Allele-Specific, Age-Dependent and BMI-Associated DNA Methylation of Human MCHR1

Background: Melanin-concentrating hormone receptor 1 (MCHR1) plays a significant role in regulation of energy balance, food intake, physical activity and body weight in humans and rodents. Several association studies for human obesity showed contrary results concerning the SNPs rs133072 (G/A) and rs133073 (T/C), which localize to the first exon of MCHR1. The variations constitute two main haplotypes (GT, AC). Both SNPs affect CpG dinucleotides, whereby each haplotype contains a potential methylation site at one of the two SNP positions. In addition, 15 CpGs in close vicinity of these SNPs constitute a weak CpG island. Here, we studied whether DNA methylation in this sequence context may contribute to population- and age-specific effects of MCHR1 alleles in obesity. \ud Principal Findings: We analyzed DNA methylation of a 315 bp region of MCHR1 encompassing rs133072 and rs133073 and the CpG island in blood samples of 49 individuals by bisulfite sequencing. The AC haplotype shows a significantly higher methylation level than the GT haplotype. This allele-specific methylation is age-dependent. In young individuals (20â\u80\u9330 years) the difference in DNA methylation between haplotypes is significant; whereas in individuals older than 60 years it is not detectable. Interestingly, the GT allele shows a decrease in methylation status with increasing BMI, whereas the methylation of the AC allele is not associated with this phenotype. Heterozygous lymphoblastoid cell lines show the same pattern of allele-specific DNA methylation. The cell line, which exhibits the highest difference in methylation levels between both haplotypes, also shows allele-specific transcription of MCHR1, which can be abolished by treatment with the DNA\ud methylase inhibitor 5-aza-2'-deoxycytidine.\ud Conclusions:We show that DNA methylation at MCHR1 is allele-specific, age-dependent, BMI-associated and affects transcription. Conceivably, this epigenetic regulation contributes to the age- and/or population specific effects reported for MCHR1 in several human obesity studies.\ud \ud doi: 10.1371/journal.pone.0017711\u

Analysis of tissue-specific & allele-specific DNA methylation

Epigenetic mechanisms such as histone modifications and DNA methylation extend the information of the underlying genomic DNA sequence. Understanding cell type-specific epigenetic codes on a global level is a major challenge after the sequencing of the human genome has been completed. In addition, differences in epigenetic patterns between individuals may contribute to phenotypic variation and disease susceptibility. However, little is known about the extent of such variation or how different epigenetic patterns are established. In the present thesis, I applied and modified the methyl-CpG immunoprecipitation (MCIp) method to globally map DNA methylation. This method is based on a recombinant, methyl-CpG-DNA binding protein (MBD2-Fc), which is used to fractionate genomic DNA depending on its methylation status. During this thesis the MCIp-method was on the one hand modified and established for a novel application. This study was designed as a pilot project to establish the MCIp approach for comparative hypomethylation analysis. To study cell-type-specific DNA methylation differences, the MCIp procedure was used in combination with oligonucleotide promoter microarrays (MCIp-on-chip) to identify differences in promoter hypomethylation of coding and non-coding genes in human tissues. Forty-four identified tissue-specifically methylated promoters were independently validated using bisulfite sequencing or single gene MCIp, confirming the results obtained by the MCIp microarray approach. A comparison of the obtained tissue methylation profiles with corresponding gene expression data indicated a significant association between tissue-specific promoter methylation and gene expression, not only in CpG-rich promoters. The data also highlighted the exceptional epigenetic status of germ line cells in testis, where many testis-specific promoters were demethylated as compared to somatic tissues. Somatic hypermethylation particularly affected many CpG-rich promoters of testis-specific genes e.g. in ampliconic areas of the Y-chromosome. In order to analyze individual (allele-specific) DNA methylation the MCIp-on-chip method was on the other hand further improved to the �mirror image� procedure. The �mirror image� approach was used to analyze the methylation profiles of immune cells from two inbred mice strains (C57BL/6 and BALB/c) that represent prototypic models for Th1- or Th2-dominated immune responses. Using MCIp the genomes of bone marrow-derived macrophages were fractionated into methylated and unmethylated genome pools and separately analyzed by microarray at 181 genomic regions (covering 28 Mb of mouse genome) that were selected based on differential gene expression between both mouse strains. The combined analysis of hypo- and hypermethylated profiles allowed the identification of several hundred differentially methylated regions, but also uncovered several copy number variations as well as many single nucleotide polymorphisms (SNPs) or micro- and macro-deletions/insertions. By using DNA sequencing and mass spectrometry it was shown for a subset of regions that specific allelic methylation patterns in somatic cells were maintained in F1- hybrid animals, regardless of their status in germ line cells. A common association with sequence polymorphisms suggests that the genomic context at these differentially methylated regions determines their developmentally regulated methylation in cis

