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

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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Dynamic Analysis of Proviral Induction and De Novo Methylation: Implications for a Histone Deacetylase-Independent, Methylation Density-Dependent Mechanism of Transcriptional Repression

Molecular and Cellular Biology ◽

10.1128/mcb.20.3.842-850.2000 ◽

2000 ◽

Vol 20 (3) ◽

pp. 842-850 ◽

Cited By ~ 86

Author(s):

Matthew C. Lorincz ◽

Dirk Schübeler ◽

Scott C. Goeke ◽

Mark Walters ◽

Mark Groudine ◽

...

Keyword(s):

Dna Methylation ◽

Histone Acetylation ◽

Histone Deacetylase ◽

Transcriptional Repression ◽

Mammalian Cells ◽

Fluorescent Protein ◽

De Novo ◽

Trichostatin A ◽

Histone Deacetylation ◽

Over Time

ABSTRACT Methylation of cytosines in the CpG dinucleotide is generally associated with transcriptional repression in mammalian cells, and recent findings implicate histone deacetylation in methylation-mediated repression. Analyses of histone acetylation in in vitro-methylated transfected plasmids support this model; however, little is known about the relationships among de novo DNA methylation, transcriptional repression, and histone acetylation state. To examine these relationships in vivo, we have developed a novel approach that permits the isolation and expansion of cells harboring expressing or silent retroviruses. MEL cells were infected with a Moloney murine leukemia virus encoding the green fluorescent protein (GFP), and single-copy, silent proviral clones were treated weekly with the histone deacetylase inhibitor trichostatin A or the DNA methylation inhibitor 5-azacytidine. Expression was monitored concurrently by flow cytometry, allowing for repeated phenotypic analysis over time, and proviral methylation was determined by Southern blotting and bisulfite methylation mapping. Shortly after infection, proviral expression was inducible and the reporter gene and proviral enhancer showed a low density of methylation. Over time, the efficacy of drug induction diminished, coincident with the accumulation of methyl-CpGs across the provirus. Bisulfite analysis of cells in which 5-azacytidine treatment induced GFP expression revealed measurable but incomplete demethylation of the provirus. Repression could be overcome in late-passage clones only by pretreatment with 5-azacytidine followed by trichostatin A, suggesting that partial demethylation reestablishes the trichostatin-inducible state. These experiments reveal the presence of a silencing mechanism which acts on densely methylated DNA and appears to function independently of histone deacetylase activity.

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The Core Element of a CpG Island Protects Avian Sarcoma and Leukosis Virus-Derived Vectors from Transcriptional Silencing

Journal of Virology ◽

10.1128/jvi.00419-08 ◽

2008 ◽

Vol 82 (16) ◽

pp. 7818-7827 ◽

Cited By ~ 19

Author(s):

Filip Šenigl ◽

Jiří Plachý ◽

Jiří Hejnar

Keyword(s):

Binding Sites ◽

Mammalian Cells ◽

Cpg Island ◽

Cpg Islands ◽

Transcriptional Silencing ◽

Retroviral Vectors ◽

Core Element ◽

Promoter Sequences ◽

Rous Sarcoma ◽

Sarcoma Virus

ABSTRACT Unmethylated CpG islands are known to keep adjacent promoters transcriptionally active. In the CpG island adjacent to the adenosine phosphoribosyltransferase gene, the protection against transcriptional silencing can be attributed to the short CpG-rich core element containing Sp1 binding sites. We report here the insertion of this CpG island core element, IE, into the long terminal repeat of a retroviral vector derived from Rous sarcoma virus, which normally suffers from progressive transcriptional silencing in mammalian cells. IE insertion into a specific position between enhancer and promoter sequences led to efficient protection of the integrated vector from silencing and gradual CpG methylation in rodent and human cells. Individual cell clones with IE-modified reporter vectors display high levels of reporter expression for a sustained period and without substantial variegation in the cell culture. The presence of Sp1 binding sites is important for the protective effect of IE, but at least some part of the entire antisilencing capacity is maintained in IE with mutated Sp1 sites. We suggest that this strategy of antisilencing protection by the CpG island core element may prove generally useful in retroviral vectors.

