Reprogramming of DNA Methylation in Pollen Guides Epigenetic Inheritance via Small RNA

Epigenetic inheritance is more widespread in plants than in mammals, in part because mammals erase epigenetic information by germline reprogramming. We sequenced the methylome of three haploid cell types from developing pollen: the sperm cell, the vegetative cell, and their precursor, the postmeiotic microspore, and found that unlike in mammals the plant germline retains CG and CHG DNA methylation. However, CHH methylation is lost from retrotransposons in microspores and sperm cells and restored by de novo DNA methyltransferase guided by 24 nt small interfering RNA, both in the vegetative nucleus and in the embryo after fertilization. In the vegetative nucleus, CG methylation is lost from targets of DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), and their homologs, which include imprinted loci and recurrent epialleles that accumulate corresponding small RNA and are premethylated in sperm. Thus genome reprogramming in pollen contributes to epigenetic inheritance, transposon silencing, and imprinting, guided by small RNA.

Geminivirus Rep Protein Interferes with the Plant DNA Methylation Machinery and Modifies the Host Epigenome

The apparent simplicity of viruses hides the complexity of their interactions with their hosts. Viruses are masters at circumventing host defenses and manipulating the cellular environment for their own benefit. The replication of the largest known family of single-stranded DNA viruses, Geminiviridae, is impaired by DNA methylation and Arabidopsis mutants affected in cytosine methylation are hypersusceptible to geminivirus infection. This implies that plants might use methylation as a defense against geminiviruses and that the viral genome is a target for plant DNA methyltransferases. We have found a novel counter-defense strategy used by geminiviruses, that reduces the expression of the plant maintenance DNA methyltransferases, MET1 and CMT3, in both, locally and systemically infected tissues. Furthermore, we demonstrated that the virus-mediated repression of these two maintenance DNA methyltransferases is widely spread among different geminivirus species. Additionally, we identified Rep as the geminiviral protein responsible for the repression of MET1 and CMT3, and another viral protein, C4, as an ancillary player in MET1 downregulation. The presence of Rep, suppresses TGS of an Arabidopsis transgene and of host loci whose expression is strongly controlled by CG methylation. Bisulfite sequencing analyses showed that the expression of Rep caused a substantial reduction in the levels of DNA methylation at CG sites. Our findings suggest that Rep, the only viral protein essential for geminiviral replication, displays TGS suppressor activity through a mechanism distinct from the one thus far described for geminiviruses.

Reprogramming of DNA Methylation in Pollen Guides Epigenetic Inheritance via Small RNA

Epigenetic inheritance is more widespread in plants than in mammals, in part because mammals erase epigenetic information by germline reprogramming. We sequenced the methylome of three haploid cell types from developing pollen: the sperm cell, the vegetative cell, and their precursor, the postmeiotic microspore, and found that unlike in mammals the plant germline retains CG and CHG DNA methylation. However, CHH methylation is lost from retrotransposons in microspores and sperm cells and restored by de novo DNA methyltransferase guided by 24 nt small interfering RNA, both in the vegetative nucleus and in the embryo after fertilization. In the vegetative nucleus, CG methylation is lost from targets of DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), and their homologs, which include imprinted loci and recurrent epialleles that accumulate corresponding small RNA and are premethylated in sperm. Thus genome reprogramming in pollen contributes to epigenetic inheritance, transposon silencing, and imprinting, guided by small RNA.

