The Krüppel-like zinc finger transcription factor, GLI-similar 1, is regulated by hypoxia-inducible factors via non-canonical mechanisms Elham Khalesi a , Hideaki Nakamura b , Kian Leong Lee c , Andika Chandra Putra d , Takahiro Fukazawa a , Yumi Kawahara a , Yuichi Makino e , Lorenz Poellinger b,c , Louis Yuge a , Keiji Tanimoto d,⇑ a Department of Bio-Environmental Adaptation Sciences, Graduate School of Health Sciences, Hiroshima University, Hiroshima, Japan b Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden c Cancer Science Institute of Singapore, National University of Singapore, Singapore d Department of Radiation Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan e Department of Internal Medicine, Asahikawa Medical University, Asahikawa, Japan article info Article history: Received 12 October 2013 Available online 25 October 2013 Keywords: GLIS1 Hypoxia HIF AP1 Gene transcription abstract GLI-similar 1 (GLIS1) is important for the reprogramming of fibroblasts into induced pluripotent stem cells (iPSCs). However, the molecular mechanisms of regulation of GLIS1 expression remain unclear. We have therefore examined GLIS1 expression in various cancer cell lines and demonstrated that GLIS1 expression was dramatically increased under hypoxic conditions. Importantly, GLIS1 expression was sig- nificantly attenuated in VHL-overexpressing renal cell carcinoma cells compared to the VHL-deficient parent control. Moreover, promoter analysis demonstrated that GLIS1 transcription was regulated by hypoxia through a hypoxia-inducible factors (HIFs)-dependent mechanism. Co-transfection experiments revealed that HIF-2a had greater potency on the GLIS1 promoter activation than HIF-1a. Subsequent studies using wild-type and mutant HIF-2a demonstrated that DNA binding activity was not necessary but TADs were critical for GLIS1 induction. Finally, co-transfection experiments indicated that HIF-2a cooperated with AP-1 family members in upregulating GLIS1 transcription. These results suggest that the hypoxic signaling pathway may play a pivotal role in regulating the reprogramming factor GLIS1, via non-canonical mechanisms involving partner transcription factor rather than by direct HIF transactivation. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction The Krüppel-like protein Gli-similar 1 (GLIS1) was reported to be both temporally and spatially regulated, suggesting that it may play a role in the regulation of embryonic developmental pro- grams at specific stages [1,2]. GLIS1 expression was increased by phorbol-12-myristate-13-acetate (PMA) or interferon c treatment and stable transfection of GLIS1DC that lacks activation domains prompted PMA-induced epidermal differentiation. This indicated a regulatory role for GLIS1 in aberrant epidermal differentiation and remodeling of tumorigenic PMA-treated psoriatic skin [3]. Cru- cially, GLIS1 markedly enhances the generation of induced plurip- otent stem cells (iPSCs) from both mouse and human fibroblasts when it is expressed together with OCT3/4 (POU5F1), SOX2 and KLF4. GLIS1 is able to replace oncogenic MYC, resulting in decreased tumorigenicity as well as improving safety and efficiency of iPS cell production, demonstrating its utility in stem cell biology [4]. Stem cells frequently reside in a specialized physiological microenvironment called the stem cell niche. The role of the niche is to maintain stem cell properties such as pluripotency and self- renewal. Recent evidence suggests that hypoxia may be an impor- tant physiological component of the microenvironment necessary for stem cell maintenance [5,6]. Consistent with this, hypoxic (low oxygen) conditions were able to improve the efficiency of iPS cell generation from mouse and human somatic cells [7]. In- deed, the master regulator of stemness Oct-4 is a direct transcrip- tional target of hypoxia-inducible factor-2-alpha (HIF-2a also known as endothelial PAS domain protein 1 or EPAS1) which is one of the main transcription factors of the hypoxia pathway further highlighting the importance of hypoxia in stem cells biology [8]. Mechanistically, hypoxia-inducible factor alpha (HIF-a) subunits are degraded through von Hippel–Lindau (VHL)- mediated ubiquitin–proteasomal degradation under normoxic conditions, and hypoxia stabilizes HIF-a thereby allowing their 0006-291X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2013.10.083 ⇑ Corresponding author. Fax: +81 82 256 7105. E-mail address: ktanimo@hiroshima-u.ac.jp (K. Tanimoto). Biochemical and Biophysical Research Communications 441 (2013) 499–506 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc