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The imprinted Dlk1-Dio3 domain comprises the developmental genes Dlk1 and Rtl1, which are silenced on the maternal chromosome in different cell types. On this parental chromosome, the domain's imprinting control region activates a polycistron that produces the lncRNA Meg3 and many miRNAs (Mirg) and C/D-box snoRNAs (Rian). Although Meg3 lncRNA is nuclear and associates with the maternal chromosome, it is unknown whether it controls gene repression in cis. We created mouse embryonic stem cells (mESCs) that carry an ectopic poly(A) signal, reducing RNA levels along the polycistron, and generated Rian-/- mESCs as well. Upon ESC differentiation, we found that Meg3 lncRNA (but not Rian) is required for Dlk1 repression on the maternal chromosome. Biallelic Meg3 expression acquired through CRISPR-mediated demethylation of the paternal Meg3 promoter led to biallelic Dlk1 repression, and to loss of Rtl1 expression. lncRNA expression also correlated with DNA hypomethylation and CTCF binding at the 5'-side of Meg3. Using Capture Hi-C, we found that this creates a Topologically Associating Domain (TAD) organization that brings Meg3 close to Dlk1 on the maternal chromosome. The requirement of Meg3 for gene repression and TAD structure may explain how aberrant MEG3 expression at the human DLK1-DIO3 locus associates with imprinting disorders., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cystic fibrosis (CF) is a chronic genetic disease that mainly affects the respiratory and gastrointestinal systems. No curative treatments are available, but the follow-up in specialized centers has greatly improved the patient life expectancy. Robust biomarkers are required to monitor the disease, guide treatments, stratify patients, and provide outcome measures in clinical trials. In the present study, we outline a strategy to select putative DNA methylation biomarkers of lung disease severity in cystic fibrosis patients. In the discovery step, we selected seven potential biomarkers using a genome-wide DNA methylation dataset that we generated in nasal epithelial samples from the MethylCF cohort. In the replication step, we assessed the same biomarkers using sputum cell samples from the MethylBiomark cohort. Of interest, DNA methylation at the cg11702988 site ( ATP11A gene) positively correlated with lung function and BMI, and negatively correlated with lung disease severity, P. aeruginosa chronic infection, and the number of exacerbations. These results were replicated in prospective sputum samples collected at four time points within an 18-month period and longitudinally. To conclude, (i) we identified a DNA methylation biomarker that correlates with CF severity, (ii) we provided a method to easily assess this biomarker, and (iii) we carried out the first longitudinal analysis of DNA methylation in CF patients. This new epigenetic biomarker could be used to stratify CF patients in clinical trials.

Twin and sibling studies have shown that lung disease severity is variable among cystic fibrosis (CF) patients and affected to the same extent by genetic and nonheritable factors. Genetic factors have been thoroughly assessed, whereas the molecular mechanisms whereby nonheritable factors contribute to the phenotypic variability of CF patients are still unknown. Epigenetic modifications may represent the missing link between nonheritable factors and phenotypic variation in CF. Herein, we review recent studies showing that DNA methylation is altered in CF and we address three possible factors responsible for these variations: (i) overproduction of reactive oxygen species, (ii) depletion of DNA methylation cofactors and (iii) susceptibility to acute and chronic bacterial infections. Also, we hypothesize that the unique DNA methylation profile of each patient can modulate the phenotype and discuss the interest of implementing integrated genomic, epigenomic and transcriptomic studies to further understand the clinical diversity of CF patients (Graphical Abstract)., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Cystic fibrosis (CF) is a rare genetic disease that affects the respiratory and digestive systems. Lung disease is variable among CF patients and associated with the development of comorbidities and chronic infections. The rate of lung function deterioration depends not only on the type of mutations in CFTR, the disease-causing gene, but also on modifier genes. In the present study, we aimed to identify genes and pathways that (i) contribute to the pathogenesis of cystic fibrosis and (ii) modulate the associated comorbidities. We profiled blood samples in CF patients and healthy controls and analyzed RNA-seq data with Weighted Gene Correlation Network Analysis (WGCNA). Interestingly, lung function, body mass index, the presence of diabetes, and chronic P. aeruginosa infections correlated with four modules of co-expressed genes. Detailed inspection of networks and hub genes pointed to cell adhesion, leukocyte trafficking and production of reactive oxygen species as central mechanisms in lung function decline and cystic fibrosis-related diabetes. Of note, we showed that blood is an informative surrogate tissue to study the contribution of inflammation to lung disease and diabetes in CF patients. Finally, we provided evidence that WGCNA is useful to analyze-omic datasets in rare genetic diseases as patient cohorts are inevitably small., Competing Interests: The authors have declared that no competing interests exist.