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LSH Cooperates with DNA Methyltransferases To Repress Transcription

Molecular and Cellular Biology ◽

10.1128/mcb.01073-07 ◽

2007 ◽

Vol 28 (1) ◽

pp. 215-226 ◽

Cited By ~ 83

Author(s):

Kevin Myant ◽

Irina Stancheva

Keyword(s):

Dna Methylation ◽

Chromatin Remodeling ◽

Transcriptional Repression ◽

Histone Deacetylases ◽

Mammalian Cells ◽

Dna Methyltransferase ◽

Dna Methyltransferases ◽

Large Protein ◽

Large Protein Complex ◽

Nuclear Extracts

ABSTRACT LSH, a protein related to the SNF2 family of chromatin-remodeling ATPases, is required for efficient DNA methylation in mammals. How LSH functions to support DNA methylation and whether it associates with a large protein complex containing DNA methyltransferase (DNMT) enzymes is currently unclear. Here we show that, unlike many other chromatin-remodeling ATPases, native LSH is present mostly as a monomeric protein in nuclear extracts of mammalian cells and cannot be detected in a large multisubunit complex. However, when targeted to a promoter of a reporter gene, LSH acts as an efficient transcriptional repressor. Using this as an assay to identify proteins that are required for LSH-mediated repression we found that LSH cooperates with the DNMTs DNMT1 and DNMT3B and with the histone deacetylases (HDACs) HDAC1 and HDAC2 to silence transcription. We show that transcriptional repression by LSH and interactions with HDACs are lost in DNMT1 and DNMT3B knockout cells but that the enzymatic activities of DNMTs are not required for LSH-mediated silencing. Our data suggest that LSH serves as a recruiting factor for DNMTs and HDACs to establish transcriptionally repressive chromatin which is perhaps further stabilized by DNA methylation at targeted loci.

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Silencing of Transposable Elements Mediated by 5-mC and Compensation of the Heterochromatin Content by Presence of B Chromosomes in Astyanax scabripinnis

Cells ◽

10.3390/cells10051162 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1162

Author(s):

Patrícia Barbosa ◽

Zelinda Schemczssen-Graeff ◽

André Marques ◽

Maelin da Silva ◽

Giovani Marino Favero ◽

...

Keyword(s):

Transposable Elements ◽

Compensation Effect ◽

Pericentromeric Region ◽

B Chromosome ◽

B Chromosomes ◽

Chromosome 1 ◽

Complex Processes ◽

Genome Methylation ◽

Regulation Mechanisms ◽

Heterochromatin Content

The way in which transcriptional activity overcomes the physical DNA structure and gene regulation mechanisms involves complex processes that are not yet fully understood. Modifications in the cytosine-guanine sequence of DNA by 5-mC are preferentially located in heterochromatic regions and are related to gene silencing. Herein, we investigate evidence of epigenetic regulation related to the B chromosome model and transposable elements in A. scabripinnis. Indirect immunofluorescence using anti-5-mC to mark methylated regions was employed along with quantitative ELISA to determine the total genomic DNA methylation level. 5-mC signals were dispersed in the chromosomes of both females and males, with preferential accumulation in the B chromosome. In addition to the heterochromatic methylated regions, our results suggest that methylation is associated with transposable elements (LINE and Tc1-Mariner). Heterochromatin content was measured based on the C-band length in relation to the size of chromosome 1. The B chromosome in A. scabripinnis comprises heterochromatin located in the pericentromeric region of both arms of this isochromosome. In this context, individuals with B chromosomes should have an increased heterochromatin content when compared to individuals that do not. Although, both heterochromatin content and genome methylation showed no significant differences between sexes or in relation to the occurrence of B chromosomes. Our evidence suggests that the B chromosome can have a compensation effect on the heterochromatin content and that methylation possibly operates to silence TEs in A. scabripinnis. This represents a sui generis compensation and gene activity buffering mechanism.

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

10.1101/2022.01.05.474142 ◽

2022 ◽

Author(s):

Andrew C. Read ◽

Trevor Weiss ◽

Peter A. Crisp ◽

Zhikai Liang ◽

Jaclyn Noshay ◽

...

Keyword(s):

Dna Methylation ◽

Transcriptional Repression ◽

Regulation Of Gene Expression ◽

Transcriptome Profiling ◽

Setaria Viridis ◽

Loss Of Function ◽

Promoter Regions ◽

Altered Expression ◽

Genome Methylation

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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Retroviral Expression in Embryonic Stem Cells and Hematopoietic Stem Cells

Molecular and Cellular Biology ◽

10.1128/mcb.20.20.7419-7426.2000 ◽

2000 ◽

Vol 20 (20) ◽

pp. 7419-7426 ◽

Cited By ~ 207

Author(s):

Sara R. Cherry ◽

D. Biniszkiewicz ◽

L. van Parijs ◽

D. Baltimore ◽

R. Jaenisch

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Transcriptional Repression ◽

