BACKGROUND: P16 inactivation is frequently accompanied by telomerase reverse transcriptase ( TERT ) amplification in human cancer genomes. P16 inactivation by DNA methylation often occurs automatically during immortalization of normal cells by TERT . However, direct evidence remains to be obtained to support the causal effect of epigenetic changes, such as P16 methylation, on cancer development. This study aimed to provide experimental evidence that P16 methylation directly drives cancer development. METHODS: A zinc finger protein-based P16 -specific DNA methyltransferase (P16-Dnmt) vector containing a "Tet-On" switch was used to induce extensive methylation of P16 CpG islands in normal human fibroblast CCD-18Co cells. Battery assays were used to evaluate cell immortalization and transformation throughout their lifespan. Cell subcloning and DNA barcoding were used to track the diversity of cell evolution. RESULTS: Leaking P16-Dnmt expression (without doxycycline-induction) could specifically inactivate P16 expression by DNA methylation. P16 methylation only promoted proliferation and prolonged lifespan but did not induce immortalization of CCD-18Co cells. Notably, cell immortalization, loss of contact inhibition, and anchorage-independent growth were always prevalent in P16-Dnmt&TERT cells, indicating cell transformation. In contrast, almost all TERT cells died in the replicative crisis. Only a few TERT cells recovered from the crisis, in which spontaneous P16 inactivation by DNA methylation occurred. Furthermore, the subclone formation capacity of P16-Dnmt&TERT cells was two-fold that of TERT cells. DNA barcoding analysis showed that the diversity of the P16-Dnmt&TERT cell population was much greater than that of the TERT cell population. CONCLUSION P16 methylation drives TERT -mediated immortalization and transformation of normal human cells that may contribute to cancer development.
Humans ; Telomerase/genetics* ; DNA Methylation/physiology* ; Fibroblasts/cytology* ; Cyclin-Dependent Kinase Inhibitor p16/metabolism* ; Cell Line ; Cell Transformation, Neoplastic/genetics*

BACKGROUND: The main cause of restenosis after percutaneous transluminal angioplasty (PTA) is the excessive proliferation and migration of vascular smooth muscle cells (VSMCs). Lin28a has been reported to play critical regulatory roles in this process. However, whether CCAAT/enhancer-binding proteins β (C/EBPβ) binds to the Lin28a promoter and drives the progression of restenosis has not been clarified. Therefore, in the present study, we aim to clarify the role of C/EBPβ-Lin28a axis in restenosis. METHODS: Restenosis and atherosclerosis rat models of type 2 diabetes ( n   =  20, for each group) were established by subjecting to PTA. Subsequently, the difference in DNA methylation status and expression of C/EBPβ between the two groups were assessed. EdU, Transwell, and rescue assays were performed to assess the effect of C/EBPβ on the proliferation and migration of VSMCs. DNA methylation status was further assessed using Methyltarget sequencing. The interaction between Lin28a and ten-eleven translocation 1 (TET1) was analysed using co-immunoprecipitation (Co-IP) assay. Student's t -test and one-way analysis of variance were used for statistical analysis. RESULTS: C/EBPβ expression was upregulated and accompanied by hypomethylation of its promoter in restenosis when compared with atherosclerosis. In vitroC/EBPβ overexpression facilitated the proliferation and migration of VSMCs and was associated with increased Lin28a expression. Conversely, C/EBPβ knockdown resulted in the opposite effects. Chromatin immunoprecipitation assays further demonstrated that C/EBPβ could directly bind to Lin28a promoter. Increased C/EBPβ expression and enhanced proliferation and migration of VSMCs were observed after decitabine treatment. Further, mechanical stretch promoted C/EBPβ and Lin28a expression accompanied by C/EBPβ hypomethylation. Additionally, Lin28a overexpression reduced C/EBPβ methylation via recruiting TET1 and enhanced C/EBPβ-mediated proliferation and migration of VSMCs. The opposite was noted in Lin28a knockdown cells. CONCLUSION Our findings suggest that the C/EBPβ-Lin28a axis is a driver of restenosis progression, and presents a promising therapeutic target for restenosis.
Animals ; Cell Proliferation/genetics* ; Cell Movement/genetics* ; Muscle, Smooth, Vascular/metabolism* ; Rats ; DNA Methylation/physiology* ; CCAAT-Enhancer-Binding Protein-beta/genetics* ; Male ; Myocytes, Smooth Muscle/cytology* ; Rats, Sprague-Dawley ; RNA-Binding Proteins/genetics* ; Cells, Cultured ; Coronary Restenosis/metabolism*

