SALL4 maintains self-renewal of porcine pluripotent stem cells through downregulation of OTX2

doi:10.15302/J-FASE-2017180

Front. Agr. Sci. Eng.

2019, Vol. 6

Issue (1) : 81-92 https://doi.org/10.15302/J-FASE-2017180

RESEARCH ARTICLE

SALL4 maintains self-renewal of porcine pluripotent stem cells through downregulation of OTX2

Ning WANG, Sile WANG, Yaxian WANG, Yuanxing CAI, Fan YANG, Huayan WANG(

)

Department of Animal Biotechnology, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China

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Abstract

Sall4 as one of the spalt family members contains several alternative splicing variants, which are differentially expressed and has a key role in maintaining pluripotent stem cells. However, the molecular features and function of SALL4 have not been well elucidated in porcine induced pluripotent stem cells (piPSCs). In this study, we identified SALL4 splice variants and found two SALL4 splicing variants through analysis of the porcine transcriptome data derived from piPSCs. SALL4A was only detected in piPSCs but SALL4B was globally expressed in porcine tissues and piPSCs. The level of SALL4B was significantly reduced when piPSCs differen-tiation occurred, however, the expression of SALL4A was not affected, indicating that SALL4B may be essential for the maintenance of piPSCs self-renewal. Overexpression of SALL4A and SALL4B in PEF cells could significantly stimulated expression of endogenous pluripotent genes, when SALL4B significantly promoted OCT4 expression. Conversely, SALL4A significantly promoted KLF4 expression. Additionally, both SALL4A and SALL4B could repress OTX2 promoter activity in a dose-dependent manner. Conversely, OTX2 also negatively regulated SALL4 expression. These observations indicate that a negative feedback regulatory mechanism may exist between SALL4 and OTX2, which is useful for the maintenance of the self-renewal of piPSCs.

Keywords OTX2 pluripotency pig SALL4 transcription regulation

Corresponding Author(s): Huayan WANG

Just Accepted Date: 22 November 2017 Online First Date: 26 December 2017 Issue Date: 25 February 2019

Cite this article:

Ning WANG,Sile WANG,Yaxian WANG, et al. SALL4 maintains self-renewal of porcine pluripotent stem cells through downregulation of OTX2[J]. Front. Agr. Sci. Eng. , 2019, 6(1): 81-92.

URL:

https://academic.hep.com.cn/fase/EN/10.15302/J-FASE-2017180
https://academic.hep.com.cn/fase/EN/Y2019/V6/I1/81

Fig.1 Identification and cloning of porcine SALL4 alternative splicing variants. (a) Transcriptome analysis of SALL4 in porcine tissues and pluripotent cells. Sequencing reads for SALL4A and SALL4B in piPS-F cells are given in the green box; (b) RT-PCR analysis of SALL4 splicing variants in piPSCs; (c) enzyme digestions (BamHI/XhoI) to confirm the constructs of pSALL4A and pSALL4B; (d) western blot analysis of fusion proteins, EGFP-SALL4A (140 kDa) and EGFP-SALL4B (95 kDa), and EGFP (27 kDa) in 293T cells; (e) vectors of pSALL4A, pSALL4B, and pEGFP-C1 were transfected into 293T cells for 48 h. EGFP-SALL4 fusion proteins were translocated into nuclei.

Fig.2 SALL4 expression in porcine tissues and pluripotent cells. (a) Dynamic DNA methylation profile of porcine SALL4. MeDIP-Seq data of SALL4 methylation in porcine longissimusdorsi muscle (LDM) and piPSCs were visualized in UCSC genome browser; (b) bisulfite genomic sequencing analysis of SALL4 in LDM and piPS-F cells. Open and filled circles represent unmethylated and methylated CpGs; (c) RT-PCR (upper) and densitometry (lower) analyses of SALL4 expression in porcine tissues and piPSCs; (d) alkaline phosphatase staining (upper) and immunofluorescence staining (lower) of SALL4, SOX2, and OCT4 in undifferentiated and differentiated piPSCs. Scale bar, 50 mm.

Fig.3 SALL4 regulates the expression of pluripotent genes. Quantitative RT-PCR analyses were applied to determine the expression of pluripotent genes in piPSCs and PEF cells. (a) Expression of SALL4 and pluripotent genes in the differentiated piPSCs (piPS^+RA) that were treated by retinoic acid (RA) for various time points; (b) expression of SALL4A and SALL4B in piPS^+RA; (c) overexpression of SALL4A (OE-4A) and SALL4B (OE-4B) in PEF cells for 48 h. Ctrl, cells were transfected by pEGFP-C1; (d) knockdown (KD) SALL4 expression by siRNAs affected the expression of pluripotent genes. Ctrl, cells were transfected with an unspecific siRNA. Data are presented as mean±SD, * P<0.05, ** P<0.01, n = 3; (e) morphology of piPSCs with (KD) and without (Ctrl) siRNA treatment. Scale bar, 100 mm.

Fig.4 SALL4 suppresses OTX2 expression. (a) Luciferase assay of OTX2 promoter activity. The pG-OTX2 only (left) and pG-OTX2 with pSALL4A and pSALL4B (right) were cotransfected into 293T cells for 36 h. Ctrl cells were cotransfected by pG-OTX2 and pGL3-basic; (b) diagram of pG-OTX2 and the truncated OTX2 promoter constructs with potential transcription binding sites (upper). Luciferase assay of OTX2 promoter activity in 293T cells (lower). Ctrl, cells were transfected by pGL3-basic; (c) SALL4A and SALL4B regulate OTX2 promoter activation in 293T cells. Ctrl, cells were transfected by pEGFP-C1; (d) luciferase assays. For time-dependent assay (left), pG-OTX2 with pSALL4 and pSALL4B were cotransfected into 293T cells for 48 h. For dose-dependent assay (right), pG-OTX2 and different amount of SALL4 constructs were cotransfected into 293T cells for 36 h. Ctrl, cells were transfected by pGL3-OTX2; (e) alkaline phosphatase staining of overexpression of OTX2 (pE-OTX2, OTX2+ ) and suppression of OTX2 (OTX2+ plus pSALL4A and pSALL4B) in piPSCs. Ctrl, cells were transfected by pEGFP-C1. Number of AP positive clones was counted in 36 h post-transfection. Scale bar, 50 mm. Data are presented as mean±SD, * P<0.05; ** P<0.01; n = 3.

Fig.5 OTX2 regulates SALL4 expression. (a) Morphology and AP staining of piPSCs that were transfected with pE-OTX2 (OTX2⁺ ). Ctrl, cells without transfection of pE-OTX2. Scale bar, 50 µm; (b, c) semiquantitative (B) and quantitative (C) RT-PCR analyses of SALL4A and SALL4B in piPSCs transfected by pE-OTX2 (OTX2⁺ ). Ctrl, cells were transfected by pEGFP-C1; (d) western blot analysis of SALL4 expression in piPSCs that were treated by OTX2 siRNAs. Ctrl, cells were treated by an unspecific siRNA; (e) constructs (left) and luciferase assay (right) of the full and truncated SALL4 promoter; (f) luciferase assay of SALL4 promoter activity. The SALL4 constructs with pE-OTX2 were cotransfected into 293T cells, respectively, for 36 h. Ctrl, cells without transfection of pE-OTX2. Data are presented as mean±SD. * P<0.05; ** P<0.01; n = 3.

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