Molecular pathogenesis of ICF syndrome

Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is characterized by frequent appearance of multiradial chromosomes, which are distinctive chromosome fusions that occur at hypomethylated pericentromeric regions comprising repetitive sequences, in activated lymphocytes. The syndrome is caused by mutations in DNMT3B, ZBTB24, CDCA7, or HELLS. Dr. Motoko Unoki, Medical Institute of Bioregulation, Kyushu University, Japan, was involved in the team to identify the CDCA7 and HELLS genes as causative genes of ICF syndrome. Recent studies including Unoki's studies suggested that de novo DNA methylation is likely defective in patients with ICF syndrome harboring mutations in DNMT3B, whereas accumulating evidence suggests that replication-uncoupled maintenance DNA methylation of late-replicating regions is impaired in patients with ICF syndrome harboring mutations in ZBTB24, CDCA7, or HELLS. ZBTB24 is a transcriptional activator of CDCA7, and CDCA7 and HELLS compose a chromatin remodeling complex and are involved in the maintenance DNA methylation through an interaction with UHRF1 in a feed-forward manner. The latest study by Unoki possibly provided the missing link between DNA hypomethylation and the formation of the abnormal chromosomes; it could occur via aberrant transcription from the hypomethylated regions, followed by pathological R-loop formation. The homologous-recombination dominant condition caused by a defect in non-homologous end joining observed in several types of ICF syndrome could facilitate the formation of multiradial chromosomes.

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CDCA7 and HELLS suppress DNA:RNA hybrid-associated DNA damage at pericentromeric repeats

Scientific Reports ◽

10.1038/s41598-020-74636-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Motoko Unoki ◽

Jafar Sharif ◽

Yuichiro Saito ◽

Guillaume Velasco ◽

Claire Francastel ◽

...

Keyword(s):

Dna Damage ◽

Autosomal Recessive ◽

Ectopic Expression ◽

Autosomal Recessive Disorder ◽

End Joining ◽

Icf Syndrome ◽

Centromeric Instability ◽

Non Homologous End Joining ◽

Genomic Regions ◽

Mutant Cells

Abstract Immunodeficiency, centromeric instability, facial anomalies (ICF) syndrome is a rare autosomal recessive disorder that is caused by mutations in either DNMT3B, ZBTB24, CDCA7, HELLS, or yet unidentified gene(s). Previously, we reported that the CDCA7/HELLS chromatin remodeling complex facilitates non-homologous end-joining. Here, we show that the same complex is required for the accumulation of proteins on nascent DNA, including the DNMT1/UHRF1 maintenance DNA methylation complex as well as proteins involved in the resolution or prevention of R-loops composed of DNA:RNA hybrids and ssDNA. Consistent with the hypomethylation state of pericentromeric repeats, the transcription and formation of aberrant DNA:RNA hybrids at the repeats were increased in ICF mutant cells. Furthermore, the ectopic expression of RNASEH1 reduced the accumulation of DNA damage at a broad range of genomic regions including pericentromeric repeats in these cells. Hence, we propose that hypomethylation due to inefficient DNMT1/UHRF1 recruitment at pericentromeric repeats by defects in the CDCA7/HELLS complex could induce pericentromeric instability, which may explain a part of the molecular pathogenesis of ICF syndrome.

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C016 Expression analysis of proteins involved in the Non Homologous End Joining DNA repair mechanism, in the bone marrow of adult de novo myelodysplastic syndromes

Leukemia Research ◽

10.1016/s0145-2126(09)70054-2 ◽

2009 ◽

Vol 33 ◽

pp. S40-S41

Author(s):

P. Economopoulou ◽

V. Pappa ◽

F. Kontsioti ◽

S. Papageorgiou ◽

E. Liakata ◽

...

