Escape from gene silencing in ICF syndrome: evidence for advanced replication time as a major determinant

Abstract

Chromosomal abnormalities associated with hypomethylation of classical satellite regions are characteristic for the ICF immunodeficiency syndrome. We, as well as others, have found that these effects derive from mutations in the DNMT3B DNA methyltransferase gene. Here we examine further the molecular phenotype of ICF cells and report several examples of extensive hypomethylation that are associated with advanced replication time, nuclease hypersensitivity and a variable escape from silencing for genes on the inactive X and Y chromosomes. Our analysis suggests that all genes on the inactive X chromosome may be extremely hypomethylated at their 5' CpG islands. Our studies of G6PD in one ICF female and SYBL1 in another ICF female provide the first examples of abnormal escape from X chromosome inactivation in untransformed human fibroblasts. XIST RNA localization is normal in these cells, arguing against an independent silencing role for this RNA in somatic cells. SYBL1 silencing is also disrupted on the Y chromosome in ICF male cells. Increased chromatin sensitivity to nuclease was found at all hypomethylated promoters examined, including those of silenced genes. The persistence of inactivation in these latter cases appears to depend critically on delayed replication of DNA because escape from silencing was only seen when replication was advanced to an active X-like pattern.

DNA replication is altered in Immunodeficiency Centromeric instability Facial anomalies (ICF) cells carrying DNMT3B mutations

“…In Drosophila, late-replicating sequences appear at the mid-blastula transition, when satellite sequences acquire heterochromatic features. 4 Similar data are not available in humans; however, this study and others 9,11,17 have shown that the DNMT3B activity is required to preserve late-replicating sequences. Since this protein starts to be expressed by 6-day epiblasts in the mouse, 25 it may contribute to settling late replication during development.…”

“…Our work is consistent with previous results that showed that other late-replicating sequences, namely satellite 2, telomeric sequences, and genes located in F-heterochromatin, replicated earlier in the S-phase in ICF cell lines. 9,11,17 However, in contrast to genes located in F-heterochromatin, 11 altered replication timing was not always correlated with activation of the repressed genes: C-hetero- chromatic genes replicated earlier in all the analyzed ICF cell lines, but escaped silencing only in some of them. 12 A correlation between loss of DNA methylation and changes in replication timing has also been reported in imprinted genes.…”

“…Non-coding repetitive sequences (ie, satellites 2 and 3, subtelomeric sequences, and Alu sequences) [8][9][10] and genes located in constitutive and facultative heterochromatin (hereafter named C-heterochromatin and F-heterochromatin, respectively) are hypomethylated. 11,12 This loss of DNA methylation is associated with chromatin decondensation and chromosome instability. ICF-specific changes in RNA levels of genes critical for immune function, development, and neurogenesis were identified through the analysis of global expression profiles.…”

“…[13][14][15][16] Finally, various late-replicating sequences (ie, satellite 2, telomeric repeats, and genes in F-heterochromatin) replicate earlier in ICF than in control cells. 9,11,17 In female ICF cells, genes located in F-heterochromatin lose DNA methylation and escape silencing, and this activation process is associated with a change in their replication timing from late to an earlier stage in the Sphase. 11 It was therefore suggested that the change in the replication timing is responsible for gene activation.…”

“…9,11,17 In female ICF cells, genes located in F-heterochromatin lose DNA methylation and escape silencing, and this activation process is associated with a change in their replication timing from late to an earlier stage in the Sphase. 11 It was therefore suggested that the change in the replication timing is responsible for gene activation.…”