CCGG Sequences Research Articles

Abstract PR12: Using whole exome sequencing of archived FFPE tissue to characterize the mutational landscape of prostate cancer in Nigerian men

Abstract Compared to other ancestral groups, men of African ancestry (MAA) have the highest incidence and mortality of prostate cancer (PCa), with African men having the highest. Multiple studies have demonstrated that genetic/biologic differences in African American (AA) tumor biology contribute to PCa development and aggressiveness; furthermore, building evidence suggests that the observed differences are population specific and form unique paths to cancer aggressiveness. Despite this, MAA continue to be underrepresented in PCa studies. This lack of adequate representation greatly diminishes the ability to identify clinical interventions to address this disparate disease. In this study, we used whole-exome sequencing of 148 Nigerian PCa FFPE samples (75 Tumor, 62 BPH, and 11 non-matched Normal) to determine the mutational landscape of PCa. Samples were collected from 6 sites in central and southwest Nigeria and quality screened. Samples passing QC were sequenced (Illumina HiSeq 4000 PE150) and read files were processed using the Tumor Only Somatic Mutation pipeline developed by the CCR Collaborative Bioinformatics Resource (CCBR). In addition to the CCBR pipeline, Exomiser was used to prioritize non-silent variants according to variant frequency, pathogenicity, quality, and model organism phenotype data. 246 genes showed significant (p ≥ 0.05) tumor mutation and high prioritization (Exomiser score ≥ 0.75). These genes included BCR, KMD1A, MSH6, TLR4, and BMPR1B with mutation rates of 17%, 13%, 12%, 11%, and 8%, respectively. These rates were higher than process matched controls and TCGA tumor samples. Mutation signature analysis showed enrichments in 4 Cosmic signatures. 38.6% of Tumor samples contained signatures similar (cosine similarity [cs] = 0.912) to Signature 1, 29.3% were similar (cs = 0.849) to Signature 4, 14.6% were similar (cs = 0.743) to Signature 5, and 17.3% were similar (cs = 0.635) to Signature 25. Groupwise comparisons of gene mutations and mutation signatures showed that 8 of the 29 tumors (p ≤ 0.06), having a Signature 1 similar signature, contained a BCR frameshift insertion (c.3275_3278dup), which duplicates a four-nucleotide CCGG sequence in exon 19. The relationship between BCR and prostate cancer is still poorly understood; however, within the TCGA PRAD cohort, low BCR expression does significantly (p ≤ 0.024) correlate with poor survival. These results suggest that mutations within BCR result in a disruption of methylation and expression patterns that could contribute to worse outcomes in Nigerian PCa patients. Without tumor/normal matched pairs, more analysis is needed to ensure the accuracy of this characterization; however, completion of this study would comprise the largest mutational analysis of Nigerian PCa to date. Understanding the genetic underpinnings of PCa in Nigerian patients will add much-needed context to the study of the disease in MAA, further illuminating clinical interventions that may prove beneficial in diminishing outcome disparities. This abstract is also being presented as Poster B079. Citation Format: Prostate Cancer Transatlantic Consortium Members, Jason White, Wei Tang, Stefan Ambs, Solomon Rotimi, Mohammed Faruuk, Folakemi Odedina, Clayton Yates. Using whole exome sequencing of archived FFPE tissue to characterize the mutational landscape of prostate cancer in Nigerian men [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr PR12.

Changes in DNA methylation patterns affect ripening time in Satsuma mandarin fruit

