DNA Methylation-dependent Manner Research Articles

PTEN regulates expression of its pseudogene in glioblastoma cells in DNA methylation-dependent manner

Glioblastoma (GBM) is the most aggressive and frequent type of primary brain cancer in adult patients. One of the key molecular features associated with GBM pathogenesis is the dysfunction of PTEN oncosuppressor. In addition to PTEN gene, humans and several primates possess processed PTEN pseudogene (PTENP1) that gives rise to long non-coding RNA lncPTENP1-S. Regulation and functions of PTEN and PTENP1 are highly interconnected, however, the exact molecular mechanism of how these two genes affect each other remains unclear. Here, we analyzed the methylation level of the CpG islands (CpGIs) in the promoter regions of PTEN and PTENP1 in patient-derived GBM neurospheres. We found that increased PTEN methylation corelates with decreased PTEN mRNA level. Unexpectedly, we showed the opposite trend for PTENP1. Using targeted methylation and demethylation of PTENP1 CpGI, we demonstrated that DNA methylation increases lncPTENP1-S expression in the presence of wild type PTEN protein but decreases lncPTENP1-S expression if PTEN protein is absent. Further experiments revealed that PTEN protein binds to PTENP1 promoter region and inhibits lncPTENP1-S expression if its CpGI is demethylated. Interestingly, we did not detect any effect of lncPTENP1-S on the level of PTEN mRNA, indicating that in GBM cells PTENP1 is a downstream target of PTEN rather than its upstream regulator. Finally, we studied the functions of lncPTENP1-S and demonstrated that it plays a pro-oncogenic role in GBM cells by upregulating the expression of cancer stem cell markers and decreasing cell adhesion.

Thymine DNA glycosylase mediates chromatin phase separation in a DNA methylation–dependent manner

Thymine DNA glycosylase (TDG) is an essential enzyme involved in numerous biological pathways, including DNA repair, DNA demethylation, and transcriptional activation. Despite these important functions, the mechanisms surrounding the actions and regulation of TDG are poorly understood. In this study, we demonstrate that TDG induces phase separation of DNA and nucleosome arrays under physiologically relevant conditions invitro and show that the resulting chromatin droplets exhibited behaviors typical of phase-separated liquids, supporting a liquid-liquid phase separation model. We also provide evidence that TDG has the capacity to form phase-separated condensates in the cell nucleus. The ability of TDG to induce chromatin phase separation is dependent on its intrinsically disordered N- and C-terminal domains, which in isolation, promote the formation of chromatin-containing droplets having distinct physical properties, consistent with their unique mechanistic roles in the phase separation process. Interestingly, DNA methylation alters the phase behavior of the disordered domains of TDG and compromises formation of chromatin condensates by full-length TDG, indicating that DNA methylation regulates the assembly and coalescence of TDG-mediated condensates. Overall, our results shed new light on the formation and physical nature of TDG-mediated chromatin condensates, which have broad implications for the mechanism and regulation of TDG and its associated genomic processes.

Dysregulation of schizophrenia-associated genes and genome-wide hypomethylation in neurons overexpressing DNMT1.

Aim: To study the effects of DNMT1 overexpression on transcript levels of genes dysregulated in schizophrenia and on genome-wide methylation patterns. Materials & methods: Transcriptome and DNA methylome comparisons were made between R1 (wild-type) and Dnmt1tet/tet mouse embryonic stem cells and neurons overexpressing DNMT1. Genes dysregulated in both Dnmt1tet/tet cells and schizophrenia patients were studied further. Results & conclusions: About 50% of dysregulated genes in patients also showed altered transcript levels in Tet/Tet neurons in a DNA methylation-independent manner. These neurons unexpectedly showed genome-wide hypomethylation, increased transcript levels of Tet1 and Apobec 1-3 genes andincreased activity and copy number of LINE-1 elements. The observed similarities between Tet/Tet neurons and schizophrenia brain samples reinforce DNMT1 overexpression as a risk factor.