Mammalian fertilization begins with the fusion of two specialized gametes, followed by major epigenetic remodeling leading to the formation of a totipotent embryo. During the development of the pre-implantation embryo, precise reprogramming progress is a prerequisite for avoiding developmental defects or embryonic lethality, but the underlying molecular mechanisms remain elusive. For the past few years, unprecedented breakthroughs have been made in mapping the regulatory network of dynamic epigenomes during mammalian early embryo development, taking advantage of multiple advances and innovations in low-input genome-wide chromatin analysis technologies. The aim of this review is to highlight the most recent progress in understanding the mechanisms of epigenetic remodeling during early embryogenesis in mammals, including DNA methylation, histone modifications, chromatin accessibility and 3D chromatin organization.
Animals ; Chromatin Assembly and Disassembly ; DNA Methylation ; DNA Transposable Elements ; Embryo, Mammalian ; Embryonic Development/genetics* ; Epigenesis, Genetic ; Epigenome ; Female ; Fertilization/physiology* ; Gene Expression Regulation, Developmental ; Histone Code ; Histones/metabolism* ; Male ; Mice ; Oocytes/metabolism* ; Spermatozoa/metabolism*

Mixed linked leukemia 4 (MLL4) is a specific methyltransferase of histone 3 position lysine 4 (H3K4). It is also one of the important members of COMPASS/Set1-like protein complex. Both MLL4 protein itself and its mediated H3K4 methylation modification can cause changes in chromatin structure and function, thus regulating gene transcription and expression. With the studies of MLL4 protein in recent years, the roles of MLL4 gene, MLL4 protein and protein complex in the development of tissues and organs, tumor diseases and other physiological and pathophysiological processes have been gradually revealed. In this paper, the research progress of MLL4 gene, MLL4 protein characteristics, biological function and its effect on disease were reviewed, in order to further understand the effect of histone methyltransferase on gene expression regulation, as well as its non-enzyme dependent function. This paper may provide new ideas for the prevention, diagnosis and treatment of related diseases.
DNA-Binding Proteins ; physiology ; Histone-Lysine N-Methyltransferase ; physiology ; Histones ; chemistry ; Humans ; Methylation

