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

2 Overview Background Experiments and results Conclusions Questions

3 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.

4 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

5 Enzymes Robertson K., Oncogene, 2002

6 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

7 Role of DNA methylation Embryonic development X chromosome inactivation in females Genomic imprinting Chromatin remodeling Tumorigenesis

8 ICF syndrome rare autosomal recessive disease defect in DNMT3B gene Immune deficiency Centromere instability Facial anomalies

9 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

10 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

11 Objectives: To gain a better understanding of the types of DNA sequences, whose methylation is established and/or maintained by DNMT3B

12 Approach Analysis of number of viral and cellular genes in 2 EBV-established well-characterized ICF cell lines Mutation in DNMT3B ICF 1Male homozygous V726G ICF 2Female heterozygous A603T intron 22 G-A  insertion of STP Cell line

13 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

14 Methods Bisulfite genomic sequencing Methylation specific PCR Semiquantative RT-PCR

15 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

16 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

17 EBV genome Tao Q., Huang H., Geiman T., Yen Lim C., Fu L., Qiu G. and Robertson K., Hum. Mol. Genetics, 2002

18 MSP analysis of Cp and Wp Wp - normally undergoes de novo methylation

19 Expression from Cp and Wp

20 EBV genome

21 BGS analysis of Rp

22 Semiquantative RT-PCR of Zp and Rp

23 RT-PCR analysis of BHRF1 and BLLF1

24 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?

25 Scheme of the MAGE-A1 and LAGE-1/2 cellular gene promoters

26 BGS analysis of MAGE-A1 and LAGE-1/2

27 Quantification of BGS data

28 Expression from MAGE-A1 and LAGE-1/2

29 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

30 Schematic of SCP-1 gene promoter

31 MSP methylation analysis of ICF and normal LCLs for SCP-1

32 SCP-1 expression monitored by RT-PCR

33 Expression of DNA methyltransferases in ICF cells

34 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

35 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?

36 Thank you!

37 References: 1.Tao Q., Huang H., Geiman T., Yen Lim C., Fu L., Qiu G and Robertson K., Hum. Mol. Genetics, 2002, 11, 18, Okano M. et al., Cell,1999, 99, Robertson K. et al., Nucleic Acids Research, 1999, 27, 11, Robertson K., Oncogene, 2002, 21, Tao Q. et al.. American Journal of Pathology, 1999, 155, 2, Frommer M. et al., PNAS, 1992, 89, Herman et al., PNAS, 1996, Scott Hansen R. et al., PNAS, 1999, 96, 25,