Spreading Of Methylation Research Articles

Identification of a novel epigenetic marker for typical and mosaic presentations of Fragile X syndrome

ABSTRACT Background Fragile X syndrome (FXS) is primarily due to CGG repeat expansions in the FMR1 gene. FMR1 alleles are classified as normal (N), intermediate (I), premutation (PM), and full mutation (FM). FXS patients often carry an FM, but size mosaicism can occur. Additionally, loss of methylation boundary upstream of repeats results in de novo methylation spreading to FMR1 promoter in FXS patients. Research design and methods This pilot study investigated the methylation boundary and adjacent regions in 66 males with typical and atypical FXS aged 1 to 30 years (10.86 ± 6.48 years). AmplideX FMR1 mPCR kit was used to discriminate allele profiles and methylation levels. CpG sites were assessed by pyrosequencing. Results 40 out of 66 FXS patients (60.6%) showed an exclusive FM (n = 40), whereas the remaining (n = 26) exhibited size mosaicism [10 PM_FM (15.15%); 10 N_FM (15.15%); 2 N_PM_FM (3%)]. Four patients (6.1%) had deletions near repeats. Noteworthy, a CpG within FMR1 intron 2 displayed hypomethylation in FXS patients and hypermethylation in controls, demonstrating remarkable specificity, sensitivity, and accuracy when a methylation threshold of 69.5% was applied. Conclusions Since intragenic methylation is pivotal in gene regulation, the intronic CpG might be a novel epigenetic biomarker for FXS diagnosis.

Progressive Spreading of DNA Methylation in the GSTP1 Promoter CpG Island across Transitions from Precursors to Invasive Prostate Cancer.

DNA hypermethylation at the GSTP1 promoter progressively spreads from being unmethylated in normal prostate to intermediate levels in precursor lesions to extensive methylation in cancer. This molecular progression of GSTP1 promoter methylation patterns in early prostate carcinogenesis could be useful for identification and interception of prostate cancer precursors.

Abstract 3734: Progressive spreading of CpG methylation in the CpG island of Glutathione S-Transferase pi 1 (GSTP1) alleles across transitions from precursor to invasive prostate cancer

Abstract Glutathione S-transferase pi 1 (GSTP1) is expressed at low levels in normal prostate luminal cells and becomes induced in most proliferative inflammatory atrophy lesions (PIA). GSTP1 becomes silenced in prostatic intraepithelial neoplasia (PIN) and prostate adenocarcinoma (CaP) via CpG island promoter hypermethylation. However, the specific methylation patterns in CaP and its precursors have not been investigated. We used bisulfite genomic sequencing to examine methylation of 39 CpG sites in the GSTP1 promoter. Radical prostatectomy specimens were selected from 32 subjects that underwent treatment for CaP. Serial sections were subjected to laser capture microdissection for enrichment of epithelial cells from benign, PIA, PIN, and CaP regions. Isolated DNA was bisulfite converted. Sequences corresponding to the GSTP1 promoter CpG island were amplified with nested PCR, and PCR products were cloned. 10 independent clones were sequenced and analyzed with a Java program, DNAMethylMap, to determine the methylation pattern of 39 CpG sites of the GSTP1 promoter in each lesion. The extent of methylation on an individual allele was classified as negative (<10% CpGs), mild (10-25%), moderate (25-50%), or high (>50%). 212 clones from 24 normal epithelium regions, 327 clones from 37 PIA regions, 167 clones from 18 PIN regions, and 202 clones from 23 CaP regions were sequenced, totaling 34863 CpG sites. Normal and PIA lesions were mostly unmethylated with 0.52% and 1.3% of total CpG sites methylated, respectively. Methylated PIA lesions were adjacent to CaP or PIN regions significantly more often than unmethylated PIA lesions (χ2, p = 0.012). PIN and CaP lesions had greater methylation with 24% and 51% of total CpG sites methylated, respectively. PIN lesions generally showed partial methylation with 28.7% of alleles in PIN having mild/moderate methylation density compared to 5.5% in PIA and 11% in CaP. PIA and PIN lesions were enriched for methylation changes at 6 CpG sites that aligned with AP1 and SP1 binding sites. Among 16 PIN and CaP lesions with an overall intermediate level of methylation, 5 of the lesions had clones that clustered into either fully methylated or unmethylated. One patient with 3 CaP lesions displayed significant heterogeneity in methylation patterns: full, moderate, or no methylation across all clones in a given lesion. The results demonstrate that methylation density in the GSTP1 CpG island from PIN was intermediate between normal prostate epithelium/PIA and CaP lesions. The observed methylation in PIA and PIN was enriched at binding sites of key transcription factors, AP1 and SP1. The results are consistent with gradual spreading of DNA methylation centered at the transcription factor binding sites in the putative precursor lesions, with subsequent spreading of methylation across the entire CpG island in transition to CaP. Citation Format: Harshath Gupta, Hitoshi Inoue, Yasutomo Nakai, Masashi Nakayama, William G. Nelson, Angelo M. De Marzo, Srinivasan Yegnasubramanian. Progressive spreading of CpG methylation in the CpG island of Glutathione S-Transferase pi 1 (GSTP1) alleles across transitions from precursor to invasive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3734.

