Functional Analysis of Sheep POU2F3 Isoforms

Guang-Wei Ma,En-Guang Rong,Yan-Kai Chu,Xiao-Hong Yan,Ning Wang,Hua Yang,Hui Li

doi:10.1007/s10528-019-09945-x

Abstract

POU domain class 2 transcription factor 3 (POU2F3) plays an important role in keratinocyte proliferation and differentiation. Our previous study identified four sheep POU2F3 transcript variants (POU2F3-1, POU2F3-2, POU2F3-3, and POU2F3-4), encoding three POU2F3 protein isoforms (POU2F3-1, POU2F3-2, and POU2F3-3). However, the functional differences among the three POU2F3 isoforms remain unknown. The objective of this study was to determine the tissue expression pattern of the four POU2F3 transcript variants in sheep and to investigate the functional differences in cell proliferation among the three POU2F3 isoforms. Quantitative RT-PCR analysis showed that the four POU2F3 transcripts were ubiquitously expressed in all tested adult sheep tissues, and POU2F3-1 exhibited higher expression level than the other three POU2F3 transcript variants in skin (P < 0.05). Cell proliferation assay showed that overexpression of any one of the three POU2F3 isoforms significantly inhibited the proliferation of sheep fetal fibroblasts and HaCaT cells at 48 and 72 h after transfection (P < 0.05). POU2F3-3 had less inhibitory effect on cell proliferation than POU2F3-1 and POU2F3-2 (P < 0.05), and POU2F3-1 and POU2F3-2 had similar inhibitory effects (P > 0.05). Dual luciferase reporter assays demonstrated that overexpression of any one of the three POU2F3 isoforms significantly inhibited the promoter activities of keratin 14 (KRT14) and matrix metalloproteinase 19 (MMP19) genes (P < 0.05). POU2F3-3 had less inhibitory effect on the promoter activities of KRT14 and MMP19 genes than POU2F3-1 and POU2F3-2 (P < 0.05), and POU2F3-1 and POU2F3-2 had similar inhibitory effects (P > 0.05). These results suggest three sheep POU2F3 isoforms have similar functional effects, but to a different extent.

Highlights

POU domain class 2 transcription factor 3 (POU2F3), a POU domain transcription factor known as Skn-1a or Oct-11, is predominantly expressed in the suprabasal layers of the epidermis (Goldsborough et al 1993; Hildesheim et al 2001)
The results showed that the four POU2F3 transcript variants were ubiquitously and differentially expressed in all tested adult sheep tissues including heart, liver, spleen, kidney, rumen, small intestine, skeletal muscle, and skin (Fig. 1)
Our results showed that four sheep POU2F3 transcript variants were widely and differentially expressed in various tissues of the Chinese Merino sheep, especially the skin (Fig. 1), suggesting that POU2F3 alternative splicing is regulated in a tissue-specific manner and that these POU2F3 isoforms might exert different functions

Summary

Introduction

POU domain class 2 transcription factor 3 (POU2F3), a POU domain transcription factor known as Skn-1a or Oct-11, is predominantly expressed in the suprabasal layers of the epidermis (Goldsborough et al 1993; Hildesheim et al 2001). A previous study demonstrated that human POU2F3 (hPOU2F3) mainly promoted keratinocyte proliferation and secondarily enhanced keratinocyte differentiation (Hildesheim et al 2001). A knockout study showed that POU2F3-knockout mice had higher proliferation rate of epidermal keratinocytes adjacent to the wound edge compared with the littermate controls (Andersen et al 1997). A colony inhibition assay showed that human POU2F3 overexpression inhibited the proliferation of cervical cancer cell lines of epithelial origin (Yutaka et al 2004). Keratin 14 gene (KRT14) and matrix metalloproteinase 19 genes (MMP19) are highly expressed in mitotically active epithelial basal cells and promote cell proliferation (Alam et al 2011; Beck et al 2007). POU2F3 overexpression inhibited the expression and promoter activity of the KRT14 gene in normal human epidermal keratinocytes (Sugihara et al 2001) and of MMP19 gene in HaCaT cells (Beck et al 2007; Sadowski et al 2003)

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