Aim: To assess whether DNA methylation levels account for the noninherited phenotypic variations observed among cystic fibrosis (CF) patients., Patients & Methods: Using the 450 K BeadChip, we profiled DNA methylation in nasal epithelial cells collected from 32 CF patients and 16 controls., Results: We detected substantial DNA methylation differences up to 55% (median β change 0.13; IQR: 0.15-0.11) between CF patients and controls. DNA methylation levels differed between mild and severe CF patients and correlated with lung function at 50 CpG sites., Conclusion: In CF samples, dynamic changes of DNA methylation occurred in genes responsible for the integrity of the epithelium and the inflammatory and immune responses, were prominent in transcriptionally active genomic regions and were over-represented in enhancers active in lung tissues. ( Clinicaltrials.gov NCT02884622).

The development of suitable Cystic Fibrosis (CF) models for preclinical bench tests of therapeutic candidates is challenging. Indeed, the validation of molecules to rescue the p.Phe508del-CFTR channel (encoded by the Cystic Fibrosis Transmembrane conductance Regulator gene carrying the p.Phe508del mutation) requires taking into account their overall effects on the epithelium. Suberoylanilide Hydroxamic Acid (SAHA), a histone deacetylase inhibitor (HDACi), was previously shown to be a CFTR corrector via proteostasis modulation in CFTR-deficient immortalized cells. Here, we tested SAHA effects on goblet cell metaplasia using an ex vivo model based on the air-liquid interface (ALI) culture of differentiated airway epithelial cells obtained by nasal scraping from CF patients and healthy controls. Ex vivo epithelium grew successfully in ALI cultures with significant rise in the expression of CFTR and of markers of airway epithelial differentiation compared to monolayer cell culture. SAHA decreased CFTR transcript and protein levels in CF and non-CF epithelia. Whereas SAHA induced lysine hyperacetylation, it did not change histone modifications at the CFTR promoter. SAHA reduced MUC5AC and MUC5B expression and inhibited goblet epithelial cell differentiation. Similar effects were obtained in CF and non-CF epithelia. All the effects were fully reversible within five days from SAHA withdrawal. We conclude that, ex vivo, SAHA modulate the structure of airway epithelia without specific effect on CFTR gene and protein suggesting that HDACi cannot be useful for CF treatment., (Copyright © 2017. Published by Elsevier Ltd.)

Background: Lung disease progression is variable among cystic fibrosis (CF) patients and depends on DNA mutations in the CFTR gene, polymorphic variations in disease modifier genes, and environmental exposure. The contribution of genetic factors has been extensively investigated, whereas the mechanism whereby environmental factors modulate the lung disease is unknown. In this project, we hypothesized that (i) reiterative stress alters the epigenome in CF-affected tissues and (ii) DNA methylation variations at disease modifier genes modulate the lung function in CF patients., Results: We profiled DNA methylation at CFTR , the disease-causing gene, and at 13 lung modifier genes in nasal epithelial cells and whole blood samples from 48 CF patients and 24 healthy controls. CF patients homozygous for the p.Phe508del mutation and ≥18-year-old were stratified according to the lung disease severity. DNA methylation was measured by bisulfite and next-generation sequencing. The DNA methylation profile allowed us to correctly classify 75% of the subjects, thus providing a CF-specific molecular signature. Moreover, in CF patients, DNA methylation at specific genes was highly correlated in the same tissue sample. We suggest that gene methylation in CF cells may be co-regulated by disease-specific trans -factors. Three genes were differentially methylated in CF patients compared with controls and/or in groups of pulmonary severity: HMOX1 and GSTM3 in nasal epithelial samples; HMOX1 and EDNRA in blood samples. The association between pulmonary severity and DNA methylation at EDNRA was confirmed in blood samples from an independent set of CF patients. Also, lower DNA methylation levels at GSTM3 were associated with the GSTM3*B allele, a polymorphic 3-bp deletion that has a protective effect in cystic fibrosis., Conclusions: DNA methylation levels are altered in nasal epithelial and blood cell samples from CF patients. Analysis of CFTR and 13 lung disease modifier genes shows DNA methylation changes of small magnitude: some of them are a consequence of the disease; other changes may result in small expression variations that collectively modulate the lung disease severity.

