Comparative (computational) analysis of the DNA methylation status of trinucleotide repeat expansion diseases

Copyright © 2013 Mohammadmersad Ghorbani et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.This article has been made available through the Brunel Open Access Publishing Fund.Previous studies have examined DNA methylation in different trinucleotide repeat diseases. We have combined this data and used a pattern searching algorithm to identify motifs in the DNA surrounding aberrantly methylated CpGs found in the DNA of patients with one of the three trinucleotide repeat (TNR) expansion diseases: fragile X syndrome (FRAXA), myotonic dystrophy type I (DM1), or Friedreich’s ataxia (FRDA). We examined sequences surrounding both the variably methylated (VM) CpGs, which are hypermethylated in patients compared with unaffected controls, and the nonvariably methylated CpGs which remain either always methylated (AM) or never methylated (NM) in both patients and controls. Using the J48 algorithm of WEKA analysis, we identified that two patterns are all that is necessary to classify our three regions CCGG* which is found in VM and not in AM regions and AATT* which distinguished between NM and VM + AM using proportional frequency. Furthermore, comparing our software with MEME software, we have demonstrated that our software identifies more patterns than MEME in these short DNA sequences. Thus, we present evidence that the DNA sequence surrounding CpG can influence its susceptibility to be de novo methylated in a disease state associated with a trinucleotide repeat.European Union Seventh Framework Programme and The Brunel University Graduate Program

VEZF1 elements mediate protection from DNA methylation

There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm β-globin HS4 insulator to block the range of enhancers or the spread of repressive histone modifications is well established. Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin stat

Protection of CpG islands against de novo DNA methylation during oogenesis is associated with the recognition site of E2f1 and E2f2

BACKGROUND: Epigenetic reprogramming during early mammalian embryonic and germ cell development is a genome-wide process. CpG islands (CGIs), central to the regulation of mammalian gene expression, are exceptional in terms of whether, when and how they are affected by epigenetic reprogramming. RESULTS: We investigated the DNA sequences of CGIs in the context of genome-wide data on DNA methylation and transcription during oogenesis and early embryogenesis to identify signals associated with methylation establishment and protection from de novo methylation in oocytes and associated with post-fertilisation methylation maintenance. We find no evidence for a characteristic DNA sequence motif in oocyte-methylated CGIs. Neither do we find evidence for a general role of regular CpG spacing in methylation establishment at CGIs in oocytes. In contrast, the resistance of most CGIs to de novo methylation during oogenesis is associated with the motif CGCGC, the recognition site of E2f1 and E2f2, transcription factors highly expressed specifically in oocytes. This association is independent of prominent known hypomethylation-associated factors: CGI promoter activity, H3K4me3, Cfp1 binding or R-loop formation potential. CONCLUSIONS: Our results support a DNA sequence-independent and transcription-driven model of de novo CGI methylation during oogenesis. In contrast, our results for CGIs that remain unmethylated are consistent with a model of protection from methylation involving sequence recognition by DNA-binding proteins, E2f1 and E2f2 being probable candidates

Genome-wide conserved consensus transcription factor binding motifs are hyper-methylated.

BACKGROUND: DNA methylation can regulate gene expression by modulating the interaction between DNA and proteins or protein complexes. Conserved consensus motifs exist across the human genome ("predicted transcription factor binding sites": "predicted TFBS") but the large majority of these are proven by chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) not to be biological transcription factor binding sites ("empirical TFBS"). We hypothesize that DNA methylation at conserved consensus motifs prevents promiscuous or disorderly transcription factor binding. RESULTS: Using genome-wide methylation maps of the human heart and sperm, we found that all conserved consensus motifs as well as the subset of those that reside outside CpG islands have an aggregate profile of hyper-methylation. In contrast, empirical TFBS with conserved consensus motifs have a profile of hypo-methylation. 40% of empirical TFBS with conserved consensus motifs resided in CpG islands whereas only 7% of all conserved consensus motifs were in CpG islands. Finally we further identified a minority subset of TF whose profiles are either hypo-methylated or neutral at their respective conserved consensus motifs implicating that these TF may be responsible for establishing or maintaining an un-methylated DNA state, or whose binding is not regulated by DNA methylation. CONCLUSIONS: Our analysis supports the hypothesis that at least for a subset of TF, empirical binding to conserved consensus motifs genome-wide may be controlled by DNA methylation.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Interactions within the mammalian DNA methyltransferase family

