- Title
- A novel method for analysis of genomic DNA methylation and transcriptional programs in single cells
- Creator
- Hunt, Kooper
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2024
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Analysis of cancer’s genetic and molecular makeup at the single-cell level has allowed for understanding of previously unappreciated levels of heterogeneity. This tumour heterogeneity is a key characteristic that drives relapse and treatment resistance in many cancers. Unravelling tumour heterogeneity provides opportunity to better personalise treatments and more effectively target the root cause of disease. Single-cell analysis technologies are advancing rapidly and currently are widely available in the research community. DNA methylation in particular is a widely studied and important epigenetic mark. The importance of DNA methylation in health and disease cannot be understated, and extensive DNA methylation heterogeneity is observed in the context of cancer. Like most single-cell analysis methods, single-cell DNA methylation analysis technologies are characterised by high costs and high levels of technicality. In this PhD project, we will develop a novel bisulfite sequencing method for paired analysis of DNA methylation and transcription in single cells, whilst addressing current limitations. By targeting high copy number transposable elements, we utilise a PCR based method to measure global DNA methylation levels in single-cell samples. This method, termed single-cell transposable element methylation sequencing, or scTEM-seq, allows for simplified and more cost effective analysis than currently available whole genome bisulfite sequencing methods. Workflows are streamlined, saving time and costs of reagents. The most significant factor in scTEM-seq’s affordability is the low sequencing demands associated. Only 20000 reads per cell is sufficient for accurate quantification of genome DNA methylation levels. When paired with transcriptome sequencing from the same cell, scTEM-seq will make study of epigenetic heterogeneity and the effects on the transcriptome accessible to labs without capacity for the high sequencing demands of currently available methods. We apply scTEM-seq to investigate the use of hypomethylating agents decitabine and azacitidine in the treatment of acute myeloid leukemia. In decitabine treated KG1a cells, our method reveals that DNA methylation heterogeneity induced by decitabine treatment is associated with altered expression of immune process genes. We also observed an increase in transposable element expression after treatment, and an increased expression of a subset of transposable elements unrelated to global methylation shifts. Building on these findings, we discovered cells that retain DNA methylation after treatment have a growth advantage over other cells, possibly causing treatment resistance and relapse. We found unique expression profiles in these methylation retaining cells including cholesterol biosynthesis pathways. By inhibiting this pathway with rosuvastatin co-treatment we reduced azacitidine resistance, as measured by reduced colony formation. This finding was tested in an induced AML mouse model, and co-treatment of decitabine and rosuvastatin was shown to significantly increase survival rates. The scTEM-seq method presented in this thesis has great untapped potential to forward research in DNA methylation heterogeneity. Not only do we present the development of the method and give examples of application, but we were able to apply scTEM-seq to make noteworthy findings that have major clinical potential to improve the use of hypomethylating agents in leukemia.
- Subject
- genomic DNA methylation; transcriptional programs; single cells; scTEM-seq; thesis by publication
- Identifier
- http://hdl.handle.net/1959.13/1513444
- Identifier
- uon:56722
- Rights
- Copyright 2024 Kooper Hunt
- Language
- eng
- Full Text
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| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | ATTACHMENT01 | Thesis | 8 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 615 KB | Adobe Acrobat PDF | View Details Download |