The Placenta Methylome: Novel Insights into DNA Modifications in the Human Placenta

Abstract

The DNA methylation profile of the placenta is unique as compared to other somatic tissues and is marked by global hypomethylation. The placental methylome has been shown to modulate developmental processes, intrauterine exposures, and placental function. However, the precise mechanisms underlying DNA methylation in regulating placental function and fetal development remains poorly studied. Aberrant DNA methylation patterns in the placenta have been associated with various pregnancy complications, such as fetal growth restriction, pre-eclampsia, perinatal death and miscarriage. Therefore, the study of the placental methylome is essential to advance our knowledge on the mechanisms underlying normal placental function. The study of DNA methylation has been traditionally focused on CpG sites, but ad- vancements in sequencing technologies and computational tools have renewed interest in the biological relevance of epigenetic modifications that occur at lower frequencies, such as non-CpG methylation (mCH). Non-CpG methylation is defined by the addition of a methyl group to cytosines that precede bases other than guanine, in a CHH and CHG context (where H = A, C or T), and has been reported in embryonic stem cells, induced pluripotent cells, somatic cell nuclear transfer stem cells, oocytes, neurons and glia. However, the distribution and role of mCH in the placenta is currently unknown. This study aimed to report the first single-base resolution map of mCH in the human placenta using whole genome oxidative bisulfite sequencing (WG(ox)BS), as well as validations using Nanopore adaptive sampling. The average genome-wide methylation across all samples was 55.8% for CpG and 1.39% for mCH (0.81% CHG and 0.58% CHH). In-silico methods were implemented to mitigate technical biases caused by bisulfite conversion. Placental mCH was found to preferentially occur in the CAC trinucleotide context. Fully methylated CAC sites were investigated within regulatory regions - genes, imprinting control regions, repetitive elements, CpG islands and enhancers - to gain potential insights into specific regulatory mechanisms underlying CAC methylation. Twenty-six regions with blocks of fully methylated CACs across all samples were found to be enriched within intronic regions and short tandem repeats ((CA)n, (GT)n, (CACACA)n). This work is the first to characterise non-CpG methylation in placentas and highlight potential novel epigenetic regulatory mechanisms. The CpG methylation profile in the placenta also undergoes dynamic changes across gestation, reflecting changes in cell type composition and functional, morphological, and physiological adaptations to support increasing fetal growth demands. The differences in mCH between the first (7-8wk), second (13-14 wk) and term (38-40wk) placentas were studied to provide insights into its potential role in modulating the placental phenotype throughout gestation. Furthermore, DNA methylation and alternative splicing are proposed as plausible regulatory mechanisms underlying the rapid and dynamic changes observed in the placenta throughout gestation. The genome-wide isoform switches from alternative splicing modes were profiled and associated with DNA methylation to understand the regulatory control underlying the transcriptomic and phenotypic changes observed between the first and second trimester placentas. The results indicate down-regulation of intron retention (IR) and up-regulation of alternative transcription termination sites and identified tissue and developmental stage specific transcripts containing retained introns, with an increase in IR-isoforms in the second trimester placentas. Of the genes that were differentially expressed, there were 23 differentially methylated CpGs sites were found within IRs that had a change in IR-isoform expression. IRs contribute to the diversity of alternatively spliced transcripts and are potential mechanisms towards fine-tuning gene expression as the placenta undergoes considerable changes to support fetal growth and development. In summary, the employment of state-of-the-art DNA methylation profiling techniques facilitated novel findings on DNA methylation in the human placenta. Understanding the epigenetic changes in the placenta could provide insights into the complex interactions of the materno-fetal interface, fetal development and potentially identify new diagnostic and therapeutic strategies. The summary of methods to interrogate DNA methylation has been published (book chapter), as well as the work in alternative splicing in the early placenta.