Possible Involvement Of DNA Methylation Research Articles

5-Methylcytosine and 5-Hydroxymethylcytosine in Scrapie-Infected Sheep and Mouse Brain Tissues.

Scrapie is a neurodegenerative disorder belonging to the group of transmissible spongiform encephalopathies or prion diseases, which are caused by an infectious isoform of the innocuous cellular prion protein (PrPC) known as PrPSc. DNA methylation, one of the most studied epigenetic mechanisms, is essential for the proper functioning of the central nervous system. Recent findings point to possible involvement of DNA methylation in the pathogenesis of prion diseases, but there is still a lack of knowledge about the behavior of this epigenetic mechanism in such neurodegenerative disorders. Here, we evaluated by immunohistochemistry the 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) levels in sheep and mouse brain tissues infected with scrapie. Expression analysis of different gene coding for epigenetic regulatory enzymes (DNMT1, DNMT3A, DNMT3B, HDAC1, HDAC2, TET1, and TET2) was also carried out. A decrease in 5mC levels was observed in scrapie-affected sheep and mice compared to healthy animals, whereas 5hmC displayed opposite patterns between the two models, demonstrating a decrease in 5hmC in scrapie-infected sheep and an increase in preclinical mice. 5mC correlated with prion-related lesions in mice and sheep, but 5hmC was associated with prion lesions only in sheep. Differences in the expression changes of epigenetic regulatory genes were found between both disease models, being differentially expressed Dnmt3b, Hdac1, and Tet1 in mice and HDAC2 in sheep. Our results support the evidence that DNA methylation in both forms, 5mC and 5hmC, and its associated epigenetic enzymes, take part in the neurodegenerative course of prion diseases.

Genome-wide alternation and effect of DNA methylation in the impairments of steroidogenesis and spermatogenesis after PM2.5 exposure

The effects of ambient fine particles on male reproductive health have raised widespread concern. The particular underlying mechanisms of the damage remain largely unclear and demand more research in new directions. Previous research has revealed that DNA methylation plays an important role in male reproductive development and is also vulnerable to environmental influences. However, there hasn't been enough investigation into the involvement of DNA methylation in PM2.5-induced male reproductive toxicity. Here, we establish a real-time PM2.5 exposure model and revealed that PM2.5 exposure could lead to testicular dysfunction including spermatogenesis impairment and steroid hormone dysfunction. In particular, the decrease in the testicular global level of 5-methylcytosine (5mC) indicated a possible association of DNA methylation with testicular injury induced by PM2.5 exposure. Further genome-wide methylation analysis revealed genomic hypomethylation of testicular DNA and identified more than 1000 differentially methylated regions in both CAP and UA versus FA, indicating that PM2.5 exposure, even low-dose, could modulate the testicular methylome. Furthermore, integrated analysis of methylome and transcriptome identified some key methylated genes and networks, which may be involved in spermatogenesis and synthesis of steroid hormone. The testicular methylation levels of key genes especially Cyp11a1 and Pax8 raised, and their consequent reduced expression may impair the testosterone and sperm production process. Our research provides fundamental knowledge as well as novel insights into the possible involvement of DNA methylation in PM2.5-induced male reproductive harm.

DNA Methylation Expression Profile of Blood Heat Syndrome and Blood Stasis Syndrome in TCM Psoriasis.

