[Show abstract][Hide abstract] ABSTRACT: Genomic imprinting is the epigenetic marking of genes that results in parent-of-origin monoallelic expression. Most imprinted domains are associated with differentially DNA methylated regions (DMRs) that originate in the gametes, and are maintained in somatic tissues after fertilization. This allelic methylation profile is associated with a plethora of histone tail modifications that orchestrates higher order chromatin interactions. The mouse chromosome 15 imprinted cluster contains multiple brain-specific maternally expressed transcripts including Ago2, Chrac1, Trappc9 and Kcnk9 and a paternally expressed gene, Peg13. The promoter of Peg13 is methylated on the maternal allele and is the sole DMR within the locus. To determine the extent of imprinting within the human orthologous region on chromosome 8q24, a region associated with autosomal recessive intellectual disability, Birk-Barel mental retardation and dysmorphism syndrome, we have undertaken a systematic analysis of allelic expression and DNA methylation of genes mapping within an approximately 2 Mb region around TRAPPC9.
Utilizing allele-specific RT-PCR, bisulphite sequencing, chromatin immunoprecipitation and chromosome conformation capture (3C) we show the reciprocal expression of the novel, paternally expressed, PEG13 non-coding RNA and maternally expressed KCNK9 genes in brain, and the biallelic expression of flanking transcripts in a range of tissues. We identify a tandem-repeat region overlapping the PEG13 transcript that is methylated on the maternal allele, which binds CTCF-cohesin in chromatin immunoprecipitation experiments and possesses enhancer-blocker activity. Using 3C, we identify mutually exclusive approximately 58 and 500 kb chromatin loops in adult frontal cortex between a novel brain-specific enhancer, marked by H3K4me1 and H3K27ac, with the KCNK9 and PEG13 promoters which we propose regulates brain-specific expression.
We have characterised the molecular mechanism responsible for reciprocal allelic expression of the PEG13 and KCNK9 transcripts. Therefore, our observations may have important implications for identifying the cause of intellectual disabilities associated with the 8q24 locus.
[Show abstract][Hide abstract] ABSTRACT: DNA methylation is a common epigenetic modification of the mammalian genome. Conflicting data regarding the possible presence of methylated cytosines within mitochondrial DNA (mtDNA) have been reported. To clarify this point, we analysed the methylation status of mtDNA control region (D-loop) on human and murine DNA samples from blood and cultured cells by bisulphite sequencing and methylated/hydroxymethylated DNA immunoprecipitation assays. We found methylated and hydroxymethylated cytosines in the L-strand of all samples analysed. MtDNA methylation particularly occurs within non-C-phosphate-G (non-CpG) nucleotides, mainly in the promoter region of the heavy strand and in conserved sequence blocks, suggesting its involvement in regulating mtDNA replication and/or transcription. We observed DNA methyltransferases within the mitochondria, but the inactivation of Dnmt1, Dnmt3a, and Dnmt3b in mouse embryonic stem (ES) cells results in a reduction of the CpG methylation, while the non-CpG methylation shows to be not affected. This suggests that D-loop epigenetic modification is only partially established by these enzymes. Our data show that DNA methylation occurs in the mtDNA control region of mammals, not only at symmetrical CpG dinucleotides, typical of nuclear genome, but in a peculiar non-CpG pattern previously reported for plants and fungi. The molecular mechanisms responsible for this pattern remain an open question.
[Show abstract][Hide abstract] ABSTRACT: In the mouse, ZFP57 contains three classical Cys2His2 zinc finger domains (ZF) and recognizes the methylated TGC(met)CGC target sequence using the first and the second ZFs. In this study, we demonstrate that the human ZFP57 (hZFP57) containing six Cys2His2 ZFs, binds the same methylated sequence through the third and the fourth ZFs, and identify the aminoacids critical for DNA interaction. In addition, we present evidences indicating that hZFP57 mutations and hypomethylation of the TNDM1 ICR both associated with Transient Neonatal Diabetes Mellitus type 1 result in loss of hZFP57 binding to the TNDM1 locus, likely causing PLAGL1 activation.