Polycomb-mediated genome architecture enables long-range spreading of H3K27 methylation

Polycomb-group proteins play critical roles in gene silencing through the deposition of histone H3 lysine 27 trimethylation (H3K27me3) and chromatin compaction. This process is essential for embryonic stem cell (ESC) pluripotency, differentiation, and development. Polycomb repressive complex 2 (PRC2) can both read and write H3K27me3, enabling progressive spreading of H3K27me3 on the linear genome. Long-range Polycomb-associated DNA contacts have also been described, but their regulation and role in gene silencing remain unclear. Here, we apply H3K27me3 HiChIP, a protein-directed chromosome conformation method, and optical reconstruction of chromatin architecture to profile long-range Polycomb-associated DNA loops that span tens to hundreds of megabases across multiple topological associated domains in mouse ESCs and human induced pluripotent stem cells. We find that H3K27me3 loop anchors are enriched for Polycomb nucleation points and coincide with key developmental genes. Genetic deletion of H3K27me3 loop anchors results in disruption of spatial contact between distant loci and altered H3K27me3 in cis, both locally and megabases away on the same chromosome. In mouse embryos, loop anchor deletion leads to ectopic activation of the partner gene, suggesting that Polycomb-associated loops control gene silencing during development. Further, we find that alterations in PRC2 occupancy resulting from an RNA binding–deficient EZH2 mutant are accompanied by loss of Polycomb-associated DNA looping. Together, these results suggest PRC2 uses RNA binding to enhance long-range chromosome folding and H3K27me3 spreading. Developmental gene loci have unique roles in Polycomb spreading, emerging as important architectural elements of the epigenome.

Transcriptional regulation of dosage compensation in Carica papaya

Sex chromosome evolution results in the disparity in gene content between heterogametic sex chromosomes and creates the need for dosage compensation to counteract the effects of gene dose imbalance of sex chromosomes in males and females. It is not known at which stage of sex chromosome evolution dosage compensation would evolve. We used global gene expression profiling in male and female papayas to assess gene expression patterns of sex-linked genes on the papaya sex chromosomes. By analyzing expression ratios of sex-linked genes to autosomal genes and sex-linked genes in males relative to females, our results showed that dosage compensation was regulated on a gene-by-gene level rather than whole sex-linked region in papaya. Seven genes on the papaya X chromosome exhibited dosage compensation. We further compared gene expression ratios in the two evolutionary strata. Y alleles in the older evolutionary stratum showed reduced expression compared to X alleles, while Y alleles in the younger evolutionary stratum showed elevated expression compared to X alleles. Reduced expression of Y alleles in the older evolutionary stratum might be caused by accumulation of deleterious mutations in regulatory regions or transposable element-mediated methylation spreading. Most X-hemizygous genes exhibited either no or very low expression, suggesting that gene silencing might play a role in maintaining transcriptional balance between females and males.

Methylated and unmethylated epialleles support variegated epigenetic silencing in Friedreich ataxia.