A number of chronic airway diseases are characterized by high inflammation and unbalanced activation of the immune response, which lead to tissue damage and progressive reduction of the pulmonary function. Because they are exposed to various environmental stimuli, lung cells are prone to epigenomic changes. Many genes responsible for the immune response and inflammation are tightly regulated by DNA methylation, which suggests that alteration of the epigenome in lung cells may have a considerable impact on the penetrance and/or the severity of airway diseases. A major hurdle in clinical epigenomic studies is to gather appropriate biospecimens. Herein, we show that nasal epithelial cells are suitable to analyze DNA methylation in human diseases primarily affecting the lower airway tract.

The genetic mechanisms that regulate CFTR, the gene responsible for cystic fibrosis, have been widely investigated in cultured cells. However, mechanisms responsible for tissue-specific and time-specific expression are not completely elucidated in vivo. Through the survey of public databases, we found that the promoter of CFTR was associated with bivalent chromatin in human embryonic stem (ES) cells. In this work, we analyzed fetal (at different stages of pregnancy) and adult tissues and showed that, in digestive and lung tissues, which expressed CFTR, H3K4me3 was maintained in the promoter. Histone acetylation was high in the promoter and in two intronic enhancers, especially in fetal tissues. In contrast, in blood cells, which did not express CFTR, the bivalent chromatin was resolved (the promoter was labeled by the silencing mark H3K27me3). Cis-regulatory sequences were associated with lowly acetylated histones. We also provide evidence that the tissue-specific expression of CFTR is not regulated by dynamic changes of DNA methylation in the promoter. Overall, this work shows that a balance between activating and repressive histone modifications in the promoter and intronic enhancers results in the fine regulation of CFTR expression during development, thereby ensuring tissue specificity.

ICF syndrome is a rare autosomal recessive disorder that is characterized by Immunodeficiency, Centromeric instability, and Facial anomalies. In all, 60% of ICF patients have mutations in the DNMT3B (DNA methyltransferase 3B) gene, encoding a de novo DNA methyltransferase. In ICF cells, constitutive heterochromatin is hypomethylated and decondensed, metaphase chromosomes undergo rearrangements (mainly involving juxtacentromeric regions), and more than 700 genes are aberrantly expressed. This work shows that DNA replication is also altered in ICF cells: (i) heterochromatic genes replicate earlier in the S-phase; (ii) global replication fork speed is higher; and (iii) S-phase is shorter. These replication defects may result from chromatin changes that modify DNA accessibility to the replication machinery and/or from changes in the expression level of genes involved in DNA replication. This work highlights the interest of using ICF cells as a model to investigate how DNA methylation regulates DNA replication in humans.

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 H3K9Me(3), 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.

We showed earlier that BAGE (B melanoma antigen) loci are hypermethylated in normal tissues and hypomethylated in 98% of human cancers. More recently, we provided evidence that hypomethylation of BAGE loci represents an informative marker for colon cancer detection. In this study, we show that hypomethylation of BAGE loci was an early event that occurred in 43% of colorectal adenomas. Interestingly, hypomethylation of BAGE loci was frequent (50%) in tubulo-villous and villous adenomas, these adenomas having a high probability of being transformed into colorectal cancers.

Epigenetics is the study of heritable changes in gene expression that occur without a change in the DNA sequence. Most constitutional defects in genes encoding components of the machinery that regulates the epigenome lead to embryonic death. Hypomorphic mutations may be compatible with life, but lead to severe developmental disorders. Their study is of great importance to our understanding of epigenetics and may clarify the interplay between different epigenetic mechanisms. This review will briefly introduce DNA methylation, post-translational histone modifications, and non-coding small RNA transcription, which are the best known epigenetic mechanisms. Then it will describe five human disorders (RETT, ATRX, ICF, Coffin-Lowry, and Rubinstein-Taybi) resulting from mutations in genes responsible for DNA methylation and in genes involved in chromatin remodeling. Finally, it will discuss how research in medical genetics can elucidate fundamental epigenetic processes.

