DNA methylation-mediated expression of zinc finger protein 615 affects embryonic development in Bombyxmori

DNA (cytosine-5) methylation represents one of the most widely used mechanisms of enduring cellular memory. Stable patterns of DNA methylation are established during development, resulting in creation of persisting cellular phenotypes. There is growing evidence that the nervous system has co-opted a number of cellular mechanisms used during development to subserve the formation of long term memory. In this study, we examined the role DNA (cytosine-5) methyltransferase (DNMT) activity might play in regulating the induction of synaptic plasticity. We found that the DNA within promoters for reelin and brain-derived neurotrophic factor, genes implicated in the induction of synaptic plasticity in the adult hippocampus, exhibited rapid and dramatic changes in cytosine methylation when DNMT activity was inhibited. Moreover, zebularine and 5-aza-2-deoxycytidine, inhibitors of DNMT activity, blocked the induction of long term potentiation at Schaffer collateral synapses. Activation of protein kinase C in the hippocampus decreased reelin promoter methylation and increased DNMT3A gene expression. Interestingly, DNMT activity is required for protein kinase C-induced increases in histone H3 acetylation. Considered together, these results suggest that DNMT activity is dynamically regulated in the adult nervous system and that DNMT may play a role in regulating the induction of synaptic plasticity in the mature CNS.

Inheritance of DNA cytosine methylation pattern during successive cell division is mediated by maintenance DNA (cytosine-5) methyltransferase 1 (DNMT1). Lysine 142 of DNMT1 is methylated by the SET domain containing lysine methyltransferase 7 (SET7), leading to its degradation by proteasome. Here we show that PHD finger protein 20-like 1 (PHF20L1) regulates DNMT1 turnover in mammalian cells. Malignant brain tumor (MBT) domain of PHF20L1 binds to monomethylated lysine 142 on DNMT1 (DNMT1K142me1) and colocalizes at the perinucleolar space in a SET7-dependent manner. PHF20L1 knockdown by siRNA resulted in decreased amounts of DNMT1 on chromatin. Ubiquitination of DNMT1K142me1 was abolished by overexpression of PHF20L1, suggesting that its binding may block proteasomal degradation of DNMT1K142me1. Conversely, siRNA-mediated knockdown of PHF20L1 or incubation of a small molecule MBT domain binding inhibitor in cultured cells accelerated the proteasomal degradation of DNMT1. These results demonstrate that the MBT domain of PHF20L1 reads and controls enzyme levels of methylated DNMT1 in cells, thus representing a novel antagonist of DNMT1 degradation.

Epigenetics in evolution and disease

Epigenetic regulation of cellular identity and function is at least partly achieved through changes in covalent modifications on DNA and histones. Much progress has been made in recent years to understand how these covalent modifications affect cell identity and function. Despite the advances, whether and how epigenetic factors contribute to memory formation is still poorly understood. In this review, we discuss recent progress in elucidating epigenetic mechanisms of learning and memory, primarily at the DNA level, and look ahead to discuss their potential implications in reward memory and development of drug addiction.

Promoter-targeted siRNAs Induce Gene Silencing of Simian Immunodeficiency Virus (SIV) Infection In Vitro

Aberrant DNA methylation is commonly observed in cancer and is characterized by genome-wide hypomethylation and gene-specific hypermethylation, which is thought to contribute to genomic instability and tumor suppressor gene silencing, respectively. The DNA methyltransferases (DNMTs) are responsible for the establishment (DNMT3A, DNMT3B, DNMT3L) and maintenance (DNMT1) of DNA methylation patterns genome-wide. The mechanism by which DNA methylation patterns are altered in cancer is not well understood. Genome-wide unique and overlapping target sites for each of the DNMTs are also unknown both in normal and cancer states. Identification of DNMT target loci is essential in order to better understand how aberrant DNA methylation occurs in cancer. To study this process, DNMT mRNA levels were depleted both individually and in a combinatorial fashion via RNAi-based techniques in embryonic carcinoma cells. Following reduction in DNMT expression, the resulting DNA and RNA was analyzed for genome-wide DNA methylation and gene expression patterns, respectively. An affinity purification method, Methyl-Binding Domain (MBD)-seq, was used to capture and enrich methylated regions of the genome by utilizing the methyl-binding domain of MBD2b. The enriched methylated DNA was then used to construct an Illumina sequencing library. Global gene expression patterns were analyzed by microarray for each RNA sample. Next generation sequencing and microarray data were analyzed using several available algorithms (e.g. MACS) and high-throughput data software packages (e.g. Partek Genomics Suite) in order to construct and evaluate the DNA methylation profiles and subsequent gene expression changes that result from DNMT depletion. Our data show that distinct changes in DNA methylation profiles occur among the various DNMT knockdown samples that permit us to identify unique and cooperative target loci for each DNMT. Further elucidation of DNMT target sites holds great promise for enhancing our understanding of mechanisms that control aberrant DNA methylation that is observed in cancer as well as provide insight and rationale for targeting specific DNMTs in cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5009. doi:1538-7445.AM2012-5009