Friedreich ataxia (FRDA) is typically caused by homozygosity for an expanded GAA triplet-repeat in intron 1 of the FXN gene, which results in transcriptional deficiency via epigenetic silencing. Most patients are homozygous for alleles containing > 500 triplets, but a subset (~20%) have at least one expanded allele with < 500 triplets and a distinctly milder phenotype. We show that in FRDA DNA methylation spreads upstream from the expanded repeat, further than previously recognized, and establishes an FRDA-specific region of hypermethylation in intron 1 (~90% in FRDA versus < 10% in non-FRDA) as a novel epigenetic signature. The hypermethylation of this differentially methylated region (FRDA-DMR) was observed in a variety of patient-derived cells; it significantly correlated with FXN transcriptional deficiency and age of onset, and it reverted to the non-disease state in isogenically corrected induced pluripotent stem cell (iPSC)-derived neurons. Bisulfite deep sequencing of the FRDA-DMR in peripheral blood mononuclear cells from 73 FRDA patients revealed considerable intra-individual epiallelic variability, including fully methylated, partially methylated, and unmethylated epialleles. Although unmethylated epialleles were rare (median = 0.33%) in typical patients homozygous for long GAA alleles with > 500 triplets, a significantly higher prevalence of unmethylated epialleles (median = 9.8%) was observed in patients with at least one allele containing < 500 triplets, less severe FXN deficiency (>20%) and later onset (>15 years). The higher prevalence in mild FRDA of somatic FXN epialleles devoid of DNA methylation is consistent with variegated epigenetic silencing mediated by expanded triplet-repeats. The proportion of unsilenced somatic FXN genes is an unrecognized phenotypic determinant in FRDA and has implications for the deployment of effective therapies.

Chromosome Structural Mechanics Dictates the Local Spreading of Epigenetic Marks

We present a theoretical model that demonstrates the integral role chromosome organization and structural mechanics play in the spreading of histone modifications involved in epigenetic regulation. Our model shows that heterogeneous nucleosome positioning, and the resulting position-dependent mechanical properties, must be included to reproduce several qualitative features of experimental data of histone methylation spreading around an artificially induced “nucleation site.” We show that our model recreates both the extent of spreading and the presence of a subdominant peak upstream of the transcription start site. Our model indicates that the spreading of epigenetic modifications is sensitive to heterogeneity in chromatin organization and the resulting variability in the chromatin’s mechanical properties, suggesting that nucleosome spacing can directly control the conferral of epigenetic marks by modifying the structural mechanics of the chromosome. It further illustrates how the physical organization of the DNA polymer may play a significant role in re-establishing the epigenetic code upon cell division.

CGGBP1-regulated cytosine methylation at CTCF-binding motifs resists stochasticity

BackgroundThe human CGGBP1 binds to GC-rich regions and interspersed repeats, maintains homeostasis of stochastic cytosine methylation and determines DNA-binding of CTCF. Interdependence between regulation of cytosine methylation and CTCF occupancy by CGGBP1 remains unknown.ResultsBy analyzing methylated DNA-sequencing data obtained from CGGBP1-depleted cells, we report that some transcription factor-binding sites, including CTCF, resist stochastic changes in cytosine methylation. By analysing CTCF-binding sites we show that cytosine methylation changes at CTCF motifs caused by CGGBP1 depletion resist stochastic changes. These CTCF-binding sites are positioned at locations where the spread of cytosine methylation in cis depends on the levels of CGGBP1.ConclusionOur findings suggest that CTCF occupancy and functions are determined by CGGBP1-regulated cytosine methylation patterns.

Epigenetic engineering of yeast reveals dynamic molecular adaptation to methylation stress and genetic modulators of specific DNMT3 family members.

Cytosine methylation is a ubiquitous modification in mammalian DNA generated and maintained by several DNA methyltransferases (DNMTs) with partially overlapping functions and genomic targets. To systematically dissect the factors specifying each DNMT’s activity, we engineered combinatorial knock-in of human DNMT genes in Komagataella phaffii, a yeast species lacking endogenous DNA methylation. Time-course expression measurements captured dynamic network-level adaptation of cells to DNMT3B1-induced DNA methylation stress and showed that coordinately modulating the availability of S-adenosyl methionine (SAM), the essential metabolite for DNMT-catalyzed methylation, is an evolutionarily conserved epigenetic stress response, also implicated in several human diseases. Convolutional neural networks trained on genome-wide CpG-methylation data learned distinct sequence preferences of DNMT3 family members. A simulated annealing interpretation method resolved these preferences into individual flanking nucleotides and periodic poly(A) tracts that rotationally position highly methylated cytosines relative to phased nucleosomes. Furthermore, the nucleosome repeat length defined the spatial unit of methylation spreading. Gene methylation patterns were similar to those in mammals, and hypo- and hypermethylation were predictive of increased and decreased transcription relative to control, respectively, in the absence of mammalian readers of DNA methylation. Introducing controlled epigenetic perturbations in yeast thus enabled characterization of fundamental genomic features directing specific DNMT3 proteins.