The De Novo DNA Methyltransferases <i>DNMT3A</i> and <i>DNMT3B</i> Initiate Erosion of Dosage Compensation in Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have proven to be valuable tools for both drug discovery and the development of cell based therapies. However, silencing of XIST expression followed by inappropriate reactivation and biallelic expression of X chromosome linked genes regularly occurs in female hPSCs, placing them in a state observed in human tumors. This gradual erosion of dosage compensation also occludes cellular phenotypes while modeling X-linked diseases and interferes with directed differentiation, greatly limiting the utility of female hPSCs. Here, we investigate the mechanisms regulating erosion of dosage compensation and whether it can be reversed or prevented. We found that changes to the X chromosome during erosion of dosage compensation both repress XIST through DNA methylation and prevent its RNA from silencing the chromosome, even if XIST expression is induced using CRISPR/Cas9 activation. Encouragingly, we found that elimination of the DNMT3A and genes, but the similarity of global transcriptional status of DNMT3A/3B deleted cell to non41 eroded cell differs in cell lines. While DNMT3A/3B deletion after erosion of dosage compensation failed to restore it. Thus, our study revealed that de novo DNA methyltransferases are primary factors responsible for the initiation of erosion of dosage compensation in female hPSCs and once DNA methylation is imposed on XIST promoterregions, the status is stably maintained to repress XIST by de novo DNA methylation independent manner.

Transcription of intragenic CpG islands influences spatiotemporal host gene pre-mRNA processing.

Alternative splicing (AS) and alternative polyadenylation (APA) generate diverse transcripts in mammalian genomes during development and differentiation. Epigenetic marks such as trimethylation of histone H3 lysine 36 (H3K36me3) and DNA methylation play a role in generating transcriptome diversity. Intragenic CpG islands (iCGIs) and their corresponding host genes exhibit dynamic epigenetic and gene expression patterns during development and between different tissues. We hypothesise that iCGI-associated H3K36me3, DNA methylation and transcription can influence host gene AS and/or APA. We investigate H3K36me3 and find that this histone mark is not a major regulator of AS or APA in our model system. Genomewide, we identify over 4000 host genes that harbour an iCGI in the mammalian genome, including both previously annotated and novel iCGI/host gene pairs. The transcriptional activity of these iCGIs is tissue- and developmental stage-specific and, for the first time, we demonstrate that the premature termination of host gene transcripts upstream of iCGIs is closely correlated with the level of iCGI transcription in a DNA-methylation independent manner. These studies suggest that iCGI transcription, rather than H3K36me3 or DNA methylation, interfere with host gene transcription and pre-mRNA processing genomewide and contributes to the spatiotemporal diversification of both the transcriptome and proteome.

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.

Intragenic DNA methylation and BORIS-mediated cancer-specific splicing contribute to the Warburg effect

Aberrant alternative splicing and epigenetic changes are both associated with various cancers, but epigenetic regulation of alternative splicing in cancer is largely unknown. Here we report that the intragenic DNA methylation-mediated binding of Brother of Regulator of Imprinted Sites (BORIS) at the alternative exon of Pyruvate Kinase (PKM) is associated with cancer-specific splicing that promotes the Warburg effect and breast cancer progression. Interestingly, the inhibition of DNA methylation, BORIS depletion, or CRISPR/Cas9-mediated deletion of the BORIS binding site leads to a splicing switch from cancer-specific PKM2 to normal PKM1 isoform. This results in the reversal of the Warburg effect and the inhibition of breast cancer cell growth, which may serve as a useful approach to inhibit the growth of breast cancer cells. Importantly, our results show that in addition to PKM splicing, BORIS also regulates the alternative splicing of several genes in a DNA methylation-dependent manner. Our findings highlight the role of intragenic DNA methylation and DNA binding protein BORIS in cancer-specific splicing and its role in tumorigenesis.

Epigenetic modulation of a miR-296-5p:HMGA1 axis regulates Sox2 expression and glioblastoma stem cells.