Objective Traditional Chinese medicine (TCM) emphasizes treatment based on syndrome differentiation. This study aimed to clarify the characteristics of DNA methylation expression profiles in peripheral blood mononuclear cells (PBMCs) in patients with psoriasis and analyze the differences in these profiles among different TCM syndromes of psoriasis in order to provide a material basis for the diversity of these syndromes. Methods Blood samples were collected from 32 participants, including 14 patients with psoriatic blood heat syndrome (BHS), 12 patients with psoriatic blood stasis syndrome (BSS), and 6 healthy controls. PBMCs were extracted and subjected to DNA quality inspection. An Illumina Human Methylation 850k chip was used to sequence each group of samples. According to gene annotation classification together with CpG island annotation classification, the differentially methylated regions between sample groups were screened, while Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were applied to perform functional analyses of DMGs. Finally, the DMGs closely correlating with psoriatic severity were screened using Spearman's correlation analysis. Results Compared with normal controls, patients with psoriasis showed an overall trend of hypermethylation. In psoriasis, the differential methylation probes were mainly distributed on gene body region on the genome, while those in CpG regions were mainly distributed in CpG islands. Compared with healthy controls, the overall trends in methylation were similar in psoriatic BHS and BSS patients compared to healthy controls. However, bioinformatic analysis revealed different functions of DMGs. We also found that the methylation levels of TRIM14 and PRDM16 were closely correlated with PASI scores and could serve as potential biomarkers to assess the severity of psoriasis. Conclusions Our study, for the first time, indicated the possible involvement of DNA methylation in regulating the characteristics of TCM syndromes of psoriasis, providing a new direction for research into TCM psoriatic syndromes.

Possible Involvement of DNA Methylation in TSC1 Gene Expression in Neuroprotection Induced by Hypoxic Preconditioning.

Background It has been reported that ischemia and ischemic preconditioning (IPC) have different effects on the expression of tuberous sclerosis complex 1 (TSC1), which may contribute to the tolerance to ischemia/hypoxia with the increase of autophagy. The mechanisms of TSC1 differential expression are still unclear under ischemia/IPC conditions in hippocampal Cornu Ammon 1 (CA1) and Cornu Ammon 3 (CA3) area neuronal cells. While we have shown that 5-Aza-CdR, a DNA methyltransferase inhibitor, can upregulate TSC1 and increase hypoxic tolerance by autophagy in vivo and in vitro, in this study, we examined whether DNA methylation was involved in the differential expression of TSC1 in the CA1 and CA3 regions induced by hypoxic preconditioning (HPC). Methods Level of rapamycin (mTOR) autophagy, a downstream molecular pathway of TSC1/TSC2 complex, was detected in HPC mouse hippocampal CA1 and CA3 areas as well as in the HPC model of mouse hippocampal HT22 cells. DNA methylation level of TSC1 promoter (-720 bp~ -360 bp) was determined in CA1 and CA3 areas by bisulfite-modified DNA sequencing (BMDS). At the same time, autophagy was detected in HT22 cells transfected with GFP-LC3 plasmid. The role of TSC1 in neuroprotection was measured by cell viability and apoptosis, and the role of TSC1 in metabolism was checked by ATP assay and ROS assay in HT22 cells that overexpressed/knocked down TSC1. Results HPC upregulated the expression of TSC1, downregulated the level of P-mTOR (Ser2448) and P-p70S6K (Thr389), and enhanced the activity of autophagy in both in vivo and in vitro. The increased expression of TSC1 in HPC may depend on its DNA hypomethylation in the promoter region in vivo. HPC also could reduce energy consumption in HT22 cells. Overexpression and knockdown of TSC1 can affect cell viability, cell apoptosis, and metabolism in HT22 cells exposed to hypoxia. Conclusion TSC1 expression induced by HPC may relate to the downregulation of its DNA methylation level with the increase of autophagy and the decrease of energy demand.

Epigenetics at the crossroads of secondary growth regulation.