DNA Methylation Analysis of Chromosome 21 Gene Promoters at Single Base Pair and Single Allele Resolution

Differential DNA methylation is an essential epigenetic signal for gene regulation, development, and disease processes. We mapped DNA methylation patterns of 190 gene promoter regions on chromosome 21 using bisulfite conversion and subclone sequencing in five human cell types. A total of 28,626 subclones were sequenced at high accuracy using (long-read) Sanger sequencing resulting in the measurement of the DNA methylation state of 580427 CpG sites. Our results show that average DNA methylation levels are distributed bimodally with enrichment of highly methylated and unmethylated sequences, both for amplicons and individual subclones, which represent single alleles from individual cells. Within CpG-rich sequences, DNA methylation was found to be anti-correlated with CpG dinucleotide density and GC content, and methylated CpGs are more likely to be flanked by AT-rich sequences. We observed over-representation of CpG sites in distances of 9, 18, and 27 bps in highly methylated amplicons. However, DNA sequence alone is not sufficient to predict an amplicon's DNA methylation status, since 43% of all amplicons are differentially methylated between the cell types studied here. DNA methylation in promoter regions is strongly correlated with the absence of gene expression and low levels of activating epigenetic marks like H3K4 methylation and H3K9 and K14 acetylation. Utilizing the single base pair and single allele resolution of our data, we found that i) amplicons from different parts of a CpG island frequently differ in their DNA methylation level, ii) methylation levels of individual cells in one tissue are very similar, and iii) methylation patterns follow a relaxed site-specific distribution. Furthermore, iv) we identified three cases of allele-specific DNA methylation on chromosome 21. Our data shed new light on the nature of methylation patterns in human cells, the sequence dependence of DNA methylation, and its function as epigenetic signal in gene regulation. Further, we illustrate genotype–epigenotype interactions by showing novel examples of allele-specific methylation

The DNA Methylome of Human Peripheral Blood Mononuclear Cells

Analysis across the genome of patterns of DNA methylation reveals a rich landscape of allele-specific epigenetic modification and consequent effects on allele-specific gene expression

DNA hypomethylation and hypermethylation in colorectal cancer initiation.

This project was focused on identification of new valuable molecular risk factors for the onset of colorectal cancer (CRC), studying both KRAS/NRAS/BRAF/PIK3CA mutations and DNA global hypomethylation in the early events of colorectal carcinogenesis. We analyzed a cohort of 52 colorectal adenomas and 11 carcinomas derived from MAP subjects, 80 sporadic adenomas and 15 carcinomas and a control set of 36 FAP/AFAP adenomas. Moreover, we characterized the L1-MET transcript induced by L1 hypomethylation. We observed that the early steps of oxidative DNA damage in MAP carcinogenesis are characterized by a specific pattern of somatic mutations. We also found that MAP adenomas and carcinomas show a decreased DNA global methylation and specific L1-MET hypomethylation. Finally, we hypothesized that DNA hypomethylation and expression of L1-MET chimeric transcript may play an early role in colorectal carcinogenesis characterizing a subset of more aggressive precursor lesions and cancers. In the second part of the thesis, we studied the promoter of MutL homolog 1 (MLH1) in order to elucidate the relationship between methylation and risk/protective allele at rs1800734 during CRC progression. We confirmed the association of rs1800734 with microsatellite instability (MSI) in our own data. In 33 normal colon biopsies, small allele-specific differences exist only in methylation, but not gene expression. In contrast, allele-specific differences in both MLH1 methylation and expression are present in 35 MSI cancers. We showed that MLH1 transcriptional repression is dependent on DNA methylation and can be reversed by a methylation inhibitor. The rs1800734 allele influences the rate of methylation loss and amount of re-expression