Fluorescent Protein ◽

De Novo ◽

Es Cells ◽

Embryonic Stem ◽

Hematopoietic Stem

ABSTRACT Achieving long-term retroviral expression in primary cells has been problematic. De novo DNA methylation of infecting proviruses has been proposed as a major cause of this transcriptional repression. Here we report the development of a mouse stem cell virus (MSCV) long terminal repeat-based retroviral vector that is expressed in both embryonic stem (ES) cells and hematopoietic stem (HS) cells. Infected HS cells and their differentiated descendants maintained long-term and stable retroviral expression after serial adoptive transfers. In addition, retrovirally infected ES cells showed detectable expression level of the green fluorescent protein (GFP). Moreover, GFP expression of integrated proviruses was maintained after in vitro differentiation of infected ES cells. Long-term passage of infected ES cells resulted in methylation-mediated silencing, while short-term expression was methylation independent. Tissues of transgenic animals, which we derived from ES cells carrying the MSCV-based provirus, did not express GFP. However, treatment with the demethylating agent 5-azadeoxycytidine reactivated the silent provirus, demonstrating that DNA methylation is involved in the maintenance of retroviral repression. Our results indicate that retroviral expression in ES cells is repressed by methylation-dependent as well as methylation-independent mechanisms.

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Aberrant DNA methylation in cancer: potential clinical interventions

Expert Reviews in Molecular Medicine ◽

10.1017/s1462399402004222 ◽

2002 ◽

Vol 4 (4) ◽

pp. 1-17 ◽

Cited By ~ 55

Author(s):

G. Strathdee ◽

R. Brown

Keyword(s):

Dna Methylation ◽

Transcriptional Repression ◽

Cpg Islands ◽

Covalent Modification ◽

Promoter Regions ◽

Cpg Dinucleotides ◽

Human Genes ◽

Dramatic Shift ◽

Aberrant Dna Methylation ◽

Gene Alterations

DNA methylation, the addition of a methyl group to the carbon-5 position of cytosine residues, is the only common covalent modification of human DNA and occurs almost exclusively at cytosines that are followed immediately by a guanine (so-called CpG dinucleotides). The bulk of the genome displays a clear depletion of CpG dinucleotides, and those that are present are nearly always methylated. By contrast, small stretches of DNA, known as CpG islands, are comparatively rich in CpG nucleotides and are nearly always free of methylation. These CpG islands are frequently located within the promoter regions of human genes, and methylation within the islands has been shown to be associated with transcriptional inactivation of the corresponding gene. Alterations in DNA methylation might be pivotal in the development of most cancers. In recent years, it has become apparent that the pattern of DNA methylation observed in cancer generally shows a dramatic shift compared with that of normal tissue. Although cancers often exhibit clear reductions throughout their genomes in the levels of DNA methylation, this goes hand-in-hand with increased methylation at the CpG islands. Such changes in methylation have a central role in tumourigenesis; in particular, methylation of CpG islands has been shown to be important in transcriptional repression of numerous genes that function to prevent tumour growth or development. Studies of DNA methylation in cancer have thus opened up new opportunities for diagnosis, prognosis and ultimately treatment of human tumours.

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Heat and Hypoxia Exposure Mediates Circadian Rhythms Response via Methylation Modification in Apostichopus japonicas

Frontiers in Marine Science ◽

10.3389/fmars.2021.721465 ◽

2021 ◽

Vol 8 ◽

Author(s):

Jiong Wu ◽

Weiwei Zhang ◽

Chenghua Li

Keyword(s):

Dna Methylation ◽

Circadian Rhythm ◽

Cpg Island ◽

Apostichopus Japonicus ◽

Combined Treatment ◽

Cpg Islands ◽

Venn Diagram ◽

Hypoxia Exposure ◽

Rate Limiting Step ◽

Genome Methylation

As global warming progresses, heat and hypoxia are gradually becoming important factors threatening the survival, reproduction, and development of marine organisms. To determine the effect of heat and hypoxia on Apostichopus japonicus, whole genome methylation of the respiratory tree was determined under heat, hypoxia, and heat-hypoxia conditions [designed as heat stress treatment (HT), hypoxia treatment (LO), and heat-hypoxia combined treatment (HL) groups]. The number of differentially methylated regions (DMRs) under three treatments was determined based on the Venn diagram. The network of the DMRs associated with promoters that were co-existed under the three conditions showed that circadian rhythm was involved based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Circadian rhythm-related genes, CRY1a, CRY1b, CLC, and TIM, decreased in LO and HL groups, while CRY1a, CRY1b, and BMAL1 increased in the HT group. Bisulfite sequencing PCR (BSP) showed that the methylation levels of CpG island regions in the promoters of CRY1a and CRY1b were upregulated in HT, LO, and HL groups, leading to the decreased promoter activity of CRY1a and CRY1b. RNAi of CRY1a and CRY1b led to increased enzyme activities of two energy-related enzymes, pyruvate kinase (PK) catalyzing the rate-limiting step in glycolysis, and ATPase hydrolyzing ATP to ADP, which were also increased under the three tested conditions. Thus, it was concluded that A. japonicus may respond to the heat, hypoxia, and heat-hypoxia stresses via the DNA methylation of heat, hypoxia, and heat-hypoxia stresses via the DNA methylation of CpG islands of circadian rhythm-related genes, which increased the activity of energy-related enzymes.