The development of plant tissues and organs during post-embryonic growth occurs through the activity of both primary and secondary meristems. While primary meristems (root and shoot apical meristems) promote axial plant growth, secondary meristems (vascular and cork cambium or phellogen) promote radial thickening and plant axes strengthening. The vascular cambium forms the secondary xylem and phloem, whereas the cork cambium gives rise to the periderm that envelops stems and roots. Periderm takes on an increasingly important role in plant survival under climate change scenarios, but it is also a forest product with unique features, constituting the basis of a sustainable and profitable cork industry. There is established evidence that epigenetic mechanisms involving histone post-translational modifications, DNA methylation, and small RNAs play important roles in the activity of primary meristem cells, their maintenance, and differentiation of progeny cells. Here, we review the current knowledge on the epigenetic regulation of secondary meristems, particularly focusing on the phellogen activity. We also discuss the possible involvement of DNA methylation in the regulation of periderm contrasting phenotypes, given the potential impact of translating this knowledge into innovative breeding programs.

Possible Involvement of DNA Methylation and Protective Effect of Zebularine on Neuronal Cell Death after Glutamate Excitotoxity.

Neuronal cell death after cerebral ischemia consists various steps including glutamate excitotoxity. Excessive Ca2+ influx through the N-methyl-D-aspartate (NMDA) receptor, which is one of the ionotropic glutamate receptors, plays a central role in neuronal cell death after cerebral ischemia. We previously reported that DNA methylation is transiently increased in neurons during ischemic injury and that this aberrant DNA methylation is accompanied by neuronal cell death. Therefore, we performed the present experiments on glutamate excitotoxicity to gain further insight into DNA methylation involvement in the neuronal cell death. We demonstrated that knockdown of DNA methyltransferase (DNMT)1, DNMT3a, or DNMT3b gene in Neuro2a cells was performed to examine which DNMTs were more important for neuronal cell death after glutamate excitotoxicity. Although we confirmed a decrease in the levels of the target DNMT protein after small interfering RNA (siRNA) transfection, the Neuro2a cells were not protected from injury by transfection with siRNA for each DNMT. We next revealed that the pharmacological inhibitor of DNMTs protected against glutamate excitotoxicity in Neuro2a cells and also in primary cultured cortical neurons. This protective effect was associated with a decrease in the number of 5-methylcytosine (5 mC)-positive cells under glutamate excitotoxicity. In addition, the increased level of cleaved caspase-3 was also reduced by a DNMT inhibitor. Our results suggest the possibility that at least 2 or all DNMTs functionally would cooperate to activate DNA methylation after glutamate excitotoxicity and that inhibition of DNA methylation in neurons after cerebral ischemia might become a strategy to reduce the neuronal injury.

Cannabidiol prevents disruptions in sensorimotor gating induced by psychotomimetic drugs that last for 24-h with probable involvement of epigenetic changes in the ventral striatum

Cannabidiol (CBD), a major non-psychotomimetic component of the Cannabis sativa plant, shows therapeutic potential in several psychiatric disorders, including schizophrenia. The molecular mechanisms underlying the antipsychotic-like effects of CBD are not fully understood. Schizophrenia and antipsychotic treatment can modulate DNA methylation in the blood and brain, resulting in altered expression of diverse genes associated with this complex disorder. However, to date, the possible involvement of DNA methylation in the antipsychotic-like effects of CBD has not been investigated. Therefore, this study aimed at evaluating in mice submitted to the prepulse inhibition (PPI) model: i) the effects of a single injection of CBD or clozapine followed by AMPH or MK-801 on PPI and global DNA methylation changes in the ventral striatum and prefrontal cortex (PFC); and ii). if the acute antipsychotic-like effects of CBD would last for 24-h. AMPH (5 mg/kg) and MK-801 (0.5 mg/kg) impaired PPI. CBD (30 and 60 mg/kg), similar to clozapine (5 mg/kg), attenuated AMPH- and MK801-induced PPI disruption. AMPH, but not MK-801, increased global DNA methylation in the ventral striatum, an effect prevented by CBD. CBD and clozapine increased, by themselves, DNA methylation in the prefrontal cortex. The acute effects of CBD (30 or 60 mg/kg) on the PPI impairment induced by AMPH or MK-801 was also detectable 24 h later. Altogether, the results show that CBD induces acute antipsychotic-like effects that last for 24-h. It also modulates DNA methylation in the ventral striatum, suggesting a new potential mechanism for its antipsychotic-like effects.