Functional Epigenomics In Insects Using Next-Generation Sequencing Methods

DNA methylation is a widespread epigenetic modification implicated in many important processes such as development, disease, and genomic imprinting. In well-studied mammalian systems, DNA methylation at gene promoters acts as a transcriptional repressor including playing a critical role in X chromosome inactivation. Despite the importance and prevalence of DNA methylation, essential model organisms such as D. melanogaster and C. elegans have experienced lineage-specific losses of genomic DNA methylation. This thesis focuses on a comprehensive epigenomics survey and investigation of the Hymenopteran insect order, a group of insects including wasps, bees and ants that have retained functional DNA methylation systems. This diverse group of insects allows us to gain new insights in to the function role of DNA methylation, especially in the context of gene expression regulation. I will first provide a general survey of the epigenetic landscape of insects, which have a completely different pattern compared to mammals, and offer a new approach to quantifying and analyzing DNA methylation in these organisms. Next, I investigate changes to DNA methylation and gene expression that accompany a bacterial infection and a drastic shift from sexual to asexual reproduction in a parasitoid wasp. I will then examine how the intricate honey bee society gives rise to allele-specific methylation and its potential relationship to allele-specific expression. Finally, I explore the importance of DNA methylation along with other promoter elements in regulating gene expression variation.Ph.D

Exploring The Role Of Tet1 In Genomic Imprinting

DNA methylation is an essential epigenetic mark crucial for normal mammalian development. This modification controls the expression of a unique class of genes, designated as imprinted, which are expressed monoallelically and in a parent-of-origin-specific manner. Proper parental allele-specific DNA methylation at imprinting control regions (ICRs) is necessary for appropriate imprinting. Processes that deregulate DNA methylation of imprinted loci cause disease in humans. DNA methylation patterns dramatically change during mammalian development: first, the majority of the genome, with the exception of ICRs, is demethylated after fertilization, and subsequently undergoes genome-wide de novo DNA methylation. Secondly, after primordial germ cells are specified in the embryo, another wave of demethylation occurs, with ICR demethylation occurring late in the process. Lastly, ICRs reacquire DNA methylation imprints in developing germ cells. Although much is known about DNA methylation establishment, DNA demethylation is less well understood. Recently, the Ten-Eleven Translocation proteins (TET1-3) have been shown to initiate DNA demethylation, with Tet1-/- mice exhibiting aberrant levels of imprinted gene expression and ICR methylation. Nevertheless, TET1’s role in demethylating ICRs in the female germline and controlling allele-specific expression remains to be determined. Here, we examined ICR-specific DNA methylation in Tet1-/- germ cells and ascertained whether abnormal ICR methylation impacted imprinted gene expression in F1 hybrid somatic tissues derived from Tet1-/- eggs or sperm. We show that Tet1 deficiency is associated with hypermethylation of a subset of ICRs in germ cells. Moreover, ICRs with defective germline reprogramming exhibit aberrant DNA methylation and biallelic expression of linked imprinted genes in somatic tissues. Thus, we define a discrete set of genomic regions that require TET1 for germline reprogramming and discuss mechanisms for stochastic imprinting defects

Synthetic DNA fragments bearing ICR cis elements become differentially methylated and recapitulate genomic imprinting in transgenic mice

BackgroundGenomic imprinting is governed by allele-specific DNA methylation at imprinting control regions (ICRs), and the mechanism controlling its differential methylation establishment during gametogenesis has been a subject of intensive research interest. However, recent studies have reported that gamete methylation is not restricted at the ICRs, thus highlighting the significance of ICR methylation maintenance during the preimplantation period where genome-wide epigenetic reprogramming takes place. Using transgenic mice (TgM), we previously demonstrated that the H19 ICR possesses autonomous activity to acquire paternal-allele-specific DNA methylation after fertilization. Furthermore, this activity is indispensable for the maintenance of imprinted methylation at the endogenous H19 ICR during the preimplantation period. In addition, we showed that a specific 5′ fragment of the H19 ICR is required for its paternal methylation after fertilization, while CTCF and Sox-Oct motifs are essential for its maternal protection from undesirable methylation after implantation.ResultsTo ask whether specific cis elements are sufficient to reconstitute imprinted methylation status, we employed a TgM co-placement strategy for facilitating detection of postfertilization methylation activity and precise comparison of test sequences. Bacteriophage lambda DNA becomes highly methylated regardless of its parental origin and thus can be used as a neutral sequence bearing no inclination for differential DNA methylation. We previously showed that insertion of only CTCF and Sox-Oct binding motifs from the H19 ICR into a lambda DNA (LCb) decreased its methylation level after both paternal and maternal transmission. We therefore appended a 478-bp 5′ sequence from the H19 ICR into the LCb fragment and found that it acquired paternal-allele-specific methylation, the dynamics of which was identical to that of the H19 ICR, in TgM. Crucially, transgene expression also became imprinted. Although there are potential binding sites for ZFP57 (a candidate protein thought to control the methylation imprint) in the larger H19 ICR, they are not found in the 478-bp fragment, rendering the role of ZFP57 in postfertilization H19 ICR methylation a still open question.ConclusionsOur results demonstrate that a differentially methylated region can be reconstituted by combining the activities of specific imprinting elements and that these elements together determine the activity of a genomically imprinted region in vivo

