Franck Court

Genome-wide parent-of-origin DNA methylation analysis reveals the intricacies of human imprinting and suggests a germline methylation-independent mechanism of establishment

Differential methylation between the two alleles of a gene has been observed in imprinted regions, where the methylation of one allele occurs on a parent-of-origin basis, the inactive X-chromosome in females, and at those loci whose methylation is driven by genetic variants. We have extensively characterized imprinted methylation in a substantial range of normal human tissues, reciprocal genome-wide uniparental disomies, and hydatidiform moles, using a combination of whole-genome bisulfite sequencing and high-density methylation microarrays. This approach allowed us to define methylation profiles at known imprinted domains at base-pair resolution, as well as to identify 21 novel loci harboring parent-of-origin methylation, 15 of which are restricted to the placenta. We observe that the extent of imprinted differentially methylated regions (DMRs) is extremely similar between tissues, with the exception of the placenta. This extra-embryonic tissue often adopts a different methylation profile compared to somatic tissues. Further, we profiled all imprinted DMRs in sperm and embryonic stem cells derived from parthenogenetically activated oocytes, individual blastomeres, and blastocysts, in order to identify primary DMRs and reveal the extent of reprogramming during preimplantation development. Intriguingly, we find that in contrast to ubiquitous imprints, the majority of placenta-specific imprinted DMRs are unmethylated in sperm and all human embryonic stem cells. Therefore, placental-specific imprinting provides evidence for an inheritable epigenetic state that is independent of DNA methylation and the existence of a novel imprinting mechanism at these loci.

Genome-Wide Allelic Methylation Analysis Reveals Disease-Specific Susceptibility to Multiple Methylation Defects in Imprinting Syndromes

Genomic imprinting is the parent‐of‐origin‐specific allelic transcriptional silencing observed in mammals, which is governed by DNA methylation established in the gametes and maintained throughout the development. The frequency and extent of epimutations associated with the nine reported imprinting syndromes varies because it is evident that aberrant preimplantation maintenance of imprinted differentially methylated regions (DMRs) may affect multiple loci. Using a custom Illumina GoldenGate array targeting 27 imprinted DMRs, we profiled allelic methylation in 65 imprinting defect patients. We identify multilocus hypomethylation in numerous Beckwith–Wiedemann syndrome, transient neonatal diabetes mellitus (TNDM), and pseudohypoparathyroidism 1B patients, and an individual with Silver–Russell syndrome. Our data reveal a broad range of epimutations exist in certain imprinting syndromes, with the exception of Prader–Willi syndrome and Angelman syndrome patients that are associated with solitary SNRPN‐DMR defects. A mutation analysis identified a 1 bp deletion in the ZFP57 gene in a TNDM patient with methylation defects at multiple maternal DMRs. In addition, we observe missense variants in ZFP57, NLRP2, and NLRP7 that are not consistent with maternal effect and aberrant establishment or methylation maintenance, and are likely benign. This work illustrates that further extensive molecular characterization of these rare patients is required to fully understand the mechanism underlying the etiology of imprint establishment and maintenance.

Long-range chromatin interactions at the mouse Igf2/H19 locus reveal a novel paternally expressed long non-coding RNA

Parental genomic imprinting at the Igf2/H19 locus is controlled by a methylation-sensitive CTCF insulator that prevents the access of downstream enhancers to the Igf2 gene on the maternal chromosome. However, on the paternal chromosome, it remains unclear whether long-range interactions with the enhancers are restricted to the Igf2 promoters or whether they encompass the entire gene body. Here, using the quantitative chromosome conformation capture assay, we show that, in the mouse liver, the endodermal enhancers have low contact frequencies with the Igf2 promoters but display, on the paternal chromosome, strong interactions with the intragenic differentially methylated regions 1 and 2. Interestingly, we found that enhancers also interact with a so-far poorly characterized intergenic region of the locus that produces a novel imprinted long non-coding transcript that we named the paternally expressed Igf2/H19 intergenic transcript (PIHit) RNA. PIHit is expressed exclusively from the paternal chromosome, contains a novel discrete differentially methylated region in a highly conserved sequence and, surprisingly, does not require an intact ICR/H19 gene region for its imprinting. Altogether, our data reveal a novel imprinted domain in the Igf2/H19 locus and lead us to propose a model for chromatin folding of this locus on the paternal chromosome.