[Show abstract][Hide abstract] ABSTRACT: a b s t r a c t 30 In the mouse, ZFP57 contains three classical Cys 2 His 2 zinc finger domains (ZF) and recognizes the 31 methylated TGC met CGC target sequence using the first and the second ZFs. In this study, we demon-32 strate that the human ZFP57 (hZFP57) containing six Cys 2 His 2 ZFs, binds the same methylated 33 sequence through the third and the fourth ZFs, and identify the aminoacids critical for DNA inter-34 action. In addition, we present evidences indicating that hZFP57 mutations and hypomethylation 35 of the TNDM1 ICR both associated with Transient Neonatal Diabetes Mellitus type 1 result in loss 36 of hZFP57 binding to the TNDM1 locus, likely causing PLAGL1 activation.
[Show abstract][Hide abstract] ABSTRACT: Paternal duplications of chromosome 6q24, a region that contains the imprinted PLAGL1 and HYMAI transcripts, are associated with transient neonatal diabetes mellitus. A common feature of imprinted genes is that they tend to cluster together, presumably as a result of sharing common cis-acting regulatory elements. To determine the extent of this imprinted cluster in human and mouse, we have undertaken a systematic analysis of allelic expression and DNA methylation of the genes mapping within an ∼1.4-Mb region flanking PLAGL1/Plagl1. We confirm that all nine neighbouring genes are biallelically expressed in both species. In human we identify two novel paternally expressed PLAGL1 coding transcripts that originate from unique promoter regions. Chromatin immunoprecipitation for CTCF and the cohesin subunits RAD21 and SMC3 reveals evolutionarily conserved binding sites within unmethylated regions ∼5 kb downstream of the PLAGL1 differentially methylated region and within the PLAGL1 3' untranslated region (UTR). Higher-order chromatin looping occurs between these regions in both expressing and non-expressing tissues, forming a non-allelic chromatin loop around the PLAGL1/Plagl1 gene. In placenta and brain tissues, we identify an additional interaction between the PLAGL1 P3/P4 promoters and the unmethylated element downstream of the PLAGL1 differentially methylated region that we propose facilitates imprinted expression of these alternative isoforms.
Nucleic Acids Research 01/2013; · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Heterogeneous molecular defects affecting the 11p15.5 imprinted gene cluster are associated with the opposite growth disorders Beckwith-Wiedemann Syndrome (BWS) and Silver Russell syndrome (SRS). Maternal deletions of the centromeric domain usually result in BWS, but paternal deletions have been so far associated with normal phenotype. Here we describe a case of recurrent severe Intra-Uterine Growth Restriction (IUGR) with paternal transmission of an 11p15.5 60 kb deletion. METHODS AND RESULTS: Chromosome microarray (CMA), PCR and DNA sequencing analyses showed that two fetuses conceived by a normal couple inherited from their father a 60 kb deletion encompassing the Imprinting Control Region of the 11p15.5 centromeric domain. The two fetuses died in utero with severe growth restriction. PCR amplification of parental DNAs indicated that the father carried the mutation in the mosaic state. DNA methylation and gene expression analyses showed that the deletion led to an imprinting alteration restricted to the centromeric domain and resulting in silencing of KCNQ1OT1 and activation of CDKN1C and PHLDA2. CONCLUSIONS: Our data demonstrate that the phenotype associated with 11p15.5 deletions is strongly influenced by the size of the region involved and indicate imprinting defects leading to CDKN1C and PHLDA2 activation as cause of severe IUGR.
Journal of Medical Genetics 12/2012; · 5.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: At chromosome 11p15.5, the Imprinting Centre 1 (IC1) controls the parent of origin-specific expression of the IGF2 and H19 genes. The 5 kb IC1 region contains multiple target sites (CTS) for the zinc-finger protein CTCF, whose binding on the maternal chromosome prevents the activation of IGF2 and allows that of H19 by common enhancers. CTCF binding helps maintaining the maternal IC1 methylation-free, whereas on the paternal chromosome gamete-inherited DNA methylation inhibits CTCF interaction and enhancer-blocking activity resulting in IGF2 activation and H19 silencing. Maternally inherited 1.4-2.2 kb deletions are associated with methylation of the residual CTSs and Beckwith-Wiedemann Syndrome (BWS), although with different penetrance and expressivity. We explored the relationship between IC1 microdeletions and phenotype by analysing a number of previously described and novel mutant alleles. We used a highly quantitative assay based on next generation sequencing to measure DNA methylation in affected families and analysed enhancer-blocking activity and CTCF binding in cultured cells. We demonstrate that the microdeletions mostly affect IC1 function and CTCF binding by changing CTS spacing. Thus, the extent of IC1 inactivation and the clinical phenotype are influenced by the arrangement of the residual CTSs. A CTS spacing similar to the wildtype allele results in moderate IC1 inactivation and is associated with stochastic DNA methylation of the maternal IC1 and incomplete penetrance. Microdeletions with different CTS spacing display severe IC1 inactivation and are associated with IC1 hypermethylation and complete penetrance. Careful characterization of the IC1 microdeletions is therefore needed to predict recurrence risks and phenotypical outcomes.