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) pathway is a critical intermediary for cell proliferation, differentiation, and survival. In the human colon cancer cell line SW1116, treatment with the DNA methyltransferase 1 (DNMT1) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) or the ERK-MAPK inhibitors PD98059 or rottlerin, or transient transfection with the MAP/ERK kinase (MEK)1/2 small interfering RNA down-regulates DNMT1 and proliferating cell nuclear antigen levels. In this report, we found that drug treatment or small interfering RNA transfection of SW1116 cells induced promoter demethylation of the p16(INK4A) and p21(WAF1) genes, which up-regulated their mRNA and protein expression levels. Flow cytometry revealed that rottlerin treatment induced cell cycle arrest at phase G(1) (p < 0.05). Thus, the ERK-MAPK inhibitor treatment or siRNA-mediated knockdown of ERK-MAPK decreases DNA methylation via down-regulating DNMT1 expression and other unknown mediator(s) in SW1116 colon cancer cells.

In 2014, Facebook and Apple announced that they would pay for female employees to have their oocytes frozen to allow them to delay having children and instead focus on their careers. Whatever motivated the companies to make their offers, the fact that they did so highlights a prevalent problem faced by many young women: Their most fertile years are also a crucial period for building a career, when time off work may disadvantage them. > … cryopreservation is known to affect cell survival after thawing, which can have an impact on the subsequent clinical applications of frozen cells. To fulfill their offers, Facebook and Apple will need to offer their employees access to cryopreservation technologies that profoundly change the dynamics of family planning. Such technologies are not new, but work over the past decades has been aimed at increasing safety and efficacy and has reduced costs to the point that companies can now offer cryopreservation as a way to attract and retain female workers. Of course, the potential of cryopreservation goes far beyond freezing the eggs or sperm of ambitious young technology workers—it is a ubiquitous technology used in research and medicine for a wide variety of applications (Fig 1). For example, cryopreservation is used to store and transport biological material, including adult stem cells or stem cells from umbilical cord blood or bone marrow—both of which can later be used to treat disease or extend lifespan in the same patient—blood donations, especially of rare blood types, tissues, and organs. It is also offered as a crucial service for cancer patients to preserve their gametes before they undergo therapy that may render them infertile and, generally used in assisted reproduction to store oocytes, fertilized eggs, or embryos. Cryopreservation can contribute to environmental preservation efforts, where it is used to conserve the …

Oxidative Damage Targets Complexes Containing DNA Methyltransferases, SIRT1, and Polycomb Members to Promoter CpG Islands

Effects of DNA Methylation on Progression to Interstitial Fibrosis and Tubular Atrophy in Renal Allograft Biopsies: A Multi-Omics Approach.

