The involvement of photoregulated DNA methylation into a choice between sexual and asexual developmental pathways in Neurospora crassa.

Genomic DNA of the filamentous fungus Neurospora crassa is largely devoid of methylation. Methylation at the 5 position of cytosine is heavy in certain areas of the genome, however. Overall, approximately 1.5% of the cytosines in N. crassa DNA are methylated (Russell et al. 1987; Foss et al. 1993; Selker et al. 1993b), but in certain regions, nearly 100% of the cytosines are methylated (Selker and Stevens 1985; Selker et al. 1993a). The total number of such methylated patches in the N. crassa genome is probably in the hundreds, based on the total amount of methylation (1.5%); the size and density of the two best-characterized, natural, methylated patches (1.6 kb for ζ–η, Selker et al. 1993b; and ~2 kb for ψ–63, B. Margolin and E. Selker, unpubl.); and the size of the genome (~4 × 10 7 bp, Orbach et al. 1988b). Genetic analyses have revealed that mutations in a number of genes can reduce the level of DNA methylation in Neurospora (Foss et al. 1993; Roberts and Selker 1995; H. Foss and E. Selker, unpubl.), and one mutant apparently lacks all DNA methylation in vegetative tissue ( dim-2, for d efective i n m ethylation; Foss et al. 1993). The cytosine residues that are methylated in Neurospora are not present exclusively, or even predominantly, in CpG dinucleotides (Bull and Wootton 1984; Selker and Stevens 1985; Selker et al. 1993a). This is unlike the situation in higher eukaryotes and some other fungi that have been studied (Selker 1993). Non-CpG methylation cannot be propagated...

Alternative splicing (AS) increases transcript and proteomic diversity of intron containing genes and has emerged as a pervasive property of eukaryotic genes. DNA methylation is a regulator of gene expression which is present in many eukaryotes. Recently, chromatin structure and histone modifications have been shown to regulate AS in humans (1), but it is unknown if this also occurs in plants. Here, I investigated the regulatory role that DNA and histone methylation have on AS in Arabidopsis thaliana. I utilized the SR protein gene family as a model system to examine changes in isoform abundance in response to chemical inhibition of DNA and histone methylation using the histone deacetylase inhibitor Tricostatin A (TSA) and the DNA demethylating agent 5-Azadeoxycytidine (Azad). RT-PCR revealed that 12 SR genes had isoforms that had altered abundance in response to TSA and Azad-C. Antagonistic effects were found when both drugs were applied since changes in splicing were only seen for 10 of the SR genes. AS patterns of the SR genes are known to change according to organ and developmental stage (2) and DNA methylation is also known to change over the lifespan of the plant (3). I investigated if organ type and developmental patterns of AS are disrupted in a DNA hypermethylation mutant. Splicing patterns of the SR genes displayed tissue and developmental specific changes in the DNA hypermethylation mutant. Computational analysis of three different DNA methylation mutants was performed using a publically available Illumina dataset (4). Widespread changes in AS was detected in each of the mutants across all types of AS. Changes in methylcytosine content within the coding region of the gene did not account for a large proportion of the novel AS events detected. Splice site sequence analysis of introns uniquely retained in each of the mutant genotypes uncovered sequence changes around the functionally important sequence elements. The results of my thesis indicate for the first time that AS in plants is regulated in part by changes in DNA methylation and histone modifications.

DNA methylation is involved in gene silencing and genome stability in organisms from fungi to mammals. Genetic studies in Neurospora crassa previously showed that the CUL4-DDB1 E3 ubiquitin ligase regulates DNA methylation via histone H3K9 trimethylation. However, the substrate-specific adaptors of this ligase that are involved in the process were not known. Here, we show that, among the 16 DDB1- and Cul4-associated factors (DCAFs) encoded in the N. crassa genome, three interacted strongly with CUL4-DDB1 complexes. DNA methylation analyses of dcaf knockout mutants revealed that dcaf26 was required for all of the DNA methylation that we observed. In addition, histone H3K9 trimethylation was also eliminated in dcaf26KO mutants. Based on the finding that DCAF26 associates with DDB1 and the histone methyltransferase DIM-5, we propose that DCAF26 protein is the major adaptor subunit of the Cul4-DDB1-DCAF26 complex, which recruits DIM-5 to DNA regions to initiate H3K9 trimethylation and DNA methylation in N. crassa.