Transposon-induced methylation of the RsMYB1 promoter disturbs anthocyanin accumulation in red-fleshed radish.

Red-fleshed radish (Raphanus sativus L.) is a unique cultivar whose taproot is rich in anthocyanins beneficial to human health. However, the frequent occurrence of white-fleshed mutants affects the purity of commercially produced radish and the underlying mechanism has puzzled breeders for many years. In this study, we combined quantitative trait location by genome resequencing and transcriptome analyses to identify a candidate gene (RsMYB1) responsible for anthocyanin accumulation in red-fleshed radish. However, no sequence variation was found in the coding and regulatory regions of the RsMYB1 genes of red-fleshed (MTH01) and white-fleshed (JC01) lines, and a 7372 bp CACTA transposon in the RsMYB1 promoter region occurred in both lines. A subsequent analysis suggested that the white-fleshed mutant was the result of altered DNA methylation in the RsMYB1 promoter. This heritable epigenetic change was due to the hypermethylated CACTA transposon, which induced the spreading of DNA methylation to the promoter region of RsMYB1. Thus, RsMYB1 expression was considerably down-regulated, which inhibited anthocyanin biosynthesis in the white-fleshed mutant. An examination of transgenic radish calli and the results of a virus-induced gene silencing experiment confirmed that RsMYB1 is responsible for anthocyanin accumulation. Moreover, the mutant phenotype was partially eliminated by treatment with a demethylating agent. This study explains the molecular mechanism regulating the appearance of white-fleshed mutants of red-fleshed radish.

Physical modeling of the spreading of epigenetic modifications through transient DNA looping

We utilize theoretical modeling to explore the physical mechanisms that govern the spreading of epigenetic modifications along chromosomal DNA. We focus on a particular modification, methylation of lysine 9 of histone H3 (H3K9), which is a representative and critical epigenetic mark that affects chromatin structure and gene expression. Our model captures transient loop formation in chromosomal DNA that enables distal segments to be in close spatial proximity and permits methyltransferase to confer methyl marks over a broad range of genomic distances. Using our exact results for the statistical behavior of a semiflexible polymer, we find the looping rate for a chromosomal segment based on a mean first-passage time process on a free-energy landscape. From this treatment, looping kinetics are predicted to be the rate-limiting process for methyl spreading at large genomic distances, which explains the considerable variability in the methyl profile at such scales. We then develop a ‘phase diagram’ for methylation spreading versus the methylation rate and the concentration of HP1, which we identify as a global regulator of the state of the chromosomal DNA.

Active DNA Demethylation in Plants.

Methylation of cytosine (5-meC) is a critical epigenetic modification in many eukaryotes, and genomic DNA methylation landscapes are dynamically regulated by opposed methylation and demethylation processes. Plants are unique in possessing a mechanism for active DNA demethylation involving DNA glycosylases that excise 5-meC and initiate its replacement with unmodified C through a base excision repair (BER) pathway. Plant BER-mediated DNA demethylation is a complex process involving numerous proteins, as well as additional regulatory factors that avoid accumulation of potentially harmful intermediates and coordinate demethylation and methylation to maintain balanced yet flexible DNA methylation patterns. Active DNA demethylation counteracts excessive methylation at transposable elements (TEs), mainly in euchromatic regions, and one of its major functions is to avoid methylation spreading to nearby genes. It is also involved in transcriptional activation of TEs and TE-derived sequences in companion cells of male and female gametophytes, which reinforces transposon silencing in gametes and also contributes to gene imprinting in the endosperm. Plant 5-meC DNA glycosylases are additionally involved in many other physiological processes, including seed development and germination, fruit ripening, and plant responses to a variety of biotic and abiotic environmental stimuli.

Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells

BackgroundMethylation of cytosines is an evolutionarily conserved epigenetic mark that is essential for the control of chromatin activity in many taxa. It acts mainly repressively, causing transcriptional gene silencing. In plants, de novo DNA methylation is established mainly by RNA-directed DNA-methylation pathway. Even though the protein machinery involved is relatively well-described, the course of the initial phases remains covert.ResultsWe show the first detailed description of de novo DNA-methylation dynamics. Since prevalent plant model systems do not provide the possibility to collect homogenously responding material in time series with short intervals, we developed a convenient system based on tobacco BY-2 cell lines with inducible production of siRNAs (from an RNA hairpin) guiding the methylation machinery to the CaMV 35S promoter controlling GFP reporter. These lines responded very synchronously, and a high level of promoter-specific siRNAs triggered rapid promoter methylation with the first increase observed already 12 h after the induction. The previous presence of CG methylation in the promoter did not affect the methylation dynamics. The individual cytosine contexts reacted differently. CHH methylation peaked at about 80% in 2 days and then declined, whereas CG and CHG methylation needed more time with CHG reaching practically 100% after 10 days. Spreading of methylation was only minimal outside the target region in accordance with the absence of transitive siRNAs. The low and stable proportion of 24-nt siRNAs suggested that Pol IV was not involved in the initial phases.ConclusionsOur results show that de novo DNA methylation is a rapid process initiated practically immediately with the appearance of promoter-specific siRNAs and independently of the prior presence of methylcytosines at the target locus. The methylation was precisely targeted, and its dynamics varied depending on the cytosine sequence context. The progressively increasing methylation resulted in a smooth, gradual inhibition of the promoter activity, which was entirely suppressed in 2 days.

Abstract LB-035: p16 epimutation cooperates with Apc mutation to promote colon cancer initiation and progression

Abstract Background: Although an extensive catalog of DNA methylation changes has been generated in patients with colorectal cancer (CRC), an important unmet need is to understand their functional contributions during the initiation and progression of CRC. Epimutation, or mitotically stable gene silencing associated with epigenetic alteration in promoter DNA methylation, is proposed to act as one of the classical Knudson’s two-hit model for loss of tumor suppressor gene function. Importantly, unlike genetic mutations, epigenetic mechanisms are intrinsically malleable. Thus, to improve clinical care of CRC patients, we aim to investigate how the epimutations frequently observed in cancer cells contribute to colonic tumorigenesis. Methods: We generated mice that replicates two common genetic and epigenetic events observed in human CRCs: Apc mutation and p16 epimutation. We used ApcMin/+ mice carrying a heterozygous germline mutation at codon 850 of the Apc gene (Moser et al. Science, 1990). Our p16 epimutation mice were created by targeted knock-in of a 140-bp DNA sequence (cis-element) that facilitates the spread of DNA methylation at promoter CpG islands in cis. (Yu et al. JCI, 2014). All mice were on a C57BL/6J background. For tumor phenotyping, we conducted survival and time-course histological analyses of tumor development and progression. To understand the regulatory mechanism of p16 epigenetic silencing, we used an unbiased, label-free mass spectrometry (MS)-based approach to capture the DNA-binding profile and identify proteins that bind to the p16 promoter in a DNA methylation-dependent manner. Finally, to determine the synergistic effects of Apc mutation and p16epimutation, we performed RNA-seq and proteomics analysis in both normal colonic epithelial cells and tumors. Results: We found that the mice with combined Apc mutation and p16 epimutation (ApcMin/+; p16cis/cis) had a significantly shortened overall survival than the ApcMin/+ mice (P=0.001), indicating that p16 epimutation promotes intestinal tumor progression. To assess the cause of early morbidity in ApcMin/+; p16cis/cis mice, we examined the intestines for tumor formation at 15 weeks of age. In the small intestines, we found that the ApcMin/+; p16cis/cis mice had a greater number of tumors than the ApcMin/+ mice. Moreover, the tumors were significantly larger than those in ApcMin/+ mice. In the colons, ApcMin/+; p16cis/cis mice also showed more than 2-fold increase in tumor number. Importantly, we found the colonic tumors in ApcMin/+; p16cis/cis mice were highly vascularized and had foci of invasive carcinomas, a feature of malignancy that was not observed in the age-matched ApcMin/+ mice. In addition, we have discovered sequence-specific transcription factors that are recruited to the methylated p16 promoter to control epigenetic silencing. Furthermore, our integrated approach combining transcriptome and proteomic data revealed a cell-cycle independent role for p16 epimutation during colon carcinogenesis. The most striking hits are genes involved in immune responses, inflammation, and epithelial-to-mesenchymal transition. Conclusion: Our work demonstrates that one driver mutation (Apc) plus one driver epimutation (p16) is sufficient for colon cancer development. Citation Format: Li Yang, Yue Chen, Matthew V. Holt, Feng Jin, Jong Min Choi, Sung Yun Jung, Anusha Mandala, Lanjing Zhang, Sayantani Goswami, Nan Gao, Yi Wang, Lanlan Shen. p16 epimutation cooperates with Apc mutation to promote colon cancer initiation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-035.

