Identification and characterisation of KZFPs directly regulated by p53

Approximately 50% of human DNA is composed of transposable elements (TEs), which in subsequent waves colonized our genome across evolutionary history. KRAB zinc finger proteins (KZFPs) are the largest group of DNA binding factors in the human genome and mostly target TEs, controlling chromatin accessibility by effecting epigenetic changes at their binding sites which result in a localized establishment of heterochromatin. Many of these bound TEs themselves contain binding sites for regulators of transcription and represent a huge source of genetic variation and regulatory potential by acting as enhancers or alternative promoters, influencing nearby genes. The KZFP family presents an interesting conundrum; the family is highly evolutionary dynamic, with new members appearing at every phylogenetic branch since their inception in tetrapods, yet their binding sites in TEs are conserved millions of years after their initial TE target has been inactivated. Furthermore, individual KZFPs have specific expression patterns in various somatic cell types, leading to the hypothesis that they participate in the epigenetic control of enhancers or alternative promoters derived from transposable elements, contributing to gene regulatory networks. So far, only a handful of the 350 human KZFPs have been functionally characterized, yet many of those studied have important and novel biological functions, such as ZFP57 and imprinting, with several examples of KZFPs and their TE targets demonstrably contributing to gene regulatory events. In this thesis, I describe the recently identified KZFPs which display inducible expression in response to direct trans-activation from the tumour suppressor protein p53, focussing on the KZFPs ZNF79 and ZNF561. ZNF79 presents an unusual case of a marsupial-conserved KZFP significantly older than the majority of the KZFP family at 160 million years old, yet strongly conserved across many phylogenetic branches with marsupial ZNF79. Furthermore, ZNF79 demonstrates an unusual intermediate TRIM28 binding status and a unique nucleolar localisation. In contrast, ZNF561 resembles a canonical KZFP but possesses intriguing characteristics given that its expression is induced by p53. First, its binding profile is enriched for specific LTR10 elements which can act as p53 driven enhancers. Second, it unusually binds to its own promoter and the promoter of its antisense RNA. Together, these characteristics suggest a complex regulatory pathway. By utilising CRISPR knock-out models and RNA-seq, I have identified interesting changes in the expression of genes implicated in the transcriptional and cellular response to p53 activation and in genes related to cell proliferation and survival. These results indicate that ZNF79 and ZNF561 may play a role in the transcriptional response to DNA damage and p53, identifying new preliminary networks of gene regulation. Ultimately, this work offers valuable insights into the relationships between KZFPs, evolution, and key cellular processes, providing models by which to explore these characteristics.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2022

Author dc:creator

• Daffern, Samuel

Advisor dc:contributor.advisor

• Imbeault, Michael

• DNA Damage
• KZFP
• p53

Licence

• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.93640

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/346220