Keyword(s):

Bone Marrow ◽

Dna Repair ◽

Expression Analysis ◽

Myelodysplastic Syndromes ◽

De Novo ◽

Repair Mechanism ◽

End Joining ◽

Non Homologous End Joining

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Persistent epigenetic memory impedes rescue of the telomeric phenotype in human ICF iPSCs following DNMT3B correction

eLife ◽

10.7554/elife.47859 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 3

Author(s):

Shir Toubiana ◽

Miriam Gagliardi ◽

Mariarosaria Papa ◽

Roberta Manco ◽

Maty Tzukerman ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

De Novo ◽

Pharmacological Intervention ◽

Icf Syndrome ◽

Genome Wide ◽

Mammalian Genomes ◽

Induced Pluripotent ◽

Methylation Patterns

DNA methyltransferase 3B (DNMT3B) is the major DNMT that methylates mammalian genomes during early development. Mutations in human DNMT3B disrupt genome-wide DNA methylation patterns and result in ICF syndrome type 1 (ICF1). To study whether normal DNA methylation patterns may be restored in ICF1 cells, we corrected DNMT3B mutations in induced pluripotent stem cells from ICF1 patients. Focusing on repetitive regions, we show that in contrast to pericentromeric repeats, which reacquire normal methylation, the majority of subtelomeres acquire only partial DNA methylation and, accordingly, the ICF1 telomeric phenotype persists. Subtelomeres resistant to de novo methylation were characterized by abnormally high H3K4 trimethylation (H3K4me3), and short-term reduction of H3K4me3 by pharmacological intervention partially restored subtelomeric DNA methylation. These findings demonstrate that the abnormal epigenetic landscape established in ICF1 cells restricts the recruitment of DNMT3B, and suggest that rescue of epigenetic diseases with genome-wide disruptions will demand further manipulation beyond mutation correction.

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Transcriptional overlap links DNA hypomethylation with DNA hypermethylation at adjacent promoters in cancer

10.1101/2021.05.21.445142 ◽

2021 ◽

Author(s):

Jean S Fain ◽

Axelle Loriot ◽

Anna Diacofotaki ◽

Aurelie Van Tongelen ◽

Charles De Smet

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

De Novo ◽

Tumor Development ◽

Epigenetic Mark ◽

Dna Hypomethylation ◽

Dna Hypermethylation ◽

Bioinformatics Analyses ◽

Genomic Regions ◽

Methylation Patterns

DNA methylation is an epigenetic mark associated with gene repression. It is now well established that tumor development involves alterations in DNA methylation patterns, which include both gains (hypermethylation) and losses (hypomethylation) of methylation marks in different genomic regions. The mechanisms underlying these two opposite, yet co-existing, alterations in tumors remain unclear. While studying the human MAGEA6/GABRA3 gene locus, we observed that DNA hypomethylation in tumor cells can lead to the activation of a long transcript (CT-GABRA3) that overlaps downstream promoters (GABRQ and GABRA3) and triggers their hypermethylation. Overlapped promoters displayed increases in H3K36me3, a histone mark known to be deposited during progression of the transcription machinery and to stimulate de novo DNA methylation. Consistent with such a processive mechanism, increases in H3K36me3 and DNA methylation were observed over the entire region covered by the CT-GABRA3 overlapping transcript. Importantly, experimental induction of CT-GABRA3 by depletion of DNMT1 DNA methyltransferase, resulted in a similar pattern of increased DNA methylation in the MAGEA6/GABRA3 locus. Bioinformatics analyses in lung cancer datasets identified other genomic loci displaying this process of coupled DNA hypo- and hypermethylation. In several of these loci, DNA hypermethylation affected tumor suppressor genes, e.g. RERG and PTPRO. Together, our work reveals that focal DNA hypomethylation in tumors can indirectly contribute to hypermethylation of nearby promoters through activation of overlapping transcription, and establishes therefore an unsuspected connection between these two opposite epigenetic alterations.