Reevaluation of FMR1 Hypermethylation Timing in Fragile X Syndrome.

Fragile X syndrome (FXS) is one of the most common heritable forms of cognitive impairment. It results from a fragile X mental retardation protein (FMRP) protein deficiency caused by a CGG repeat expansion in the 5′-UTR of the X-linked FMR1 gene. Whereas in most individuals the number of CGGs is steady and ranges between 5 and 44 units, in patients it becomes extensively unstable and expands to a length exceeding 200 repeats (full mutation). Interestingly, this disease is exclusively transmitted by mothers who carry a premutation allele (55–200 CGG repeats). When the CGGs reach the FM range, they trigger the spread of abnormal DNA methylation, which coincides with a switch from active to repressive histone modifications. This results in epigenetic gene silencing of FMR1 presumably by a multi-stage, developmentally regulated process. The timing of FMR1 hypermethylation and transcription silencing is still hotly debated. There is evidence that hypermethylation varies considerably between and within the tissues of patients as well as during fetal development, thus supporting the view that FMR1 silencing is a post-zygotic event that is developmentally structured. On the other hand, it may be established in the female germ line and transmitted to the fetus as an integral part of the mutation. This short review summarizes the data collected to date concerning the timing of FMR1 epigenetic gene silencing and reassess the evidence in favor of the theory that gene inactivation takes place by a developmentally regulated process around the 10th week of gestation.

Evolutionary Epigenomics of Retrotransposon-Mediated Methylation Spreading in Rice.

Plant genomes contain numerous transposable elements (TEs), and many hypotheses on the evolutionary drivers that restrict TE activity have been postulated. Few models, however, have focused on the evolutionary epigenomic interaction between the plant host and its TE. The host genome recruits epigenetic factors, such as methylation, to silence TEs but methylation can spread beyond the TE sequence and influence the expression of nearby host genes. In this study, we investigated this epigenetic trade-off between TE and proximal host gene silencing by studying the epigenomic regulation of repressing long terminal repeat (LTR) retrotransposons (RTs) in Oryza sativa. Results showed significant evidence of methylation spreading originating from the LTR-RT sequences, and the extent of spreading was dependent on five factors: 1) LTR-RT family, 2) time since the LTR-RT insertion, 3) recombination rate of the LTR-RT region, 4) level of LTR-RT sequence methylation, and 5) chromosomal location. Methylation spreading had negative effects by reducing host gene expression, but only on host genes with LTR-RT inserted in its introns. Our results also suggested high levels of LTR-RT methylation might have a role in suppressing TE-mediated deleterious ectopic recombination. In the end, despite the methylation spreading, no strong epigenetic trade-off was detected and majority of LTR-RT may have only minor epigenetic effects on nearby host genes.

Targeted DNA methylation in human cells using engineered dCas9-methyltransferases

Mammalian genomes exhibit complex patterns of gene expression regulated, in part, by DNA methylation. The advent of engineered DNA methyltransferases (MTases) to target DNA methylation to specific sites in the genome will accelerate many areas of biological research. However, targeted MTases require clear design rules to direct site-specific DNA methylation and minimize the unintended effects of off-target DNA methylation. Here we report a targeted MTase composed of an artificially split CpG MTase (sMTase) with one fragment fused to a catalytically-inactive Cas9 (dCas9) that directs the functional assembly of sMTase fragments at the targeted CpG site. We precisely map RNA-programmed DNA methylation to targeted CpG sites as a function of distance and orientation from the protospacer adjacent motif (PAM). Expression of the dCas9-sMTase in mammalian cells led to predictable and efficient (up to ~70%) DNA methylation at targeted sites. Multiplexing sgRNAs enabled targeting methylation to multiple sites in a single promoter and to multiple sites in multiple promoters. This programmable de novo MTase tool might be used for studying mechanisms of initiation, spreading and inheritance of DNA methylation, and for therapeutic gene silencing.