H19 Antisense RNA Can Up-Regulate Igf2 Transcription by Activation of a Novel Promoter in Mouse Myoblasts

It was recently shown that a long non-coding RNA (lncRNA), that we named the 91H RNA (i.e. antisense H19 transcript), is overexpressed in human breast tumours and contributes in trans to the expression of the Insulin-like Growth Factor 2 (IGF2) gene on the paternal chromosome. Our preliminary experiments suggested that an H19 antisense transcript having a similar function may also be conserved in the mouse. In the present work, we further characterise the mouse 91H RNA and, using a genetic complementation approach in H19 KO myoblast cells, we show that ectopic expression of the mouse 91H RNA can up-regulate Igf2 expression in trans despite almost complete unmethylation of the Imprinting-Control Region (ICR). We then demonstrate that this activation occurs at the transcriptional level by activation of a previously unknown Igf2 promoter which displays, in mouse tissues, a preferential mesodermic expression (Pm promoter). Finally, our experiments indicate that a large excess of the H19 transcript can counteract 91H-mediated Igf2 activation. Our work contributes, in conjunction with other recent findings, to open new horizons to our understanding of Igf2 gene regulation and functions of the 91H/H19 RNAs in normal and pathological conditions.

Stability of Genomic Imprinting and Gestational-Age Dynamic Methylation in Complicated Pregnancies Conceived Following Assisted Reproductive Technologies

ABSTRACT For the past three decades, assisted reproductive technologies (ART) have revolutionized infertility treatments. The use of ART is thought to be safe. However, early investigations suggested that children born as a result of ART had higher risk of diseases with epigenetic etiologies, including imprinting disorders caused by a lack of maternal methylation at imprinting control elements. In addition, large epidemiology studies have highlighted an increased risk of obstetric complications, including severe intrauterine growth restriction (IUGR) in babies conceived using ART. It is plausible that the increased frequency of IUGR may be due to abnormal imprinting because these transcripts are key for normal fetal growth and development. To address this, we have collected a large cohort of placenta and cord blood samples from ART conceptions and compared the imprinting status with appropriate non-ART population. Using a custom DNA methylation array that simultaneously quantifies 25 imprinted differentially methylated regions, we observed similar epigenetic profiles between groups. A multiplex Sequenom iPLEX allelic expression assay revealed monoallelic expression for 11 imprinted transcripts in our placenta cohort. We also observe appropriate gestational age-dependent methylation dynamics at retrotransposable elements and promoters associated with growth genes in ART placental biopsies. This study confirms that children conceived by ART do not show variability in imprinted regulation and that loss-of-imprinting is not commonly associated with nonsyndromic IUGR or prematurity.

Myod and H19-Igf2 locus interactions are required for diaphragm formation in the mouse

The myogenic regulatory factor Myod and insulin-like growth factor 2 (Igf2) have been shown to interact in vitro during myogenic differentiation. In order to understand how they interact in vivo, we produced double-mutant mice lacking both the Myod and Igf2 genes. Surprisingly, these mice display neonatal lethality due to severe diaphragm atrophy. Alteration of diaphragm muscle development occurs as early as 15.5 days post-coitum in the double-mutant embryos and leads to a defect in the terminal differentiation of muscle progenitor cells. A negative-feedback loop was detected between Myod and Igf2 in embryonic muscles. Igf2 belongs to the imprinted H19-Igf2 locus. Molecular analyses show binding of Myod on a mesodermal enhancer (CS9) of the H19 gene. Chromatin conformation capture experiments reveal direct interaction of CS9 with the H19 promoter, leading to increased H19 expression in the presence of Myod. In turn, the non-coding H19 RNA represses Igf2 expression in trans. In addition, Igf2 also negatively regulates Myod expression, possibly by reducing the expression of the Srf transcription factor, a known Myod activator. In conclusion, Igf2 and Myod are tightly co-regulated in skeletal muscles and act in parallel pathways in the diaphragm, where they affect the progression of myogenic differentiation. Igf2 is therefore an essential player in the formation of a functional diaphragm in the absence of Myod.