Synthetic DNA fragments bearing ICR cis elements become differentially methylated and recapitulate genomic imprinting in transgenic mice

Background: Genomic imprinting is governed by allele-specific DNA methylation at imprinting control regions (ICRs), and the mechanism controlling its differential methylation establishment during gametogenesis has been a subject of intensive research interest. However, recent studies have reported that gamete methylation is not restricted at the ICRs, thus highlighting the significance of ICR methylation maintenance during the preimplantation period where genome-wide epigenetic reprogramming takes place. Using transgenic mice (TgM), we previously demonstrated that the H19 ICR possesses autonomous activity to acquire paternal-allele-specific DNA methylation after fertilization. Furthermore, this activity is indispensable for the maintenance of imprinted methylation at the endogenous H19 ICR during the preimplantation period. In addition, we showed that a specific 5′ fragment of the H19 ICR is required for its paternal methylation after fertilization, while CTCF and Sox-Oct motifs are essential for its maternal protection from undesirable methylation after implantation. Results: To ask whether specific cis elements are sufficient to reconstitute imprinted methylation status, we employed a TgM co-placement strategy for facilitating detection of postfertilization methylation activity and precise comparison of test sequences. Bacteriophage lambda DNA becomes highly methylated regardless of its parental origin and thus can be used as a neutral sequence bearing no inclination for differential DNA methylation. We previously showed that insertion of only CTCF and Sox-Oct binding motifs from the H19 ICR into a lambda DNA (LCb) decreased its methylation level after both paternal and maternal transmission. We therefore appended a 478-bp 5′ sequence from the H19 ICR into the LCb fragment and found that it acquired paternal-allele-specific methylation, the dynamics of which was identical to that of the H19 ICR, in TgM. Crucially, transgene expression also became imprinted. Although there are potential binding sites for ZFP57 (a candidate protein thought to control the methylation imprint) in the larger H19 ICR, they are not found in the 478-bp fragment, rendering the role of ZFP57 in postfertilization H19 ICR methylation a still open question. Conclusions: Our results demonstrate that a differentially methylated region can be reconstituted by combining the activities of specific imprinting elements and that these elements together determine the activity of a genomically imprinted region in vivo

Genomic landscape of human allele-specific DNA methylation

DNA methylation mediates imprinted gene expression by passing an epigenomic state across generations and differentially marking specific regulatory regions on maternal and paternal alleles. Imprinting has been tied to the evolution of the placenta in mammals and defects of imprinting have been associated with human diseases. Although recent advances in genome sequencing have revolutionized the study of DNA methylation, existing methylome data remain largely untapped in the study of imprinting. We present a statistical model to describe allele-specific methylation (ASM) in data from high-throughput short-read bisulfite sequencing. Simulation results indicate technical specifications of existing methylome data, such as read length and coverage, are sufficient for fullgenome ASM profiling based on our model. We used our model to analyze methylomes for a diverse set of human cell types, including cultured and uncultured differentiated cells, embryonic stem cells and induced pluripotent stem cells. Regions of ASM identified most consistently across methylomes are tightly connected with known imprinted genes and precisely delineate the boundaries of several known imprinting control regions. Predicted regions of ASM common to multiple cell types frequently mark noncoding RNA promoters and represent promising starting points for targeted validation. More generally, our model provides the analytical complement to cutting-edge experimental technologies for surveying ASM in specific cell types and across species