PARP1 is essential to maintain the epigenetic hallmarks of imprinting control regions

The cells of our body contain two copies of every gene, one inherited by the mother and one by the father. For most genes, both of these copies are active. However, there is a small number of genes (~100) that are exquisitely regulated and expressed either only from the maternal or the paternal copy. These genes are known as 'imprinted' genes; they are regulated by a stretch of DNA sequence that carries an imprint which identifies its parent-of-origin. Imprinted genes are fundamentally important for normal development of the baby and its placenta during pregnancy, as well as for health after birth and of the adult. De-regulation of imprinted genes causes a number of severe disease syndromes commonly associated with developmental and growth defects, mental retardation, behavioural defects, physiological problems such as blood sugar imbalance and an increased rate of tumour development. Often, imprinted gene disorders are more frequently observed in babies conceived by assisted reproductive technologies such as IVF, either because of the underlying infertility problems or as a result of the procedure.

The regulation of imprinted genes has taught us key principles about how the activity state of our entire genome is controlled. As such the study of imprinted genes has been instrumental for our general understanding of how genes are switched on or off in the most principal biological processes. This area of research is commonly referred to as 'Epigenetics' as it deals with modifications imposed onto the DNA sequence that regulate DNA compaction and therefore gene activity state without changing the DNA sequence itself.

We have identified an entirely novel player in the control of imprinted gene regulation, a DNA-associated protein with enzymatic function called Parp1. Unlike any other factor previously investigated, Parp1 affects all imprinting control elements, and we therefore believe that it constitutes a most central player in imprinted gene regulation.

Parp1 is a factor that has been associated with many different functions, some of which depend on its binding capacity to DNA, others to its enzymatic activity, and yet others to combinations of both. Intriguingly, one of the best-studied roles of Parp1 is in DNA repair processes, and it is this function that has made it a prominent target in cancer therapies. Indeed, chemical inhibitors of Parp1 are currently tested in clinical trials for breast cancer.

The current proposal aims at investigating the precise mechanism of Parp1 function in imprinted gene regulation. This includes a detailed molecular dissection of how Parp1 maintains the normal parent-of-origin defining mark at imprinting control regions, what factors it interacts with and in which precise capacity it exerts this role. In addition, a comprehensive analysis will be performed that investigates the significance and consequences of Parp1 deletion in a developmental and physiological context.

This work will provide fundamental insights into our understanding of the epigenetic regulation of imprinted genes. As such, it is of high medical relevance for imprinting disorders, human infertility and its treatment procedures, and epigenetic contributions to cancer development. This knowledge opens up new avenues towards prospective therapies in these areas. Due to the multi-facetted functions of Parp1 in the cell, a most detailed understanding of its role in epigenetic gene regulation is of utmost importance to devise targeted intervention strategies with minimal side effects in order to exploit these clinical avenues in the future.

The focus of this proposal is to elucidate the molecular mechanism of Parp1 function in epigenetic gene regulation and imprinting control, and its physiological significance. Using ES cells as a powerful tool, we will assess the progression of epigenetic reprogramming at ICRs upon Parp1 deletion and the factors involved. In a series of intercalated experiments, we will elucidate whether Parp1 is an integral component of the ICR chromatin protein complex, or whether it is the conferred post-translational modification of ICR complex members that is critical for imprint maintenance. We will also test whether the few genomic regions that are hypomethylated in Parp1-/- ES cells in addition to known ICRs demarcate yet unknown imprinting control regions that may identify novel imprinted genes.

In parallel, the impact of Parp1 deficiency on imprinted gene regulation in vivo will be scrutinized. We will assess existing knockout models (of mild severity level) of Parp1, the highly related Parp2, and double mutants. The analyses will include to determine, in candidate and genome-wide approaches, the effect on DNA methylation, histone modifications (H3K9me3) and imprinted gene expression during gestation and postnatally. This will be complemented by a detailed analysis of developmental progression, postnatal and adult health, including assessment of the putative link to obesity that has been previously noted. Germ line mutants will serve to determine whether Parp1 (& Parp2) play a role in the establishment as well as the maintenance of gametic imprints, in epigenome surveillance and protection from transgenerational inheritance of epimutations.

This research is pertinent to our understanding of imprinting disorders and how they might arise for example in infertility treatments. Thereby, it will identify novel avenues for potential therapeutic intervention. It also provides direct links between epigenome (dys)regulation and cancer biology, in which Parp1 is a major player.

The central aim of this project is to gain novel insights into the epigenetic regulation of imprinted genes. Dysregulation of imprinted genes causes severe developmental disruptions and underlies multiple diseases such as Prader-Willi and Angelman syndromes, often associated with childhood cancers and lifelong debilitating conditions. Thus, advances in understanding the regulation of this unique class of genes, which represent a paradigm of epigenetic gene regulation, lies at the heart of MRC's mission to improve human health. Specifically, it falls centrally into Strategic Aim 1, "Further investment in epigenetics and its clinical applications", including "Investing in high throughput technologies to explore genetic variation".

This project will have substantial impact on many levels, from academic research with significant translational potential to ultimately benefit society as a whole. On the level of basic research, this study will be of great benefit to our immediate and wider research communities, including in the areas of epigenetics, genomic imprinting, X inactivation, developmental and stem cell biology, and nuclear organization and chromatin biology. Here, our work will open up new perspectives towards understanding the faithful propagation of epigenetic hallmarks, thus creating new knowledge and scientific advancement worldwide.

Our work will also be of great relevance to the wider field of biomedical and clinical academic scientists, in particular in the areas of imprinting disorders, obstetrics & gynaecology, paediatrics, and clinical genetics, as well as cancer epigenetics. Another area of high impact is in regenerative medicine and in assisted reproductive technologies, which are more commonly linked to imprinting disorders.

On the level of translational output, we expect that insights gained from this study may have significant impact on ART procedures and regenerative medicine within the foreseeable future (~ 5 yrs). We anticipate that our study may, for example, lead to improved embryo culture conditions that minimize epigenetic disruptions. Equally, our insights apply to regenerative medicine approaches by helping to maintain epigenomic health of stem and progenitor cells, thereby avoiding aberrations that may lead to incomplete differentiation or tumour development. We can well envisage that our insights will lead to adaptations in operating procedures and policies for the in vitro culture of early embryos or cells destined for a person's life. As a result of these advances, the project will benefit the public on the whole with significant economic and societal impacts, enhancing health and the quality of life, which is at the heart of the remit of 'Research Changes Lives'.

Ongoing advances of the project will be presented at national and international conferences, and the results of this work published in peer-reviewed open access journals or deposited in open access databases. The integrated genome-wide transcriptional and epigenomic network data will be made available on open-access servers, which will enable us and other researchers to expand the dataset in an interactive manner. Opportunities for intellectual property development and commercial exploitation are provided by novel insights into mechanisms of epigenome regulation, methodological advances and in the longer term potentially cell and embryo culture protocols. The lay audience will benefit from presentations, press interviews and/or public lectures, and through the social impact of the results of the study.

We further actively engage in training the next generation and fostering scientific enthusiasm through visits at schools, participating in the Researchers in Residence Scheme, and School's Day Projects. The project involves in-depth training of the staff, which will make them highly desirable for subsequent employment in the academic, health care and commercial research sector, and on scientific advisory boards.