Whole-genome methylation scan in ICF syndrome: hypomethylation of non-satellite DNA repeats D4Z4 and NBL2

Abstract

The ICF (immunodeficiency, centromeric instability and facial abnormalities) syndrome is a rare recessive disease characterized by immunodeficiency, extraordinary instability of certain heterochromatin regions and mutations in the gene encoding DNA methyltransferase 3B. In this syndrome, chromosomes 1 and 16 are demethylated in their centromere-adjacent (juxtacentromeric) heterochromatin, the same regions that are highly unstable in mitogen-treated ICF lymphocytes and B cell lines. We investigated the methylation abnormalities in CpG islands of B cell lines from four ICF patients and their unaffected parents. Genomic DNA digested with a CpG methylation-sensitive restriction enzyme was subjected to two-dimensional gel electrophoresis. Most of the restriction fragments were identical in the digests from the patients and controls, indicating that the methylation abnormality in ICF is restricted to a small portion of the genome. However, ICF DNA digests prominently displayed multicopy fragments absent in controls. We cloned and sequenced several of the affected DNA fragments and found that the non-satellite repeats D4Z4 and NBL2 were strongly hypomethylated in all four patients, as compared with their unaffected parents. The high degree of methylation of D4Z4 that we observed in normal cells may be related to the postulated role of this DNA repeat in position effect variegation in facio- scapulohumeral muscular dystrophy and might also pertain to abnormal gene expression in ICF. In addition, our finding of consistent hypomethylation and overexpression of NBL2 repeats in ICF samples suggests derangement of methylation-regulated expression of this sequence in the ICF syndrome.

Global analysis of DNA methylation and transcription of human repetitive sequences.

“…28,32 Consistent with D4Z4 demethylation observed in naturally occuring DNMT3b mutants, we also found hypomethylation of this repeat in DKO. 53 The significant reduced rDNA promoter methylation observed in DKO cells confirms previous finding that both DNMT1 and DNMT3b are essential to maintain methylation profile of this multiple-copy locus. 46 In contrast, we could not detect a strong hypomethylation of Alu repeats in DKO cells compared to the parental cell line as previously described.…”

Epigenetic regulation of telomeres in human cancer

“…The CpG methylation status of the subtelomeric repeat D4Z4 (chromosome 4q35) and the pericentric repeat NBL2 (chromosomes 9, 13, 14 and 21; Kondo et al, 2000) was established after bisulfite modification of the genomic DNA (Herman et al, 1996). Bisulfite genomic sequencing of multiple clones was then carried out as described elsewhere .…”

Role of DNMT3B in the regulation of early neural and neural crest specifiers

“…21,23 Although global levels of DNA methylation are only slightly reduced in ICF patients, 24 pericentromeric satellite repeat sequences on chromosomes 1, 9 and 16 are highly decondensed and hypomethylated. 15,[25][26][27][28][29] Other non-satellite repeat regions 30 and a number of genes on inactive X chromosome in female ICF patients [31][32][33][34][35] are also hypomethylated. However, in-depth genome-wide analyses of the methylation effects of DNMT3B deficiency have not been previously performed.…”