Limited consequences for loss of RNA-directed DNA methylation in Setaria viridis domains rearranged methyltransferase (DRM) mutants

The Domains Rearranged Methyltransferases (DRMs) are crucial for RNA-directed DNA methylation (RdDM) in plant species. Setaria viridis is a model monocot species with a relatively compact genome that has limited transposable element content. CRISPR-based genome editing approaches were used to create loss-of-function alleles for the two putative functional DRM genes in S. viridis to probe the role of RdDM. The analysis of drm1ab double mutant plants revealed limited morphological consequences for the loss of RdDM. Whole-genome methylation profiling provided evidence for wide-spread loss of methylation in CHH sequence contexts, particularly in regions with high CHH methylation in wild-type plants. There is also evidence for locus-specific loss of CG and CHG methylation, even in some regions that lack CHH methylation. Transcriptome profiling identified a limited number of genes with altered expression in the drm1ab mutants. The majority of genes with elevated CHH methylation directly surrounding the transcription start site or in nearby promoter regions do not have altered expression in the drm1ab mutant even when this methylation is lost, suggesting limited regulation of gene expression by RdDM. Detailed analysis of the expression of transposable elements identified several transposons that are transcriptionally activated in drm1ab mutants. These transposons likely require active RdDM for maintenance of transcriptional repression.

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MBD5 and MBD6 couple DNA methylation to gene silencing through the J-domain protein SILENZIO

Science ◽

10.1126/science.abg6130 ◽

2021 ◽

pp. eabg6130

Author(s):

Lucia Ichino ◽

Brandon A. Boone ◽

Luke Strauskulage ◽

C. Jake Harris ◽

Gundeep Kaur ◽

...

Keyword(s):

Dna Methylation ◽

Gene Silencing ◽

Molecular Chaperone ◽

Transcriptional Repression ◽

Transcriptional Repressor ◽

Loss Of Function ◽

Eukaryotic Genes ◽

J Domain ◽

Domain Protein

DNA methylation is associated with transcriptional repression of eukaryotic genes and transposons, but the downstream mechanism of gene silencing is largely unknown. Here we describe two Arabidopsis methyl-CpG binding domain proteins, MBD5 and MBD6, that are recruited to chromatin by recognition of CG methylation, and redundantly repress a subset of genes and transposons without affecting DNA methylation levels. These methyl-readers recruit a J-domain protein, SILENZIO, that acts as a transcriptional repressor in loss-of-function and gain-of-function experiments. J-domain proteins often serve as co-chaperones with HSP70s. Indeed, we found that SILENZIO’s conserved J-domain motif was required for its interaction with HSP70s and for its silencing function. These results uncover an unprecedented role of a molecular chaperone J-domain protein in gene silencing downstream of DNA methylation.

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Abstract TMP25: Long Non-Coding RNA Mediates Stroke-Induced Neurogenesis

Stroke ◽

10.1161/str.51.suppl_1.tmp25 ◽

2020 ◽

Vol 51 (Suppl_1) ◽

Author(s):

Baoyan Fan ◽

Wanlong Pan ◽

Xinli Wang ◽

Michael Chopp ◽

Zheng Gang Zhang ◽

...

Keyword(s):