Cytosine methylation at CpCpG sites triggers accumulation of non-CpG methylation in gene bodies.

Methylation of cytosine is an epigenetic mark involved in the regulation of transcription, usually associated with transcriptional repression. In mammals, methylated cytosines are found predominantly in CpGs but in plants non-CpG methylation (in the CpHpG or CpHpH contexts, where H is A, C or T) is also present and is associated with the transcriptional silencing of transposable elements. In addition, CpG methylation is found in coding regions of active genes. In the absence of the demethylase of lysine 9 of histone 3 (IBM1), a subset of body-methylated genes acquires non-CpG methylation. This was shown to alter their expression and affect plant development. It is not clear why only certain body-methylated genes gain non-CpG methylation in the absence of IBM1 and others do not. Here we describe a link between CpG methylation and the establishment of methylation in the CpHpG context that explains the two classes of body-methylated genes. We provide evidence that external cytosines of CpCpG sites can only be methylated when internal cytosines are methylated. CpCpG sites methylated in both cytosines promote spreading of methylation in the CpHpG context in genes protected by IBM1. In contrast, CpCpG sites remain unmethylated in IBM1-independent genes and do not promote spread of CpHpG methylation.

The DNA demethylase ROS1 targets genomic regions with distinct chromatin modifications.

The Arabidopsis ROS1/DEMETER family of 5mC DNA glycosylases are the first genetically characterized DNA demethylases in eukaryotes. However, the features of ROS1 targeted genomic loci are not well-understood. In this study, we characterized ROS1 target loci in Arabidopsis Col-0 and C24 ecotypes. We found that ROS1 preferentially targets transposable elements (TEs) and intergenic regions. Compared to most TEs, ROS1-targeted TEs are closer to protein coding genes, suggesting that ROS1 may prevent DNA methylation spreading from TEs to nearby genes. ROS1-targeted TEs are specifically enriched for H3K18Ac and H3K27me3, and depleted of H3K27me and H3K9me2. Importantly, we identified thousands of previously unknown RNA-directed DNA methylation (RdDM) targets upon depletion of ROS1, suggesting that ROS1 strongly antagonizes RdDM at these loci. In addition, we show that ROS1 also antagonizes RdDM-independent DNA methylation at some loci. Our results provide important insights into the genome-wide targets of ROS1 and the crosstalk between DNA methylation and ROS1-mediated active DNA demethylation.

Methyl-CpG-binding protein MBD2 plays a key role in maintenance and spread of DNA methylation at CpG islands and shores in cancer

Cancer is characterised by DNA hypermethylation and gene silencing of CpG island-associated promoters, including tumour-suppressor genes. The methyl-CpG-binding domain (MBD) family of proteins bind to methylated DNA and can aid in the mediation of gene silencing through interaction with histone deacetylases and histone methyltransferases. However, the mechanisms responsible for eliciting CpG island hypermethylation in cancer, and the potential role that MBD proteins play in modulation of the methylome remain unclear. Our previous work demonstrated that MBD2 preferentially binds to the hypermethylated GSTP1 promoter CpG island in prostate cancer cells. Here, we use functional genetic approaches to investigate if MBD2 plays an active role in reshaping the DNA methylation landscape at this locus and genome-wide. First, we show that loss of MBD2 results in inhibition of both maintenance and spread of de novo methylation of a transfected construct containing the GSTP1 promoter CpG island in prostate cancer cells and Mbd2-/- mouse fibroblasts. De novo methylation was rescued by transient expression of Mbd2 in Mbd2-/- cells. Second, we show that MBD2 depletion triggers significant hypomethylation genome-wide in prostate cancer cells with concomitant loss of MBD2 binding at promoter and enhancer regulatory regions. Finally, CpG islands and shores that become hypomethylated after MBD2 depletion in LNCaP cancer cells show significant hypermethylation in clinical prostate cancer samples, highlighting a potential active role of MBD2 in promoting cancer-specific hypermethylation. Importantly, co-immunoprecipiation of MBD2 shows that MBD2 associates with DNA methyltransferase enzymes 1 and 3A. Together our results demonstrate that MBD2 has a critical role in 'rewriting' the cancer methylome at specific regulatory regions.