AbstractDifferences in DNA methylation patterns might lead to differences in fruit maturation time in the citrus Satsuma, but the underlying mechanism is unclear. Here, we explored the differences in methylation patterns during fruit ripening in Satsuma among 20 early‐ and late‐maturing Satsuma varieties and compared DNA methylation patterns in two Satsuma cultivars (Ooita wase and Aoshima unshiu) at different developmental stages using methylation‐sensitive fragment length polymorphism (MSAP) analysis. We cloned and sequenced DNA from different bands to identify differentially methylated genes involved in fruit ripening. The internal methylation of CCGG sequences occurred more frequently in the early‐maturing Satsuma cultivars (8.47%) than the late‐maturing cultivars (6.74%), whereas the external methylation of CCGG sequences occurred less frequently in the early‐maturing cultivars (24.98%) compared to the late‐maturing types (27.27%). Genes encoding the G protein α subunit and zinc‐binding protein of the histidine triad (HIT) family and two unknown genes homologous to expressed sequence tag (EST) sequences (CX052976.1 and CX071135.1) were found to be modified by methylation during the fruit ripening process. These findings suggest that DNA methylation might play important roles in regulating fruit ripening in Satsuma. Our results lay the foundation for analysing the molecular mechanism underlying fruit ripening in this important citrus crop.

24-Epibrassinolide alters DNA cytosine methylation of Linum usitatissimum L. under salinity stress

Salinity is a common environmental challenge limiting worldwide agricultural crop yield. Plants employ epigenetic regulatory strategies, such as DNA methylation which relatively allows rapid adaptation to new conditions in response to environmental stresses. Brassinosteroids (BRs) are a novel group of phytohormones recognized as transcription and translation regulators which are able to mitigate the impact of environmental stresses on the plants. In the current investigation, the influence of salinity and 24-epibrassinolide (24-epiBL) was investigated on the extent and pattern of cytosine DNA methylation using methylation-sensitive amplified polymorphisms (MSAP) technique in flax. Upon NaCl (150 mM) exposure, total methylation of CCGG sequences was decreased in comparison to control plants, while 24-epiBL (10−8 M) induced total methylation under salinity stress. Sequencing and analysis of six randomly selected MSAP fragments detected genes involved in various biological and molecular processes such as vitamine B1 biosynthesis, protein targeting and localization, post-translational modification and gene regulation. In conclusion, 24-epiBL seed priming could play critical role in regulation of cellular and biological processes in response to salt stress by epigenetic modification and induction of methylation.

Unique mechanism of target recognition by PfoI restriction endonuclease of the CCGG-family

Restriction endonucleases (REs) of the CCGG-family recognize a set of 4–8 bp target sequences that share a common CCGG or CCNGG core and possess PD…D/ExK nuclease fold. REs that interact with 5 bp sequence 5′-CCNGG flip the central N nucleotides and ‘compress’ the bound DNA to stack the inner base pairs to mimic the CCGG sequence. PfoI belongs to the CCGG-family and cleaves the 7 bp sequence 5′-T|CCNGGA ("|" designates cleavage position). We present here crystal structures of PfoI in free and DNA-bound forms that show unique active site arrangement and mechanism of sequence recognition. Structures and mutagenesis indicate that PfoI features a permuted E…ExD…K active site that differs from the consensus motif characteristic to other family members. Although PfoI also flips the central N nucleotides of the target sequence it does not ‘compress’ the bound DNA. Instead, PfoI induces a drastic change in DNA backbone conformation that shortens the distance between scissile phosphates to match that in the unperturbed CCGG sequence. Our data demonstrate the diversity and versatility of structural mechanisms employed by restriction enzymes for recognition of related DNA sequences.

A relative quantitative Methylation-Sensitive Amplified Polymorphism (MSAP) method for the analysis of abiotic stress