Possible involvement of DNA methylation in hippocampal synaptophysin gene expression during postnatal development of mice

Synaptophysin (Syp) is an integral membrane protein of synaptic vesicles, and is ubiquitously expressed in neurons throughout the brain. As Syp expression is correlated with synaptogenesis during development of the central nervous system, the expression of Syp is considered to be a critical aspect of neuronal maturation and circuit formation. However, little information is available concerning the regulatory mechanisms of Syp gene expression during postnatal development of the brain. In the present study, we investigated changes in Syp mRNA in the hippocampus of mice during postnatal development, and examined the gene regulation mechanisms, focusing on DNA methylation. We found that hippocampal Syp expression involving both mRNA and protein levels increased during the first two weeks of life, and that this increase was accompanied by a transition from hypermethylation to hypomethylation at the CpG sites of the Syp gene upstream region. In addition, DNA demethylating agent 5-Aza-2′-deoxycytidine (5-aza-dC) de-repressed Syp gene expression both in vitro in Neuro-2a mouse neuronal cells and in vivo in the hippocampus of early postnatal mice. Furthermore, the methylation levels at upstream region of Syp gene in the hippocampus of developing mice was decreased by intraperitoneal injection of 5-aza-dC. These results suggest that Syp gene regulation, at least during postnatal brain development, could be mediated by DNA methylation. Our findings promote understanding of the molecular basis of synaptogenesis during postnatal brain development, and provide novel insight into therapeutic aspects of neurodevelopmental disorders involving synaptic dysfunction.

Possible involvement of DNA methylation by DNMT3a in ischemia-induced neuronal injury

Possible involvement of DNA methylation by DNMT3a in ischemia-induced neuronal injury

Epigenome-wide DNA methylation regulates cardinal pathological features of psoriasis

BackgroundPsoriasis is a chronic inflammatory autoimmune skin disorder. Several studies suggested psoriasis to be a complex multifactorial disease, but the exact triggering factor is yet to be determined. Evidences suggest that in addition to genetic factors, epigenetic reprogramming is also involved in psoriasis development. Major histopathological features, like increased proliferation and abnormal differentiation of keratinocytes, and immune cell infiltrations are characteristic marks of psoriatic skin lesions. Following therapy, histopathological features as well as aberrant DNA methylation reversed to normal levels. To understand the role of DNA methylation in regulating these crucial histopathologic features, we investigated the genome-wide DNA methylation profile of psoriasis patients with different histopathological features.ResultsGenome-wide DNA methylation profiling of psoriatic and adjacent normal skin tissues identified several novel differentially methylated regions associated with psoriasis. Differentially methylated CpGs were significantly enriched in several psoriasis susceptibility (PSORS) regions and epigenetically regulated the expression of key pathogenic genes, even with low-CpG promoters. Top differentially methylated genes overlapped with PSORS regions including S100A9, SELENBP1, CARD14, KAZN and PTPN22 showed inverse correlation between methylation and gene expression. We identified differentially methylated genes associated with characteristic histopathological features in psoriasis. Psoriatic skin with Munro’s microabscess, a distinctive feature in psoriasis including parakeratosis and neutrophil accumulation at the stratum corneum, was enriched with differentially methylated genes involved in neutrophil chemotaxis. Rete peg elongation and focal hypergranulosis were also associated with epigenetically regulated genes, supporting the reversible nature of these characteristic features during remission and relapse of the lesions.ConclusionOur study, for the first time, indicated the possible involvement of DNA methylation in regulating the cardinal pathophysiological features in psoriasis. Common genes involved in regulation of these pathologies may be used to develop drugs for better clinical management of psoriasis.

