Influence of assisted reproductive technologies on imprinting centers and functional characterization of conserved elements in Beckwith Wiedemann Syndrome (BWS) region

Abstract

The epigenetic mark such as DNA methylation, at imprinted centers is often disrupted in imprinting syndromes in human. Recently these epigenetic alterations have also been implicated in assisted reproduction technology (ART) derived syndromes. The DMRs (sometimes also called imprinting centers) are indispensable for gene regulation in the imprinting domains. However the details of tissue and allele specific gene regulation in imprinted regions are poorly understood. In this study I investigated the Beckwith Wiedemann syndrome Region (BWS) in order to gain more knowledge on the influence of ART on maintance of imprinting in cattle and on the regulatory role of conserved elements in the vicinity of KvDMR1, one of the imprinting centers in the BWS region.

For investigation of epigenetic aberrations caused after the usage of ART, cattle was used as a model organism because of the easy accessibility of sample material and also frequent occurrence of overgrowth phenotypes similar to BWS syndrome. In cattle, the putative imprinting center regulating BWS and PWS /AS imprinting clusters are well conserved in genome in location when compared to human and mouse sequences. These centers also faithfully maintain differential methylation imprints in the germ lines as well as in embryo proper and in placental tissues. On comparative methylation analysis between foetuses derived from different fertilization procedures, i.e. AI derived (control animals) and IVF derived foetuses (day 80 of gestation), a substantial loss of methylation in placental tissues was detected at imprinting centers in BWS region i.e. H19 DMR and KvDMR1. In contrast the embryo proper showed no methylation defect in IVF derived foetuses. It was also observed that not all imprinting centers were affected by the usage of ART. Imprints at the Snrpn DMR, in the PWS / AS region, were well maintained in IVF derived and control AI derived foetuses.

The functional regulation of the cis acting conserved elements was analyzed at KvDMR1 imprinting sub-domain. The KvDMR1 sub-domain is characterized by the presence of high sequence conservation in intron 10 of the Kcnq1 gene (Paulsen, Khare et al. 2005). When 4 of highly conserved elements were linked to a reporter gene and tested in transient transfection assays, they showed varying influence on the KvDMR1 promoter. When tandem repeats were deleted from the KvDMR1 promoter, release of the influence of conserved elements on promoter activity was observed. This is the first report showing that tandem repeats in KvDMR1 promoter modulate its promoter activity by interacting with conserved elements in the BWS domain. Interestingly, co-expression of the placental and heart specific transcriptional factor Hand1 has resulted in enhanced KvDMR1 promoter activity only in presence of conserved elements. In particular potential functioning of one conserved element in placenta is also indicated by an unusual allele specific DNA methylation pattern.

In conclusion, the presented data on methylation in bovine embryo and placenta and to some extent the functional analysis on cis acting conserved elements support the hypothesis that the KvDMR1 sub-domain behaves differently in the embryo proper and in placental tissues.

Der Mechanismus des Imprinting ist eine in Säugetieren bekannte Erscheinung. Genomisches Imprinting beinhaltet multiple Faktoren. Es ist bekannt, dass fehlreguliertes Imprinting im Menschen verschiedene Syndrome verursacht. Gegenwärtige Untersuchungen zeigen einen Zusammenhang zwischen künstlicher Befruchtung und Imprinting-Syndromen im Menschen. In der vorliegenden Arbeit wurden Föten von Rindern untersucht, um den Einfluß künstlicher Befruchtungstechniken (ART) auf Imprinting-Regionen zu überprüfen. Es wurden verschiedene Imprinting-Regionen gewählt und deren Imprinting-Zentren durch Sequenzabgleich mit Mensch und Maus identifiziert. An allen untersuchten Imprinting-Zentren waren die Imprints (DNA-Methylierung) vorhanden. Beim Vergleich der Imprints von Föten einer Kontrollgruppe (AI) mit denen von IVF-generierten Föten, zeigte sich ein substanzieller Verlust paternaler Methylierung in der H19DMR, wohingegen sich maternale Methylierung der KvDMR1 nur in plazentalen Geweben IVF-generierter Föten (ART) fand.

Der zweite Fokus der vorliegenden Arbeit lag darin, das regulatorische Potenzial hoch-konservierter, kurzer Sequenzen (NICE), die das KvDMR1-Zentrum flankieren, zu untersuchen. Vier der hochkonservierten Elemente wurden getestet und zeigten einen Einfluß auf die Promotor-Aktivität. Dabei zeigte sich, dass dieser Effekt durch die Anwesenheit von Tandem Repeats in der KvDMR1 moduliert wurde. Dies ist die erste Untersuchung, die eine mögliche Rolle der konservierten NICE-Elemente und der Tandem Repeats auf die Promotor-Aktivität des KvDMR1-Imprinting-Zentrums aufzeigt.