BackgroundWe present a new methylation-sensitive amplified polymorphism (MSAP) approach for the evaluation of relative quantitative characteristics such as demethylation, de novo methylation, and preservation of methylation status of CCGG sequences, which are recognized by the isoschizomers HpaII and MspI. We applied the technique to analyze aluminum (Al)-tolerant and non-tolerant control and Al-stressed inbred triticale lines. The approach is based on detailed analysis of events affecting HpaII and MspI restriction sites in control and stressed samples, and takes advantage of molecular marker profiles generated by EcoRI/HpaII and EcoRI/MspI MSAP platforms.MethodsFive Al-tolerant and five non-tolerant triticale lines were exposed to aluminum stress using the physiologicaltest. Total genomic DNA was isolated from root tips of all tolerant and non-tolerant lines before and after Al stress following metAFLP and MSAP approaches. Based on codes reflecting events affecting cytosines within a given restriction site recognized by HpaII and MspI in control and stressed samples demethylation (DM), de novo methylation (DNM), preservation of methylated sites (MSP), and preservation of nonmethylatedsites (NMSP) were evaluated. MSAP profiles were used for Agglomerative hierarchicalclustering (AHC) based on Squared Euclidean distance and Ward’s Agglomeration method whereas MSAP characteristics for ANOVA.ResultsRelative quantitative MSAP analysis revealed that both Al-tolerant and non-tolerant triticale lines subjected to Al stress underwent demethylation, with demethylation of CG predominating over CHG. The rate of de novo methylation in the CG context was ~3-fold lower than demethylation, whereas de novo methylation of CHG was observed only in Al-tolerant lines.ConclusionsOur relative quantitative MSAP approach, based on methylation events affecting cytosines within HpaII–MspI recognition sequences, was capable of quantifying de novo methylation, demethylation, methylation, and non-methylated status in control and stressed Al-tolerant and non-tolerant triticale inbred lines. The method could also be used to analyze methylation events affecting CG and CHG contexts, which were differentially methylated under Al stress. We cannot exclude that the methylation changes revealed among lines as well as between Al-tolerant and non-tolerant groups of lines were due to some experimental errors or that the number of lines was too small for ANOVA to prove the influence of Al stress. Nevertheless, we suspect that Al tolerance in triticale could be partly regulated by epigenetic factors acting at the level of DNA methylation. This method provides a valuable tool for studies of abiotic stresses in plants.

DNA methylation patterns of banana leaves in response to Fusarium oxysporum f. sp. cubense tropical race 4

Fusarium wilt of banana, which is caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), is a serious soil-borne fungal disease. Now, the epigenetic molecular pathogenic basis is elusive. In this study, with methylation-sensitive amplification polymorphism (MSAP) technique, DNA methylation was compared between the leaves inoculated with Foc TR4 and the mock-inoculated leaves at different pathogenic stages. With 25 pairs of primers, 1 144 and 1 255 fragments were amplified from the infected and mock-inoculated leaves, respectively. DNA methylation was both changed and the average methylated CCGG sequences were 34.81 and 29.26% for the infected and the mock-inoculated leaves. And DNA hypermethylation and hypomethylation were induced by pathogen infection during all pathogenic stages. Further, 69 polymorphic fragments were sequenced and 29 of them showed sequence similarity to genes with known functions. And RT-PCR results of four genes indicated that their expression patterns were consistent with their methylation patterns. Our results suggest that DNA methylation plays important roles in pathogenic response to Foc TR4 for banana.

Mapping of unmethylated sites in rDNA repeats in barley NOR deletion line

Extensive cytosine methylation is characteristic of plant rDNA. Evidence exists, however, that the active rRNA genes are less methylated. In this work we report on the mapping of unmethylated CCGG sites in Hordeum vulgare rDNA repeats by digestion with methylation sensitive restriction enzyme HpaII and indirect end-labeling of the generated fragments. For mapping we used genomic DNA from barley deletion line with a single NOR on chromosome 5H. This NOR is more active in order to compensate for the missing NOR 6H. The enhanced NOR 5H activity in the deletion mutant is not due to higher multiplicity of the rRNA genes or, as sequencing showed, to changes in the subunit structure of the intergenic spacer. The HpaII sites in barley rDNA are heavily methylated. Nevertheless, a fraction of the rDNA repeats is hypomethylated with unmethylated CCGG sites at various positions. One unmethylated CCGG sequence is close to the transcription start site, downstream of the 135bp subrepeats. Unmethylated sites are also present in the external transcribed spacer and in the genes coding mature rRNAs. The patterns of unmethylated sites in the barley deletion line and in lines with two NORs were compared. It is hypothesized that the occurrence of unmethylated sites on a fixed subset of rDNA repeats correlates with their transcriptional activity.

