Comparative methylome analysis of ICF patients identifies heterochromatin loci that require ZBTB24, CDCA7 and HELLS for their methylated state

Abstract

Alterations of DNA methylation landscapes and machinery are a hallmark of many human diseases. A prominent case is the ICF syndrome, a rare autosomal recessive immunological/neurological disorder diagnosed by the loss of DNA methylation at (peri)centromeric repeats and its associated chromosomal instability. It is caused by mutations in the de novo DNA methyltransferase DNMT3B in about half of the patients (ICF1). In the remainder, the striking identification of mutations in factors devoid of DNA methyltransferase activity, ZBTB24 (ICF2), CDCA7 (ICF3) or HELLS (ICF4), raised key questions about common or distinguishing DNA methylation alterations downstream of these mutations and hence, about the functional link between the four factors. Here, we established the first comparative methylation profiling in ICF patients with all four genotypes and we provide evidence that, despite unifying hypomethylation of pericentromeric repeats and a few common loci, methylation profiling clearly distinguished ICF1 from ICF2, 3 and 4 patients. Using available genomic and epigenomic annotations to characterize regions prone to loss of DNA methylation downstream of ICF mutations, we found that ZBTB24, CDCA7 and HELLS mutations affect CpG-poor regions with heterochromatin features. Among these, we identified clusters of coding and non-coding genes mostly expressed in a monoallelic manner and implicated in neuronal development, consistent with the clinical spectrum of these patients' subgroups. Hence, beyond providing blood-based biomarkers of dysfunction of ICF factors, our comparative study unveiled new players to consider at certain heterochromatin regions of the human genome.

Genetics meets DNA methylation in rare diseases

“…ZBTB24, CDCA7 and HELLS could indeed be involved in the recruitment of DNMT3B at pericentromeric repeats, whose hypomethylation is a hallmark of all ICF patients. In contrast, DNA hypomethylation at germ line gene promoters and subtelomeric repeats is a signature of ICF1, and is not altered in ICF2‐4 patients, suggesting that ZBTB24, CDCA7 and HELLS are not required to direct DNMT3B to these regions. In fact, the transcriptional repressor E2F6 was shown to be required for DNMT3B recruitment at a subset of germ line gene promoters and maintenance of their methylated state although no mutation in this factor could be found in ICF patients …”

“…Characterization of DNAme defects in ICF patients shows that, in addition to Sat‐2 and Sat‐3 repeats, reduced DNAme markedly affects non‐satellite NBL2 (now called SST1) and D4Z4 DNA repeats, subtelomeric regions, CpG islands of the inactive X chromosome in females, and a subset of germ line gene promoters . Hypomethylation of these germ line genes, which are maintained silent in all normal somatic cells, represents a specific pathological signature of DNMT3B dysfunction that could serve as a powerful biomarker for diagnosis, although the extent of its contribution to disease phenotype is unclear .…”

“…In fact, this syndrome, which groups patients under the same diagnosis, is molecularly and phenotypically heterogeneous: a subset of patients without mutation in DNMT3B exhibit additional hypomethylation at centromeric alpha-satellite repeats and a higher incidence of ID. 36 patients, 42,43 suggesting that ZBTB24, CDCA7 and HELLS are not required to direct DNMT3B to these regions. In fact, the transcriptional repressor E2F6 was shown to be required for DNMT3B recruitment at a subset of germ line gene promoters and maintenance of their methylated state although no mutation in this factor could be found in ICF patients.…”

Genome-wide identification of genes regulating DNA methylation using genetic anchors for causal inference

“…Mutations in CDCA7 have been shown to cause ICF, a rare primary immunodeficiency characterized by epigenetic abnormalities (Thijssen et al, 2015). Previous research showed that CDCA7-mutated ICF patients show decreased DNA methylation levels at pericentromeric repeats and heterochromatin regions and, similarly, CDCA7 depletion in mouse embryonic fibroblasts leads to decreased DNA methylation at centromeric repeats (Thijssen et al, 2015;Velasco et al, 2018). In line with this we found that increased expression of CDCA7 is associated with increased methylation levels at 79 CpG-sites that were distributed across chromosomes (figure 4a) and were enriched in low-activity regions (e.g.…”

Zbtb24 binding protects promoter activity by antagonizing DNA methylation in mESCs

“…Recessive mutations in ZBTB24 cause Immunodeficiency, Centromeric instability, Facial anomalies (ICF) syndrome (de Greef et al 2011;Cerbone et al 2012;Nitta et al 2013), a disorder characterized by hypomethylation of repetitive DNA . Aberrant DNA methylation has been reported in ICF patients carrying ZBTB24 nonsense mutations (Velasco et al 2018), upon shRNA-mediated ZBTB24 depletion in HCT116 cells, a human colon cancer cell line (Thompson et al 2018), and in a zebrafish zbtb24 knock out model (Rajshekar et al 2018). A combined function of ZBTB24 and DNMT3B has been suggested for mediating some gene body methylation in HCT116 cells (Thompson et al 2018), but the mechanisms by which ZBTB24 influences DNA methylation at other genomic regions remain unclear.…”