Transcription Is Required to Establish Maternal Imprinting at the Prader-Willi Syndrome and Angelman Syndrome Locus

University of Cambridge, United Kingdom
PLoS Genetics (Impact Factor: 7.53). 12/2011; 7(12):e1002422. DOI: 10.1371/journal.pgen.1002422
Source: PubMed


The Prader-Willi syndrome (PWS [MIM 17620]) and Angelman syndrome (AS [MIM 105830]) locus is controlled by a bipartite imprinting center (IC) consisting of the PWS-IC and the AS-IC. The most widely accepted model of IC function proposes that the PWS-IC activates gene expression from the paternal allele, while the AS-IC acts to epigenetically inactivate the PWS-IC on the maternal allele, thus silencing the paternally expressed genes. Gene order and imprinting patterns at the PWS/AS locus are well conserved from human to mouse; however, a murine AS-IC has yet to be identified. We investigated a potential regulatory role for transcription from the Snrpn alternative upstream exons in silencing the maternal allele using a murine transgene containing Snrpn and three upstream exons. This transgene displayed appropriate imprinted expression and epigenetic marks, demonstrating the presence of a functional AS-IC. Transcription of the upstream exons from the endogenous locus correlates with imprint establishment in oocytes, and this upstream exon expression pattern was conserved on the transgene. A transgene bearing targeted deletions of each of the three upstream exons exhibited loss of imprinting upon maternal transmission. These results support a model in which transcription from the Snrpn upstream exons directs the maternal imprint at the PWS-IC.