CpG methylated ribosomal RNA genes in relation to wing polymorphism in the rice pest Sogatella furcifera

Sogatella furcifera is a major rice pest that exhibits wing dimorphism. The methylation-sensitive representational difference analysis (MS-RDA) is widely used in plants and some animals to identify methylation differences in genomic DNA regions. However the method has been applied to insects very less. The objective of the current research was to monitor differential cytosine methylations at CCGG sequences in macropterous and brachypterous adults of female S. furcifera to determine the association between gene methylation and wing phenotypes using MS-RDA. After the second subtractive hybridization, two differentially methylated DNA fragments were obtained. These DNA fragments were then cloned into the pGEM-T vector and sequenced. One fragment from the positive hybridization was 464 bp, and highly similar to the 18S ribosomal RNA (rRNA) genes from some animals. Another fragment from the reverse hybridization was 453 bp, and homologous to the 28S rRNA gene of S. furcifera with a similarity rate as high as 99%. These results provide potential evidence that DNA methylation of rRNA genes may be related to wing phenotype variations in S. furcifera, and will facilitate future studies on the epigenetic mechanisms of insect wing polymorphism.

Differentially methylated genomic fragments related with sexual dimorphism of rice pests, Sogatella furcifera.

Sogatella furcifera (Hovarth) is a major rice pest with sexual dimorphism. The objective of the current research was to monitor differentially cytosine methylation at CCGG sequences in male and female adults of S. furcifera to determine the association between gene methylation and sexual phenotypes using methylation-sensitive representational difference analysis. After the second subtractive hybridization, four differentially methylated DNA bands were obtained and sequenced. Ten different fragments were found. One fragment from the positive hybridization was 120 bp, and highly similar to the tramtrack genes from Nasonia vitripennis. Another fragment from the reverse hybridization was 414 bp, and homologous to the 28S rRNA gene of S. furcifera with a similarity rate as high as 99%. We also discussed how DNA methylation of tramtrack and 28S rRNA genes produced effects on sexual differentiation and development. These results provide potential evidence that DNA methylation of some genes may be related to sexual phenotype variations in S. furcifera and will facilitate future studies on the epigenetic mechanisms of insect sexual dimorphism.

Different genomic DNA methylation patterns between male and female adults of white-backed planthoppers Sogatella furcifera

DNA methylation plays a key role in gene regulation and phenotype variation in many organisms. The aim of this study was to survey the frequency and variation of cytosine methylation at CCGG sequences in adult male and female planthoppers Sogatella furcifera, a major rice pest in Asia, and to determine the occurrence of methylation changes associated with sexual dimorphism using methylation-sensitive amplification polymorphism. 1131 DNA fragments including CCGG sites were amplified using 36 pairs of selective primers: about 191 methylated bands were identified. In male planthoppers, we got a total of 581 bands, including 40 fully-methylated bands, 65 hemi-methylated bands and 476 none-methylated bands, so the fully-methylated ratio, hemi-methylated ratio and total methylated ratio were 6.88%, 11.19% and 18.07%, respectively. In the female planthopper, there were a total of 550 bands, including 44 fully-methylated bands, 42 hemi-methylated bands and 464 none-methylated bands. The fully-methylated ratio was 7.64% in female planthoppers, which was slightly higher than in the male planthoppers, however, the hemi-methylated ratio was lower (8.00%) in the female compared with the male planthopper. Altogether, 46 DNA bands displayed variable cytosine methylation patterns between male and female samples: 20 of them occurred only in male samples and 26 only in female samples. Thus, the genome methylation patterns are different between male and female adults. The results suggest that DNA methylation might be related to sexual differentiation and development in S. furcifera.

Oligonucleotide treatment causes flax β-glucanase up-regulation via changes in gene-body methylation.

