Attwood, Yung & Richardson (2002) DNA methylation review

Q1. What percentage of cytosines are methylated in the human genome, and in what sequence context does methylcytosine almost exclusively occur?

~4% of cytosines are methylated; almost exclusively at 5'-cytosine-guanine-3' (CG dinucleotides).

Q2. What is a CpG island, and what percentage of the genome do they constitute?

CG-rich regions (CG/GC ratio ~1 vs. 0.2 for bulk DNA) with higher CpG frequency than expected by chance; constitute 1-2% of the total genome.

Q3. What is the methylation status of CpG islands vs. non-CpG island CG dinucleotides in normal cells?

CpG islands are largely unmethylated; non-CpG island CG dinucleotides are ~70-80% methylated.

Q4. List the three mammalian DNA methyltransferases (DNMTs) and their primary function.

Dnmt1 (maintenance methylation), Dnmt3a (de novo methylation), Dnmt3b (de novo methylation, especially of satellite repeats).

Q5. What is the catalytic preference of Dnmt1, and what is the consequence of homozygous Dnmt1 deletion in mice?

5-30-fold preference for hemimethylated DNA over unmethylated DNA; homozygous deletion causes embryonic lethality prior to mid-gestation.

Q6. What evidence suggested the existence of de novo methyltransferases despite Dnmt1 knockout?

Dnmt1-deficient ES cells are viable, maintain low DNA methylation levels, and still methylate newly integrated proviral DNA at normal rates.

Q7. What congenital disease is associated with mutations in DNMT3B, and what are its key features?

ICF syndrome (immunodeficiency, centromere instability, facial anomalies); characterized by hypomethylation of juxtacentromeric satellite repeats on chromosomes 1, 9, and 16.

Q8. Name the four MBD family members that bind methylated DNA, and which one is mutated in Rett syndrome?

MeCP2, MBD1, MBD2, MBD3, MBD4; MeCP2 mutations cause Rett syndrome.

Q9. How does MeCP2 repress transcription?

Binds methylated CpGs via its methyl-CpG binding domain, then recruits Sin3A histone deacetylase complex (HDAC1/HDAC2) → histone deacetylation → chromatin condensation → transcriptional repression.

Q10. What are the two general mechanisms by which DNA methylation suppresses gene transcription?

1) Direct inhibition of transcription factor binding to methylated recognition sequences; 2) Indirect via methylcytosine-binding proteins recruiting histone deacetylase complexes, causing chromatin condensation.

Q11. What is the effect of 5-azacytidine treatment on DNA methyltransferases?

5-azacytidine incorporated into DNA forms covalent bonds with DNMTs, trapping them and causing loss of enzyme activity (DNMT depletion) → DNA hypomethylation.

Q12. What was the first evidence that DNA methylation is involved in X chromosome inactivation?

Treatment with 5-azacytidine reactivated the HGPRT gene on the inactive X chromosome (Mohandas et al., 1981).

Q13. What happens to DNA methylation patterns in cancer cells?

Simultaneous global hypomethylation (of repetitive sequences and some gene bodies) and focal CpG island hypermethylation (at tumor suppressor gene promoters).

Q14. Name three genes that become hypermethylated and silenced in cancers, and their functions.

RB1 (tumor suppressor/retinoblastoma), VHL (von Hippel-Lindau/renal carcinoma), MLH1 (DNA mismatch repair/colorectal cancer).

Q15. How does age-related CpG island hypermethylation potentially increase cancer risk?

Progressive age-related hypermethylation of CpG islands (e.g., estrogen receptor gene in colon mucosa) can silence genes in normal tissue, creating a field defect that predisposes to malignancy.

Q16. What is the difference between passive and active DNA demethylation?

Passive = occurs during DNA replication when maintenance methylation is blocked; Active = independent of replication, involves demethylase enzymes (e.g., 5-methylcytosine DNA glycosylase).

Q17. Which DNA methyltransferase is dispensable for viability based on knockout studies?

Dnmt2 – knockout ES cells are normal, have no detectable methyltransferase activity, and maintain normal methylation of viral sequences.

Q18. How does DNA methylation regulate T helper cell differentiation regarding the IFNγ gene?

IFNγ promoter is methylated in naïve T cells and Th2 cells (non-expressing); demethylation of specific CG dinucleotides occurs in Th1 cells (expressing); 5-azacytidine treatment of Th2 clones induces IFNγ production.

Q19. What is the connection between Dnmt1, proliferating cell nuclear antigen (PCNA), and DNA replication?

Dnmt1 associates with PCNA at replication foci during S phase, targeting maintenance methylation to newly synthesized hemimethylated DNA.

Q20. What do the methylcytosine-binding proteins MBD2 and MBD4 do besides binding methylated DNA?

MBD2 is a transcriptional repressor in MeCP1, Sin3A, and Mi-2/NuRD complexes; MBD4 has G/T mismatch and 5-methylcytosine DNA glycosylase activity (DNA repair/demethylatio