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Studying Physiology and Pathology of Imprinted Genes to understand the role of Epigenetic Mutations in Human Disease

Final Report Summary - INGENIUM (Studying Physiology and Pathology of Imprinted Genes to understand the role of Epigenetic Mutations in Human Disease)

As a consequence of differential epigenetic modifications established during female and male gametogenesis, a subset of mammalian genes known as imprinted is expressed from only its maternal or its paternal allele. Failure in the normal establishment, maintenance and erasure of these marks results in gene dosage dysregulation and developmental anomalies that in the human correspond to a group of specific syndromes collectively called Imprinting Disorders (IDs). The focus of INGENIUM was a highly integrated approach to study the functional interactions between expression of imprinted genes and epigenetic mechanisms in physiological and pathological contexts with the final goal of formulating novel diagnostic/prognostic markers and therapeutic approaches for IDs. To reach this goal INGENIUM has brought together 14 research groups (10 beneficiaries and 4 associated partners) derived from both public and private sectors and from 7 European and one extra-European countries, and enrolled 15 Fellows of 8 different nationalities. The strength of INGENIUM resided in a multidisciplinary and integrated expertise and cutting-edge methodologies that were provided by a complementary and cooperating group of world-class biologists and clinicians and were made available to the fellows. Besides research, an integrated training program of scientific and complementary activities has been offered to INGENIUM Fellows to complement and strengthen their career development. INGENIUM has led to major advances in the knowledge of the molecular mechanisms governing normal and pathological aspects of genomic imprinting and epigenetic reprogramming, with the identification of novel cis-acting elements and trans-acting factors that constitute candidate targets for the development of new diagnostic markers and therapeutic approaches to treat IDs. INGENIUM was articulated in five workpackages dealing with (1) the mechanisms for epigenetic reprogramming and establishment of genomic imprinting in germ cells, (2) the role of histone methyltransferases and demethylases in imprinting control, (3) the regulatory mechanisms for maintenance of imprinting in somatic cells, (4) the identification of the genetic, epigenetic and environmental factors affecting genomic imprinting in human disease and (5) the role of genomic imprinting in stem cells. Concerning the mechanisms of epigenetic reprogramming in early embryogenesis, we demonstrated that 5-formyl-cytosine (5fC) is a stable epigenetic modification of the mammalian genome, identified 5fC-binding proteins suggesting functions for this DNA modification in transcription and chromatin regulation, and demonstrated that an impairment of DNA methylation maintenance is the main cause of the global demethylation observed in naive embryonic stem cells (BI). In addition, by obtaining DNA methylation profiles of oocytes, we demonstrated that histone remodeling and in particular removal of H3K4 methylation is necessary for proper methylation establishment in the female gametes (BI). Concerning the role of chromatin in imprinting maintenance in somatic cells, IGMM demonstrated that enhancer RNA-like ncRNAs control imprinted gene expression and differential histone modification and DNA replication at specific Imprinting Control Regions (ICRs). In addition, the relevance of histone H3K4me3 and H3K9me3 demethylases in imprinting control was evaluated (BRIC). The mechanisms by which KRAB-Zinc-Finger Proteins (KRAB-ZFPs) with their cofactor KAP1 control repressive marks at transposable elements (TE) and imprinted genes in somatic cells were also studied in details. In particular, EPFL identified two KRAB-ZFPs that use TE-based platforms to regulate neighboring genes in adult tissues and IGB demonstrated that ZFP57 recognizes clusters of the [TG]GCmCGC motif to maintain repressive epigenetic marks at ICRs and other non-imprinted target sites. INGENIUM partners also performed comprehensive genetic and epigenetic analyses of cohorts of patients affected by IDs. In particular, UB performed a genome-wide DNA methylation analysis to establish the frequency and phenotypic associations of Multi-Locus Imprinting Disturbances (MLID) in the Beckwith-Wiedemann syndrome (BWS). In addition, several novel molecular defects have been identified, including a CDKN1C activating mutation, maternal Uni-Parental Disomies of chromosomes 16 and 20, and loss of methylation of the DLK1/GTL2 ICRs in the Silver-Russell syndrome (SRS), and mutations in an OCT4/SOX2 binding motif associated with gain of methylation at the IGF2/H19 ICR in BWS (INSERM). Extensive (epi)genotype-phenotype correlation studies were also perfomed to identify phenotypic differences within molecular subgroups and propose new clinical scoring systems for BWS and SRS (UB, IGB, INSERM). A collaborative effort of IGB and UPENN allowed to develop a mouse model reproducing the molecular and phenotypic characteristics of BWS and SRS on maternal and paternal transmission that is now ready for in depth phenotypic analysis and testing novel therapeutic approaches. Another intensely investigated topic in INGENIUM was the role of imprinted genes in stem cells. In this context, a close collaboration between UCAM and INSERM have allowed to uncover novel roles of Dlk1, Rtl1 and Peg3/Wt1 in the development and regeneration of the muscle fibers. The role of CRG was instrumental to evaluate the relevance of the molecular pathways involved in cell reprogramming (e.g. WNT/b-CATENIN) in imprinting maintenance. Finally, the company participating to our network as beneficiary (DIAGENODE) developed an optimized protocol for Chromatin Immuno-precipitation of transcription factors which represented the basis for the preparation of a kit to be commercialized. The success of INGENIUM is demonstrated by the several scientific articles published or in preparation with the participation of the Fellows, who were all enrolled in PhD Programs at the participating institutions and will all be awarded a PhD title within the end of 2016. Our website (www.ingenium-itn.eu) is active since 2012 and has contributed to disseminate INGENIUM research and to promote MSCA actions to a very wide audience. Our Fellows had the opportunity to develop solid bases for their future career and to build up a scientific network of relationships in the field of the European research on epigenetics that will last for many years. INGENIUM has organized five scientific conferences including a final meeting that was co-sponsored by the COST action BM1208 on Congenital Imprinting Disorders and that allowed to meet clinicians who are responsible for ID diagnosis and management in main European medical centers.