BackgroundNowadays, the challenge for biotechnology is to develop tools for agriculture and industry to provide plants characterized by productivity and quality that will satisfy the growing demand for different kinds of natural products. To meet the challenge, the generation and application of genetically modified plants is justified. However, the strong social resistance to genetically modified organisms and restrictive regulations in European Union countries necessitated the development of a new technology for new plant types generation which uses the knowledge resulting from analysis of genetically modified plants to generate favourably altered plants while omitting the introduction of heterologous genes to their genome. Four-year experiments led to the development of a technology inducing heritable epigenetic gene activation without transgenesis.ResultsThe method comprises the induction of changes in methylation/demethylation of the endogenous gene by the plant’s treatment with short oligodeoxynucleotides antisense to the coding region. In vitro cultured plants and F3 generation flax plants overproducing the β-1,3-glucanase gene (EMO-βGlu flax) were characterized by up-regulation of β-glucanase and chitinase genes, decreases in the methylation of CCGG sequences in the β-glucanase gene and in total DNA methylation and, more importantly, reasonable resistance against Fusarium infection. In addition, EMO-βGlu flax obtained by this technology showed similar features as those obtained by genetic engineering.ConclusionTo our best knowledge, this is the first report on plant gene activation by treatment with oligodeoxynucleotides homologous to the coding region of the gene. Apart from the evident effectiveness, the most important issue is that the EMO method allows generation of favourably altered plants, whose cultivation makes the plant producer independent from the complicated procedure of obtaining an agreement on GMO release into the environment and whose products might be more easily introduced to the global market.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0261-z) contains supplementary material, which is available to authorized users.

Active coping toward predatory stress is associated with lower corticosterone and progesterone plasma levels and decreased methylation in the medial amygdala vasopressin system

An active coping style displayed under stress – which involves proactive investigatory responses toward environmental threats – has been associated with reduced vulnerability to psychiatric illness. However, the neurobiological determinants of coping styles are not well understood. When rats are exposed to a naturalistic stressor (cat fur) in a group, some individuals in the group show robust active investigation of the stimulus while others show a passive response involving retreat, immobility and close aggregation with conspecifics. Here we explored endocrine and epigenetic correlates of these contrasting coping styles. Male Wistar rats (n=48) were exposed to cat fur in groups of 4 and the passive and active responders were identified and assessed for endocrine and epigenetic differences. Three days after the final cat fur exposure, active responders had substantially lower plasma levels of corticosterone and progesterone than passive responders. Plasma and testicular testosterone levels did not differ between active and passive responders. Active responders had markedly less methylation of the AVP CGCG promoter region located at base 4970 in the posterodorsal region of the medial amygdala but did not differ in the methylation status of the CCGG sequence located at base 2243. This is in agreement with prior research suggesting that AVP and progesterone act in opposition within the medial amygdala to modulate stress-related behaviors. The present study reports striking endocrine and epigenetic differences between active and passive responders, providing insight into potential systems involved in the manifestation of differing coping styles.

Differential DNA methylation between two wing phenotypes adults of Sogatella furcifera

Sogatella furcifera is a major rice pest with wing dimorphism. DNA methylation is an important epigenetic modification that plays a role in gene regulation and phenotype variation in most organisms. The objective of the current research was to survey the frequencies and variation of cytosine methylation at CCGG sequences in macropterous female adults (MFA) and brachypterous female adults (BFA) of S. furcifera, and to determine the occurrence of methylation changes associated with wing phenotypes by using methylation-sensitive amplification polymorphism (MSAP). No differences were found in the average proportions of methylated CCGG sites between MFA and BFA, but there were significant differences for methylation patterns between MFA and BFA. The fully methylated ratio was 5.81% in BFA, much higher than 2.40% in MFA; while the hemi-methylated ratio was 4.35% in BFA, much lower than 8.35% in MFA. These results provide circumstantial evidence that DNA methylation might be related to wing phenotype variation in S. furcifera. We also cloned and got 14 satisfactory sequences, which displayed variable cytosine methylation patterns between MFA and BFA. All these data will facilitate the researches on the epigenetic mechanisms of insect wing polymorphism.