DNA methylation naturally happens at the fifth carbon position of cytosine base within the CpG dinucleotides, plays a significant role in the numerous biological events, such as gene expression, cellular proliferation, embryonic developments, and chromosome instability. Aberrancies in DNA methylation pattern can lead to the genomic instability, resulting in the development of various human diseases including cancer, considered as one of the promising epigenetic (diagnostic and prognostic) biomarkers. Current research shows that abnormality in DNA methylation pattern presents a signature for disease diagnosis, therapeutic interventions, and prognosis of outcome. Therefore, current DNA methylation research has a major focus and needs for the development of easy, reliable and sensitive detection strategies. Throughout the last few decades, extensive research has been reported towards the quantification of DNA methylation in the mammalian genome. However, having their respective advantages in analytical performance and reliability, most of these strategies are confined to laboratory-based molecular biology techniques, such as real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), microarrays, and sequence-based methods. These methods largely require either bisulfite treatment (BT) or specific restriction enzyme digestion followed by a subsequence DNA amplification or sequencing. BT converts unmethylated cytosine to uracils leaving methylated cytosine unchanged, leveraging the methylated fragments distinguishable for detection. Low conversion, non-specific responses, false reading, longer assay time, amplification biased and complex chemistries significantly reducing the practice of BT and PCR amplification in current DNA methylation analysis. Hence, development of new analytical techniques without the BT steps and PCR amplification could be a valuable and out-of-bench tool for sensitive, low-cost and rapid platform to accomplish the emerging demand in genome-wide DNA methylation analysis in clinics. In this regard, bisulfite-free electrochemical biosensors have gained much attention in recent years as electrochemistry offers sensitive, cost-effective, portable, and simple biomolecules recognition readout. Parallel with electrochemistry, biosensors with optical readout enable direct real-time detection of biological molecules and are easily adaptable to multiplexing. Incorporation of electrochemical and optical readouts to bisulfite-free DNA methylation analysis is paving the way for translation of this important biomarker to standard patient care even resource-poor settings. This PhD thesis explores various electrochemistry along with colorimetric approach based sensitive, specific, rapid and inexpensive biosensor platform for bisulfitefree global DNA methylation analysis. Moreover, a series of commercial and in-house synthesised novel superparamagnetic nanomaterials were integrated to enhance the sensitivity and portability of the detection platform. A comprehensive literature review entailing detailed mechanism in DNA methylation pattern, association of DNA methylation with various human diseases, progress in DNA methylation biosensors techniques with a special emphasis on electrochemical and optical detection platforms have been reported. The challenges associated with current strategies have been outlined, and a great deal of recommendations also addressed to overcome the existing techniques. In the following chapters, four novel DNA methylation analysis strategies have reported based on colorimetry, electrochemistry and engineered nanostructures based inorganic enzymes (nanozymes) for the sensitive, rapid, and inexpensive analysis of global DNA methylation. First, based on the nucleic-acid affinity with the gold surface and methylation site-specific 5-mC antibody conjugated with horseradish peroxidase (HRP, natural enzyme), an amplification-free electrochemical and colorimetric assay was developed for the detection of global DNA methylation. In this strategy, the methylationsites to 5-mC antibody recognition were carried out on a screen-printed electrode surface and HRP catalysed 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation were employed to read out the recognition event. The widely used colorimetry and ultra-sensitive chronoamperometry (i-t) were used to readout the DNA methylation pattern. Subsequent to the development of this proof-of-concept sensor, we replaced the commercial HRP enzyme with an in-house specially designed mesoporous iron-oxide for reading the methylation site recognition events in genomic DNA obtained from the oesophageal cancer cell lines. In this DNA biosensor, both the BT and PCR amplification were avoided to overcome the challenges associated with them and to achieve relatively simple and rapid DNA methylation detection. Prior to integration of mesoporous iron-oxides into the sensor platform, the peroxidase-like activity was explored and a colorimetric glucose sensor was developed. This intrinsic property was then combined with DNA methylation site-5-mC antibody immunocomplex and hence a sensitive colorimetric and electrochemical readout were reported for global DNA methylations. In the following chapter, a more sensitive assay was stated by utilising the coupling of enzymatic and electrocatalytic strengths of another natural (glucose oxidase) enzyme. In this assay, chronocoulometric (CC) charge measurement from redox mediator [Ru(NH3)6]3+ in the presence of glucose subtract via glucose oxidase enzyme was used for DNA methylation analysis. In our final readout strategies, we extended our approach towards an inexpensive and portable assay platform which enabled naked-eye, colorimetric and electrochemical interrogation of in-house synthesised porous graphene oxide-loaded iron oxide (GOFe2O3) via (TMB)/Nanozyme)-based colorimetric assay. All the readout platforms reported herein have shown excellent analytical performance with high sensitivity (limit of detection-LOD for global DNA methylation 5%) and specificity. The applicability of the assays was also demonstrated in different cancer cell line samples with high reproducibility. We envisage that this research efforts will contribute to the on-going development of simple, inexpensive and sensitive technology for global DNA methylation analysis towards commercial aspect of both the laboratory settings and resource-poor clinical settings.

DNMT3 proteins are de novo DNA methyltransferases that are responsible for the establishment of DNA methylation patterns in mammalian genomes. Here, we have determined the crystal structures of the ATRX-DNMT3-DNMT3L (ADD) domain of DNMT3A in an unliganded form and in a complex with the amino-terminal tail of histone H3. Combined with the results of biochemical analysis, the complex structure indicates that DNMT3A recognizes the unmethylated state of lysine 4 in histone H3. This finding indicates that the recruitment of DNMT3A onto chromatin, and thereby de novo DNA methylation, is mediated by recognition of the histone modification state by its ADD domain. Furthermore, our biochemical and nuclear magnetic resonance data show mutually exclusive binding of the ADD domain of DNMT3A and the chromodomain of heterochromatin protein 1alpha to the H3 tail. These results indicate that de novo DNA methylation by DNMT3A requires the alteration of chromatin structure.

S-adenosylmethionine Levels Regulate the Schwann Cell DNA Methylome

DNA methylation, an epigenetic mechanism connecting folate to healthy embryonic development and aging

Epigenetics in Male Germ Cells