Unravelling gene regulation in the epidermis during differentiation
Anisa Banu Abdul Rahim
Date of Issue2018-07-12
School of Biological Sciences
A*STAR Institute of Medical Biology (IMB)
Polyamines are small polycationic molecules that are essential for many cellular processes including DNA replication, RNA modification and protein synthesis. The major polyamines in eukaryotic cells are putrescine (Put), spermidine (Spd) and spermine (Spm). Polyamine levels vary between tissues and are tightly regulated. They are differentially distributed throughout the epidermis and dermis of normal human skin with higher levels being found in the epidermis. The rate-limiting enzyme adenosyl methionine decarboxylase (AMD1) promotes the synthesis of Spd and Spm from Put and changes in AMD1 levels directly influence the ratio and levels of all three polyamines. We find that AMD1 is highly enriched in the suprabasal layers of the human epidermis and is translationally upregulated during in vitro differentiation of immortalized and human primary keratinocytes. Polyamine measurements in undifferentiated and differentiated keratinocytes showed that Put levels were significantly reduced while Spm levels were elevated during differentiation. Knockdown or inhibition of AMD1 in cultured immortalized keratinocytes and 3-dimentional (3-D) organotypic skin equivalents reduced the expression of early and late differentiation markers and impeded keratinocyte stratification. The phenotype was rescued with extracellular addition of Spd and Spm suggesting that AMD1 plays a role during keratinocyte differentiation. The role of AMD1 during keratinocyte differentiation was determined by microarray analysis, to detect its potential downstream targets. We discovered that AMD1 is upstream of several key regulators of keratinocyte differentiation such as JUNB, KLF4, NOTCH1 and ZNF750. Additionally, we found CPEB4, an RNA binding protein to be downstream of AMD1 and is a novel marker of keratinocyte differentiation. While CPEB4 strongly stained the granular layer of the human epidermis, it also depicted a possible centrosomal staining in cells at the basal and suprabasal layers by confocal microscopy. This study has paved ways to understand the critical role of polyamines in the epidermis and the complexity of the gene regulatory network that drives keratinocyte differentiation.