Defective de novo methylation of viral and cellular DNA sequences in ICF syndrome cells Robertson K....
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![Page 1: Defective de novo methylation of viral and cellular DNA sequences in ICF syndrome cells Robertson K. et al. Human Molecular Genetics, 2002 Gergana Ugrinova.](https://reader031.fdocuments.us/reader031/viewer/2022032800/56649d4b5503460f94a297e8/html5/thumbnails/1.jpg)
Defective de novo methylation of viral and cellular DNA sequences in
ICF syndrome cells
Robertson K. et al.
Human Molecular Genetics, 2002
Gergana UgrinovaUniversity of Notre Dame October 11, 2002
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Overview
• Background
• Experiments and results
• Conclusions
• Questions
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DNA methylation
• covalent modification at 5' position of the cytosine ring
• occurs predominantly within the context of CpG dinucleotide
Molecular Biology of the Cell. 3rd ed., Alberts, B. et al.
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CpG distribution
• Majority of mammalian genome is very CG poor• 5'-promoter regions of all housekeeping genes and
some tissue-specific genes contain clusters of CpG dinucleotides, termed CpG islands
• Most parasitic and repetitive DNA sequences (satellite DNA) are very rich in CpG dinucleotides
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Enzymes
Robertson K., Oncogene, 2002
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Enzymes
• Three catalytically active methyltransferases
– DNMT1 - maintenance methyltransferase• localizes to DNA replication foci
– DNMT3A and DNMT3B - de novo methyltransferases• highly active in ES cells and early embryos
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Role of DNA methylation
• Embryonic development• X chromosome inactivation in females• Genomic imprinting• Chromatin remodeling• Tumorigenesis
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ICF syndrome
• rare autosomal recessive disease• defect in DNMT3B gene
• Immune deficiency• Centromere instability• Facial anomalies
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ICF syndrome phenotype
• At the cytogenetic level – anomalous chromosome decondensation
– centromeric breakage
– multiradial chromosomes
– hypomethylation of the juxtacentromeric repeat sequences on chromosomes 1, 9 and 16
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What is known up to now?
• BGS revealed a 50% decrease in methylation of satellite 2 repeats (on chromosomes 1 and 16)
• The overall reduction in cellular 5-methylcytosine levels was about 7%
• A number of genes on the inactive X chromosome have been found to be hypomethylated in ICF cells
• Genes whose expression was aberrantly up- or down-regulated in ICF do not have detectable methylation changes
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Objectives:
• To gain a better understanding of the types of DNA sequences, whose methylation is established and/or maintained by DNMT3B
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Approach
• Analysis of number of viral and cellular genes in 2 EBV-established well-characterized ICF cell lines
Mutation in DNMT3B
ICF 1 Male homozygous V726G
ICF 2 Female heterozygous A603T
intron 22 G-A insertion of STP
Cell line
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Advantages of EBV-based system
• EBV minichromosome is completely sequenced and gene expression patterns upon infection have been extensively studied
• methylation status is well characterized and it is known that it undergoes defined de novo methylation events during the establishment of LCLs in vitro
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Methods
• Bisulfite genomic sequencing
• Methylation specific PCR
• Semiquantative RT-PCR
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Bisulfite genomic sequencing
• Bisulfite treatment of DNA - convert unmethylated cytosine to uracil
• PCR with strand specific primers • cloning of PCR products• sequencing of the cloned PCR products
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Methylation specific PCR
• Bisulfite treatment of DNA - convert unmethylated cytosine to uracil
• PCR with methylation specific primers– primers for methylated C (C-G)
– primers for unmethylated C (U-A)
• Agarose gel electrophoresis of the resulting PCR products
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EBV genome
Tao Q., Huang H., Geiman T., Yen Lim C., Fu L., Qiu G. and Robertson K., Hum. Mol. Genetics, 2002
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MSP analysis of Cp and Wp
Wp - normally undergoes de novo methylation
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Expression from Cp and Wp
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EBV genome
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BGS analysis of Rp
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Semiquantative RT-PCR of Zp and Rp
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RT-PCR analysis of BHRF1 and BLLF1
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Summary of analysis of EBV regions
• All promoters subject to de novo methylation in normal LCLs are highly hypomethylated in ICF cells
• What about cellular genes?
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Scheme of the MAGE-A1 and LAGE-1/2 cellular gene promoters
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BGS analysis of MAGE-A1 and LAGE-1/2
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Quantification of BGS data
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Expression from MAGE-A1 and LAGE-1/2
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Summary of the analysis of C-T cellular genes
• MAGE-A1 CpG island promoter was heavily methylated in all cell line and expression of the gene was not detectable
• LAGE-1/2 CpG island promoter was heavily methylated in ICF 1 and normal cells
• LAGE-1/2 in ICF 2 cell line showed 2 fold decrease in methylation and the gene LAGE-1 was detected by RT-PCR
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Schematic of SCP-1 gene promoter
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MSP methylation analysis of ICF and normal LCLs for SCP-1
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SCP-1 expression monitored by RT-PCR
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Expression of DNA methyltransferases in ICF cells
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Conclusions
• These studies provide first direct evidence for defective de novo methylation in ICF cells
• C-T gene family may represent a new class of genes that are reliant on DNMT3B for proper de novo methylation
• Utility of EBV-based system for examining the complex and poorly understood process of de novo methylation
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Questions
• How the specific mutation in DNMT3B gene plays a role in severity of the phenotype?
• Are the interactions between DNM3B and other DNA-binding proteins (HDACs, chromatin remodeling proteins) impaired and if yes -in what way?
• Have all of the DNA methylating activities in mammalian cells been defined?
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Thank you!
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References:
1. Tao Q., Huang H., Geiman T., Yen Lim C., Fu L., Qiu G and Robertson K., Hum. Mol. Genetics, 2002, 11, 18, 2091-2102
2. Okano M. et al., Cell,1999, 99, 245-257
3. Robertson K. et al., Nucleic Acids Research, 1999, 27, 11, 2291-2298
4. Robertson K., Oncogene, 2002, 21, 5361-5379
5. Tao Q. et al.. American Journal of Pathology, 1999, 155, 2, 619-625
6. Frommer M. et al., PNAS, 1992, 89, 1827-1831
7. Herman et al., PNAS, 1996, 93. 9821-9826
8. Scott Hansen R. et al., PNAS, 1999, 96, 25, 14412-14417