotting, Western
- Carcinogenesis
- Cell Line
- Cell Line, Tumor
- Cell Survival
- Cellular Senescence
- Cyclin-Dependent Kinase Inhibitor p21
- Enhancer of Zeste Homolog 2 Protein
- Female
- Gene Expression Regulation
- HEK293 Cells
- Hep G2 Cells
- High Mobility Group Proteins
- Humans
- Mice, Nude
- Protein Binding
- Proto-Oncogene Proteins c-mdm2
- RNA Interference
- Repressor Proteins
- Reverse Transcriptase Polymerase Chain Reaction
- Signal Transduction
- Transcription Factor 4
- Transcription Factors
- Transplantation, Heterologous
- Tumor Suppressor Protein p53
Keywords
- HBP1
- Wnt signaling
- histone methylation
- p21
- p53
- senescence
- tumorigenesis
- ubiquitylation (ubiquitination)
Cholangiocarcinoma, a malignant tumor arising in the hepatobiliary system, presents with poor prognosis because of difficulty in its early detection/diagnosis. Recent progress revealed that cellular senescence may be involved in the pathophysiology of cholangiocarcinoma. Cellular senescence is defined as permanent growth arrest caused by several cellular injuries, such as oncogenic mutations and oxidative stress. "Oncogene-induced" and/or stress-induced senescence may occur in the process of multi-step cholangiocarcinogenesis, and overexpression of a polycomb group protein EZH2 may play a role in the escape from, and/or bypassing of, senescence. Furthermore, senescent cells may play important roles in tumor development and progression via the production of senescence-associated secretory phenotypes. Cellular senescence may be a new target for the prevention, early diagnosis, and therapy of cholangiocarcinoma in the near future.
MeSH Terms
- Animals
- Bile Duct Neoplasms
- Bile Ducts
- Biomarkers, Tumor
- Cellular Senescence
- Cholangiocarcinoma
- Genetic Predisposition to Disease
- Humans
- Mutation
- Phenotype
- Signal Transduction
Keywords
- Cellular senescence
- EZH2
- cholangiocarcinoma
- oncogene-induced senescence
- p16
SIRT1 is likely to play a role in the extension in healthspan induced by dietary restriction. Actions of SIRT1 are pleiotropic, and effects on healthspan may include effects on DNA methylation. Polycomb group protein target genes (PCGTs) are suppressed by epigenetic mechanisms in stem cells, partly through the actions of the polycomb repressive complexes (PRCs), and have been shown previously to correspond with loci particularly susceptible to age-related changes in DNA methylation. We hypothesised that SIRT1 would affect DNA methylation particularly at PCGTs. To map the sites in the genome where SIRT1 affects DNA methylation, we altered SIRT1 expression in human intestinal (Caco-2) and vascular endothelial (HuVEC) cells by transient transfection with an expression construct or with siRNA. DNA was enriched for the methylated fraction then sequenced (HuVEC) or hybridised to a human promoter microarray (Caco-2). The profile of genes where SIRT1 manipulation affected DNA methylation was enriched for PCGTs in both cell lines, thus supporting our hypothesis. SIRT1 knockdown affected the mRNA for none of seven PRC components nor for DNMT1 or DNMT3b. We thus find no evidence that SIRT1 affects DNA methylation at PCGTs by affecting the expression of these gene transcripts. EZH2, a component of PRC2 that can affect DNA methylation through association with DNA methyltransferases (DNMTs), did not co-immunoprecipitate with SIRT1, and SIRT1 knockdown did not affect the expression of EZH2 protein. Thus, it is unlikely that the effects of SIRT1 on DNA methylation at PCGTs are mediated through direct intermolecular association with EZH2 or through effects in its expression. SIRT1 affects DNA methylation across the genome, but particularly at PCGTs. Although the mechanism through which SIRT1 has these effects is yet to be uncovered, this action is likely to contribute to extended healthspan, for example under conditions of dietary restriction.