Transformation of eukaryotic cells can be used to test potential signals for DNA methylation. This approach is not always reliable, however, because of chromosomal position effects and because integration of multiple and/or rearranged copies of transforming DNA can influence DNA methylation. We developed a robust system to evaluate the potential of DNA fragments to function as signals for de novo methylation in Neurospora crassa. The requirements of the system were (i) a location in the N. crassa genome that becomes methylated only in the presence of a bona fide methylation signal and (ii) an efficient gene replacement protocol. We report here that the am locus fulfills these requirements, and we demonstrate its utility with the identification of a 2.7-kb fragment from the psi 63 locus as a new portable signal for de novo methylation.

Transformation of eukaryotic cells can be used to test potential signals for DNA methylation. This approach is not always reliable, however, because of chromosomal position effects and because integration of multiple and/or rearranged copies of transforming DNA can influence DNA methylation. We developed a robust system to evaluate the potential of DNA fragments to function as signals for de novo methylation in Neurospora crassa. The requirements of the system were (i) a location in the N. crassa genome that becomes methylated only in the presence of a bona fide methylation signal and (ii) an efficient gene replacement protocol. We report here that the am locus fulfills these requirements, and we demonstrate its utility with the identification of a 2.7-kb fragment from the psi 63 locus as a new portable signal for de novo methylation.

Background:Osteoarthritis (OA) remains one of the most common osteopathy for centuries, which can be attributed to multiple risk factors including mechanical and biochemical ones. More and more studies verified that inflammatory cytokines play important roles in the progression of OA, such as tumor necrosis factor-alpha (TNF-α). In this study, we aimed to investigate the relationship between epigenetic manifestations of TNF-? and the pathogenesis of OA.Methods:Totally, 37 OA patients’ cartilage was collected through the knee joint and 13 samples of articular cartilage as healthy control was collected through traumatic amputation. Real-time PCR, Western blot and ELISA analysis were performed to observe the expression of target genes and proteins in collected samples.Results:Compared with the healthy control group, TNF-? was over-expressing in cartilage which was collected from OA patients. DNA hypomethylation, histone hyperacetylation and histone methylation were observed in the TNF-? promoter in OA compared with normal patients, and we also studied series of enzymes associated with epigenetics. The results showed that by increasing DNA methylation and decreasing histone acetylation in the TNF-? promoter, and TNF-? over-expression in OA cartilage was suppressed, histone methylation has no significant correlation with OA.Conclusion:In conclusion, the changes of epigenetic status regulate TNF-α expression in the cells, which are pivotal to the OA disease process. These results may give us a better understanding of OA and may provide new therapeutic options.

DNA methylation is an epigenetic mark that plays an important role in genetic regulation in eukaryotes. Major progress has been made in dissecting the molecular pathways that regulate DNA methylation. Yet, little is known about DNA methylation variation over evolutionary time. Here we present an investigation of the variation of DNA methylation and transposable element (TE) content in species of the filamentous ascomycetes Neurospora. We generated genome-wide DNA methylation data at single-base resolution, together with genomic TE content and gene expression data, of 10 individuals representing five closely related Neurospora species. We found that the methylation levels were low (ranging from 1.3% to 2.5%) and varied among the genomes in a species-specific way. Furthermore, we found that the TEs over 400 bp long were targeted by DNA methylation, and in all genomes, high methylation correlated with low GC, confirming a conserved link between DNA methylation and Repeat Induced Point (RIP) mutations in this group of fungi. Both TE content and DNA methylation pattern showed phylogenetic signal, and the species with the highest TE load (N. crassa) also exhibited the highest methylation level per TE. Our results suggest that DNA methylation is an evolvable trait and indicate that the genomes of Neurospora are shaped by an evolutionary arms race between TEs and host defence.