Cell Cycle ◽

Regulation Of Gene Expression ◽

Artery Occlusion ◽

Loss Of Function ◽

Bioinformatics Analyses ◽

Guide Rna ◽

Non Coding Rna ◽

Cerebral Artery Occlusion ◽

Long Non Coding Rna

Background and Purpose: Adult neurogenesis contributes to functional recovery after stroke. Long non-coding RNAs (lncRNAs) regulate stem cell self-renewal and differentiation. However, the role of lncRNAs in stroke-induced neurogenesis remains unknown. Methods and Results: Using lncRNA array and in situ hybridization, we analyzed lncRNA profiles of adult neural stem cells (NSCs) isolated from the subventricular zone neurogenic region in rats subjected to middle cerebral artery occlusion. We found that H19 was the most highly upregulated lncRNA (19 fold) in ischemic NSCs compared with non-ischemic NSCs. Reduction of endogenous H19 in NSCs by CRISPR-Cas9 genome editing significantly decreased the proliferation and increased the apoptosis of ischemic NSCs, as assayed by the number of BrdU + cells (56±5% vs 22±3%, p<0.01, n=3) and Caspase-3/7 activity compared to NSCs transfected with scrambled small guide RNA (sgRNA). Knockdown of H19 significantly decreased the number of Tuj1 + neuroblasts (8±2% vs 5±0.4%, p<0.01, n=3) and NG 2 + oliogodendrocyte progenitor cells (10±1% vs 5±0.3%, p<0.01, n=3), suggesting that deletion of H19 suppresses the proliferation and survival and blocks the differentiation of NSCs into neurons and oligodendrocytes. Additional RNA-sequencing and bioinformatics analyses revealed that genes deregulated by H19 knockdown were involved in transcription, apoptosis, proliferation, cell cycle and response to hypoxia. Western blot analysis validated that loss-of-function and gain-of-function of H19 significantly increased and reduced, respectively, the transcription of cell cycle-related genes including p27. Using ChIRP assay, we found that upregulated H19 in NSCs was physically associated with EZH2 which catalyzes the repressive H3K27me3 histone marker. Knockdown of H19 significantly reduced the enrichment of H3K27me3 at the promoter of p27, leading to the upregulation of p27 expression and consequently inhibition of NSC proliferation. Conclusions: H19 mediates stroke-induced neurogenesis by regulating genes involved in cell cycle and survival through the interaction with chromatin remodeling proteins. Our data provide novel insights into epigenetic regulation of gene expression by lncRNA in neurogenesis.

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The Emerging Role of Epigenetic Methylation in Kidney Disease

Current Medicinal Chemistry ◽

10.2174/1573405617666211213110222 ◽

2021 ◽

Vol 28 ◽

Author(s):

Li Wen ◽

Hong-liu Yang ◽

Lin Lin ◽

Liang Ma ◽

Ping Fu

Keyword(s):

Dna Methylation ◽

Kidney Disease ◽

Histone Methylation ◽

Kidney Diseases ◽

Therapeutic Approaches ◽

Promoter Regions ◽

Recent Advances ◽

Epigenetic Methylation

: Kidney disease has complex and multifactorial pathophysiology and pathogenesis. Recent studies have revealed that epigenetic methylation changes, namely DNA methylation, histone methylation and non-histone methylation, are strongly implicated in various forms of kidney diseases. This review provides a perspective on the emerging role of epigenetic methylation in kidney disease, including the effects of DNA methylation in diverse promoter regions, regulation and implication of histone methylation, and recent advances and potential directions related to non-histone methylation. Monitoring or targeting epigenetic methylation has potential to contribute to development of therapeutic approaches for multiple kidney diseases.

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Fetal and Neonatal Exposure to the Endocrine Disruptor Methoxychlor Causes Epigenetic Alterations in Adult Ovarian Genes

Endocrinology ◽

10.1210/en.2009-0499 ◽

2009 ◽

Vol 150 (10) ◽

pp. 4681-4691 ◽

Cited By ~ 91

Author(s):

Aparna Mahakali Zama ◽

Mehmet Uzumcu

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Ovarian Function ◽

Endocrine Disrupting Chemicals ◽

Neonatal Exposure ◽

Dna Hypermethylation ◽

Promoter Regions ◽

Altered Expression ◽

Arbitrarily Primed Pcr ◽

Methylation Patterns

Abstract Exposure to endocrine-disrupting chemicals during development could alter the epigenetic programming of the genome and result in adult-onset disease. Methoxychlor (MXC) and its metabolites possess estrogenic, antiestrogenic, and antiandrogenic activities. Previous studies showed that fetal/neonatal exposure to MXC caused adult ovarian dysfunction due to altered expression of key ovarian genes including estrogen receptor (ER)-β, which was down-regulated, whereas ERα was unaffected. The objective of the current study was to evaluate changes in global and gene-specific methylation patterns in adult ovaries associated with the observed defects. Rats were exposed to MXC (20 μg/kg·d or 100 mg/kg·d) between embryonic d 19 and postnatal d 7. We performed DNA methylation analysis of the known promoters of ERα and ERβ genes in postnatal d 50–60 ovaries using bisulfite sequencing and methylation-specific PCRs. Developmental exposure to MXC led to significant hypermethylation in the ERβ promoter regions (P < 0.05), whereas the ERα promoter was unaffected. We assessed global DNA methylation changes using methylation-sensitive arbitrarily primed PCR and identified 10 genes that were hypermethylated in ovaries from exposed rats. To determine whether the MXC-induced methylation changes were associated with increased DNA methyltransferase (DNMT) levels, we measured the expression levels of Dnmt3a, Dnmt3b, and Dnmt3l using semiquantitative RT-PCR. Whereas Dnmt3a and Dnmt3l were unchanged, Dnmt3b expression was stimulated in ovaries of the 100 mg/kg MXC group (P < 0.05), suggesting that increased DNMT3B may cause DNA hypermethylation in the ovary. Overall, these data suggest that transient exposure to MXC during fetal and neonatal development affects adult ovarian function via altered methylation patterns.