BACKGROUND: In mammals, epigenetic information is established and maintained via the postreplicative methylation of cytosine residues by the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 is required for maintenance methylation whereas Dnmt3a and Dnmt3b are responsible for de novo methylation. Contrary to Dnmt3a or Dnmt3b, the isolated C-terminal region of Dnmt1 is catalytically inactive, despite the presence of the sequence motifs typical of active DNA methyltransferases. Deletion analysis has revealed that a large part of the N-terminal domain is required for enzymatic activity. RESULTS: The role played by the N-terminal domain in this regulation has been investigated using the yeast two-hybrid system. We show here the presence of an intra-molecular interaction in Dnmt1 but not in Dnmt3a or Dnmt3b. This interaction was confirmed by immunoprecipitation and was localized by deletion mapping. Furthermore, a systematic analysis of interactions among the Dnmt family members has revealed that DNMT3L interacts with the C-terminal domain of Dnmt3a and Dnmt3b. CONCLUSIONS: The lack of methylating ability of the isolated C-terminal domain of Dnmt1 could be explained in part by a physical interaction between N- and C-terminal domains that apparently is required for activation of the catalytic domain. Our deletion analysis suggests that the tertiary structure of Dnmt1 is important in this process rather than a particular sequence motif. Furthermore, the interaction between DNMT3L and the C-terminal domains of Dnmt3a and Dnmt3b suggests a mechanism whereby the enzymatically inactive DNMT3L brings about the methylation of its substrate by recruiting an active methylase

The Influence of cis-Regulatory Elements on DNA Methylation Fidelity

It is now established that, as compared to normal cells, the cancer cell genome has an overall inverse distribution of DNA methylation (“methylome”), i.e., predominant hypomethylation and localized hypermethylation, within “CpG islands” (CGIs). Moreover, although cancer cells have reduced methylation “fidelity” and genomic instability, accurate maintenance of aberrant methylomes that underlie malignant phenotypes remains necessary. However, the mechanism(s) of cancer methylome maintenance remains largely unknown. Here, we assessed CGI methylation patterns propagated over 1, 3, and 5 divisions of A2780 ovarian cancer cells, concurrent with exposure to the DNA cross-linking chemotherapeutic cisplatin, and observed cell generation-successive increases in total hyper- and hypo-methylated CGIs. Empirical Bayesian modeling revealed five distinct modes of methylation propagation: (1) heritable (i.e., unchanged) high- methylation (1186 probe loci in CGI microarray); (2) heritable (i.e., unchanged) low-methylation (286 loci); (3) stochastic hypermethylation (i.e., progressively increased, 243 loci); (4) stochastic hypomethylation (i.e., progressively decreased, 247 loci); and (5) considerable “random” methylation (582 loci). These results support a “stochastic model” of DNA methylation equilibrium deriving from the efficiency of two distinct processes, methylation maintenance and de novo methylation. A role for cis-regulatory elements in methylation fidelity was also demonstrated by highly significant (p<2.2×10−5) enrichment of transcription factor binding sites in CGI probe loci showing heritably high (118 elements) and low (47 elements) methylation, and also in loci demonstrating stochastic hyper-(30 elements) and hypo-(31 elements) methylation. Notably, loci having “random” methylation heritability displayed nearly no enrichment. These results demonstrate an influence of cis-regulatory elements on the nonrandom propagation of both strictly heritable and stochastically heritable CGIs

DNA methylation and trinucleotide repeat expansion diseases

Copyright @ 2012 InTechThis article has been made available through the Brunel Open Access Publishing Fund.This article is made available through the Brunel Open Access Publishing Fund

