Characterisation of monoallelic regulation variation during human development

Daskeviciute, Dagne (2024) Characterisation of monoallelic regulation variation during human development. Doctoral thesis, University of East Anglia.

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Abstract

Genomic imprinting is the parent-of-origin monoallelic expression of genes. It is an epigenetic process in which chromosomal regions from both parents become differentially marked, primarily by DNA methylation. Several research groups, including ours, have previously found that the human placenta contains many unique differentially methylated regions (DMRs) that are not present in other somatic tissues. A more extensive characterisation of these placenta-specific DMRs revealed that they are derived from human oocytes and are maintained throughout pre-implantation development. I refer to these regions as placenta-specific maternal DMRs (mDMRs). Many of these placenta-specific mDMRs were identified by screening whole-genome bisulphite sequencing (WGBS) datasets from human gametes, blastocysts, and somatic tissues, including the term placenta. Curiously, some mDMRs were found to be highly polymorphic in the human population, and only some regulate monoallelic expression. The role of these placenta-specific mDMRs during development remains unclear, and many of the previously identified regions have yet to be fully characterised.

In addition, several groups have identified a novel form of imprinting, initially mediated by histone post-translational modifications (PTMs) in rodent pre-implantation embryos. These histone PTMs are later replaced by secondary DMRs (sDMRs), often at endogenous retroviral elements (ERVs) in rodent extra-embryonic tissues. This type of imprinting is referred to as non-canonical imprinting. Non-canonical imprinting has been shown to be critical for imprinted X chromosome inactivation in rodent embryos and plays an important role in normal placental development. A few studies have attempted to investigate whether non-canonical imprinting is conserved in human embryos, but the findings have been inconsistent. Their status in the human placenta remains uninvestigated.

During my PhD, I revisited placenta-specific mDMRs discovered by our group and others, which led to the identification of two promising placenta-specific mDMRs located at the CpG island promoters of G0S2 and PIK3R1 isoform 3. I applied various molecular biology techniques, including methylation-sensitive genotyping, bisulphite PCR, and allelic RT-PCR, followed by Sanger sequencing, in a large placental cohort to characterise their allelic usage. I demonstrated that the placenta-specific mDMRs of G0S2 and PIK3R1 isoform 3 are highly polymorphic, exhibiting maternal allele-specific methylation and monoallelic expression. Bisulphite-converted DNA from placental trophoblast and stromal cells, isolated using magnetic cell separation, revealed cell type-specific imprinting of these two genes.

Additionally, I applied the same techniques to investigate non-canonical imprinted genes, primarily on the human term placenta and human pre-implantation embryos. I screened human orthologs of mouse and rat non-canonical imprinted genes, non-canonical imprinted genes previously reported in human embryos, genes with primate-specific ERV LTR elements, and genes harbouring potential placental sDMRs. The results provided no evidence of non-canonical imprinting in the human placenta. However, further research is needed to investigate non-canonical imprints in human pre-implantation embryos. During this screen, I also demonstrated that XIST non-coding RNA, which is required for X chromosome inactivation in females, is not imprinted in human placental samples. Additionally, I identified several novel placenta-specific mDMRs that may regulate placenta-specific imprinting in the human placenta.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Chris White
Date Deposited:	13 Oct 2025 12:04
Last Modified:	13 Oct 2025 12:04
URI:	https://ueaeprints.uea.ac.uk/id/eprint/100707
DOI:

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