Solid malignancies contain subsets of multipotent cells that grow as spheres and efficiently propagate tumors in xenograft models, reflecting a stem-like, self-renewing and tumor-propagating phenotype. These cancer 'stem cells (SCs)' have been shown to maintain tumor growth, contribute to resistance and drive tumor recurrence. Cancer cell stemness is dynamically influenced by epigenetic mechanisms and differentially regulated coding and noncoding RNAs. How these mechanisms specifically contribute to the generation and/or maintenance of cancer SCs remains unclear. This study identifies a novel epigenetically regulated circuit that integrates microRNA, chromatin remodeling and the reprogramming transcription factor Sox2 to regulate glioblastoma (GBM)-propagating SCs. We show that miR-296-5p expression is repressed in a DNA methylation-dependent manner under conditions that promote GBM cell stemness and that miR-296-5p inhibits GBM cell stemness and their capacity to self-renew as spheres and propagate glioma xenografts in vivo. We show that the chromatin remodeling protein HMGA1 functions as a downstream effector of these biological responses to miR-296-5p and regulates Sox2 expression, a master driver of cell stemness, by modifying chromatin architecture at the Sox2 promoter. These results show for the first time that miR-296-5p inhibits transcriptional mechanisms that support GBM SCs and identify a miR-296-5p:HMGA1:Sox2 axis as a novel regulator of GBM SCs and candidate pathway for targeting therapies directed at depleting tumors of their tumor-propagating stem cell subsets.

EPIG-12EPIGENETIC MODULATION OF A miR-296-5p:HMGA1 AXIS REGULATES THE GLIOBLASTOMA STEM CELL PHENOTYPE

The long term goal of our research is to understand the cellular and molecular mechanisms that regulate brain tumor malignancy. Cell subsets isolated from glioblastoma (GBM) have the capacity to grow as spheres (i.e. neurospheres) and efficiently propagate tumors in xenograft models, reflecting a stem-like, self-renewing and tumor-propagating phenotype. Furthermore, GBM stem-like cells have been shown to maintain tumor growth, contribute to resistance, and there is strong evidence suggesting their role in tumor recurrence. Characterizing the basic mechanisms driving this phenotype will allow us to identify new therapeutic targets and treatment strategies. Cell fate determination and phenotype reprogramming are complex and dynamic processes that involve epigenetic mechanisms and differentially regulated coding and non-coding RNAs. We recently showed that the coordinated actions of Oct4 and Sox2 induce a tumor-propagating stem-like state in GBM cells via a mechanism involving direct DNMT promoter transactivation, DNA methylation, and methylation-dependent repression of multiple miRNAs. However, how specific epigenetic changes contribute to the acquisition and/or maintenance of the cancer stem-like phenotype remains unclear. This study focuses on understanding the cross-talk between specific DNA methylation events, miRNA expression and regulation, and the GBM-propagating stem cell phenotype. We show that miR-296-5p expression is repressed in a DNA methylation-dependent manner under conditions that promote GBM cell stemness and that forced expression of miR-296-5p inhibits self-renewal capacity of GBM neurosphere cells in vitro and the growth of neurosphere-derived glioma xenografts in vivo. Additionally, we identify the chromatin remodeling protein HMGA1 as downstream effector of miR-296-5p action in the context of GBM stem-like cells. These results show that miR-296-5p regulate chromatin architecture by targeting HMGA1 and that the miR-296-5p:HMGA1 axis as a novel regulator of the GBM stem cell phenotype and a candidate pathway for targeted therapies directed at depleting tumors of their tumor-propagating stem cell subsets.

DNA methyltransferase 3A promotes cell proliferation by silencing CDK inhibitor p18INK4C in gastric carcinogenesis