MeSH Terms
- Aging
- Caco-2 Cells
- DNA (Cytosine-5-)-Methyltransferase 1
- DNA (Cytosine-5-)-Methyltransferases
- DNA Methylation
- DNA-Binding Proteins
- Enhancer of Zeste Homolog 2 Protein
- Epigenesis, Genetic
- Gene Expression Regulation
- Humans
- Polycomb Repressive Complex 2
- Polycomb-Group Proteins
- Promoter Regions, Genetic
- Sirtuin 1
Polycomb group proteins are epigenetic transcriptional repressors that function through recognition and modification of histone methylation and chromatin structure. As a member of PcG proteins, enhancer of zeste homolog 2 (EZH2) targets cell cycle regulatory proteins which govern cell cycle progression and cellular senescence. In previous work, we reported that EZH2 depletion functionally induced cellular senescence in human gastric cancer cells with mutant p53. However, whether EZH2 expression contributes to the change of key cell cycle regulators and the mechanism involved are still unclear. To address this issue, we investigated the effects of EZH2 depletion on alteration of histone methylation pattern. In gastric cancer cells, INK4/ARF locus was activated to certain extent in consequence of a decrease of H3K27me3 along it caused by EZH2 silence, which contributed substantially to an increase in the expression of p15(INK4b), p14(ARF) and p16(INK4a) and resulted in cellular senescence ultimately. Furthermore, MKN28 cells, which did not express p16(INK4a) and p21(cip), could be induced to senescence via p15(INK4b) activation and suppression of p15(INK4b) reversed senescence progression induced by EZH2 downregulated. These data unravel a crucial role of EZH2 in the regulation of INK4/ARF expression and senescence procedure in gastric cancer cells, and show that the cellular senescence could just depend on the activation of p15(INK4b)/Rb pathway, suggesting the cell-type and species specificity involved in the mechanisms of senescence inducement.
MeSH Terms
- Cell Cycle
- Cell Line, Tumor
- Cell Proliferation
- Cellular Senescence
- Cyclin-Dependent Kinase Inhibitor p15
- Cyclin-Dependent Kinase Inhibitor p16
- Enhancer of Zeste Homolog 2 Protein
- Histones
- Humans
- Methylation
- Polycomb Repressive Complex 2
- Stomach Neoplasms
- Tumor Suppressor Protein p14ARF
Keywords
- Cell cycle
- Enhancer of zeste homolog 2
- Gastric cancer
- Histone H3 Lys27 trimethylation
- INK4/ARF
- Senescence
Although age-associated gene expression and methylation changes have been reported throughout the literature, the unifying epigenomic principles of aging remain poorly understood. Recent explosion in availability and resolution of functional/regulatory genome annotation data (epigenomic data), such as that provided by the ENCODE and Roadmap Epigenomics projects, provides an opportunity for the identification of epigenomic mechanisms potentially altered by age-associated differentially methylated regions (aDMRs) and regulatory signatures in the promoters of age-associated genes (aGENs). In this study we found that aDMRs and aGENs identified in multiple independent studies share a common Polycomb Repressive Complex 2 signature marked by EZH2, SUZ12, CTCF binding sites, repressive H3K27me3, and activating H3K4me1 histone modification marks, and a "poised promoter" chromatin state. This signature is depleted in RNA Polymerase II-associated transcription factor binding sites, activating H3K79me2, H3K36me3, H3K27ac marks, and an "active promoter" chromatin state. The PRC2 signature was shown to be generally stable across cell types. When considering the directionality of methylation changes, we found the PRC2 signature to be associated with aDMRs hypermethylated with age, while hypomethylated aDMRs were associated with enhancers. In contrast, aGENs were associated with the PRC2 signature independently of the directionality of gene expression changes. In this study we demonstrate that the PRC2 signature is the common epigenomic context of genomic regions associated with hypermethylation and gene expression changes in aging.