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Vitamin C Transporters and Their Implications in Carcinogenesis

Nutrients ◽

10.3390/nu12123869 ◽

2020 ◽

Vol 12 (12) ◽

pp. 3869

Author(s):

Kinga Linowiecka ◽

Marek Foksinski ◽

Anna A. Brożyna

Keyword(s):

Vitamin C ◽

Cancer Biology ◽

Redox Homeostasis ◽

Regulation Of Gene Expression ◽

Dna Demethylation ◽

Altered Expression ◽

Tet Proteins ◽

Active Dna Demethylation ◽

Hypoxic State

Vitamin C is implicated in various bodily functions due to its unique properties in redox homeostasis. Moreover, vitamin C also plays a great role in restoring the activity of 2-oxoglutarate and Fe2+ dependent dioxygenases (2-OGDD), which are involved in active DNA demethylation (TET proteins), the demethylation of histones, and hypoxia processes. Therefore, vitamin C may be engaged in the regulation of gene expression or in a hypoxic state. Hence, vitamin C has acquired great interest for its plausible effects on cancer treatment. Since its conceptualization, the role of vitamin C in cancer therapy has been a controversial and disputed issue. Vitamin C is transferred to the cells with sodium dependent transporters (SVCTs) and glucose transporters (GLUT). However, it is unknown whether the impaired function of these transporters may lead to carcinogenesis and tumor progression. Notably, previous studies have identified SVCTs’ polymorphisms or their altered expression in some types of cancer. This review discusses the potential effects of vitamin C and the impaired SVCT function in cancers. The variations in vitamin C transporter genes may regulate the active transport of vitamin C, and therefore have an impact on cancer risk, but further studies are needed to thoroughly elucidate their involvement in cancer biology.

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Epigenetic Gene Regulation in the Bacterial World

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.00016-06 ◽

2006 ◽

Vol 70 (3) ◽

pp. 830-856 ◽

Cited By ~ 349

Author(s):

Josep Casadesús ◽

David Low

Keyword(s):

Dna Methylation ◽

Dna Replication ◽

Transcriptional Activation ◽

Transcriptional Repression ◽

Cytosine Methylation ◽

Regulation Of Gene Expression ◽

Phase Variation ◽

E Coli ◽

Adenine Methylation ◽

Methylation Patterns

SUMMARY Like many eukaryotes, bacteria make widespread use of postreplicative DNA methylation for the epigenetic control of DNA-protein interactions. Unlike eukaryotes, however, bacteria use DNA adenine methylation (rather than DNA cytosine methylation) as an epigenetic signal. DNA adenine methylation plays roles in the virulence of diverse pathogens of humans and livestock animals, including pathogenic Escherichia coli, Salmonella, Vibrio, Yersinia, Haemophilus, and Brucella. In Alphaproteobacteria, methylation of adenine at GANTC sites by the CcrM methylase regulates the cell cycle and couples gene transcription to DNA replication. In Gammaproteobacteria, adenine methylation at GATC sites by the Dam methylase provides signals for DNA replication, chromosome segregation, mismatch repair, packaging of bacteriophage genomes, transposase activity, and regulation of gene expression. Transcriptional repression by Dam methylation appears to be more common than transcriptional activation. Certain promoters are active only during the hemimethylation interval that follows DNA replication; repression is restored when the newly synthesized DNA strand is methylated. In the E. coli genome, however, methylation of specific GATC sites can be blocked by cognate DNA binding proteins. Blockage of GATC methylation beyond cell division permits transmission of DNA methylation patterns to daughter cells and can give rise to distinct epigenetic states, each propagated by a positive feedback loop. Switching between alternative DNA methylation patterns can split clonal bacterial populations into epigenetic lineages in a manner reminiscent of eukaryotic cell differentiation. Inheritance of self-propagating DNA methylation patterns governs phase variation in the E. coli pap operon, the agn43 gene, and other loci encoding virulence-related cell surface functions.

