How does ageing-related loss of basement membrane collagen regulate epidermal barrier homeostasis?

Aim 1 Establish 3D skin equivalents incorporating immortalised keratinocytes with shRNA knockdown of Col7 or Col17. Integrin receptors, actin, nucleocytoskeleton and differentiation proteins will be examined using immunofluorescence. Existing RNAseq data from keratinocytes with knockdown (KD) of Col7 and Col17 will be mined to direct further analyses. The skin barrier will be characterized using dye exclusion, Nile Red staining and transglutaminase activity assays. Experiments will be replicated using primary (1o) keratinocytes with KD of Col7/Col17.
Aim 2 Apply mechanical/UV stress to the Aim 1 3D skin equivalents. The student will develop the 3D models on a stretchable porous membrane (Gautrot lab) to apply mechanical stretch. An alternative stressor is UV irradiation. In parallel, glycolysis and oxidative phosphorylation will be measured using Seahorse technology in monolayer cultures with KD of Col7/Col17 . Immunostaining will be performed for oxidative stress, proliferation and DNA damage markers. Changes in nuclear morphology and chromatin remodelling will be analysed by immunostaining of histone marks and high-resolution confocal microscopy.
Aim 3 Use epigenomic methods to identify regulators of the identified transcriptional response from BM loss. Keratinocytes from Aim 2 will be subjected to chromatin immunoprecipitation (ChIP) using antibodies for selected transcriptional regulators followed by Next Generation Sequencing (NGS). The student will integrate these data with the existing RNAseq data to identify how the BM is regulating epidermal homeostasis. Findings can be compared in young and aged skin by immunostaining.
Aim 4 Identify and test compounds to mitigate effects of BM alterations on epidermal homeostasis
At Unilever, bioinformatic and chemi-informatic tools will be used to identify compounds via a combination of RNA-based signature analysis, pathway-driven and target/lead ID approaches. Proof of principle options will be tested in aged keratinocyte models to examine if the barrier and basement membrane can be improved.