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Dppa2/4 Counteract De Novo Methylation to Establish a Permissive Epigenome for Development

10.1101/2020.03.11.987701 ◽

2020 ◽

Author(s):

Kristjan H. Gretarsson ◽

Jamie A. Hackett

Keyword(s):

Dna Methylation ◽

De Novo ◽

Developmental Competence ◽

Epigenetic Memory ◽

Mammalian Development ◽

Dna Hypomethylation ◽

Lineage Specification ◽

Regulatory Pathways ◽

Genome Wide ◽

Early Mammalian Development

ABSTRACTEarly mammalian development entails genome-wide epigenome remodeling, including DNA methylation erasure and reacquisition, which facilitates developmental competence. To uncover the mechanisms that orchestrate DNA methylation (DNAme) dynamics, we coupled a single-cell ratiometric DNAme reporter with unbiased CRISPR screening in ESC. We identify key genes and regulatory pathways that drive global DNA hypomethylation, and characterise roles for Cop1 and Dusp6. We also identify Dppa2 and Dppa4 as essential safeguards of focal epigenetic states. In their absence, developmental genes and evolutionary-young LINE1 elements, which DPPA2 specifically binds, lose H3K4me3 and gain ectopic de novo DNA methylation in pluripotent cells. Consequently, lineage-associated genes (and LINE1) acquire a repressive epigenetic memory, which renders them incompetent for activation during future lineage-specification. Dppa2/4 thereby sculpt the pluripotent epigenome by facilitating H3K4me3 and bivalency to counteract de novo methylation; a function co-opted by evolutionary young LINE1 to evade epigenetic decommissioning.

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Sequence and Structure Characteristics of 22 Deletion Breakpoints in Intron 44 of the DMD Gene Based on Long-Read Sequencing

Frontiers in Genetics ◽

10.3389/fgene.2021.638220 ◽

2021 ◽

Vol 12 ◽

Author(s):

Chang Geng ◽

Yuanren Tong ◽

Siwen Zhang ◽

Chao Ling ◽

Xin Wu ◽

...

Keyword(s):

Becker Muscular Dystrophy ◽

Repetitive Elements ◽

Loop Formation ◽

End Joining ◽

Origin Firing ◽

Large Deletions ◽

Long Read ◽

Non Homologous End Joining ◽

Dmd Gene ◽

Palindromic Sequences

Purpose: Exon deletions make up to 80% of mutations in the DMD gene, which cause Duchenne and Becker muscular dystrophy. Exon 45-55 regions were reported as deletion hotspots and intron 44 harbored more than 25% of deletion start points. We aimed to investigate the fine structures of breakpoints in intron 44 to find potential mechanisms of large deletions in intron 44.Methods: Twenty-two dystrophinopathy patients whose deletion started in intron 44 were sequenced using long-read sequencing of a DMD gene capture panel. Sequence homology, palindromic sequences, and polypyrimidine sequences were searched at the breakpoint junctions. RepeatMasker was used to analyze repetitive elements and Mfold was applied to predict secondary DNA structure.Results: With a designed DMD capture panel, 22 samples achieved 2.25 gigabases and 1.28 million reads on average. Average depth was 308× and 99.98% bases were covered at least 1×. The deletion breakpoints in intron 44 were scattered and no breakpoints clustered in any region less than 500 bp. A total of 72.7% of breakpoints located in distal 100 kb of intron 44 and more repetitive elements were found in this region. Microhomologies of 0–1 bp were found in 36.4% (8/22) of patients, which corresponded with non-homologous end-joining. Microhomologies of 2–20 bp were found in 59.1% (13/22) of patients, which corresponded with microhomology-mediated end-joining. Moreover, a 7 bp insertion was found in one patient, which might be evidence of aberrant replication origin firing. Palindromic sequences, polypyrimidine sequences, and small hairpin loops were found near several breakpoint junctions. No evidence of large hairpin loop formation in deletion root sequences was observed.Conclusion: This study was the first to explore possible mechanisms underlying exon deletions starting from intron 44 of the DMD gene based on long-read sequencing. Diverse mechanisms might be associated with deletions in the DMD gene.