Impairing DNA methylation obstructs memory enhancement for at least 24 hours in Lymnaea

ABSTRACTStressor-induced memory enhancement has previously been shown to involve DNA methylation in the mollusc Lymnaea stagnalis. Specifically, injection of the DNA methylation inhibitor 5-AZA one hour before exposure to a memory-enhancing stressor obstructs memory augmentation. However, the duration of the influence of 5-AZA on this memory enhancement has not yet been examined. In this study, 2 memory-enhancing stressors (a thermal stress and exposure to the scent of a predator) were used to examine whether injection of the DNA methylation inhibitor 5-AZA 24 hours before stress exposure would still impair memory enhancement. Indeed, it was observed that memory is still obstructed when 5-AZA is injected 24 hours before exposure to either of these stressors in Lymnaea. Understanding that 5-AZA still effectively impairs memory enhancement after a period of 24 hours is valuable because it indicates that experimental manipulations do not need to be made within one hour after the injection of this DNA methylation inhibitor and can instead be made within one day (i.e. 24 hours). These results will allow for a future examination of the possible involvement of DNA methylation in memory enhancement related to longer-term stressors or environmental changes. This study further elucidates the involvement of epigenetic changes in memory enhancement in Lymnaea, providing insight into the process of memory formation in this mollusc.

Epigenetics Reprogramming of Autophagy is involved in Childhood Acute Lymphatic Leukemi

Cancer disease has been considered as a result of progressive genetic alterations such as mutations in tumor suppressor genes and oncogenes, chromosomal abnormality and epigenetics reprogramming. Epigenetic modification plays a crucial role in the tumor initiation and tumor evaluation via DNA methylation activity and chromatin alternation mechanism. Noteworthy, autophagy is a fundamental, conserved physiological process by which cells target long-lived cytosolic proteins and organelles for lysosomal degradation. Recently, autophagy has been showed to play critical role in cancer evaluation and therapeutical strategy. On the other hand, lymphatic leukaemia is a type of cancer which contains two major conditions, acute and chronic leukaemia. Acute lymphatic leukemia (ALL) is the most common cancer in the pediatric population. Therefore, in the current work we were interested to investigate the possible involvement of DNA methylation of cellular genes in blood samples that derived from children with ALL. Interestingly, the relative expression of DNA methyltransferase 1 (DNMT1) has been increased in ALL samples compared to healthy individual samples. Meanwhile, the relative expression of cellular autophagy genes (LC3B and Atg5) has been strongly decreased in ALL samples suggesting the possible involvement of DNA methylation of related autophagy molecules. Accordingly, digestion of LC3B gene segment with restriction enzyme (HpaII) that specifically targeted CpG position showed inactive digestion on ALL samples in comparison with control samples indicating the possible methyl alternation of cytosine nucleotides. Further, we found another methylation activity on promoter regions of Atg5 and LC3B in ALL derived samples. These data strongly suggest that the expression of cellular autophagy genes LC3B and Atg5 are reduced and subsequently autophagic machinery is interrupted in ALL cases most likely via DNA methylation activity.

Possible involvement of DNA methylation in regulating ZmGRP3 tissue-specific expression in maize

Cytosine methylation (C meth) is a ubiquitous regulator of gene transcription. The maize gene ZmGRP3 is a notable marker for root initiation, but how its root-specific transcription is regulated is not understood. Here, bisulfite sequencing and a C meth sensitive Southern blot assay were used to show that the transcription of ZmGRP3 was promoted by a reduction in the extent of C meth both in its promoter and in the vicinity of its translation start site. The result was validated by revealing the effect of 5-aza-2-deoxycytidine on the C meth status in various seedling organs. An analysis of methylation patterns indicates that the C meth of cytosine-guanine dinucleotides was the most important determinant of transcription, however, the C meth of CHG or CHH trinucleotides had little influence.

Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells.