UV -B Exposure of Indoor-Grown Picea abies Seedlings Causes an Epigenetic Effect and Selective Emission of Terpenes

Terpenoids are involved in various defensive functions in plants, especially conifers. Epigenetic mechanisms, for example DNA methylation, can influence plant defence systems. The purpose of the present study was to investigate the influence of UV-B exposure on the release of terpenoids from spruce seedlings and on needle DNA methylation. Ten-week-old seedlings grown indoors were exposed to UV-B radiation during 4 h, and the volatile compounds emitted from the seedlings were analysed. Analysis of the volatiles 1, 3, and 22 d after this UV-B exposure showed that bornyl acetate, borneol, myrcene, and limonene contents increased during the first 3 days, while at day 22 the level of emission had returned to the control level. UV-B exposure decreased the level of DNA methylation in needles of young seedlings, reflected in methylation changes in CCGG sequences. Exposure of young seedlings to UV-B radiation might be a way to potentiate the general defensive capacity, improving their ability to survive in outdoor conditions. UV-B-induced defence is discussed in the light of epigenetic mechanisms.

UV-B Exposure of Indoor-Grown Picea abies Seedlings Causes an Epigenetic Effect and Selective Emission of Terpenes

Terpenoids are involved in various defensive functions in plants, especially conifers. Epigenetic mechanisms, for example DNA methylation, can influence plant defence systems. The purpose of the present study was to investigate the influence of UV-B exposure on the release of terpenoids from spruce seedlings and on needle DNA methylation. Ten-week-old seedlings grown indoors were exposed to UV-B radiation during 4 h, and the volatile compounds emitted from the seedlings were analysed. Analysis of the volatiles 1, 3, and 22 d after this UV-B exposure showed that bornyl acetate, borneol, myrcene, and limonene contents increased during the first 3 days, while at day 22 the level of emission had returned to the control level. UV-B exposure decreased the level of DNA methylation in needles of young seedlings, reflected in methylation changes in CCGG sequences. Exposure of young seedlings to UV-B radiation might be a way to potentiate the general defensive capacity, improving their ability to survive in outdoor conditions. UV-B-induced defence is discussed in the light of epigenetic mechanisms.

The Mammalian de Novo DNA Methyltransferases DNMT3A and DNMT3B Are Also DNA 5-Hydroxymethylcytosine Dehydroxymethylases

For cytosine (C) demethylation of vertebrate DNA, it is known that the TET proteins could convert 5-methyl C (5-mC) to 5-hydroxymethyl C (5-hmC). However, DNA dehydroxymethylase(s), or enzymes able to directly convert 5-hmC to C, have been elusive. We present in vitro evidence that the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B, but not the maintenance enzyme DNMT1, are also redox-dependent DNA dehydroxymethylases. Significantly, intactness of the C methylation catalytic sites of these de novo enzymes is also required for their 5-hmC dehydroxymethylation activity. That DNMT3A and DNMT3B function bidirectionally both as DNA methyltransferases and as dehydroxymethylases raises intriguing and new questions regarding the structural and functional aspects of these enzymes and their regulatory roles in the dynamic modifications of the vertebrate genomes during development, carcinogenesis, and gene regulation.

Tissue culture-induced alteration in cytosine methylation in new rice recombinant inbred lines

Zizania DNA introgression could induce a large number of genetic and epigenetic changes of the new rice recombinant inbred lines genome. In this present study, we employed inter-simple sequence repeat (ISSR) to further study the genetic and epigenetic changes that are induced by tissue culture. Changes induced by tissue culture are mostly epigenetic. One kind of methylation alterations is that the unmethylated CCGG sequence was transformed into external hemimethylated cytosines. This type change, however, did not transmit to progenies of the plant. Another kind of methylation alterations is tissue culture-induced occurrence of de novo methylation at the external cytosines of the CCGG sites that are originally with hyper-methylated internal cytosines. Interestingly, many of these methylated external cytosines became demethylated again upon plant regeneration, implying that the hypomethylated state of these loci is required for successful plant regeneration. Because the changed methylation patterns are highly similar among calli subcultured for different intervals, and because apparent concordance exists between randomly selected individual regenerants, we are tempted to deduce that the DNA methylation changes did not occur randomly, rather, the methylation events likely to have hot spots, and/or occur non-randomly. Sequence analysis indicates that the loci underwent methylation alterations are predominantly genic, sharing significant homology to known-function genes or expressed sequence tag (EST) sequences. This finding implies that the epigenetic changes induced by tissue culture could potentially affect the expression of the relevant genes.