Genomic Matrix Attachment Region and Chromosome Conformation Capture Quantitative Real Time PCR Assays Identify Novel Putative Regulatory Elements at the Imprinted Dlk1/Gtl2 Locus

We previously showed that genomic imprinting regulates matrix attachment region activities at the mouse Igf2 (insulin-like growth factor 2) locus and that these activities are functionally linked to neighboring differentially methylated regions (DMRs). Here, we investigate the similarly structured Dlk1/Gtl2 imprinted domain and show that in the mouse liver, the G/C-rich intergenic germ line-derived DMR, a sequence involved in domain-wide imprinting, is highly retained within the nuclear matrix fraction exclusively on the methylated paternal copy, reflecting its differential function on that chromosome. Therefore, not only “classical” A/T-rich matrix attachment region (MAR) sequences but also other important regulatory DNA elements (such as DMRs) can be recovered from genomic MAR assays following a high salt treatment. Interestingly, the recovery of one A/T-rich sequence (MAR4) from the “nuclear matrix” fraction is strongly correlated with gene expression. We show that this element possesses an intrinsic activity that favors transcription, and using chromosome conformation capture quantitative real time PCR assays, we demonstrate that the MAR4 interacts with the intergenic germ line-derived DMR specifically on the paternal allele but not with the Dlk1/Gtl2 promoters. Altogether, our findings shed a new light on gene regulation at this locus.

Human Oocyte-Derived Methylation Differences Persist in the Placenta Revealing Widespread Transient Imprinting

Thousands of regions in gametes have opposing methylation profiles that are largely resolved during the post-fertilization epigenetic reprogramming. However some specific sequences associated with imprinted loci survive this demethylation process. Here we present the data describing the fate of germline-derived methylation in humans. With the exception of a few known paternally methylated germline differentially methylated regions (DMRs) associated with known imprinted domains, we demonstrate that sperm-derived methylation is reprogrammed by the blastocyst stage of development. In contrast a large number of oocyte-derived methylation differences survive to the blastocyst stage and uniquely persist as transiently methylated DMRs only in the placenta. Furthermore, we demonstrate that this phenomenon is exclusive to primates, since no placenta-specific maternal methylation was observed in mouse. Utilizing single cell RNA-seq datasets from human preimplantation embryos we show that following embryonic genome activation the maternally methylated transient DMRs can orchestrate imprinted expression. However despite showing widespread imprinted expression of genes in placenta, allele-specific transcriptional profiling revealed that not all placenta-specific DMRs coordinate imprinted expression and that this maternal methylation may be absent in a minority of samples, suggestive of polymorphic imprinted methylation.

The PEG13 -DMR and brain-specific enhancers dictate imprinted expression within the 8q24 intellectual disability risk locus

BackgroundGenomic 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.ResultsUtilizing 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.ConclusionsWe 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.

Corneal Transduction by Intra-Stromal Injection of AAV Vectors In Vivo in the Mouse and Ex Vivo in Human Explants

The cornea is a transparent, avascular tissue that acts as the major refractive surface of the eye. Corneal transparency, assured by the inner stroma, is vital for this role. Disruption in stromal transparency can occur in some inherited or acquired diseases. As a consequence, light entering the eye is blocked or distorted, leading to decreased visual acuity. Possible treatment for restoring transparency could be via viral-based gene therapy. The stroma is particularly amenable to this strategy due to its immunoprivileged nature and low turnover rate. We assayed the potential of AAV vectors to transduce keratocytes following intra-stromal injection in vivo in the mouse cornea and ex vivo in human explants. In murine and human corneas, we transduced the entire stroma using a single injection, preferentially targeted keratocytes and achieved long-term gene transfer (up to 17 months in vivo in mice). Of the serotypes tested, AAV2/8 was the most promising for gene transfer in both mouse and man. Furthermore, transgene expression could be transiently increased following aggression to the cornea.