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
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Date
2023
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Massey University
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Abstract
EP400 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.
Description
All 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.
Keywords
Chromatin, Enzymes, Genetic transcription, Genetic regulation