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DNA damage in transcriptional regulation

Stranger, Ane (2023) DNA damage in transcriptional regulation. Doctor of Philosophy (PhD) thesis, University of Kent,. (doi:10.22024/UniKent/01.02.104623) (KAR id:104623)


The genome is continuously exposed to DNA damage, the most genotoxic of which are double-strand DNA breaks (DSBs). Their presence is signalled to the cell through phosphorylation of the histone variant H2AX at ser139, forming γH2AX, which recruits DNA repair factors. γH2AX has been extensively studied using ChIP-seq; here, a novel ChIP-based technique, CUT&Tag, was used to detect γH2AX formation in DNA damage-inducible cells and the results were analysed alongside publicly available γH2AX ChIP-seq datasets. CUT&Tag required fewer cells and sequencing reads than ChIP-seq, and yet exhibited improved signal:noise. H2AX can also be phosphorylated at tyr142, which is lost upon DNA damage. It is thought that H2AX phosphorylated at ser139 and tyr142 (di-γH2AX) exists transiently in cells, however, that a sustained di-γH2AX signal is pro-apoptotic. Di-γH2AX was investigated using a newly developed antibody by immunofluorescence, western blot and ChIP-qPCR; however, it was later discovered that the antibody was non-specific. DNA damage also plays a role in gene regulation. Estrogen receptor (ER) is bound by its ligand, estrogen, upon which it activates its target genes. In the ER+ breast cancer cell line MCF7, ER recruits the cytidine deaminase APOBEC3B (A3B), which deaminates cytosine, leading to DSBs, repair of which facilitates activation of target genes. To investigate A3B binding further, in the presence and absence of ER, existing ChIP-seq datasets were reanalysed; sites of recruitment were categorised based on ER coincidence and each category characterised in detail, based on the enriched motifs, targeted genes and gene ontology. Sites occupied by both A3B and ER were strongly enriched for the ER

motif and estrogen-regulated genes. Interestingly, sites at which A3B binds without ER were also enriched for the ER motif and estrogen-associated genes, suggesting that even sites at which A3B binds independently of ER are associated with the estrogen response.

Item Type: Thesis (Doctor of Philosophy (PhD))
Thesis advisor: Ellis, Peter
DOI/Identification number: 10.22024/UniKent/01.02.104623
Uncontrolled keywords: DNA damage; transcriptional regulation; estrogen receptor; H2AX; gH2AX; APOBEC3B
Subjects: Q Science > QR Microbiology
Divisions: Divisions > Division of Natural Sciences > Biosciences
Funders: University of Kent (
SWORD Depositor: System Moodle
Depositing User: System Moodle
Date Deposited: 15 Jan 2024 16:10 UTC
Last Modified: 16 Jan 2024 14:12 UTC
Resource URI: (The current URI for this page, for reference purposes)

University of Kent Author Information

Stranger, Ane.

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