Claxton, Michael
(2023)
Epigenetic features, allelic expression and transcriptional regulatory elements at the Peg13, Trappc9, Ago2 imprinted gene cluster in brain and neural cells.
PhD thesis, University of Liverpool.
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Abstract
Genomic imprinting is a molecular mechanism that causes genes to be expressed in a parent-of-origin-specific manner due to epigenetic modifications to the genome. This results in mono-allelic or heavily biased expression of one allele, while the other remains inactive. For certain genes, imprinted allelic expression may be tissue-specific and reliant on CTCF-influenced enhancer-promoter interactions. The Peg13 imprinted cluster, located on chromosome 15qD3 in the mouse genome, is associated with neurodevelopmental disorders such as Birk-Barel syndrome and non-syndromic intellectual disability. It consists of canonical imprinted genes that are conserved between mouse and human and brain-specific imprinted genes in the mouse. The former consists of Peg13, a conserved imprinted gene that exhibits a strong, non-tissue-specific, paternal expression bias, while the latter consists of Trappc9, Chrac1 and Ago2, which display a maternal allelic expression bias of ~75% in the murine brain but exhibit biallelic expression in peripheral mouse tissues and in humans. The regulation of allele-specific expression of these genes is thought to result from a differentially methylated CpG region near the promoter of Peg13. To date, most imprinted expression studies have been conducted on bulk tissue data, which seeks to identify tissue-specificity for multiple imprinted clusters, novel imprinted genes and conservation of imprinting between species. However, the finding of such allelic expression biases generalised on the tissue lysate level raises the fundamental question of whether these patterns are conserved in each cell or whether there is variability and mosaicism in allelic expression between individual cells of the imprinted tissue. To address this, I characterised the allelic expression of some of these imprinted genes in both bulk tissues and at a single-cell level using newborn C57BL/6J x cast/EiJ hybrid mice to generate identifiable SNPs and determine allelic expression ratios. In Chapter 3, I discuss my use of three tissue samples: whole-brain lysates, cultured neural stem cells (NSCs) isolated from hippocampus tissue, and kidney, to confirm previously identified brain-specific imprinted expression for genes within the Peg13 cluster. Using a combination of pyrosequencing and Sanger sequencing, I identified that Trappc9, Ago2, and Chrac1 all display brain-specific imprinted expression, with varying degrees of strength, but all preferentially expressed from the maternal allele in brain tissue lysate, whereas kidney samples exhibited expected biallelic expression. Furthermore, I confirmed that both Kcnk9 and Peg13 do not display tissue-specific imprinting, with both whole-brain lysates and kidney samples showing preferential expression from the maternal and paternal allele, respectively. Interestingly, the data also showed that Trappc9 and Ago2 are not imprinted in hippocampus derived NSCs, while Peg13 retains its strong bias of paternal allele expression in these samples. Additionally, in Chapter 3, I discuss methylation analysis of bulk tissue samples for CpG islands (CGIs) located proximal to the promoter region of each gene within the cluster. This analysis revealed that methylation patterns were consistent with current literature, displaying hypomethylation of all CGI regions within the cluster, with the exception of the CGIs located at Trappc9 exon 2 and the Peg13 differentially methylated region (DMR), which both exhibited high levels of methylation. Chapter 4 focuses on answering whether single cells within a known imprinted tissue exhibit the same allele-specific expression pattern or show variability between cells. To achieve this, I cultured both neural progenitor (neurosphere) cells (NPCs) and in vitro differentiated neurons derived from newborn C57BL/6J x cast/EiJ hybrid mice. I used the single-cell genotyping, expression, and methylation (GEM) technique, combined with Sanger sequencing, to determine allelic expression patterns for Peg13, Trappc9, and Ago2. The results indicated that single-cell imprinted expression was not uniform, but instead contained several variable states of allelic expression in individual NPCs and neurons. Regarding Peg13, while the majority of cells were in line with bulk tissue data, a small proportion of cells deviated from this expected paternal allele bias, exhibiting equal biallelic or even mono-allelic maternal expression. Furthermore, for Trappc9 and Ago2, I identified a spectrum of expression states ranging from mono-allelic maternal, mono-allelic paternal, and varying levels of preferential biallelic expression. However, while the presence of multiple imprinting expression patterns within a cell population highlights an increased complexity of imprinting regulation, as a whole, the ratio of variable allele-specific expression was reflective of the bulk tissue data. In addition to analysing imprinted expression, by utilising the methylation-sensitive restriction enzyme BstUI, I also attempted to identify the presence of methylation at the promoter-proximal CGIs in single cells for genes within this cluster. The results of this analysis identified that the majority of single cells appear to be in line with the bulk tissue methylation data. Finally, in Chapter 5, I discuss a potential molecular mechanism for the regulation of imprinted expression in this cluster. Through interrogation of the ENCODE3, UCSC and Ensembl genome browsers, I identified the location of candidate brain-specific regulatory elements relative to a CTCF site located proximal to Peg13 in mouse that showed potential for regulating gene expression. To investigate this, I transfected promoter-reporter gene constructs into primary neurons isolated from newborn C57BL/6 mice and fibroblasts. Results showed that several of these regulatory elements exhibit either tissue-specific or general silencer activity, specific to the individual element, which may potentially contribute to the regulation of imprinted expression bias of the Trappc9 gene. In conclusion, the expression of tissue-specific imprinted genes is likely more complex and molecularly dynamic than initially believed within single cells at any specific time point. Single cells within an imprinted tissue appear to exhibit a wide range of allele-specific expression patterns that can deviate from the bulk-tissue standard. The mechanism behind this variation is yet to be elucidated. However, further investigation of the driving factors behind this regulation will establish a deeper understanding of imprinted single-cell transcriptomics.
| Item Type: | Thesis (PhD) |
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| Divisions: | Faculty of Health and Life Sciences |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 29 Nov 2023 12:28 |
| Last Modified: | 29 Nov 2023 12:28 |
| DOI: | 10.17638/03172743 |
| Supervisors: |
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| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3172743 |
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