Little is known about the roles of DNA methyltransferase 3A (DNMT3A) in gastric carcinogenesis. Here, we reported that the exogenous expression of DNMT3A promoted gastric cancer (GC) cell proliferation by accelerating the G1/S transition. Subsequently, p18INK4C was identified as a downstream target of DNMT3A. The elevated expression of DNMT3A suppressed p18INK4C at least at the transcriptional level. Depletion of p18INK4C expression in GC cells induced cell cycle progression, whereas its re-expression alleviated the effect of DNMT3A overexpression on G1/S transition. Furthermore, we found that DNMT3A modulated p18INK4C by directly binding to and silencing the p18INK4C gene via promoter hypermethylation. In clinical GC tissue specimens analyzed, the level of methylation of p18INK4C detected in tumor tissues was significantly higher than that in paired non-tumor tissues. Moreover, elevated level of DNMT3A expression was associated with the differentiation of GC tissues and was negatively correlated with the p18INK4C expression level. Taken together, our results found that DNMT3A contributes to the dysregulation of the cell cycle by repressing p18INK4C in a DNA methylation-dependent manner, suggesting that DNMT3A-p18INK4C axis involved in GC. These findings provide new insights into gastric carcinogenesis and a potential therapeutic target for GC that may be further investigated in the future.

Methyl supplementation attenuates cocaine-seeking behaviors and cocaine-induced c-Fos activation in a DNA methylation-dependent manner.

Epigenetic mechanisms, such as histone modifications, regulate responsiveness to drugs of abuse, such as cocaine, but relatively little is known about the regulation of addictive-like behaviors by DNA methylation. To investigate the influence of DNA methylation on the locomotor-activating effects of cocaine and on drug-seeking behavior, rats receiving methyl supplementation via chronic l-methionine (MET) underwent either a sensitization regimen of intermittent cocaine injections or intravenous self-administration of cocaine, followed by cue-induced and drug-primed reinstatement. MET blocked sensitization to the locomotor-activating effects of cocaine and attenuated drug-primed reinstatement, with no effect on cue-induced reinstatement or sucrose self-administration and reinstatement. Furthermore, upregulation of DNA methyltransferase 3a and 3b and global DNA hypomethylation were observed in the nucleus accumbens core (NAc), but not in the medial prefrontal cortex (mPFC), of cocaine-pretreated rats. Glutamatergic projections from the mPFC to the NAc are critically involved in the regulation of cocaine-primed reinstatement, and activation of both brain regions is seen in human addicts when reexposed to the drug. When compared with vehicle-pretreated rats, the immediate early gene c-Fos (a marker of neuronal activation) was upregulated in the NAc and mPFC of cocaine-pretreated rats after cocaine-primed reinstatement, and chronic MET treatment blocked its induction in both regions. Cocaine-induced c-Fos expression in the NAc was associated with reduced methylation at CpG dinucleotides in the c-Fos gene promoter, effects reversed by MET treatment. Overall, these data suggest that drug-seeking behaviors are, in part, attributable to a DNA methylation-dependent process, likely occurring at specific gene loci (e.g., c-Fos) in the reward pathway.

Deregulation between miR-29b/c and DNMT3A is associated with epigenetic silencing of the CDH1 gene, affecting cell migration and invasion in gastric cancer.

The de-regulation of the miR-29 family and DNA methyltransferase 3A (DNMT3A) is associated with gastric cancer (GC). While increasing evidence indicates miR-29b/c could regulate DNA methylation by targeting DNMT3A, it is currently unknown if epigenetic silencing of miR-29b/c via promoter hypermethylation in GC is caused by abnormal expression of DNMT3A. Thus, we aimed to evaluate whether cross-talk regulation exists between miR-29b/c and DNMT3A and whether it is associated with a malignant phenotype in GC. First, wound healing and Transwell assays revealed that miR-29b/c suppresses tumor metastasis in GC. A luciferase reporter assay demonstrated that DNMT3A is a direct target of miR-29b/c. We used bisulfite genomic sequencing to analyze the DNA methylation status of miR-29b/c. The percentage of methylated CpGs was significantly decreased in DNMT3A-depleted cells compared to the controls. Furthermore, the involvement of DNMT3A in promoting GC cell migration was associated with the promoter methylation-mediated repression of CDH1. In 50 paired clinical GC tissue specimens, decreased miR-29b/c was significantly correlated with the degree of differentiation and invasion of the cells and was negatively correlated with DNMT3A expression. Together, our preliminary results suggest that the following process may be involved in GC tumorigenesis. miR-29b/c suppresses the downstream gene DNMT3A, and in turn, miR-29b/c is suppressed by DNMT3A in a DNA methylation-dependent manner. The de-regulation of both of miR-29b/c and DNMT3A leads to the epigenetic silencing of CDH1 and contributes to the metastasis phenotype in GC. This finding reveals that DNA methylation-associated silencing of miR-29b/c is critical for GC development and thus may be a therapeutic target.