BackgroundThe GBGT1 gene encodes the globoside alpha-1,3-N-acetylgalactosaminyltransferase 1. This enzyme catalyzes the last step in the multi-step biosynthesis of the Forssman (Fs) antigen, a pentaglycosyl ceramide of the globo series glycosphingolipids. While differential GBGT1 mRNA expression has been observed in a variety of human tissues being highest in placenta and ovary, the expression of GBGT1 and the genes encoding the glycosyltransferases and glycosidases involved in the biosynthesis of Fs as well as the possible involvement of DNA methylation in transcriptional regulation of GBGT1 expression have not yet been investigated.ResultsRT-qPCR profiling showed high GBGT1 expression in normal ovary surface epithelial (HOSE) cell lines and low GBGT1 expression in all (e.g. A2780, SKOV3) except one (OVCAR3) investigated ovarian cancer cell lines, a finding that was confirmed by Western blot analysis. Hierarchical cluster analysis showed that GBGT1 was even the most variably expressed gene of Fs biosynthesis-relevant glycogenes and among the investigated cell lines, whereas NAGA which encodes the alpha-N-acetylgalactosaminidase hydrolyzing Fs was not differentially expressed. Bisulfite- and COBRA-analysis of the CpG island methylation status in the GBGT1 promoter region demonstrated high or intermediate levels of GBGT1 DNA methylation in all ovarian cancer cell lines (except for OVCAR3) but marginal levels of DNA methylation in the two HOSE cell lines. The extent of DNA methylation inversely correlated with GBGT1 mRNA and protein expression. Bioinformatic analysis of GBGT1 in The Cancer Genome Atlas ovarian cancer dataset demonstrated that this inverse correlation was also found in primary ovarian cancer tissue samples confirming our cell line-based findings. Restoration of GBGT1 mRNA and protein expression in low GBGT1-expressing A2780 cells was achieved by 5-aza-2’-deoxycytidine treatment and these treated cells exhibited increased helix pomatia agglutinin-staining, reflecting the elevated presence of Fs disaccharide on these cells.ConclusionsGBGT1 expression is epigenetically silenced through promoter hypermethylation in ovarian cancer. Our findings not only suggest an involvement of DNA methylation in the synthesis of Fs antigen but may also explain earlier studies showing differential GBGT1 expression in various human tissue samples and disease stages.

Breed-specific patterns of hepatic gluconeogenesis and glucocorticoid action in pigs

Abstract. In the present study, Erhualian and Pietrain pigs were employed to investigate the breed-specific patterns of hepatic gluconeogenesis by detecting the related enzyme mRNA expression, and to analyse the relationship with the hepatic glucocorticoid receptors and 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) expression. Furthermore, hepatic DNA methyltransferase 1 (DNMT1) expression was determined to detect DNA methyltransferase state difference between the two breeds. The results demonstrated that the Erhualian pigs exhibited significantly lower plasma lactate acid concentration, but higher liver lactate acid content than the Pietrain pigs. A significantly higher expression of pyruvate carboxylase (PC), fructose-1, 6-bisphosphatase (FBP) and the mitochondria phosphoenolpyruvate carboxykinase (PCK2) mRNA were observed in Erhualian pigs. The Erhualian pigs demonstrated a significantly higher expression of glucocorticoid receptor and 11β-HSD1 mRNA than the Pietrain pigs, though there was no difference in the hepatic cortisol content between the two breeds. The hepatic DNMT1 mRNA expression in the Erhualian pigs was significantly lower, and the DNMT1 protein content in Erhualian pigs tended to be decreased compared with Pietrain pigs (P=0.066). The results suggest that the Erhualian pigs demonstrated higher hepatic gluconeogenesis capacity in comparison to the Pietrain pigs. The up-regulation of hepatic glucocorticoid receptor and 11β-HSD1 expression may be involved in the enhanced hepatic gluconeogenesis in Erhualian pigs. Moreover, the down-regulation of DNMT1 in Erhualian pigs implies possible involvement of DNA methylation in a breed-specific pattern of hepatic gluconeogenesis.