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Methylation-Mediated Transcriptional Silencing in Euchromatin by Methyl-CpG Binding Protein MBD1 Isoforms

Molecular and Cellular Biology ◽

10.1128/mcb.19.9.6415 ◽

1999 ◽

Vol 19 (9) ◽

pp. 6415-6426 ◽

Cited By ~ 131

Author(s):

Naoyuki Fujita ◽

Shin-ichiro Takebayashi ◽

Katsuzumi Okumura ◽

Shinichi Kudo ◽

Tsutomu Chiba ◽

...

Keyword(s):

Dna Methylation ◽

Transcriptional Repression ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Cpg Islands ◽

Transcriptional Silencing ◽

Pericentromeric Region ◽

Chromosome 1 ◽

Reverse Transcription Pcr ◽

Genome Methylation

ABSTRACT DNA methylation of promoter-associated CpG islands is involved in the transcriptional repression of vertebrate genes. To investigate the mechanisms underlying gene inactivation by DNA methylation, we characterized a human MBD1 protein, one of the components of MeCP1, which possesses a methyl-CpG binding domain (MBD) and cysteine-rich (CXXC) domains. Four novel MBD1 isoforms (MBD1v1, MBD1v2, MBD1v3, and MBD1v4) were identified by the reverse transcription-PCR method. We found that these transcripts were alternatively spliced in the region of CXXC domains and the C terminus. Green fluorescent protein-fused MBD1 was localized to multiple foci on the human genome, mostly in the euchromatin regions, and particularly concentrated in the pericentromeric region of chromosome 1. Both the MBD sequence and genome methylation were required for proper localization of the MBD1 protein. We further investigated whether MBD1 isoforms are responsible for transcriptional repression of human genes. A bacterially expressed MBD1 protein bound preferentially to methylated DNA fragments containing CpG islands from the tumor suppressor genes p16,VHL, and E-cadherin and from an imprintedSNRPN gene. All MBD1 isoforms inhibited promoter activities of these genes via methylation. Interestingly, MBD1 isoforms v1 and v2 containing three CXXC domains also suppressed unmethylated promoter activities in mammalian cells. These effects were further manifested inDrosophila melanogaster cells, which lack genome methylation. Sp1-activated transcription of methylated p16and SNRPN promoters was inhibited by all of the MBD1 isoforms, whereas the isoforms v1 and v2 reduced Sp1-activated transcription from unmethylated promoters as well. These findings suggested that the MBD1 isoforms have different roles in methylation-mediated transcriptional silencing in euchromatin.

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TET-mediated epimutagenesis of the Arabidopsis thaliana methylome

10.1101/151027 ◽

2017 ◽

Author(s):

Lexiang Ji ◽

William T. Jordan ◽

Xiuling Shi ◽

Lulu Hu ◽

Chuan He ◽

...

Keyword(s):

Dna Methylation ◽

Arabidopsis Thaliana ◽

Transcriptional Repression ◽

Dna Methyltransferase ◽

Developmental Defects ◽

Altered Expression ◽

Differentially Methylated Genes ◽

Plant Genes ◽

Novel Method ◽

Phenotypic Variations

DNA methylation in the promoters of plant genes sometimes leads to transcriptional repression, and the wholesale removal of DNA methylation as seen in methyltransferase mutants results in drastic changes in gene expression and severe developmental defects. However, many cases of naturally-occurring DNA methylation variations have been reported, whereby the altered expression of differentially methylated genes is responsible for agronomically important traits. The ability to manipulate plant methylomes to generate populations of epigenetically distinct individuals could provide invaluable resources for breeding and research purposes. Here we describe “epimutagenesis”, a novel method to rapidly generate variation of DNA methylation through random demethylation of the Arabidopsis thaliana genome. This method involves the expression of a human Ten-eleven translocation (TET) enzyme, and results in widespread hypomethylation that can be inherited to subsequent generations, mimicking mutants in the maintenance DNA methyltransferase met1. Application of TET-mediated epimutagenesis to agriculturally significant plants may result in differential expression of alleles normally silenced by DNA methylation, uncovering previously hidden phenotypic variations.

