Heterochromatic Genes Undergo Epigenetic Changes and Escape Silencing in Immunodeficiency, Centromeric Instability, Facial Anomalies (ICF) Syndrome

Abstract

Immunodeficiency, Centromeric Instability, Facial Anomalies (ICF) syndrome is a rare autosomal recessive disorder that is characterized by a marked immunodeficiency, severe hypomethylation of the classical satellites 2 and 3 associated with disruption of constitutive heterochromatin, and facial anomalies. Sixty percent of ICF patients have mutations in the DNMT3B (DNA methyltransferase 3B) gene, encoding a de novo DNA methyltransferase.In the present study, we have shown that, in ICF lymphoblasts and peripheral blood, juxtacentromeric heterochromatic genes undergo dramatic changes in DNA methylation, indicating that they are bona fide targets of the DNMT3B protein. DNA methylation in heterochromatic genes dropped from about 80% in normal cells to approximately 30% in ICF cells. Hypomethylation was observed in five ICF patients and was associated with activation of these silent genes. Although DNA hypomethylation occurred in all the analyzed heterochromatic genes and in all the ICF patients, gene expression was restricted to some genes, every patient having his own group of activated genes. Histone modifications were preserved in ICF patients. Heterochromatic genes were associated with histone modifications that are typical of inactive chromatin: they had low acetylation on H3 and H4 histones and were slightly enriched in H3K9Me3, both in ICF and controls. This was also the case for those heterochromatic genes that escaped silencing. This finding suggests that gene activation was not generalized to all the cells, but rather was restricted to a clonal cell population that may contribute to the phenotypic variability observed in ICF syndrome. A slight increase in H3K27 monomethylation was observed both in heterochromatin and active euchromatin in ICF patients; however, no correlation between this modification and activation of heterochromatic genes was found.

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

“…12 The aberrant activation of these genes was variable, each patient having his own group of expressed genes. Herein, we have provided evidence that at least two of these C-heterochromatic genes replicated earlier in the S-phase in ICF cell DNA hypomethylation affects DNA replication E Lana et al lines.…”

“…12 Briefly, ICF3 is a male patient carrying the K770E homozygous mutation. ICF4 is a female patient carrying the D817G homozygous mutation.…”

“…Both genes are highly expressed in peripheral blood and lymphoblast cell lines and their promoters are associated with histone marks that are specific of an open chromatin. 12 In addition to these four juxtacentromeric genes, we analyzed CAV2, a euchromatic gene located in 7q31.1 that was silent in all lymphoblast cells. To assess the replication timing, we immunoprecipitated BrdU-labeled DNA and quantified the bound fraction by real-time PCR (Supplementary Table 2).…”

“…12 DNA hypomethylation was found in all the analyzed C-heterochromatic genes and in all ICF cell lines, whereas expression was restricted to some genes, every patient having his own group of activated genes. In this work, we asked whether (i) similar to genes located in F-heterochromatin, genes located in C-heterochromatin change their replication timing and (ii) if this change is responsible for aberrant transcription.…”

“…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.…”