Download Defective de novo methylation of viral and cellular DNA sequences

Defective de novo methylation of
viral and cellular DNA sequences in
ICF syndrome cells
Robertson K. et al.
Human Molecular Genetics, 2002
Gergana Ugrinova
University of Notre Dame
October 11, 2002
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
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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
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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 downregulated 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
Cell line
Mutation in DNMT3B
ICF 1
Male homozygous V726G
ICF 2
Female heterozygous A603T
intron 22 G-A  insertion of
STP
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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:
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Tao Q., Huang H., Geiman T., Yen Lim C., Fu L., Qiu G and Robertson K., Hum.
Mol. Genetics, 2002, 11, 18, 2091-2102
Okano M. et al., Cell,1999, 99, 245-257
Robertson K. et al., Nucleic Acids Research, 1999, 27, 11, 2291-2298
Robertson K., Oncogene, 2002, 21, 5361-5379
Tao Q. et al.. American Journal of Pathology, 1999, 155, 2, 619-625
Frommer M. et al., PNAS, 1992, 89, 1827-1831
Herman et al., PNAS, 1996, 93. 9821-9826
Scott Hansen R. et al., PNAS, 1999, 96, 25, 14412-14417
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