Early detection of colorectal cancer is a decisive step in the successful and complete cure of the disease. Epigenetic markers, in particular, those based on aberrant DNA methylation, can be used to diagnose cancer. B melanoma antigens (BAGE) are a family of genes and truncated genes located in the heterochromatic regions of several human chromosomes. Our previous work showed that BAGE loci (i.e., genes and truncated genes) were hypermethylated in normal tissues and hypomethylated in 98% of human cancers. In the present study, we analyzed DNA methylation of the BAGE loci in 54 colon cancers and in neighboring histopathologic normal tissue samples. Using a combined bisulfite restriction assay, we showed that BAGE loci were hypomethylated in 81% of carcinoma samples. Colon cancer could be diagnosed with 94% specificity, 83% sensitivity, and 89% accuracy. No correlation was found between DNA methylation of BAGE loci and age, gender of patients, nor with the tumor stage or site. Based on the hypothesis that during neoplastic transformation, hypomethylation occurs in juxtacentromeric CpG islands, we suggest that other genes located in the heterochromatic compartment should be tested. These new markers enrich the list of currently studied epigenetic alterations in colon cancer and could be associated with hypermethylation markers to develop reliable diagnostic tests.

Euchromatin and heterochromatin are functional compartments of the genome. However, little is known about the structure and the precise location of the heterochromatin-euchromatin boundaries in higher eukaryotes. Constitutive heterochromatin in centromeric regions is associated with (1) specific histone methylation patterns, (2) high levels of DNA methylation, (3) low recombination frequency, and (4) the repression of transcription. All of this contrasts with the permissive structure of euchromatin found along chromosome arms. On the sequence level, the transition between these two domains consists most often of patchworks of segmental duplications. We present here a comprehensive analysis of gene expression, DNA methylation in CpG islands, distribution of histone isoforms, and recombination activity for the juxtacentromeric (or pericentromeric) region of the long arm of human chromosome 21. We demonstrate that most HapMap data are reliable within this region. We show that high linkage disequilibrium between pairs of SNPs extends 719-737 kb from the centromeric alpha-satellite. In the same region we find a peak of histone isoforms H3K9Me3 and H3K27Me (715-822 kb distal to the alpha-satellite). In normal somatic cells, CpG islands proximal to this peak are highly methylated, whereas distal CpG islands are not or very little methylated. This methylation profile undergoes dramatic changes in cancer cells and during spermatogenesis. As a consequence, transcription from heterochromatic genes is activated in the testis, and aberrant gene activation can occur during neoplastic transformation. Our data indicate that the frontier between the juxtacentromeric heterochromatic domain and euchromatic domain of the long arm of chromosome 21 is marked by a heterochromatic peak located approximately 750 kb distal to the alpha-satellite.

We characterized the mouse ortholog of the human MLL3 gene and a 10.6 kb-Mll3 transcript. The mouse Mll3 gene comprises 60 exons that encompass 226 kb in chromosome 5. The predicted protein of 3464 amino acids contains two PHD domains, an ATPase alpha_beta signature, an HMG, and a SET domain. We analyzed the expression of the Mll3 gene during the embryonic development of the mouse by whole-mount in situ hybridization. Low levels of expression throughout the embryo were first detected at 8.0 dpc. At this stage, the signal was already stronger in the forebrain neuroepithelium and absent in the heart. Next, expression outlined the ventral neural tube, the somites, the limbs, and the eye lens remaining at low levels throughout the embryo. By 13.0 dpc, expression became stronger in the spinal cord, in hand/foot plates, and in gonads. RT-PCR confirmed that Mll3 is expressed early during gametogenesis. We suggest that Mll3 is expressed early in pre-spermatogonia and then in spermatogonia.

The BAGE (B melanoma antigens) sequence family contains 15 nearly identical sequences that are in the juxtacentromeric regions of chromosomes 9, 13, 18, and 21. BAGE loci are expressed in male germ tissue and in a high percentage of cancers and cancer cell lines. We analyzed the DNA methylation state of the sequences in or near the promoters of the BAGE loci by a quantitative bisulfite and PCR-based assay (multiplex COBRA) using MboI and HphI in 18 somatic tissue samples, 4 testis and 4 sperm samples, and 48 tumors and tumor cell lines. In 94% of the control somatic tissue samples, DNA was highly methylated in the analyzed regions. In contrast, 98% of tumor DNA samples displayed hypomethylation. Also, DNA from testes and sperm was hypomethylated in at least one of the BAGE loci. BAGE transcripts were observed in only 47% of the analyzed tumor samples. Consequently, we propose BAGE hypomethylation as a new, highly informative epigenetic biomarker for the diagnosis of cancer, whose hypomethylation in cancer may be causally related to that of juxtacentromeric satellite DNA., (Copyright 2005 Wiley-Liss, Inc.)