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    • "Several imprinted retrogenes, such as U2af1- rs1 and Nap1l5, have integrated into the introns of larger transcription units and have their own maternal gDMRs, their intragenic location seeming to be a characteristic (Smith et al., 2003; Wood et al., 2007). A role for transcription in gDMR methylation in oocytes has now been demonstrated genetically for the Gnas and SNRPN imprinted loci (Chotalia et al., 2009; Smith et al., 2011); in many cases, the transcription events required for gDMR methylation appear to initiate from upstream, oocyte-specific promoters. "
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    ABSTRACT: At the heart of genomic imprinting in mammals are imprinting control regions (ICRs), which are the discrete genetic elements that confer imprinted monoallelic expression to several genes in imprinted gene clusters. A characteristic of the known ICRs is that they acquire different epigenetic states, exemplified by differences in DNA methylation, in the sperm and egg, and these imprint marks remain on the sperm- and oocyte-derived alleles into the next generation as a lifelong memory of parental origin. Although there has been much focus on gametic marking of ICRs as the point of imprint specification, recent mechanistic studies and genome-wide DNA methylation profiling do not support the existence of a specific imprinting machinery in germ cells. Rather, ICRs are part of more widespread methylation events that occur during gametogenesis. Instead, a decisive component in the specification of imprints is the choice of which sites of gamete-derived methylation to maintain in the zygote and preimplantation embryo at a time when much of the remainder of the genome is being demethylated. Among the factors involved in this selection, the zinc-finger protein Zfp57 can be regarded as an imprint-specific, sequence-specific DNA binding factor responsible for maintaining methylation at most ICRs. The recent insights into the balance of gametic and zygotic contributions to imprint specification should help understand mechanistic opportunities and constraints on the evolution of imprinting in mammals.Heredity advance online publication, 18 June 2014; doi:10.1038/hdy.2014.54.
    Heredity 06/2014; 113(2). DOI:10.1038/hdy.2014.54 · 3.81 Impact Factor
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    • "Distally located DNA elements communicate with the promoter to regulate gene expression. Recombined bacterial artificial chromosome (BAC) transgenes in which distal elements are deleted, have proved useful for studying the influence of these elements on DNA methylation in cis [10]. Here we report the characterization of GH promoter DNA methylation in which the putative mouse LCR was deleted. "
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    ABSTRACT: Regulatory elements for the mouse growth hormone (GH) gene are located distally in a putative locus control region (LCR) in addition to key elements in the promoter proximal region. The role of promoter DNA methylation for GH gene regulation is not well understood. Pit-1 is a POU transcription factor required for normal pituitary development and obligatory for GH gene expression. In mammals, Pit-1 mutations eliminate GH production resulting in a dwarf phenotype. In this study, dwarf mice illustrated that Pit-1 function was obligatory for GH promoter hypomethylation. By monitoring promoter methylation levels during developmental GH expression we found that the GH promoter became hypomethylated coincident with gene expression. We identified a promoter differentially methylated region (DMR) that was used to characterize a methylation-dependent DNA binding activity. Upon DNA affinity purification using the DMR and nuclear extracts, we identified structural maintenance of chromosomes hinge domain containing -1 (SmcHD1). To better understand the role of SmcHD1 in genome-wide gene expression, we performed microarray analysis and compared changes in gene expression upon reduced levels of SmcHD1 in human cells. Knock-down of SmcHD1 in human embryonic kidney (HEK293) cells revealed a disproportionate number of up-regulated genes were located on the X-chromosome, but also suggested regulation of genes on non-sex chromosomes. Among those, we identified several genes located in the protocadherin β cluster. In addition, we found that imprinted genes in the H19/Igf2 cluster associated with Beckwith-Wiedemann and Silver-Russell syndromes (BWS & SRS) were dysregulated. For the first time using human cells, we showed that SmcHD1 is an important regulator of imprinted and clustered genes.
    PLoS ONE 05/2014; 9(5):e97535. DOI:10.1371/journal.pone.0097535 · 3.23 Impact Factor
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    • "This suggests that KDM1B controls imprinting of those genes that acquire DNA methylation imprints relatively late during oocyte growth. Transcription through DMRs at Snrpn and Gnas was observed in growing oocytes, and transcript truncation upstream of the DMRs led to a loss of DNA methylation at these loci (28,29). However, KDM1B and transcription through DMRs account for genomic imprinting at defined regions and are not common to most imprinted regions. "
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    ABSTRACT: In mammals, genomic imprinting governed by DNA methyltransferase DNMT3A and its cofactor DNMT3L is essential for functional gametes. Oocyte-specific methylation imprints are established during oocyte growth concomitant with DNMT3A/DNMT3L expression, although the mechanisms of oocyte-specific imprinting are not fully understood. To determine whether the presence of DNMT3A/DNMT3L in oocytes is sufficient for acquisition of methylation imprints, we produced transgenic mice to induce DNMT3A/DNMT3L expression prematurely in oogenesis and analysed DNA methylation imprints. The results showed that two- to four-fold greater expression of DNMT3A/DNMT3L was achieved in non-growing oocytes versus fully grown oocytes derived from wild-type mice, but the analysed imprint domains were not methylated. Thus, the presence of DNMT3A/DNMT3L in non-growing oocytes is insufficient for methylation imprints and imprinted regions are resistant to DNMT3A/DNMT3L in non-growing oocytes. In contrast, excess DNMT3A/DNMT3L accelerated imprint acquisition at Igf2r, Lit1, Zac1, and Impact but not Snrpn and Mest in growing oocytes. Therefore, DNMT3A/DNMT3L quantity is an important factor for imprint acquisition. Transcription at imprinted domains is proposed to be involved in de novo methylation; however, transcription at Lit1, Snrpn, and Impact was observed in non-growing oocytes. Thus, transcription cannot induce DNMT3A catalysis at imprinted regions even if DNMT3A/DNMT3L is present. However, the accelerated methylation imprints in oocytes, with the exception of Igf2r, were erased during embryogenesis. In conclusion, a sufficient amount of DNMT3A/DNMT3L and a shift from the resistant to permissive state are essential to establish oocyte-specific methylation imprints and that maintenance of the acquired DNA methylation imprints is essential for functional imprinting.
    Human Molecular Genetics 03/2014; 23(14). DOI:10.1093/hmg/ddu100 · 6.39 Impact Factor
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