Identifying unique regulatory elements related to polymorphic imprinting and gestational aging in the placenta

The placenta grows during pregnancy and is attached to the wall of the womb. It links the mother to the baby via the umbilical cord. Its main purpose is to supply the baby with all nutrients and oxygen required for growth, whilst removing waste products and carbon dioxide from the baby's blood. If the placenta does not develop or function correctly, this can lead to pregnancy complications that have huge consequences for the baby, not immediately following delivery, but later in life. Cells are the fundamental unit of complex tissues such as the placenta. Cell phenotype and function are very different between the different cell types. It is the code and activity (referred to as expression) of our genes that are ultimately responsible for defining which cells are produced and how they behave. This is achieved by 'epigenetic' modifications to the DNA. One form of epigenetic regulation is achieved by marking certain regions of the genome with methyl groups that tend to act as a semi-permanent block for gene expression. However, DNA methylation does not act alone, there are different interactive mechanisms that help in epigenetic regulation. In recent years a class of genes, known as imprinted genes, have been shown to be essential for placenta development and function. These uniquely regulated genes are epigenetically turned "on" and expressed solely from the maternal or paternal copy, but not both. The placenta is fascinating as it has a unique epigenetic profile having significantly less DNA methylation compared to all other tissues, which subtly changes during pregnancy. The hierarchical consequences of this unique hypomethylated state on the addition layers of epigenetic information have not been investigated. Furthermore, the placenta harbours hundreds of unique imprinted genes not found in other tissues, the expression of which generally decrease with gestation in a DNA methylation independent manner. This suggests other epigenetic mechanisms are responsible for the observed downregulation. Our understanding of this phenomenon has been hindered by the fact we do not know the mechanisms responsible for healthy aging within the placenta. In this application, we seek to understand how methylation in placenta influences other epigenetic marks at different time point in pregnancy and how this ultimately impacts on imprinted gene expression (Objectives 1.1-1.2). To achieve this, we will utilize, and in some cases improve, the latest technologies to identify which area of the genome are associated with different epigenetic signatures and how this influence development (Objectives 2.1 & 3). Furthermore, since we anticipate variability and cell-type specific signatures within our samples, we will profile isolated single-cells, allowing for us to identify patterns which would not be evident in "bulk" placenta biopsies (Objective 2.2). To ensure maximum chance of success building of our previous experiences, we have established a team that includes researchers responsible for technical development at the UK National Capability in Genomics at the Earlham Institute and computational experts for data analysis. Following the analysis using extremely powerful computers and specialised computer programs (Objective 2.3) we will compare our placenta-derived data across time, and within difference cells- types, to identify regions important for gestation-age related expression changes, especially for imprinted genes, which we will subsequently be remove or alter to determine functionality (Objective 4).

In this application we propose a series of experiments to better understand the hierarchical placental epigenome across gestation (by assessing two different developmental time points) to determine intra-sample variability and to gain widespread functional insights of potential cis-acting regulatory elements with unique placenta profiles. The data will be generated and assessed in a genome-wide unbiased fashion, allowing us to monitor gestational-dynamics in imprinted gene expression and identify cis-regulatory usage that would account for the widespread down-regulation of expression previously reported.

There is an inter-relationship between DNA methylation and additional layers of epigenetic information essential for genome function. We have previously shown that the placenta is uniquely hypomethylated and that this methylation is dynamic through pregnancy. However, addition epigenetic tiers have largely been uncharacterised in this essential tissue and mechanisms responsible for the global increase in methylation (on the unique hypomethylated background) are unknown. Fascinatingly, transcriptional down-regulation associated with imprinted transcripts at term is independent of methylation, since all imprinted differentially methylated regions maintain faithful allelic profiles, suggesting other epigenetic mechanisms must be responsible.

Main deliverables: (1) We will produced consensus epigenetic maps for early and late placenta samples. Through exploitation of these datasets, we will ultimately identify functional regulatory elements, many of which we anticipate will change during gestation. (2) By performing bioinformatic analyses using trio-SNP information to discriminate alleles, the temporal epigenetic and expression profiles for imprinted genes will be revealed. Since the epigenetic datasets will be produced in an unbiased genome-wide fashion, this will allow novel cis-regulatory elements to be defined and functionally characterised.