FOXH1 Is Regulated by NANOG and LIN28 for Early-stage Reprogramming

Ling Wang,Chang Huang,Jiaqi Zhu,Young Tang,Alexander Chu,Alec Knupp,Yexuan Yin,Yue Su

doi:10.1038/s41598-019-52861-8

Ling Wang, Chang Huang + Show 6 more

Open Access

https://doi.org/10.1038/s41598-019-52861-8

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Journal: Scientific Reports	Publication Date: Nov 11, 2019
Citations: 11	License type: open-access

Affiliation: University of Connecticut

Abstract

FOXH1 is a primitive-streak specifier and ACTIVIN co-effector that plays an important role in development, and positively regulates the generation of human induced pluripotent stem cells (iPSCs) from somatic cells by OCT4, SOX2, KLF4, and MYC (OSKM) transduction. However, the mechanism and upstream regulation for FOXH1 expression in reprogramming are unclear. We found FOXH1 expression plays a significant role to enhance epithelial marker and suppress mesenchymal gene expression in OSKM-mediated human cell reprogramming. Furthermore, NANOG and LIN28 (NL) co-stimulate FOXH1 expression, which correlates with the enhanced reprogramming efficiency by NL-factors. FOXH1 expression is also stimulated by a specific inhibitor for H3K79 methyltransferase DOT1L (iDOT1L) but not by inhibition of the canonical WNT signaling. We further show that blocking endogenous FOXH1 expression eliminates the enhanced reprogramming effect by NL and iDOT1L. However, overexpressing FOXH1 in NL plus iDOT1L condition results in significantly reduced TRA-1-60 positively expressed cells and decreases pluripotent marker expression in reprogramming. Our study elucidated an essential role for properly stimulated FOXH1 expression by NANOG, LIN28, and H3K79 demethylation for dramatic enhancement of reprograming.

Highlights

Two gene cocktails including OCT4, SOX2, KLF4, c-MYC (OSKM)[1,2] and OCT4, SOX2, NANOG, LIN28A (OSNL)[3] can reprogram somatic cells to embryonic stem cell (ESC)-like, induced pluripotent stem cells
Quantitative reverse transcription-PCR on reprogramming day 14 cell RNAs showed that the expression of endogenous pluripotent genes OCT4, SOX2, and NANOG was further enhanced by addition of FOXH1 (Fig. 1C)
We found that FOXH1 expression significantly stimulated the epithelial markers E-CADHERIN (E-CAD) and OCLN (Fig. 1D), EPCAM expression was not stimulated by FOXH1 as previously reported in reprogramming human fibroblasts[7] (Fig. 1D)

Summary

Introduction

Two gene cocktails including OCT4, SOX2, KLF4, c-MYC (OSKM)[1,2] and OCT4, SOX2, NANOG, LIN28A (OSNL)[3] can reprogram somatic cells to embryonic stem cell (ESC)-like, induced pluripotent stem cells (iPSCs). We further found endogenous FOXH1 expression is necessary for the enhanced reprogramming effect of NL and inhibition of DOT1L, but overexpressing FOXH1 in NL and iDOT1L condition greatly reduces TRA-1-60 positive (+) cell population in reprogramming. Quantitative reverse transcription-PCR (qRT-PCR) on reprogramming day 14 cell RNAs showed that the expression of endogenous pluripotent genes OCT4, SOX2, and NANOG was further enhanced by addition of FOXH1 (Fig. 1C).

Results

Conclusion