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Evidence for mitochondrial localization of a novel human sialidase (NEU4)

Biochemical Journal ◽

10.1042/bj20050017 ◽

2005 ◽

Vol 390 (1) ◽

pp. 85-93 ◽

Cited By ~ 102

Author(s):

Kazunori Yamaguchi ◽

Keiko Hata ◽

Koichi Koseki ◽

Kazuhiro Shiozaki ◽

Hirotoshi Akita ◽

...

Keyword(s):

Amino Acid ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Short Form ◽

Cell Types ◽

Culture Cell ◽

Reverse Transcription Pcr ◽

Long Form ◽

Biochemical Fractionation ◽

Green Fluorescent

Based on the human cDNA sequence predicted to represent the NEU4 sialidase gene in public databases, a cDNA covering the entire coding sequence was isolated from human brain and expressed in mammalian cells. The cDNA encodes two isoforms: one possessing an N-terminal 12-amino-acid sequence that is predicted to be a mitochondrial targeting sequence, and the other lacking these amino acids. Expression of the isoforms is tissuespecific, as assessed by reverse transcription–PCR. Brain, muscle and kidney contained both isoforms; liver showed the highest expression, and the short form was predominant in this organ. In transiently transfected COS-1 cells, enzyme activity was markedly increased with gangliosides as well as with glycoproteins and oligosaccharides as substrates compared with the control levels. This differs from findings with other human sialidases. Although the isoforms were not distinguishable with regard to substrate specificity, they exhibited differential subcellular localizations. Immunofluorescence microscopy and biochemical fractionation demonstrated that an exogenously expressed haemagglutinin-tagged long form of NEU4 was concentrated in mitochondria in several human culture cell types, whereas the short form was present in intracellular membranes, indicating that the sequence comprising the N-terminal 12 amino acid residues acts as a targeting signal for mitochondria. Co-localization of the long form to mitochondria was further supported by efficient targeting of the N-terminal region fused to enhanced green fluorescent protein, and by the targeting failure of a mutant with an amino acid substitution in this region. NEU4 is possibly involved in regulation of apoptosis by modulation of ganglioside GD3, which accumulates in mitochondria during apoptosis and is the best substrate for the sialidase.

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Data Mining Technique Applied To DNA Sequencing

International Research Journal of Management IT and Social Sciences ◽

10.21744/irjmis.v2i7.70 ◽

2015 ◽

Vol 2 (7) ◽

pp. 18

Author(s):

R. Jamuna

Keyword(s):

Data Mining ◽

Dna Methylation ◽

Markov Chain ◽

Human Genome ◽

Transition Probabilities ◽

Markov Chain Model ◽

Effective Means ◽

Cpg Islands ◽

Chain Model ◽

The Given

CpG islands (CGIs) play a vital role in genome analysis as genomic markers. Identification of the CpG pair has contributed not only to the prediction of promoters but also to the understanding of the epigenetic causes of cancer. In the human genome [1] wherever the dinucleotides CG occurs the C nucleotide (cytosine) undergoes chemical modifications. There is a relatively high probability of this modification that mutates C into a T. For biologically important reasons the mutation modification process is suppressed in short stretches of the genome, such as ‘start’ regions. In these regions [2] predominant CpG dinucleotides are found than elsewhere. Such regions are called CpG islands. DNA methylation is an effective means by which gene expression is silenced. In normal cells, DNA methylation functions to prevent the expression of imprinted and inactive X chromosome genes. In cancerous cells, DNA methylation inactivates tumor-suppressor genes, as well as DNA repair genes, can disrupt cell-cycle regulation. The most current methods for identifying CGIs suffered from various limitations and involved a lot of human interventions. This paper gives an easy searching technique with data mining of Markov Chain in genes. Markov chain model has been applied to study the probability of occurrence of C-G pair in the given gene sequence. Maximum Likelihood estimators for the transition probabilities for each model and analgously for the model has been developed and log odds ratio that is calculated estimates the presence or absence of CpG is lands in the given gene which brings in many facts for the cancer detection in human genome.

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