The roles of microRNAs (miRNAs) and the miRNA processing machinery in the regulation of stem cell biology are not well understood. Here, we show that the p53 family member and p63 isoform, ΔNp63, is a critical transcriptional activator of a co-factor pivotal for miRNA processing, DGCR8. This regulation is important in the terminal differentiation of adult keratinocytes and likely of other cells. Loss of ΔNp63 or DGCR8 induces pluripotency in these cells.
Generation of a conditional ΔNp63 knock-out mouse model with the help of a Cre-LoxP system led to the development of mice with single layered fragile and disorganized epidermis. Analysis of the skin with immunohistochemistry and immmunofluorescence exhibit aberrant expression of differentiation markers like keratin 5,14,10, loricrin and filaggrin, suggesting a defect in terminal differentiation and BrdU incorporation assay suggested hyperproliferation and self-renewal. Further characterization of stem cell marker expression in the epidermal cells from these embryos was found to be high. Chromatin immunoprecipitation assay and luciferase assays demonstrated the transcriptional activation of the DGCR8 promoter by ΔNp63. Overexpression of DGCR8 in the epidermal cells with the help of a lentiviral doxycycline inducible DGCR8 construct forced these cells to undergo differentiation as validated by in vitro differentiation assay and teratoma formation assay. These cells were also capable of generating chimeras when injected into blastocysts. Lentiviral knock-down of ΔNp63 or DGCR8 in normal human epidermal keratinocytes led to the development of induced pluripotent stem cells capable of forming teratomas as analyzed by differentiation markers AFP, MyoD and Nestin for various lineages like endoderm, mesoderm and ectoderm respectively.
Low expression of key miRNAs in ΔNp63 deficient cells results in the failure to repress genes required for stem cell pluripotency, Oct4, Sox2, and Nanog. Strikingly, ΔNp63-/- epidermal cells display profound defects in terminal differentiation and express markers and miRNAs of pluripotency at levels comparable to those in embryonic stem cells. Moreover, these cells can differentiate into multiple cell fates in vitro and in vivo. We found that human primary keratinocytes depleted of ΔNp63 or DGCR8 become pluripotent. For the first time we demonstrate a novel role for ΔNp63 in the transcriptional regulation of DGCR8 to maintain stem cell pluripotency. Importantly, our findings identify a simple method of generating human induced pluripotent stem cells through the knockdown of a single gene.
Citation Format: Deepavali Chakravarti, Xiaohua Su, Minsoon Cho, Young Jin Gi, Ashley Benham, Cristian Coarfa, Avinashnarayan Venkatanarayan, Jennifer Alana, Chad Creighton, weimin Xiao, Marco Leung, Harina Vin, Io Long Chan, Arianexys Aquino, Nicole Müller, Jan Parker-Thornburg, Kenneth Y. Tsai, Preethi H. Gunarante, Elsa R. Flores. Induced pluripotency in adult keratinocytes through downregulation of ΔNp63 or DGCR8. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1119. doi:10.1158/1538-7445.AM2013-1119