Human Molecular Genetics 10/2012; · 7.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Loss-of-function mutations in the gene encoding the cyclin-dependent kinase inhibitor CDKN1C cause Beckwith-Wiedemann syndrome and cancer. A new study now identifies potentially gain-of-function missense mutations in CDKN1C that cause the undergrowth-associated IMAGe syndrome.
[Show abstract][Hide abstract] ABSTRACT: Beckwith-Wiedemann syndrome (BWS), an overgrowth disorder with several congenital abnormalities, encompasses nephrourological anomalies. The objective of the report is to analyze the latter and related genotype-phenotype correlations. The study was a retrospective review of nephrourological investigations and genotype in 67 BWS patients. Imaging and laboratory studies have been correlated with the molecular anomalies typical of BWS. Thirty-eight (56.7%) patients had a total of 61 nonmalignant nephrourological findings, including nephromegaly (n = 24), collecting system abnormalities (n = 14), cryptorchidism (n = 11), nephrolithiasis (n = 5), cysts (n = 5), and dysplasia (n = 1). Four patients had Wilms' tumor, all associated with renal hyperplasia. Renal findings were almost consistent in the BWS(IC1) group, with nephromegaly in all patients and collecting system abnormalities in half of them. BWS(UPD) and negative patients also had frequent anomalies (63.6% and 61.9% respectively), whereas only 36.0% of BWS(IC2) had renal findings (p = 0.003). Cryptorchidism was associated with abdominal wall defects (p < 0.001) appearing more frequently in BWS(IC2) (p = 0.028). Urinary tract infections were observed in 17.9% of patients, with two resulting in life-threatening sepsis. Hypercalciuria was present in 10% of cases. 55.5% of BWS patients have renal findings. Although variegate, these anomalies disclose a genotype-phenotype correlation.
[Show abstract][Hide abstract] ABSTRACT: 22q11.2 deletion syndrome is mainly characterized by conotruncal congenital heart defects, velopharyngeal insufficiency, hypocalcemia and a characteristic craniofacial appearance. The etiology in the majority of patients is a 3-Mb recurrent deletion in region 22q11.2. Nevertheless, recently some cases of infrequent deletions with various sizes have been reported with a different phenotype. We report on a patient with congenital heart disease (truncus arteriosus type 2) in whom a de novo 1.3-Mb 22q11.2 deletion was detected by array comparative genomic hybridization. The deletion described corresponds to an atypical and distal deletion which spans low copy repeat (LCR) 4 and is associated with breakpoint sites that do not correspond to known LCRs of 22q11.2. We examine the clinical phenotype of our case and compare our findings with those published in the literature. The most prevalent clinical features in this type of deletion are a history of prematurity, pre-natal and post-natal growth retardation, slight facial dysmorphic features, microcephaly and developmental delay, with a speech defect in particular. These are clearly different from those found in the classic 22q11.2 deletion syndrome, and we believe that the main differential diagnosis should be with Silver-Russel syndrome. In our case we observe the cardiac phenotype with truncus arteriosus communis usually seen in the classic 22q11.2 deletion syndrome, and so far associated with the TBX1 gene. Significantly, however, TBX1 is not included in our patient's deletion. The possible roles of a position effect or other genes are discussed.
[Show abstract][Hide abstract] ABSTRACT: The maintenance of H3K9 and DNA methylation at imprinting control regions (ICRs) during early embryogenesis is key to the regulation of imprinted genes. Here, we reveal that ZFP57, its cofactor KAP1, and associated effectors bind selectively to the H3K9me3-bearing, DNA-methylated allele of ICRs in ES cells. KAP1 deletion induces a loss of heterochromatin marks at ICRs, whereas deleting ZFP57 or DNMTs leads to ICR DNA demethylation. Accordingly, we find that ZFP57 and KAP1 associated with DNMTs and hemimethylated DNA-binding NP95. Finally, we identify the methylated TGCCGC hexanucleotide as the motif that is recognized by ZFP57 in all ICRs and in several tens of additional loci, several of which are at least ZFP57-dependently methylated in ES cells. These results significantly advance our understanding of imprinting and suggest a general mechanism for the protection of specific loci against the wave of DNA demethylation that affects the mammalian genome during early embryogenesis.