Insufficient maintenance DNA methylation is associated with abnormal embryonic development

BackgroundEarly pregnancy loss (EPL) is a frustrating clinical problem, whose mechanisms are not completely understood. DNA methylation, which includes maintenance methylation and de novo methylation directed by DNA methyltransferases (DNMTs), is important for embryo development. Abnormal function of these DNMTs may have serious consequences for embryonic development.MethodsTo evaluate the possible involvement of DNA methylation in human EPL, the expression of DNMT proteins and global methylation of DNA were assessed in villous or decidua from EPL patients. The association of maintenance methylation with embryo implantation and development was also examined.ResultsWe found that DNMT1 and DNMT3A were both expressed in normal human villous and decidua. DNMT1 expression and DNA global methylation levels were significantly down-regulated in villous of EPL. DNMT3A expression was not significantly changed in the EPL group compared to controls in either villous or decidua. We also found that disturbance of maintenance methylation with a DNMT1 inhibitor may result in a decreased global DNA methylation level and impaired embryonic development in the mouse model, and inhibit in vitro embryo attachment to endometrial cells.ConclusionsOur results demonstrate that defects in DNA maintenance methylation in the embryo, not in the mother, are associated with abnormal embryonic implantation and development. The findings of the current study provide new insights into the etiology of EPL.

Possible involvement of DNA methylation in NKCC1 gene expression during postnatal development and in response to ischemia

In CNS, GABA(A) receptor-mediated responses switch from depolarization to hyperpolarization during postnatal development. This switch is mediated by developmental down-regulation of inwardly directed Na(+)-K(+)-2Cl(-) co-transporter type 1 (NKCC1) and up-regulation of outwardly directed K(+)-Cl(-) co-transporter type 2. While several factors have been shown to regulate K(+)-Cl(-) co-transporter type 2 expression, little is known about the mechanisms by which the expression of NKCC1 is regulated during postnatal development. Here, we report a novel epigenetic mechanism underlying the developmental regulation of NKCC1 gene expression in the rat cerebral cortex. In vitro DNA methylation of the NKCC1 promoter region, which contains a high density of cytosine-phosphodiester-guanine islands, significantly decreased the expression of NKCC1 mRNA, and the degree of methylation of the NKCC1 promoter region significantly increased during postnatal development. In addition, treatment with 5-aza-2'-deoxycytidine, a specific DNA methyltransferase inhibitor, elicited an increase in the expression of NKCC1 mRNA and protein in cortical slice cultures. Focal ischemic injury induced by the occlusion of the middle cerebral artery led to the re-expression of NKCC1 mRNA and protein even in the mature rat cortex. The re-expression of NKCC1 mRNA and protein in the injured cerebral cortex was related to a decrease in the methylation status of the NKCC1 promoter region. Our results indicate that epigenetic mechanisms, such as DNA methylation, might be involved in the regulation of NKCC1 gene expression during postnatal development as well as under pathological conditions.

Possible involvement of DNA methylation on expression regulation of carrot LEC1 gene in its 5′-upstream region