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Transcriptional overlap links DNA hypomethylation with DNA hypermethylation at adjacent promoters in cancer

Scientific Reports ◽

10.1038/s41598-021-96844-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jean S. Fain ◽

Axelle Loriot ◽

Anna Diacofotaki ◽

Aurélie Van Tongelen ◽

Charles De Smet

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

De Novo ◽

Tumor Development ◽

Transcriptional Elongation ◽

Dna Hypomethylation ◽

Dna Hypermethylation ◽

Bioinformatics Analyses ◽

Genomic Regions ◽

Methylation Patterns

AbstractTumor development involves alterations in DNA methylation patterns, which include both gains (hypermethylation) and losses (hypomethylation) in different genomic regions. The mechanisms underlying these two opposite, yet co-existing, alterations in tumors remain unclear. While studying the human MAGEA6/GABRA3 gene locus, we observed that DNA hypomethylation in tumor cells can lead to the activation of a long transcript (CT-GABRA3) that overlaps downstream promoters (GABRQ and GABRA3) and triggers their hypermethylation. Overlapped promoters displayed increases in H3K36me3, a histone mark deposited during transcriptional elongation and known to stimulate de novo DNA methylation. Consistent with such a processive mechanism, increases in H3K36me3 and DNA methylation were observed over the entire region covered by the CT-GABRA3 overlapping transcript. Importantly, experimental induction of CT-GABRA3 by depletion of DNMT1 DNA methyltransferase, resulted in a similar pattern of regional DNA hypermethylation. Bioinformatics analyses in lung cancer datasets identified other genomic loci displaying this process of coupled DNA hypo/hypermethylation, and some of these included tumor suppressor genes, e.g. RERG and PTPRO. Together, our work reveals that focal DNA hypomethylation in tumors can indirectly contribute to hypermethylation of nearby promoters through activation of overlapping transcription, and establishes therefore an unsuspected connection between these two opposite epigenetic alterations.

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Loss of ZBTB24 impairs nonhomologous end-joining and class-switch recombination in patients with ICF syndrome

Journal of Experimental Medicine ◽

10.1084/jem.20191688 ◽

2020 ◽

Vol 217 (11) ◽

Cited By ~ 2

Author(s):

Angela Helfricht ◽

Peter E. Thijssen ◽

Magdalena B. Rother ◽

Rashmi G. Shah ◽

Likun Du ◽

...

Keyword(s):

Autosomal Recessive ◽

Class Switch Recombination ◽

Nonhomologous End Joining ◽

Dna Breaks ◽

End Joining ◽

Immunoglobulin Production ◽

Icf Syndrome ◽

Class Switch ◽

Centromeric Instability ◽

Gene Defects

The autosomal recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a genetically heterogeneous disorder. Despite the identification of the underlying gene defects, it is unclear how mutations in any of the four known ICF genes cause a primary immunodeficiency. Here we demonstrate that loss of ZBTB24 in B cells from mice and ICF2 patients affects nonhomologous end-joining (NHEJ) during immunoglobulin class-switch recombination and consequently impairs immunoglobulin production and isotype balance. Mechanistically, we found that ZBTB24 associates with poly(ADP-ribose) polymerase 1 (PARP1) and stimulates its auto-poly(ADP-ribosyl)ation. The zinc-finger in ZBTB24 binds PARP1-associated poly(ADP-ribose) chains and mediates the PARP1-dependent recruitment of ZBTB24 to DNA breaks. Moreover, through its association with poly(ADP-ribose) chains, ZBTB24 protects them from degradation by poly(ADP-ribose) glycohydrolase (PARG). This facilitates the poly(ADP-ribose)-dependent assembly of the LIG4/XRCC4 complex at DNA breaks, thereby promoting error-free NHEJ. Thus, we uncover ZBTB24 as a regulator of PARP1-dependent NHEJ and class-switch recombination, providing a molecular basis for the immunodeficiency in ICF2 syndrome.

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