DNA methyltransferase expression in the mouse germ line during periods of de novo methylation

DNA methyltransferase (DNMT) 3A and DNMT3B are both active de novo DNA methyltransferases required for development, whereas DNMT3L, which has no demonstrable methyltransferase activity, is required for methylation of imprinted genes in the oocyte. We show here that different mechanisms are used to restrict access by these proteins to their targets during germ cell development. Transcriptional control of the Dnmt3l promoter guarantees that message is low or absent except during periods of de novo activity. Use of an alternative promoter at the Dnmt3a locus produces the shorter Dnmt3a2 transcript in the germ line and postimplantation embryo only, whereas alternative splicing of the Dnmt3b transcript ensures that Dnmt3b1 is absent in the male prospermatogonia. Control of subcellular protein localization is a common theme for DNMT3A and DNMT3B, as proteins were seen in the nucleus only when methylation was occurring. These mechanisms converge to ensure that the only time that functional products from each locus are present in the germ cell nuclei is around embryonic day 17.5 in males and after birth in the growing oocytes in females.

Distinct methylation patterns in genes that affect mitochondrial function are associated with kidney disease in blood-derived DNA from individuals with Type 1 diabetes

AIMS: Epigenetic modifications, such as DNA methylation, can influence the risk of developing kidney disease. We studied methylation profiles in genes related to mitochondrial function to assess whether differences in these epigenetic features were associated with diabetic kidney disease in people with Type 1 diabetes.

METHODS: A case-control association study was undertaken (n = 196 individuals with diabetic kidney disease vs. n = 246 individuals without renal disease). Participants were White and diagnosed with Type 1 diabetes before 31 years of age. Genes that encode mitochondrial proteins (n = 780) were downloaded from mitoproteome. org. DNA methylation profiles from blood-derived DNA were generated using the Illumina Infinium HumanMethylation450 (262 samples) and Illumina Infinium HumanMethylation27 (192 samples) arrays. Beta values (β) were calculated and quality control was conducted, including evaluating blind duplicate DNA samples.

RESULTS: Fifty-four Cytosine-phosphate-Guanine probes across 51 unique genes were significantly associated (P ≤ 10(-8) ) with diabetic kidney disease across both the 450K and the 27K methylation arrays. A subanalysis, employing the 450K array, identified 755 Cytosine-phosphate-Guanine probes in 374 genes that were significantly associated (P ≤ 10(-8) ) with end-stage renal disease. Forty-six of the top-ranked variants for diabetic kidney disease were also identified as being differentially methylated in individuals with end-stage renal disease. The largest change in methylation (Δβ = 0.2) was observed for cg03169527 in the TAMM41 gene, chromosome 3p25.2. Three genes, PMPCB, TSFM and AUH, were observed with differential methylation at multiple Cytosine-phosphate-Guanine sites each (P < 10(-12) ).

CONCLUSIONS: Differential methylation in genes that influence mitochondrial function are associated with kidney disease in individuals with Type 1 diabetes. 

Comparative DNA sequence and methylation analyses of orthologous genes in humans and non-human primates: Genetic and epigenetic footprints of evolution