Targeting and dynamics of gene repression during stem cell differentiation

The identity and function of different cellular subtypes critically depend on their unique set of expressed genes. Gene expression programs and their changes during development are mainly controlled by sequence-specific DNA binding factors. It has recently become clear that chromatin modifications are important regulators of these processes. While there are several chromatin-based pathways that correlate with gene repression, their exact role in silencing remains elusive. Moreover, for many repressive chromatin modifications a complete picture of the genomic distribution and its dynamics during development is lacking. Finally, it is still unclear how these genomic patterns of repressive chromatin marks are established. For my PhD work, I set out to address these questions by studying the targeting of H3K9me2 and DNA methylation during cellular differentiation. Our analysis revealed that H3K9me2 is highly abundant in embryonic stem cells and occurs in large domains that occupy more than half of the genome. H3K9me2 marks chromatin outside of transcribed, active or polycomb regulated sites, possibly keeping it in a repressed state. Importantly, abundance of H3K9me2 increases only slightly during neuronal differentiation, with a localized gain occurring at gene bodies of transcribed genes. By gene expression profiling we further show that the transcriptome complexity is very similar in stem cells and derived post-mitotic neurons. These data are in contrast to a previously suggested model which states that the pluripotent state of stem cells is accompanied by a global reduction in heterochromatin and a concomitant higher proportion of transcription. Together with results from other groups our data rather indicate that repressive chromatin is abundant in stem cells and upon differentiation gets redistributed only locally and not globally. It has been suggested that such a localized increase of repression at gene regulatory regions helps stabilizing lineage choices and differentiation processes. In order to investigate how chromatin-based repression pathways are targeted to gene regulatory sites, we focused on DNA methylation, a modification whose catalysis and epigenetic propagation are well understood. By site-specific sequence integration experiments we show that 1 kb promoter elements are sufficient to recapitulate endogenous DNA methylation patterns in stem cells and their dynamic changes upon differentiation, in a process that is independent of transcription. In stem cells, promoters are protected from DNA methylation by small sequence elements that we termed methylation determining regions (MDRs). Protection from DNA methylation by MDRs depends on a combination of DNA binding motifs, which get recognized by transcription factors such as RFX2. It has been speculated before that establishment of an unmethylated promoter state is facilitated by proteins that recognize unmethylated CpGs. While not excluding a role in maintenance, our data suggest that CpG-richness alone is not sufficient for initiation of this chromatin state. Remarkably, no additional sequence besides an MDR is needed to recapitulate differentiation-induced de novo methylation. Moreover, MDRs are able to protect neighboring sequences from DNA methylation in stem cells and from de novo methylation during differentiation. These results imply that one possible way of differentiation-induced de novo methylation could involve reduced binding of factors that protect from DNA methylation. In summary, H3K9me2 and DNA methylation occupy per default most the genome, even in cells with a high developmental potential. Accordingly, cellular differentiation is accompanied by focal, rather than global changes in repressive chromatin modifications. In the case of DNA methylation, such local changes at gene regulatory sites are determined by the underlying sequence and likely involve binding of transcription factors that protect from DNA methylation

Spatial and Functional Relationships Among Pol V-Associated Loci, Pol IV-Dependent siRNAs, and Cytosine Methylation in the \u3cem\u3eArabidopsis\u3c/em\u3e Epigenome

Multisubunit RNA polymerases IV and V (Pols IV and V) mediate RNA-directed DNA methylation and transcriptional silencing of retrotransposons and heterochromatic repeats in plants. We identified genomic sites of Pol V occupancy in parallel with siRNA deep sequencing and methylcytosine mapping, comparing wild-type plants with mutants defective for Pol IV, Pol V, or both Pols IV and V. Approximately 60% of Pol V-associated regions encompass regions of 24-nucleotide (nt) siRNA complementarity and cytosine methylation, consistent with cytosine methylation being guided by base-pairing of Pol IV-dependent siRNAs with Pol V transcripts. However, 27% of Pol V peaks do not overlap sites of 24-nt siRNA biogenesis or cytosine methylation, indicating that Pol V alone does not specify sites of cytosine methylation. Surprisingly, the number of methylated CHH motifs, a hallmark of RNA-directed de novo methylation, is similar in wild-type plants and Pol IV or Pol V mutants. In the mutants, methylation is lost at 50%–60% of the CHH sites that are methylated in the wild type but is gained at new CHH positions, primarily in pericentromeric regions. These results indicate that Pol IV and Pol V are not required for cytosine methyltransferase activity but shape the epigenome by guiding CHH methylation to specific genomic sites