OBJECTIVE: To investigate the effects of Zhizi Chuanxiong Capsule (ZCC, ) on abnormal DNA methylation in a rabbit model of atherosclerosis (AS). METHODS: After 1 week of adaptive feeding, 48 New Zealand white rabbits were randomly divided into 4 groups: a control group (n=12) fed with normal diet for 22 weeks; a model group (n=12) fed with high fat diet for 14 weeks followed by 8 weeks of normal diet feeding; a low-dose ZCC group (n=12) fed with high fat diet and low-dose ZCC for 14 weeks, followed by 8 weeks of normal diet and low-dose drug; a high-dose ZCC group (n=12) fed with high fat diet and high-dose drug for 14 weeks, followed by 8 weeks of normal diet and high-dose drug. After 22 weeks of feeding, blood samples were taken from the rabbit ear vein, and the genomic DNA was extracted for methylation immunoprecipitation sequencing (Medip-seq). The aorta tissues were collected for hematoxylin-eosin (HE) staining. RESULTS Eight rabbits died during the feeding process. HE staining showed that the size of the lipid deposition on vessel wall and atherosclerotic plaque formation were reduced in both low- and high-dose group. The Medip-seq results showed that there were 146 abnormally methylated genes (including both hypermethylated gene and hypomethylated genes) in the model group, compared with the control group. Gene Ontology (GO) and Pathway analysis showed that these abnormally methylated genes were found to be involved in multiple AS-related functions and pathways, such as protein kinase C activity, cholesterol transport, mitogen-activated protein kinase (MAPK) signaling pathway, peroxisome proliferater-activated receptor signaling pathway, vascular smooth muscle contraction, inflammation and so on. The abnormal methylated genes in AS model group were altered in both low- and high-dose groups: low-dose ZCC could change 72 of the 146 abnormally methylated genes, high-dose ZCC could change 71. Through GO and Pathway analysis, these altered methylated genes were involved in protein kinase C activity, inflammatory pathway, MAPK signaling pathway, vascular endothelial growth factor signaling pathway, etc. CONCLUSION: ZCC could treat AS through regulating the abnormal hypermethylated and hypomethylated genes in AS rabbit model.
Animals ; Atherosclerosis ; drug therapy ; genetics ; Capsules ; DNA Methylation ; drug effects ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; MAP Kinase Signaling System ; drug effects ; Male ; Rabbits ; Vascular Endothelial Growth Factor A ; physiology

The study illustrate the inner correlation between global DNA methylation variation and different birth weights. Infant birth weight was used to identify cases and controls. Cord blood and placentas were collected. We performed DNA methylation profiling of bisulphite-converted DNA. We have identified many differentially methylated CpG sites in experimental groups; these sites involved in hundreds of signalings. Among these, more than ten pathways were referred to the glucose and lipid metabolism. Methylation changes in the insulin-signaling pathway (ISP), adipocytokine signaling pathway (ASP) and MAPK signaling pathway are involved in the fetal programming of diabetes..
Birth Weight ; DNA Methylation ; Female ; Gene Expression Regulation, Developmental ; physiology ; Genome-Wide Association Study ; Humans ; Infant, Newborn ; Male ; Organ Size ; Placenta ; anatomy & histology ; Pregnancy ; Signal Transduction

Human physiology and pathology can be affected by different nutritional conditions. At cellular level, the availability of a nutritional component not only mediates metabolic reactions but also transmits signals for diverse biological activities. Epigenetic regulation such as DNA methylation and histone post-translational modifications is considered as one of the nutrient-mediated signaling receivers as almost all of the epigenetic enzyme activities require intermediary metabolites as cofactors. The gut microbiome as “forgotten organ” has been suggested as a metabolite generator as well as a nutrient sensor for its host organism, affecting human health and diseases. Given the metabolite-dependent activities of epigenetic regulators, the gut microbiome has a high potential to influence the epigenetics in human physiology. Here, I review the involvement of gut microbiome in diverse human diseases and the mechanisms of epigenetic regulation by different metabolites. Thereafter, I discuss how the gut microbiome-generated metabolites affect host epigenetics, raising a possibility to develop a therapeutic intervention based on the interaction between the microbiome and epigenetics for human health.
DNA Methylation ; Epigenomics* ; Gastrointestinal Microbiome* ; Histones ; Humans* ; Metabolism ; Microbiota ; Pathology ; Physiology ; Protein Processing, Post-Translational

Epigenetic factors play an important role in male infertility though about 60%－65% of the disease is idiopathic and its underlying causes are not yet clear. Many studies have indicated that epigenetic modifications, including DNA methylation, histone tail modifications, chromatin remodeling, and non-coding RNAs, may be involved in idiopathic male infertility. Abnormal methylation is associated with decreased sperm quality and fertility. It is known that 1 881 miRNAs are related to male fertility and such non-coding RNAs as piRNA, IncRNA, and circRNA play a regulating role in male reproduction. This review focuses on the value of epigenetics in the etiology and pathogenesis of male infertility, aiming to provide some evidence for the establishment of some strategies for the treatment and prediction of the disease.
DNA Methylation ; Epigenesis, Genetic ; Fertility ; Humans ; Infertility, Male ; genetics ; Male ; MicroRNAs ; physiology ; RNA, Small Interfering ; Spermatozoa