MeSH Terms
- Aging
- Binding Sites
- CCCTC-Binding Factor
- Cell Line
- Chromatin
- DNA Methylation
- Enhancer of Zeste Homolog 2 Protein
- Epigenomics
- Gene Expression Regulation
- Histones
- Humans
- Polycomb Repressive Complex 2
- Promoter Regions, Genetic
- Protein Binding
- Protein Structure, Tertiary
- Repressor Proteins
Keywords
- ENCODE
- ENCODE, Encyclopedia of DNA elements
- GenomeRunner
- PRC2
- PRC2, Polycomb repressive complex 2
- TFBS, transcription factor binding site
- aDMR, age-associated differentially methylated region
- aGEN, promoter of an age-associated gene
- aging
- epigenetics
- epigenomics
- methylation
- polycomb
A series of novel quinazolinone hybrids were synthesized by employing click chemistry and evaluated for anti-proliferative activities against MCF-7, HeLa and K562 cell lines. Among these cell lines, HeLa cells were found to respond effectively to these quinazolinone hybrids with IC50 values ranging from 5.94 to 16.45 μM. Some of the hybrids (4q, 4r, 4e, 4k, 4t, 4w) with promising anti-cancer activity were further investigated for their effects on the cell cycle distribution. FACS analysis revealed the G1 cell cycle arrest nature of these hybrids. Further to assess the senescence inducing ability of these compounds, a senescence associated β-gal assay was performed. The senescence inducing nature of these compounds was supported by the effect of hybrid (4q) on p16 promoter activity, the marker for senescence. Moreover, cells treated with most effective compound (4q) show up-regulation of p53, p21 and down-regulation of HDAC-1, HDAC-2, HDAC-5 and EZH2 mRNA levels. Docking results suggest that, the triazole nitrogen showed Zn( 2) mediated interactions with the histidine residue of HDACs.
MeSH Terms
- Apoptosis
- Cell Cycle
- Cellular Senescence
- Click Chemistry
- Enhancer of Zeste Homolog 2 Protein
- HeLa Cells
- Histone Deacetylase 1
- Histone Deacetylase 2
- Histone Deacetylase Inhibitors
- Histone Deacetylases
- Humans
- Inhibitory Concentration 50
- K562 Cells
- MCF-7 Cells
- Molecular Docking Simulation
- Polycomb Repressive Complex 2
- Pyrroles
- Quinazolinones
- Quinones
- Tumor Suppressor Protein p53
- Tumor Suppressor Proteins
Keywords
- Click chemistry
- Docking studies
- HDACs
- Quinazolinone hybrids
- Senescence
Inadequate functional β cell mass underlies both type 1 and type 2 diabetes. β Cell growth and regeneration also decrease with age through mechanisms that are not fully understood. Age-dependent loss of enhancer of zeste homolog 2 (EZH2) prevents adult β cell replication through derepression of the gene encoding cyclin-dependent kinase inhibitor 2a (INK4a). We investigated whether replenishing EZH2 could reverse the age-dependent increase of Ink4a transcription. We generated an inducible pancreatic β cell-specific Ezh2 transgenic mouse model and showed that transgene expression of Ezh2 was sufficient to increase β cell replication and regeneration in young adult mice. In mice older than 8 months, induction of Ezh2 was unable to repress Ink4a. Older mice had an enrichment of a trithorax group (TrxG) protein complex at the Ink4a locus. Knockdown of TrxG complex components, in conjunction with expression of Ezh2, resulted in Ink4a repression and increased replication of β cells in aged mice. These results indicate that combined modulation of polycomb group proteins, such as EZH2, along with TrxG proteins to repress Ink4a can rejuvenate the replication capacity of aged β cells. This study provides potential therapeutic targets for expansion of adult β cell mass.