Under the conditions of nitrogen starvation, illumination by blue light of wc-1 and wc-2 mutants of the ascomycete Neurospora crassa failed to stimulate the formation of protoperithecia and inhibit conidiation (contrary to what was observed in the mycelium of the wild-type fungus). The data obtained indicate that wc-1 and wc-2 genes of N. crassa are involved in light-dependent formation of protoperithecia and conidia. The effects of 5-azacytidine (an inhibitor of DNA methylation) under the same experimental conditions suggest that the balance between the formation of sexual and asexual reproductive structures, maintained in N. crassa, depends on genome methylation processes sensitive to the action of light, which is mediated by the photoreceptor complex of WC proteins.

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.

A broad range of factors have been associated with the development of adolescent loneliness. In the family context, a lack of parental support and high levels of parental psychological control have systematically been linked to loneliness. On the biological level, DNA methylation (which is an epigenetic process that suppresses gene expression) is believed to play a role in the development of loneliness. Specifically, high levels of DNA methylation in genes that play an important role in the functioning of the human stress response system are believed to elevate the risk of loneliness. Moreover, DNA methylation levels in these stress-related genes can be influenced by stressful environmental factors, suggesting a potential mediating role of DNA methylation in the association between parenting behaviors and loneliness. The current 3-year longitudinal study is the first study to examine the potential bidirectional longitudinal associations between loneliness, DNA methylation in stress-related genes, and both perceived parental support and psychological control. Furthermore, we explored the potential mediating role of DNA methylation in stress-related genes in the associations between perceived parenting and loneliness. The sample comprised 622 early adolescents (55% girls, Mage T1 = 10.77 years, SDage T1 = 0.48) who were followed from Grade 5 to 7. Parental support, psychological control, and loneliness were assessed annually by adolescent self-report questionnaires and DNA methylation was determined from saliva samples. Cross-Lagged Panel Models (CLPM) revealed that higher levels of loneliness predicted lower perceived parental support and higher perceived psychological control over time, as well as higher DNA methylation in some stress-related genes, that is, the glucocorticoid receptor gene (NR3C1) and the brain-derived neurotrophic factor (BDNF). In addition, higher NR3C1 methylation was predictive of lower perceived parental support and higher psychological control over time. No evidence was found for a mediating role of DNA methylation. Overall, our longitudinal findings challenge the current focus on DNA methylation and parenting behaviors as risk factors for adolescent loneliness. Instead, they suggest that the less considered direction of effects, which implies that loneliness predicts DNA methylation and aspects of parenting such as support and psychological control, should receive greater attention in future research.

BackgroundThe RIPper (http://theripper.hawk.rocks) is a set of web-based tools designed for analyses of Repeat-Induced Point (RIP) mutations in the genome sequences of Ascomycota. The RIP pathway is a fungal genome defense mechanism that is aimed at identifying repeated and duplicated motifs, into which it then introduces cytosine to thymine transition mutations. RIP thus serves to deactivate and counteract the deleterious consequences of selfish or mobile DNA elements in fungal genomes. The occurrence, genetic context and frequency of RIP mutations are widely used to assess the activity of this pathway in genomic regions of interest. Here, we present a bioinformatics tool that is specifically fashioned to automate the investigation of changes in RIP product and substrate nucleotide frequencies in fungal genomes.ResultsWe demonstrated the ability of The RIPper to detect the occurrence and extent of RIP mutations in known RIP affected sequences. Specifically, a sliding window approach was used to perform genome-wide RIP analysis on the genome assembly of Neurospora crassa. Additionally, fine-scale analysis with The RIPper showed that gene regions and transposable element sequences, previously determined to be affected by RIP, were indeed characterized by high frequencies of RIP mutations. Data generated using this software further showed that large proportions of the N. crassa genome constitutes RIP mutations with extensively affected regions displaying reduced GC content. The RIPper was further useful for investigating and visualizing changes in RIP mutations across the length of sequences of interest, allowing for fine-scale analyses.ConclusionThis software identified RIP targeted genomic regions and provided RIP statistics for an entire genome assembly, including the genomic proportion affected by RIP. Here, we present The RIPper as an efficient tool for genome-wide RIP analyses.