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Zebrafish Ski7 tunes RNA levels during the oocyte-to-embryo transition

10.1101/2020.03.19.998716 ◽

2020 ◽

Cited By ~ 1

Author(s):

Luis Enrique Cabrera Quio ◽

Alexander Schleiffer ◽

Karl Mechtler ◽

Andrea Pauli

Keyword(s):

Regulation Of Gene Expression ◽

Physiological Role ◽

Transcriptome Profiling ◽

Time Frame ◽

Rna Degradation ◽

Developmental Transitions ◽

Mrna Targets ◽

Go Enrichment ◽

Normal Adults

AbstractPost-transcriptional mechanisms are crucial for the regulation of gene expression. These mechanisms are particularly important during rapid developmental transitions such as the oocyte-to-embryo transition, which is characterized by dramatic changes to the developmental program in the absence of nuclear transcription. Under these conditions, changes to the RNA content are solely dependent on RNA degradation. Although several mechanisms that promote RNA decay during embryogenesis have been identified, it remains unclear which cellular machineries contribute to remodeling the maternal transcriptome during the oocyte-to-embryo transition. Here, we focused on the auxiliary 3’-to-5’ degradation factor Ski7 in zebrafish as its mRNA peaks during this time frame. Homozygous ski7 mutant fish were viable and developed into morphologically normal adults, yet they had decreased fertility. Consistent with the idea that Ski7 participates in remodeling the transcriptome during the oocyte-to-embryo transition, transcriptome profiling identified stage-specific mRNA targets of Ski7. Genes upregulated in ski7 mutants were generally lowly expressed in wild type, suggesting that Ski7 maintains low transcript levels for this subset of genes. GO enrichment analyses of genes mis-regulated in ski7 mutants implicated Ski7 in the regulation of redox processes. This was confirmed experimentally by an increased resistance of ski7 mutant embryos to reductive stress. Overall, our results provide first insights into the physiological role of vertebrate Ski7 as an important post-transcriptional regulator during the oocyte-to-embryo transition.

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The impact of Piscirickettsia Salmonis infection on genome-wide DNA methylation profile in Atlantic Salmon

10.1101/2021.12.20.473279 ◽

2021 ◽

Author(s):

Robert Mukiibi ◽

Carolina Peñaloza ◽

Alejandro Gutierrez ◽

José M. Yáñez ◽

Ross D. Houston ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Atlantic Salmon ◽

Negative Correlation ◽

Regulation Of Gene Expression ◽

Head Kidney ◽

Epigenetic Mechanism ◽

Piscirickettsia Salmonis ◽

The Impact

Salmon rickettsial septicaemia (SRS), caused by the intracellular bacteria Piscirickettsia Salmonis, generates significant mortalities to farmed Atlantic salmon, particularly in Chile. Due to its economic importance, a wealth of research has focussed on the biological mechanisms underlying pathogenicity of P. salmonis, the host response, and genetic variation in host resistance. DNA methylation is a fundamental epigenetic mechanism that influences almost every biological process via the regulation of gene expression and plays a key role in the response of an organism to stimuli. In the current study, the role of head kidney and liver DNA methylation in the response to P. salmonis infection was investigated in a commercial Atlantic salmon population. A total of 66 salmon were profiled using reduced representation bisulphite sequencing (RRBS), with head kidney and liver methylomes compared between infected animals (3 and 9 days post infection) and uninfected controls. These included groups of salmon with divergent (high or low) breeding values for resistance to P. salmonis infection, to examine the influence of genetic resistance. Head kidney and liver showed organ-specific global methylation patterns, but with similar distribution of methylation across gene features. Integration of methylation with RNA-Seq data revealed that methylation levels predominantly showed a negative correlation with gene expression, although positive correlations were also observed. Methylation within the first exon showed the strongest negative correlation with gene expression. A total of 911 and 813 differentially methylated CpG sites were identified between infected and control samples in the head kidney at 3 and 9 days respectively, whereas only 30 and 44 sites were differentially methylated in the liver. Differential methylation in the head kidney was associated with immunological processes such as actin cytoskeleton regulation, phagocytosis, endocytosis and pathogen associated pattern receptor signaling. We also identified 113 and 48 differentially methylated sites between resistant and susceptible fish in the head kidney and liver respectively. Our results contribute to the growing understanding of the role of methylation in regulation of gene expression and response to infectious diseases, and in particular reveal key immunological functions regulated by methylation in Atlantic salmon in response to P. salmonis.

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