MeSH Terms
- Aging
- Animals
- Cell Proliferation
- Cyclin-Dependent Kinase Inhibitor p16
- Diabetes Mellitus
- Enhancer of Zeste Homolog 2 Protein
- Gene Expression
- Gene Knockdown Techniques
- Histone-Lysine N-Methyltransferase
- Humans
- Insulin-Secreting Cells
- Jumonji Domain-Containing Histone Demethylases
- Mice
- Mice, Inbred C57BL
- Mice, Inbred DBA
- Mice, Transgenic
- Myeloid-Lymphoid Leukemia Protein
- Polycomb Repressive Complex 2
- Polycomb-Group Proteins
- RNA, Messenger
With increasing age, T cells gain expression of killer immunoglobulin-like receptors (KIRs) that transmit negative signals and dampen the immune response. KIR expression is induced in CD4 and CD8 T cells by CpG DNA demethylation suggesting epigenetic control. To define the mechanisms that underlie the age-associated preferential KIR expression in CD8 T cells, we examined KIR2DL3 promoter methylation patterns. With age, CD8 T cells developed a patchy and stochastic promoter demethylation even in cells that did not express the KIR2DL3-encoded CD158b protein; complete demethylation of the minimal KIR2DL3 promoter was characteristic for CD158b-expressing cells. In contrast, the promoter in CD4 T cells was fully methylated irrespective of age. The selectivity for CD8 T cells correlated with lower DNMT1 recruitment to the KIR2DL3 promoter which further diminished with age. In contrast, binding of the polycomb protein EZH2 known to be involved in DNMT1 recruitment was not different. Our data suggest that CD8 T cells endure increasing displacement of DNMT1 from the KIR promoter with age, possibly because of an active histone signature. The ensuing partial demethylation lowers the threshold for transcriptional activation and renders CD8 T cells more susceptible to express KIR, thereby contributing to the immune defect in the elderly.
MeSH Terms
- Adult
- Aged
- Aged, 80 and over
- Aging
- CD4-Positive T-Lymphocytes
- CD8-Positive T-Lymphocytes
- CpG Islands
- DNA (Cytosine-5-)-Methyltransferase 1
- DNA (Cytosine-5-)-Methyltransferases
- DNA Methylation
- DNA-Binding Proteins
- Enhancer of Zeste Homolog 2 Protein
- Epigenesis, Genetic
- Female
- Humans
- Male
- Polycomb Repressive Complex 2
- Promoter Regions, Genetic
- Receptors, KIR2DL3
- Transcription Factors
Cellular senescence, an irreversible growth arrest, is considered to play as safeguard against malignant progression, though such a mechanism is speculative in human carcinogenesis. In gallbladder carcinoma, cholecystolithiasis and pancreaticobiliary maljunction (PBM) are major risk factors. Here, by using 113 surgically resected gallbladders and cultures of human gallbladder epithelial cells (HGECs) and gallbladder carcinoma cell line (TGBC2TKB), we examined carcinogenesis with respect to cellular senescence. Among 15 cases of PBM in which carcinoma was found in 4 cases, nonneoplastic gallbladder mucosa showed diffuse papillary hyperplasia (PHP). PHP was not found in gallbladders with cholecystolithiasis. Interestingly, PHP exhibited senescent features such as expression of p16(INK4A) and low cell proliferative activity. In contrast, EZH2, a polycomb group protein, was overexpressed in intraepithelial neoplasm and carcinoma in gallbladders with cholecystolithiasis. In PBM, EZH2 was expressed only in carcinoma foci but not in PHP. Cultured HGECs treated with lysolecithin, the level of which is elevated in gallbladder bile of PBM, showed increased expression of p16(INK4A) and senescence-associated beta-galactosidase. Conversely, enforced overexpression of EZH2 in senescent HGECs reduced p16(INK4A) expression. A knockdown of EZH2 in cultured TGBC2TKB cells increased p16(INK4a) expression. In conclusion, PHP in PBM may act as a barrier to malignant transformation for decades. EZH2 may be responsible for the escape from cellular senescence followed by malignant transformation in the gallbladder of PBM.
MeSH Terms
- Adolescent
- Adult
- Aged
- Aged, 80 and over
- Aging
- Carcinoma in Situ
- Cell Line, Tumor
- Cell Proliferation
- Cell Transformation, Neoplastic
- Cholecystolithiasis
- Common Bile Duct
- Common Bile Duct Diseases
- Cyclin-Dependent Kinase Inhibitor p16
- DNA-Binding Proteins
- Enhancer of Zeste Homolog 2 Protein
- Epithelial Cells
- Female
- Gallbladder
- Gallbladder Neoplasms
- Gene Silencing
- Humans
- Hyperplasia
- Lysophosphatidylcholines
- Male
- Middle Aged
- Pancreatic Diseases
- Pancreatic Ducts
- Polycomb Repressive Complex 2
- Transcription Factors
- Young Adult