We have analysed the genomic structure and transcriptional activity of a 2.3-Mb genomic sequence in the juxtacentromeric region of human chromosome 21. Our work shows that this region comprises two different chromosome domains. The 1.5-Mb proximal domain: (i) is a patchwork of chromosome duplications; (ii) shares sequence similarity with several chromosomes; (iii) contains several gene fragments (truncated genes having an intron/exon structure) intermingled with retrotransposed pseudogenes; and (iv) harbours two genes (TPTE and BAGE2) that belong to gene families and have a cancer and/or testis expression profile. The TPTE gene family was generated before the branching of Old World monkeys from the great ape lineage, by intra- and interchromosome duplications of the ancestral TPTE gene mapping to phylogenetic chromosome XIII. By contrast, the 0.8-Mb distal domain: (i) is devoid of chromosome duplications; (ii) has a chromosome 21-specific sequence; (iii) contains no gene fragments and only one retrotransposed pseudogene; and (iv) harbours six genes including housekeeping genes. G-rich sequences commonly associated with duplication termini cluster at the boundary between the two chromosome domains. These structural and transcriptional features lead us to suggest that the proximal domain has heterochromatic properties, whereas the distal domain has euchromatic properties.

In this paper, we show that the BAGE (B melanoma antigen) gene family was generated by chromosome rearrangements that occurred during the evolution of hominoids. An 84-kb DNA fragment derived from the phylogenetic 7q36 region was duplicated in the juxtacentromeric region of either chromosome 13 or chromosome 21. The duplicated region contained a fragment of the MLL3 gene, which, after juxtacentromeric reshuffling, generated the ancestral BAGE gene. Then, this ancestral gene gave rise to several independent genes through successive rounds of inter- and intrachromosome duplications. Comparison of synonymous and nonsynonymous mutations in putative coding regions shows that BAGE genes, but not the BAGE gene fragments, are under selective pressure. Our data strongly suggest that BAGE proteins have a function and that juxtacentromeric regions, whose plasticity is now largely proved, are not a simple junkyard of gene fragments, but may be the birth site of novel genes.

A first BAGE (B melanoma antigen) gene, BAGE1, was identified because it encodes a human tumour antigen recognised by a cytolytic T lymphocyte. Here, we characterised five new BAGE genes mapping to the juxtacentromeric regions of human chromosomes 13 and 21 and nine BAGE gene fragments mapping to the juxtacentromeric regions of chromosomes 9, 13, 18, and 21. Genes and gene fragments share extensive regions of 90-99% nucleotide identity. We analysed the expression of BAGE genes on 215 tumours of various histological types and on nine normal tissues. Similar to BAGE1, the new BAGE genes are expressed in melanomas, bladder and lung carcinomas and in a few tumours of other histological types. All the normal tissues were negative, with the exception of testis. Our results show that human juxtacentromeric regions harbour genes, which are transcribed and translated, in addition to gene fragments that are generally not expressed. We suggest that the pattern of expression restricted to cancer/testis is a feature of the few genes mapping to juxtacentromeric regions.

We characterized MLL3, a new human member of the TRX/MLL gene family. MLL3 is expressed in peripheral blood, placenta, pancreas, testes, and foetal thymus and is weakly expressed in heart, brain, lung, liver, and kidney. It encodes a predicted protein of 4911 amino acids containing two plant homeo domains (PHD), an ATPase alpha_beta signature, a high mobility group, a SET (Suppressor of variegation, Enhancer of zeste, Trithorax) and two FY (phenylalanine tyrosine)-rich domains. The amino acid sequence of the SET domain was used to obtain a phylogenetic tree of human MLL genes and their homologues in different species. MLL3 is closely related to human MLL2, Fugu mll2, a Caenorhabditis elegans predicted protein, and Drosophila trithorax-related protein. Interestingly, PHD and SET domains are frequently found in proteins encoded by genes that are rearranged in different haematological malignancies and MLL3 maps to 7q36, a chromosome region that is frequently deleted in myeloid disorders. Partial duplications of the MLL3 gene are found in the juxtacentromeric region of chromosomes 1, 2, 13, and 21.