Welche genetische Unterschiede machen uns verschieden von unseren nächsten Verwandten, den Schimpansen, und andererseits so ähnlich zu den Schimpansen? Was wir untersuchen und auch verstehen wollen, ist die komplexe Beziehung zwischen den multiplen genetischen und epigenetischen Unterschieden, deren Interaktion mit diversen Umwelt- und Kulturfaktoren in den beobachteten phänotypischen Unterschieden resultieren. Um aufzuklären, ob chromosomale Rearrangements zur Divergenz zwischen Mensch und Schimpanse beigetragen haben und welche selektiven Kräfte ihre Evolution geprägt haben, habe ich die kodierenden Sequenzen von 2 Mb umfassenden, die perizentrischen Inversionsbruchpunkte flankierenden Regionen auf den Chromosomen 1, 4, 5, 9, 12, 17 und 18 untersucht. Als Kontrolle dienten dabei 4 Mb umfassende kollineare Regionen auf den rearrangierten Chromosomen, welche mindestens 10 Mb von den Bruchpunktregionen entfernt lagen. Dabei konnte ich in den Bruchpunkten flankierenden Regionen im Vergleich zu den Kontrollregionen keine höhere Proteinevolutionsrate feststellen. Meine Ergebnisse unterstützen nicht die chromosomale Speziationshypothese für Mensch und Schimpanse, da der Anteil der positiv selektierten Gene (5,1% in den Bruchpunkten flankierenden Regionen und 7% in den Kontrollregionen) in beiden Regionen ähnlich war. Durch den Vergleich der Anzahl der positiv und negativ selektierten Gene per Chromosom konnte ich feststellen, dass Chromosom 9 die meisten und Chromosom 5 die wenigsten positiv selektierten Gene in den Bruchpunkt flankierenden Regionen und Kontrollregionen enthalten. Die Anzahl der negativ selektierten Gene (68) war dabei viel höher als die Anzahl der positiv selektierten Gene (17). Eine bioinformatische Analyse von publizierten Microarray-Expressionsdaten (Affymetrix Chip U95 und U133v2) ergab 31 Gene, die zwischen Mensch und Schimpanse differentiell exprimiert sind. Durch Untersuchung des dN/dS-Verhältnisses dieser 31 Gene konnte ich 7 Gene als negativ selektiert und nur 1 Gen als positiv selektiert identifizieren. Dieser Befund steht im Einklang mit dem Konzept, dass Genexpressionslevel unter stabilisierender Selektion evolvieren. Die meisten positiv selektierten Gene spielen überdies eine Rolle bei der Fortpflanzung. Viele dieser Speziesunterschiede resultieren eher aus Änderungen in der Genregulation als aus strukturellen Änderungen der Genprodukte. Man nimmt an, dass die meisten Unterschiede in der Genregulation sich auf transkriptioneller Ebene manifestieren. Im Rahmen dieser Arbeit wurden die Unterschiede in der DNA-Methylierung zwischen Mensch und Schimpanse untersucht. Dazu wurden die Methylierungsmuster der Promotor-CpG-Inseln von 12 Genen im Cortex von Menschen und Schimpansen mittels klassischer Bisulfit-Sequenzierung und Bisulfit-Pyrosequenzierung analysiert. Die Kandidatengene wurden wegen ihrer differentiellen Expressionsmuster zwischen Mensch und Schimpanse sowie wegen Ihrer Assoziation mit menschlichen Krankheiten oder dem genomischen Imprinting ausgewählt. Mit Ausnahme einiger individueller Positionen zeigte die Mehrzahl der analysierten Gene keine hohe intra- oder interspezifische Variation der DNA-Methylierung zwischen den beiden Spezies. Nur bei einem Gen, CCRK, waren deutliche intraspezifische und interspezifische Unterschiede im Grad der DNA-Methylierung festzustellen. Die differentiell methylierten CpG-Positionen lagen innerhalb eines repetitiven Alu-Sg1-Elements. Die Untersuchung des CCRK-Gens liefert eine umfassende Analyse der intra- und interspezifischen Variabilität der DNA-Methylierung einer Alu-Insertion in eine regulatorische Region. Die beobachteten Speziesunterschiede deuten darauf hin, dass die Methylierungsmuster des CCRK-Gens wahrscheinlich in Adaption an spezifische Anforderungen zur Feinabstimmung der CCRK-Regulation unter positiver Selektion evolvieren. Der Promotor des CCRK-Gens ist anfällig für epigenetische Modifikationen durch DNA-Methylierung, welche zu komplexen Transkriptionsmustern führen können. Durch ihre genomische Mobilität, ihren hohen CpG-Anteil und ihren Einfluss auf die Genexpression sind Alu-Insertionen exzellente Kandidaten für die Förderung von Veränderungen während der Entwicklungsregulation von Primatengenen. Der Vergleich der intra- und interspezifischen Methylierung von spezifischen Alu-Insertionen in anderen Genen und Geweben stellt eine erfolgversprechende Strategie dar.

