Hockney, Sean (2019) The Importance of the Transcription Factor p63 in Epidermal Development and Disease: Isoform-based analysis of p63 in keratinocytes. Masters thesis, University of Huddersfield.

The transcription factor p63 is crucial for the maintenance and development of the epidermis. The p63 protein is encoded by the TP63 gene, which contains two distinct promoters giving rise to two major protein isoforms, termed TAp63, and ΔNp63. Mutations in the TP63 gene can lead to severe epidermal disease such as ectrodactyly/ecto-dermal dysplasia and cleft lip/palate (EEC) and ankyloblepharon/ectodermal defects and cleft lip/palate (AEC) syndromes. Within the literature there is conflict as to spatial and temporal expression of the p63 isoforms in developing and differentiating epidermis, with TAp63 appearing to play roles in the early stages of ectodermal development with ΔNp63 thought to be the dominant isoform during the maturation and maintenance of the epidermis. Despite this, recent work has suggested a role for TAp63 in regulating the steps within terminal differentiation of keratinocytes.

To examine this possible role for TAp63 isoforms in terminal differentiation we induced differentiation in 2D cell culture of primary and immortalized keratinocyte lines. Isoform expression, alongside other differentiation markers, were quantified via qRT-PCR. Furthermore, siRNA knockdown systems were developed to knockdown total p63, all TAp63 isoforms and all Beta forms of p63 and qRT-PCR was used to assess knockdown efficiency and effects on isoforms expression and expression of other linked genes.

Results from calcium–induced differentiation of keratinocyte lines from basal cells demonstrated a mirroring expression pattern for both the p63 isoforms with ΔNp63 expression being higher during the early stages of differentiation with its expression decreasing beyond day 8. In comparison, TAp63 expression began to increase at day 8 of differentiation, with its peak expression after day 12 and 13 suggesting a role for this isoform during the terminal stages of differentiation. This was seen in one HaCaT cell line and two primary keratinocyte lines. Expression of other differentiation markers such as FLG, KLF4 and ETS-1 followed expected trends.

Knockdown of total p63, TAp63 isoforms and Beta isoforms provided varying results in two keratinocyte lines. When total p63 was targeted in basal cells, we saw 50-75% knockdown of p63 isoforms suggesting that ΔNp63 was the most prominent isoform at this stage. When TAp63 was targeted, we saw a 60% knockdown efficiency. When Beta isoforms were targeted, we saw between 60 and 80% knockdown efficiency for ΔNp63 and a 30% knockdown efficiency for TAp63. We also demonstrated that expression of ETS-1 was consistent through two keratinocyte lines when each of the siRNA knockdowns were introduced.

We have demonstrated in vitro that the expression of the p63 isoforms shows a clear temporal difference in differentiating keratinocytes. We have further shown that when p63 isoform expression is interfered with, downstream marker expression changes depending on the target. These findings are important for understanding the distinguished roles of each isoform in differentiating keratinocytes.

FINAL THESIS - Hockney.pdf - Accepted Version
Available under License Creative Commons Attribution Non-commercial No Derivatives.

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