Frequencies and variation in cytosine methylation patterns in diploid and tetraploid cytotypes of Paspalum notatum

Paspalum notatum Flugge is a grass species organized as an agamic complex. The objective of the current research was to survey the frequencies and variation of cytosine methylation at CCGG sequences in diploid and tetraploid genotypes, and to determine the occurrence of methylation changes associated with tetraploidization by using methylation-sensitive amplification polymorphism (MSAP) markers. No differences were found in the average proportions of methylated CCGG sites between cytotypes, but methylation patterns were significantly more variable in tetraploids. In both groups of plants, epigenetic and non-epigenetic variation correlated significantly when compared by Mantel tests. The evaluation of 159 common MSAP markers showed that 18.86 % of them differed in their methylation status in the different ploidies. Dendrogram analysis, reflecting epigenetic distances, showed that the four diploids and one experimentally-obtained sexually-reproducing tetraploid, grouped together. MSAP analysis performed on a diploid plant and its autotetraploid derivative showed that new epialleles emerged after tetraploidization. Sequencing of several MASP markers showed homologies with low copy genes, non-coding sequences and transposon/retrotransposon elements.

Frequencies and variation in cytosine methylation patterns in diploid and tetraploid cytotypes of Paspalum notatum

Paspalum notatum FlĂźgge is a grass species organized as an agamic complex. The objective of the current research was to survey the frequencies and variation of cytosine methylation at CCGG sequences in diploid and tetraploid genotypes, and to determine the occurrence of methylation changes associated with tetraploidization by using methylation-sensitive amplification polymorphism (MSAP) markers. No differences were found in the average proportions of methylated CCGG sites between cytotypes, but methylation patterns were significantly more variable in tetraploids. In both groups of plants, epigenetic and non-epigenetic variation correlated significantly when compared by Mantel tests. The evaluation of 159 common MSAP markers showed that 18.86 % of them differed in their methylation status in the different ploidies. Dendrogram analysis, reflecting epigenetic distances, showed that the four diploids and one experimentally-obtained sexually-reproducing tetraploid, grouped together. MSAP analysis performed on a diploid plant and its autotetraploid derivative showed that new epialleles emerged after tetraploidization. Sequencing of several MASP markers showed homologies with low copy genes, non-coding sequences and transposon/retrotransposon elements.

Tissue-specific Distribution and Dynamic Changes of 5-Hydroxymethylcytosine in Mammalian Genomes

Cytosine residues in the vertebrate genome are enzymatically modified to 5-methylcytosine, which participates in transcriptional repression of genes during development and disease progression. 5-Methylcytosine can be further enzymatically modified to 5-hydroxymethylcytosine by the TET family of methylcytosine dioxygenases. Analysis of 5-methylcytosine and 5-hydroxymethylcytosine is confounded, as these modifications are indistinguishable by traditional sequencing methods even when supplemented by bisulfite conversion. Here we demonstrate a simple enzymatic approach that involves cloning, identification, and quantification of 5-hydroxymethylcytosine in various CCGG loci within murine and human genomes. 5-Hydroxymethylcytosine was prevalent in human and murine brain and heart genomic DNAs at several regions. The cultured cell lines NIH3T3 and HeLa both displayed very low or undetectable amounts of 5-hydroxymethylcytosine at the examined loci. Interestingly, 5-hydroxymethylcytosine levels in mouse embryonic stem cell DNA first increased then slowly decreased upon differentiation to embryoid bodies, whereas 5-methylcytosine levels increased gradually over time. Finally, using a quantitative PCR approach, we established that a portion of VANGL1 and EGFR gene body methylation in human tissue DNA samples is indeed hydroxymethylation.