The effects of DNA cytosine methylation on H -ras promoter structure and function

The effect of DNA cytosine methylation on H-ras promoter activity was assessed using a transient expression system employing the plasmid H-rasCAT (NaeI H-ras promoter linked to the chloramphenicol acetyltransferase (CAT) gene). This 551 bp promoter is 80% GC rich, enriched with 168 CpG dinucleotides, and contains six functional GC box elements which represent major DNA methylation target sites. Prokaryotic methyltransferases HhaI (CGm$\sp5$CG) and HpaII (Cm$\sp5$CGG) alone or in combination with a human placental methyltransferase (HP MTase) were used to introduce methyl groups at different CpG sites within the promoter. To test for functional promoter activity, the methylated plasmids were introduced into CV-1 cells and CAT activity assessed 48 h post-transfection. Methylation at specific HhaI and HpaII sites reduced CAT expression by 70%, whereas more extensive methylation at generalized CpG sites with HP MTase inactivated the promoter $>$95%. The inhibition of H-ras promoter activity was not attributable to methylation-induced differences in DNA uptake or stability in the cell, topological form of the plasmid, or methylation effects in nonpromoter regions. We also observed that DNA cytosine methylation of a 360 bp promoter fragment by HP MTase induced a local change in DNA conformation. Using three independent methodologies (nitrocellulose filter binding assays, gel mobility shifts, and Southwestern blots), we determined that this change in promoter conformation affected the interaction of nuclear proteins with cis-regulatory sequences residing in the promoter region. The results provide evidence to suggest that DNA methylation may regulate gene expression by inducing changes in local promoter conformation which in turn alters the interactions between DNA and protein factors required for transcription. The results provide supportive evidence for the hypothesis of Cedar and Riggs, who postulated that DNA methylation may regulate gene expression by altering the binding affinities of proteins for DNA. ^

Chromosomal insertion of foreign (adenovirus type 12, plasmid, or bacteriophage lambda) DNA is associated with enhanced methylation of cellular DNA segments.

Insertion of foreign DNA into an established mammalian genome can extensively alter the patterns of cellular DNA methylation. Adenovirus type 12 (Ad12)-transformed hamster cells, Ad12-induced hamster tumor cells, or hamster cells carrying integrated DNA of bacteriophage lambda were used as model systems. DNA methylation levels were examined by cleaving cellular DNA with Hpa II, Msp I, or Hha I, followed by Southern blot hybridization with 32P-labeled, randomly selected cellular DNA probes. For several, but not all, cellular DNA segments investigated, extensive increases in DNA methylation were found in comparison with the methylation patterns in BHK21 or primary Syrian hamster cells. In eight different Ad12-induced hamster tumors, moderate increases in DNA methylation were seen. Increased methylation of cellular genes was also documented in two hamster cell lines with integrated Ad12 DNA without the Ad12-transformed phenotype, in one cloned BHK21 cell line with integrated plasmid DNA, and in at least three cloned BHK21 cell lines with integrated lambda DNA. By fluorescent in situ hybridization, the cellular hybridization probes were located to different hamster chromosomes. The endogenous intracisternal A particle genomes showed a striking distribution on many hamster chromosomes, frequently on their short arms. When BHK21 hamster cells were abortively infected with Ad12, increases in cellular DNA methylation were not seen. Thus, Ad12 early gene products were not directly involved in increasing cellular DNA methylation. We attribute the alterations in cellular DNA methylation, at least in part, to the insertion of foreign DNA. Can alterations in the methylation profiles of hamster cellular DNA contribute to the generation of the oncogenic phenotype?

Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

BACKGROUND: Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.

METHODS: CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.

RESULTS: Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2'-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2.

CONCLUSION: These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.