BackgroundThe aim of this study was to evaluate whether global levels of DNA methylation status were associated with albuminuria and progression of diabetic nephropathy in a case-control study of 123 patients with type 2 diabetes- 53 patients with albuminuria and 70 patients without albuminuria.MethodsThe 5-methyl cytosine content was assessed by reverse phase high pressure liquid chromatography (RP-HPLC) of peripheral blood mononuclear cells to determine individual global DNA methylation status in two groups.ResultsGlobal DNA methylation levels were significantly higher in patients with albuminuria compared with those in normal range of albuminuria (p = 0.01). There were significant differences in global levels of DNA methylation in relation to albuminuria (p = 0.028) and an interesting pattern of increasing global levels of DNA methylation in terms of albuminuria severity.In patients with micro- and macro albuminuria, we found no significant correlations between global DNA methylation levels and duration of diabetes (p > 0.05). In both sub groups, there were not significant differences between global DNA methylation levels with good and poor glycaemic control (p > 0.05). In addition, in patients with albuminuria, no differences in DNA methylation levels were observed between patients with and without other risk factors including age, gender, hypertension, dyslipidaemia and obesity.ConclusionsThese data may be helpful in further studies to develop novel biomarkers and new strategies for clinical care of patients at risk of diabetic nephropathy.

Application of lateral flow and microfluidic bio-assay and biosensing towards identification of DNA-methylation and cancer detection: Recent progress and challenges in biomedicine

DNA methylation (DNAm) suppresses gene expression and contributes to dosage compensation in mammals but whether DNAm plays a similar role in female ZW chromosome heterogametic species remains unresolved. We assessed chromosome-level DNAm using whole genome bisulphite sequencing in two avian species, zebra finches and jackdaws. Dosage compensation by DNAm would result in higher and more variable DNAm level in males relative to females on the Z chromosome. However, we found that the level of DNAm and its variance on the Z chromosome was lower in males. Moreover, male Z chromosome-based gene promoters were more frequently hypomethylated compared to females, indicating absence of upregulation on a gene-by-gene basis across the female Z chromosome. We suggest our findings reveal mitigation of an intra-genomic sexual conflict, with females suppressing expression of Z chromosome-based genes that benefit male but not female fitness. W was the most methylated chromosome, but hypermethylation on the W chromosome was mostly confined to intergenic regions, presumably resulting in the downregulation of transposable elements known to comprise a large part of the W chromosome. Thus, DNAm is involved in the development of sex-dependent phenotypes, but dosage compensation is achieved through other mechanisms.

Since the cloning and discovery of DNA methyltransferases (DNMT), there has been a growing interest in DNA methylation, its role as an epigenetic modification, how it is established and removed, along with the implications in development and disease. In recent years, it has become evident that dynamic DNA methylation accompanies the circadian clock and is found at clock genes in Neurospora, mice and cancer cells. The relationship among the circadian clock, cancer and DNA methylation at clock genes suggests a correlative indication that improper DNA methylation may influence clock gene expression, contributing to the etiology of cancer. The molecular mechanism underlying DNA methylation at clock loci is best studied in the filamentous fungi, Neurospora crassa, and recent data indicate a mechanism analogous to the RNA-dependent DNA methylation (RdDM) or RNAi-mediated facultative heterochromatin. Although it is still unclear, DNA methylation at clock genes may function as a terminal modification that serves to prevent the regulated removal of histone modifications. In this capacity, aberrant DNA methylation may serve as a readout of misregulated clock genes and not as the causative agent. This review explores the implications of DNA methylation at clock loci and describes what is currently known regarding the molecular mechanism underlying DNA methylation at circadian clock genes.