Characterisation of pseudogene-like EP400NL in chromatin remodelling and transcriptional regulation : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Ph.D.) in Biochemistry at Massey University, Manawatū, New Zealand

Simple item page
dc.confidentialEmbargo : Noen_US
dc.contributor.advisorCooper, Timothy
dc.contributor.authorLi, Zidong
dc.date.accessioned2023-06-19T05:20:14Z
dc.date.available2023-06-19T05:20:14Z
dc.date.issued2023
dc.descriptionAll the work included in this thesis was performed by Zidong (Andy) Li. A publication based on this research can be found at https://doi.org/10.1016/j.bbagrm.2022.194889 published under a Creative Commons Attribution 4.0 International (CC BY 4.0) license.en
dc.description.abstractEP400 is an ATP-dependent chromatin remodelling enzyme that has been implicated in DNA double-strand break repair and transcription regulation including Myc-dependent gene expression. It was previously shown that the ectopic expression of the N-terminal domain of EP400 increases the efficacy of chemotherapeutic drugs against cancer cells. This prompted the question of whether the EP400 N-terminal-Like (EP400NL) gene, which resides next to the EP400 gene locus, also plays a similar role in epigenetic transcriptional regulation to the full-length EP400 protein. To characterize the function of the EP400NL nuclear complex, a stable cell line expressing TAP-tagged EP400NL was established, and the EP400NL complex was affinity purified and analyzed by mass spectrometry. EP400NL was found to form a human NuA4-like chromatin remodelling complex that lacks both the TIP60 histone acetyltransferase and EP400 ATPase. However, despite no histone acetyltransferase activity being detected, the EP400NL complex displayed H2A.Z deposition activity on a chromatin template comparable to the human NuA4 complex, suggesting another associated ATPase such as BRG1 or RuvBL1/RuvBL2 catalyses the reaction. In addition to a role in H2A.Z deposition, it was also determined that the transcriptional coactivator function of EP400NL is required for serum and IFNγ- mediated transcriptional activation of the immune checkpoint gene PD-L1. EP400NL, cMyc and multiple identified ATPases such as BRG1, RuvBL1/RuvBL2 were shown to be recruited to the promoter region of PD-L1. To further demonstrate the importance of EP400NL in regulating Myc and IFNγ-mediated PD-L1 expression, CRISPR/Cas9 mediated EP400NL indels were introduced in H1299, a human non-small cell lung carcinoma cell line. These EP400NL indel cell lines show compromised gene induction profiles with significantly decreased PD-L1 expression from both Myc and IFNγ stimulation experiments. In contrast to full-length EP400NL, two deletion mutants (Δ246- 260 and Δ360-419) lacked the ability to enhance the expression level of PD-L1 mRNA or protein, indicating that these regions are important for coactivator activity. Collectively, these data show that EP400NL plays a role as a transcription coactivator for cMyc-mediated gene expression and provides a potential target to modulate PD-L1 expression in cancer immunotherapy.en_US
dc.identifier.urihttp://hdl.handle.net/10179/17460
dc.publisherMassey Universityen_US
dc.rightsThe Authoren_US
dc.subjectChromatinen
dc.subjectEnzymesen
dc.subjectGenetic transcriptionen
dc.subjectGenetic regulationen
dc.subject.anzsrc310508 Genome structure and regulationen
dc.titleCharacterisation of pseudogene-like EP400NL in chromatin remodelling and transcriptional regulation : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Ph.D.) in Biochemistry at Massey University, Manawatū, New Zealanden_US
dc.typeThesisen_US
massey.contributor.authorLi, Zidongen_US
thesis.degree.disciplineBiochemistry and Molecular Biologyen_US
thesis.degree.grantorMassey Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophy (PhD)en_US
Files
Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
Li_ZidongPhDThesis.pdf
Size:
12.78 MB
Format:
Adobe Portable Document Format
Description:
Download
Collections
Theses and Dissertations