Gemcitabine reactivates epigenetically silenced genes and functions as a DNA methyltransferase inhibitor

Gemcitabine is indicated in combination with cisplatin as first-line therapy for solid tumours including non-small cell lung cancer (NSCLC), bladder cancer and mesothelioma. Gemcitabine is an analogue of pyrimidine cytosine and functions as an anti-metabolite. Structurally, however, gemcitabine has similarities to 5-aza-2-deoxycytidine (decitabine/Dacogen®), a DNA methyltransferase inhibitor (DNMTi). NSCLC, mesothelioma and prostate cancer cell lines were treated with decitabine and gemcitabine. Reactivation of epigenetically silenced genes was examined by RT-PCR/qPCR. DNA methyltransferase activity in nuclear extracts and recombinant proteins was measured using a DNA methyltransferase assay, and alterations in DNA methylation status were examined using methylation-specific PCR (MS-PCR) and pyrosequencing. We observe a reactivation of several epigenetically silenced genes including GSTP1, IGFBP3 and RASSF1A. Gemcitabine functionally inhibited DNA methyltransferase activity in both nuclear extracts and recombinant proteins. Gemcitabine dramatically destabilised DNMT1 protein. However, DNA CpG methylation was for the most part unaffected by gemcitabine. In conclusion, gemcitabine both inhibits and destabilises DNA methyltransferases and reactivates epigenetically silenced genes having activity equivalent to decitabine at concentrations significantly lower than those achieved in the treatment of patients with solid tumours. This property may contribute to the anticancer activity of gemcitabine.

Computational analysis of epigenetic information in human DNA sequences

Over the last few years, investigations of human epigenetic profiles have identified key elements of change to be Histone Modifications, stable and heritable DNA methylation and Chromatin remodeling. These factors determine gene expression levels and characterise conditions leading to disease. In order to extract information embedded in long DNA sequences, data mining and pattern recognition tools are widely used, but efforts have been limited to date with respect to analyzing epigenetic changes, and their role as catalysts in disease onset. Useful insight, however, can be gained by investigation of associated dinucleotide distributions. The focus of this paper is to explore specific dinucleotides frequencies across defined regions within the human genome, and to identify new patterns between epigenetic mechanisms and DNA content. Signal processing methods, including Fourier and Wavelet Transformations, are employed and principal results are reported.

Mapping the regulatory sequences controlling 93 breast cancer-associated miRNA genes leads to the identification of two functional promoters of the Hsa-mir-200b cluster, methylation of which is associated with metastasis or hormone receptor status in advanced breast cancer

MicroRNAs (miRNAs) are small non-coding RNAs of 20 nt in length that are capable of modulating gene expression post-transcriptionally. Although miRNAs have been implicated in cancer, including breast cancer, the regulation of miRNA transcription and the role of defects in this process in cancer is not well understood. In this study we have mapped the promoters of 93 breast cancer-associated miRNAs, and then looked for associations between DNA methylation of 15 of these promoters and miRNA expression in breast cancer cells. The miRNA promoters with clearest association between DNA methylation and expression included a previously described and a novel promoter of the Hsa-mir-200b cluster. The novel promoter of the Hsa-mir-200b cluster, denoted P2, is located 2 kb upstream of the 5′ stemloop and maps within a CpG island. P2 has comparable promoter activity to the previously reported promoter (P1), and is able to drive the expression of miR-200b in its endogenous genomic context. DNA methylation of both P1 and P2 was inversely associated with miR-200b expression in eight out of nine breast cancer cell lines, and in vitro methylation of both promoters repressed their activity in reporter assays. In clinical samples, P1 and P2 were differentially methylated with methylation inversely associated with miR-200b expression. P1 was hypermethylated in metastatic lymph nodes compared with matched primary breast tumours whereas P2 hypermethylation was associated with loss of either oestrogen receptor or progesterone receptor. Hypomethylation of P2 was associated with gain of HER2 and androgen receptor expression. These data suggest an association between miR-200b regulation and breast cancer subtype and a potential use of DNA methylation of miRNA promoters as a component of a suite of breast cancer biomarkers.