The occurrence and development of acute myeloid leukemia (AML) is not only related to gene mutations, but also influenced by abnormal epigenetic regulation, in which DNA methylation is one of the most important mechanisms. Abnormal DNA methylation may lead to the activation of oncogene and the inactivation of tumor suppressor gene, resulting in the occurrence of leukemia. The mutations of DNA methylation enzymes associated with AML may have certain characteristics. The AML with recurrent cytogenetic abnormalities is also related to abnormal methylation. Some fusion genes can alter DNA methylation status to participate in the pathogenesis of leukemia. In addition, chemotherapy drug resistance in patients with AML is associated with the change of gene methylation status. Considering the reversibility of the epigenetic modification, targeted methylation therapy has become a hotspot of AML research.
DNA Methylation ; drug effects ; genetics ; physiology ; DNA Modification Methylases ; genetics ; physiology ; Drug Resistance, Neoplasm ; genetics ; Epigenesis, Genetic ; genetics ; physiology ; Humans ; Leukemia, Myeloid, Acute ; etiology ; genetics ; pathology ; Mutation ; genetics

BACKGROUNDThe acute myeloid leukemia 1 (AML1)-eight-twenty-one (ETO) fusion protein generated by the t(8;21)(q22;q22) translocation is considered to display a crucial role in leukemogenesis in AML. By focusing on the anti-leukemia effects of eyes absent 4 (EYA4) gene on AML cells, we investigated the biologic and molecular mechanism associated with AML1-ETO expressed in t(8;21) AML.

METHODSQualitative polymerase chain reaction (PCR), quantitative reverse transcription PCR (RT-PCR), and Western blotting analysis were used to observe the mRNA and protein expression levels of EYA4 in cell lines. Different plasmids (including mutant plasmids) of dual luciferase reporter vector were built to study the binding status of AML1-ETO to the promoter region of EYA4. Chromatin immunoprecipitation assay was used to study the epigenetic silencing mechanism of EYA4. Bisulfite sequencing was applied to detect the methylation status in EYA4 promoter region. The influence of EYA4 gene in the cell proliferation, apoptosis, and cell clone-forming ability was detected by the technique of Cell Counting Kit-8, flow cytometry, and clonogenic assay.

RESULTSEYA4 gene was hypermethylated in AML1-ETO+ patients and its expression was down-regulated by 6-fold in Kasumi-1 and SKNO-1 cells, compared to HL-60 and SKNO-1-siA/E cells, respectively. We demonstrated that AML1-ETO triggered the epigenetic silencing of EYA4 gene by binding at AML1-binding sites and recruiting histone deacetylase 1 and DNA methyltransferases. Enhanced EYA4 expression levels inhibited cellular proliferation and suppressed cell colony formation in AML1-ETO+ cell lines. We also found EYA4 transfection increased apoptosis of Kasumi-1 and SKNO-1 cells by 1.6-fold and 1.4-fold compared to negative control, respectively.

CONCLUSIONSOur study identified EYA4 gene as targets for AML1-ETO and indicated it as a novel tumor suppressor gene. In addition, we provided evidence that EYA4 gene might be a novel therapeutic target and a potential candidate for treating AML1-ETO+ t (8;21) AML.

Apoptosis ; genetics ; physiology ; Blotting, Western ; Cell Line, Tumor ; Cell Proliferation ; genetics ; physiology ; Chromatin Immunoprecipitation ; Core Binding Factor Alpha 2 Subunit ; genetics ; metabolism ; DNA Methylation ; genetics ; Epigenesis, Genetic ; genetics ; Gene Silencing ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; pathology ; Oncogene Proteins, Fusion ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; RUNX1 Translocation Partner 1 Protein ; Radioimmunoprecipitation Assay ; Trans-Activators ; genetics ; metabolism