DNA methylation plays important roles in various developmental processes in many organisms. In carrots, the treatment of embryogenic cells (ECs) with DNA methylation inhibitors induces hypomethylation and blocks somatic embryogenesis. CARROT-LEAFY COTYLEDON 1 ( C- LEC1) is an important transcription factor for embryo development that shows embryo-specific expression in ECs and somatic and zygotic embryos. However, the regulation of embryo-specific transcription factor genes such as C-LEC1 in plants is not well understood. In this study, we used embryogenic carrot cells ( Daucus carota L. cv. US-Harumakigosun) to investigate the DNA methylation status of the embryogenesis-related genes C-LEC1, Carrot ABA INSENSITIVE 3 ( C-ABI3), and Daucus carota Embryogenic cell protein 31 ( DcECP 31) during the transition from embryogenesis to vegetative growth. The C-LEC1 promoter region showed a reduced level of DNA methylation during somatic embryogenesis followed by an increase during the transition from embryonic to vegetative growth. To test whether the increased level of DNA methylation down-regulates C-LEC1 expression, RNA-directed DNA methylation (RdDM) was used to induce the hypermethylation of two segments of the C-LEC1 5′-upstream region: Regions 1 and 2, corresponding to nucleotides − 1,904 to − 1,272 and − 896 to − 251, respectively. When the hypermethylation of Region 1 was induced by RdDM, C-LEC1 expression was reduced in the transgenic ECs, indicating a negative correlation between DNA methylation and C-LEC1 expression. In contrast, the hypermethylation of Region 2 did not greatly affect C-LEC1 expression. Based on these results, we hypothesize that DNA methylation may be involved in the control of C-LEC1 expression during carrot embryogenesis.

DNA Methylation, Chondrogenesis, and Cartilage Degeneration

In the last few years there has been an explosion of research into epigenetics and, in particular, the roles of DNA methylation in the normal functioning of the mammalian organism as well as whether changes in methylation status contribute to or cause aberrant gene expression in diseases. While abnormal patterns of DNA methylation in cancer cells have been intensively investigated, little attention has so far been paid to the role of DNA methylation in cartilage and cartilage degeneration. This review summarizes the current knowledge of the mechanism of methylation, its association with transcriptional silencing, possible mechanisms of hyper- and hypomethylation as well as age- and disease related changes in methylation pattern. We discuss the possible involvement of DNA methylation in chondrogenesis as well as its potential importance for cartilage degradation. Overall, epigenetic gene regulation has largely been neglected in cartilage research, but is likely to be an important issue in future. There is increasing evidence that besides cytokines, growth factors and changes in matrix composition, variations in the genetic methylation pattern might also be important determinators of the complex gene expression pattern pathognomically observed in osteoarthritic cartilage tissue.

Regulation of the human secretin gene is controlled by the combined effects of CpG methylation, Sp1/Sp3 ratio, and the E-box element.

To unravel the mechanisms that regulate the human secretin gene expression, in this study, we have used secretin-expressing (HuTu-80 cells, human duodenal adenocarcinoma) and non-secretin-expressing [PANC-1 (human pancreatic ductile carcinoma) and HepG2 (human hepatocellular carcinoma) cells] cell models for in vitro and in vivo analyses. By transient transfection assays, within the promoter region (-11 to -341 from ATG, relative to the ATG initiation codon), we have initially identified several functional motifs including an E-box and 2 GC-boxes. Results from gel mobility shift and chromatin immunoprecipitation assays confirmed further that NeuroD, E2A, Sp1, and Sp3 bind to these E- and GC-boxes in HuTu-80 cells in vitro and in vivo, whereas only high levels of Sp3 is observed to bind the promoter in HepG2 cells. In addition, overexpression of Sp3 resulted in a dose-dependent repression of the Sp1-mediated transactivation. Collectively, these data suggest that the Sp1/Sp3 ratio is instrumental to controlling secretin gene expression in secretin-producing and non-secretin-producing cells. The functions of GC-box and Sp proteins prompted us to investigate the possible involvement of DNA methylation in regulating this gene. Consistent with this idea, we found a putative CpG island (-336 to 262 from ATG) that overlaps with the human secretin gene promoter. By methylation-specific PCR, all the CpG dinucleo-tides (26 of them) within the CpG island in HuTu-80 cells are unmethylated, whereas all these sites are methylated in PANC-1 and HepG2 cells. The expressions of secretin in PANC-1 and HepG2 cells were subsequently found to be significantly activated by a demethylation agent, 5'-Aza-2' deoxycytidine. Taken together, our data indicate that the human secretin gene is controlled by the in vivo Sp1/Sp3 ratio and the methylation status of the promoter.