Role of histidine residues in EcoP15I DNA methyltransferase activity as probed by chemical modification and site-directed mutagenesis

Towards understanding the catalytic mechanism of M.EcoP15I [EcoP15I MTase (DNA methyltransferase); an adenine methyltransferase], we investigated the role of histidine residues in catalysis. M.EcoP15I, when incubated with DEPC (diethyl pyrocarbonate), a histidine-specific reagent, shows a time- and concentration-dependent inactivation of methylation of DNA containing its recognition sequence of 5'-CAGCAG-3'. The loss of enzyme activity was accompanied by an increase in absorbance at 240 nm. A difference spectrum of modified versus native enzyme shows the formation of N-carbethoxyhistidine that is diminished by hydroxylamine. This, along with other experiments, strongly suggests that the inactivation of the enzyme by DEPC was specific for histidine residues. Substrate protection experiments show that pre-incubating the methylase with DNA was able to protect the enzyme from DEPC inactivation. Site-directed mutagenesis experiments in which the 15 histidine residues in the enzyme were replaced individually with alanine corroborated the chemical modification studies and established the importance of His-335 in the methylase activity. No gross structural differences were detected between the native and H335A mutant MTases, as evident from CD spectra, native PAGE pattern or on gel filtration chromatography. Replacement of histidine with alanine residue at position 335 results in a mutant enzyme that is catalytically inactive and binds to DNA more tightly than the wild-type enzyme. Thus we have shown in the present study, through a combination of chemical modification and site-directed mutagenesis experiments, that His-335 plays an essential role in DNA methylation catalysed by M.EcoP15I.

Epigenetics Underpinning DNA Damage Repair

Epigenetics is the study of heritable changes in gene expression that are not the result of genetic alterations. These changes include DNA methylation, histone modifications, or indeed microRNA expression. Chromatin is a tightly compacted DNA–protein complex that allows approximately two meters of DNA to be packaged inside a cell, only a few micrometers across. Although the resulting DNA structure is very stable, it is not very amiable to DNA-dependent processes, so mechanisms have to exist to allow processes such as transcription, replication, and DNA repair to occur. This chapter will look at how a cell responds to and deals with genomic instability at the epigenetic level and highlight how critical chromatin remodeling is for correct DNA repair and cell survival following DNA damage. This chapter will initially look at the DNA repair pathways that function in human cells and then at how the repair of DNA damage is controlled by epigenetics.

Studies on hypomethylation of liver DNA during early stages of chemical carcinogenesis in rat liver

Our finding that the inhibitors of DNA methylation, 5-azacytidine, 5-azadeoxycytidine or adenosine dialdehyde, given after a carcinogen all potentiated initiation suggested that hypomethylation of DNA during repair synthesis of DNA might play a role in the initiation of the carcinogenic process. To examine this aspect further, we have asked the question, do the nodules which develop from initiated cells after promotion with 1% orotic acid exhibit an altered methylation pattern in their DNA? The methylation status of the DNA from nodules has been examined using the restriction endonucleases HpaII/MspI and HhaI which distinguish between methylated and unmethylated cytosines in their nucleotide recognition DNA 5'-CCGG and 5'-GCGC respectively. The proto-oncogenes, c-myc, c-fos and c-Ha-ras, in the DNA were primarily studied in this investigation because of their possible involvement in cell proliferation and/or in cell transformation and tumorigenesis. The results indicate that in the nodule DNA, c-myc and c-fos are hypomethylated in the sequence of CCGG while the c-Ha-ras shows hypomethylation in the alternating GCGC sequence. This methylation pattern seen in the nodule DNA is not found in the DNA of the non-nodular surrounding liver or liver tissue after exposure to promoter or carcinogen alone. It is also not found in the DNA of regenerating liver. It is particularly significant that the methylation patterns in the c-myc and c-Ha-ras regions are similar to those found in several cancer tissues. The results suggest that this methylation pattern is acquired early in the carcinogenic process and raises the question whether it has any bearing on the process.