Involvement of p53 Mutation and Mismatch Repair Proteins Dysregulation in NNK-Induced Malignant Transformation of Human Bronchial Epithelial Cells

Genome integrity is essential for normal cellular functions and cell survival. Its instability can cause genetic aberrations and is considered as a hallmark of most cancers. To investigate the carcinogenesis process induced by tobacco-specific carcinogen NNK, we studied the dynamic changes of two important protectors of genome integrity, p53 and MMR system, in malignant transformation of human bronchial epithelial cells after NNK exposure. Our results showed that the expression of MLH1, one of the important MMR proteins, was decreased early and maintained the downregulation during the transformation in a histone modification involved and DNA methylation-independent manner. Another MMR protein PMS2 also displayed a declined expression while being in a later stage of transformation. Moreover, we conducted p53 mutation analysis and revealed a mutation at codon 273 which led to the replacement of arginine by histidine. With the mutation, DNA damage-induced activation of p53 was significantly impaired. We further reintroduced the wild-type p53 into the transformed cells, and the malignant proliferation can be abrogated by inducing cell cycle arrest and apoptosis. These findings indicate that p53 and MMR system play an important role in the initiation and progression of NNK-induced transformation, and p53 could be a potential therapeutic target for tobacco-related cancers.

Integrative analysis of tissue-specific methylation and alternative splicing identifies conserved transcription factor binding motifs

The exact role of intragenic DNA methylation in regulating tissue-specific gene regulation is unclear. Recently, the DNA-binding protein CTCF has been shown to participate in the regulation of alternative splicing in a DNA methylation-dependent manner. To globally evaluate the relationship between DNA methylation and tissue-specific alternative splicing, we performed genome-wide DNA methylation profiling of mouse retina and brain. In protein-coding genes, tissue-specific differentially methylated regions (T-DMRs) were preferentially located in exons and introns. Gene ontology and evolutionary conservation analysis suggest that these T-DMRs are likely to be biologically relevant. More than 14% of alternatively spliced genes were associated with a T-DMR. T-DMR-associated genes were enriched for developmental genes, suggesting that a specific set of alternatively spliced genes may be regulated through DNA methylation. Novel DNA sequences motifs overrepresented in T-DMRs were identified as being associated with positive and/or negative regulation of alternative splicing in a position-dependent context. The majority of these evolutionarily conserved motifs contain a CpG dinucleotide. Some transcription factors, which recognize these motifs, are known to be involved in splicing. Our results suggest that DNA methylation-dependent alternative splicing is widespread and lay the foundation for further mechanistic studies of the role of DNA methylation in tissue-specific splicing regulation.

The SET-Domain Protein SUVR5 Mediates H3K9me2 Deposition and Silencing at Stimulus Response Genes in a DNA Methylation–Independent Manner

In eukaryotic cells, environmental and developmental signals alter chromatin structure and modulate gene expression. Heterochromatin constitutes the transcriptionally inactive state of the genome and in plants and mammals is generally characterized by DNA methylation and histone modifications such as histone H3 lysine 9 (H3K9) methylation. In Arabidopsis thaliana, DNA methylation and H3K9 methylation are usually colocated and set up a mutually self-reinforcing and stable state. Here, in contrast, we found that SUVR5, a plant Su(var)3–9 homolog with a SET histone methyltransferase domain, mediates H3K9me2 deposition and regulates gene expression in a DNA methylation–independent manner. SUVR5 binds DNA through its zinc fingers and represses the expression of a subset of stimulus response genes. This represents a novel mechanism for plants to regulate their chromatin and transcriptional state, which may allow for the adaptability and modulation necessary to rapidly respond to extracellular cues.

Genome-wide analyses of Zta binding to the Epstein-Barr virus genome reveals interactions in both early and late lytic cycles and an epigenetic switch leading to an altered binding profile.

