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

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 1 Male homozygous V726G

ICF 2 Female 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, 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