[Show abstract][Hide abstract] ABSTRACT: A cluster of imprinted genes at chromosome 11p15.5 is associated with the growth disorders, Silver-Russell syndrome (SRS) and Beckwith-Wiedemann syndrome (BWS). The cluster is divided into two domains with independent imprinting control regions (ICRs). We describe two maternal 11p15.5 microduplications with contrasting phenotypes. The first is an inverted and in cis duplication of the entire 11p15.5 cluster associated with the maintenance of genomic imprinting and with the SRS phenotype. The second is a 160 kb duplication also inverted and in cis, but resulting in the imprinting alteration of the centromeric domain. It includes the centromeric ICR (ICR2) and the most 5' 20 kb of the non-coding KCNQ1OT1 gene. Its maternal transmission is associated with ICR2 hypomethylation and the BWS phenotype. By excluding epigenetic mosaicism, cell clones analysis indicated that the two closely located ICR2 sequences resulting from the 160 kb duplication carried discordant DNA methylation on the maternal chromosome and supported the hypothesis that the ICR2 sequence is not sufficient for establishing imprinted methylation and some other property, possibly orientation-dependent, is needed. Furthermore, the 1.2 Mb duplication demonstrated that all features are present for correct imprinting at ICR2 when this is duplicated and inverted within the entire cluster. In the individuals maternally inheriting the 160 kb duplication, ICR2 hypomethylation led to the expression of a truncated KCNQ1OT1 transcript and to down-regulation of CDKN1C. We demonstrated by chromatin RNA immunopurification that the KCNQ1OT1 RNA interacts with chromatin through its most 5' 20 kb sequence, providing a mechanism likely mediating the silencing activity of this long non-coding RNA.
Human Molecular Genetics 09/2011; 21(1):10-25. · 7.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The overgrowth disorder Beckwith-Wiedemann syndrome (BWS) is associated with dysregulation of imprinted genes at chromosome 11p15.5. The molecular defects are heterogeneous but most of the cases are associated with defective DNA methylation at either one of two Imprinting Control Regions (IC1 and IC2) or Uniparental paternal Disomy (UPD) at 11p15.5. In rare cases, the BWS phenotype has been found associated with maternal transmission of IC1 microdeletions. We describe a family with a novel 1.8 kb deletion that is associated with hypermethylation at IC1. The mutation results from recombination between highly homologous sequences containing target sites for the zinc-finger protein CTCF (CTSs). This finding supports the hypothesis that the function of IC1 and the penetrance of the clinical phenotype depend on the spacing of the CTSs resulting from recombination in the mutant allele.
European journal of medical genetics 05/2011; 54(4):e451-4. · 1.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hyper- and hypomethylation at the IGF2-H19 imprinting control region (ICR) result in reciprocal changes in IGF2-H19 expression and the two contrasting growth disorders, Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS). DNA methylation of the ICR controls the reciprocal imprinting of IGF2 and H19 by preventing the binding of the insulator protein, CTCF. We here show that local changes in histone modifications and CTCF--cohesin binding at the ICR in BWS and SRS together with DNA methylation correlate with the higher order chromatin structure at the locus. In lymphoblastoid cells from control individuals, we found the repressive histone H3K9me3 and H4K20me3 marks associated with the methylated paternal ICR allele and the bivalent H3K4me2/H3K27me3 mark together with H3K9ac and CTCF--cohesin associated with the non-methylated maternal allele. In patient-derived cell lines, the mat/pat asymmetric distribution of these epigenetic marks was lost with H3K9me3 and H4K20me3 becoming biallelic in the BWS and H3K4me2, H3K27me3 and H3K9ac together with CTCF-cohesin becoming biallelic in the SRS. We further show that in BWS and SRS cells, there is opposing chromatin looping conformation mediated by CTCF--cohesin binding sites surrounding the locus. In normal cells, lack of CTCF--cohesin binding at the paternal ICR is associated with monoallelic interaction between two CTCF sites flanking the locus. CTCF--cohesin binding at the maternal ICR blocks this interaction by associating with the CTCF site downstream of the enhancers. The two alternative chromatin conformations are differently favoured in BWS and SRS likely predisposing the locus to the activation of IGF2 or H19, respectively.
Human Molecular Genetics 02/2011; 20(7):1363-74. · 7.69 Impact Factor