The Epstein-Barr virus (EBV) genome sustains substantial epigenetic modification involving chromatin remodelling and DNA methylation during lytic replication. Zta (ZEBRA, BZLF1), a key regulator of the EBV lytic cycle, is a transcription and replication factor, binding to Zta response elements (ZREs) in target promoters and EBV lytic origins of replication. In vitro, Zta binding is modulated by DNA methylation; a subset of CpG-containing Zta binding sites (CpG ZREs) is bound only in a DNA methylation-dependent manner. The question of how the dynamic epigenetic environment impacts Zta interaction during the EBV lytic cycle is unknown. To address this, we used chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-Seq) to identify Zta binding sites across the EBV genome before and after viral DNA replication. Replication did not alter the association of Zta across many regions of the EBV genome, but a striking reduction in Zta binding occurred at some loci that contain CpG ZREs. Separating Zta-bound DNA into methylated and nonmethylated fractions, we found that promoters that contain CpG ZREs were enriched in the methylated fraction but that Zta binding to promoters lacking CpG ZREs was not reduced. We hypothesize that the loss of DNA methylation on the EBV genome during the lytic cycle causes the reduced binding to CpG ZREs; this may act as a lytic cycle epigenetic switch. However, the epigenetic changes associated with the replicated EBV genome do not affect the interaction of Zta with many loci that are rich in non-CpG ZREs; this leads to sustained binding at these regions.

Tu1908 HIF-1a Regulates CEACAM6 Expression in Response to Crohn's Disease-Associated E. coli Infection in a DNA Methylation Dependent Manner

Tu1908 HIF-1a Regulates CEACAM6 Expression in Response to Crohn's Disease-Associated E. coli Infection in a DNA Methylation Dependent Manner

Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells

BackgroundDNA methylation, histone modifications and nucleosome occupancy act in concert for regulation of gene expression patterns in mammalian cells. Recently, G9a, a H3K9 methyltransferase, has been shown to play a role in establishment of DNA methylation at embryonic gene targets in ES cells through recruitment of de novo DNMT3A/3B enzymes. However, whether G9a plays a similar role in maintenance of DNA methylation in somatic cells is still unclear.ResultsHere we show that G9a is not essential for maintenance of DNA methylation in somatic cells. Knockdown of G9a has no measurable effect on DNA methylation levels at G9a-target loci. DNMT3A/3B remain stably anchored to nucleosomes containing methylated DNA even in the absence of G9a, ensuring faithful propagation of methylated states in cooperation with DNMT1 through somatic divisions. Moreover, G9a also associates with nucleosomes in a DNMT3A/3B and DNA methylation-independent manner. However, G9a knockdown synergizes with pharmacologic inhibition of DNMTs resulting in increased hypomethylation and inhibition of cell proliferation.ConclusionsTaken together, these data suggest that G9a is not involved in maintenance of DNA methylation in somatic cells but might play a role in re-initiation of de novo methylation after treatment with hypomethylating drugs, thus serving as a potential target for combinatorial treatments strategies involving DNMTs inhibitors.

Distinct modes of DNA accessibility in plant chromatin

The accessibility of DNA to regulatory proteins is a major property of the chromatin environment that favours or hinders transcription. Recent studies in flies reported that H3K9me2-marked heterochromatin is accessible while H3K27me3-marked chromatin forms extensive domains of low accessibility. Here we show that plants regulate DNA accessibility differently. H3K9me2-marked heterochromatin is the least accessible in the Arabidopsis thaliana genome, and H3K27me3-marked chromatin also has low accessibility. We see that very long genes without H3K9me2 or H3K27me3 are often inaccessible and generated significantly lower amounts of antisense transcripts than other genes, suggesting that reduced accessibility is associated with reduced recognition of alternative promoters. Low accessibility of H3K9me2-marked heterochromatin and long genes depend on cytosine methylation, explaining why chromatin accessibility differs between plants and flies. Together, we conclude that restriction of DNA accessibility is a local property of chromatin and not necessarily a consequence of microscopically visible compaction.

Putting muscle in DNA methylation

Putting muscle in DNA methylation