Targeting of SET/I2PP2A oncoprotein inhibits Gli1 transcription revealing a new modulator of Hedgehog signaling

Iliana Serifi,Thomais Papamarcaki,Panagiota Giardoglou,Simoni Besta,Dimitris Beis,Zoe Karetsou,Pawel Niewiadomski

doi:10.1038/s41598-021-93440-0

Abstract

The Hedgehog (Hh)/Gli signaling pathway controls cell proliferation and differentiation, is critical for the development of nearly every tissue and organ in vertebrates and is also involved in tumorigenesis. In this study, we characterize the oncoprotein SET/I2PP2A as a novel regulator of Hh signaling. Our previous work has shown that the zebrafish homologs of SET are expressed during early development and localized in the ciliated organs. In the present work, we show that CRISPR/Cas9-mediated knockdown of setb gene in zebrafish embryos resulted in cyclopia, a characteristic patterning defect previously reported in Hh mutants. Consistent with these findings, targeting setb gene using CRISPR/Cas9 or a setb morpholino, reduced Gli1-dependent mCherry expression in the Hedgehog reporter zebrafish line Tg(12xGliBS:mCherry-NLS). Likewise, SET loss of function by means of pharmacological inhibition and gene knockdown prevented the increase of Gli1 expression in mammalian cells in vitro. Conversely, overexpression of SET resulted in an increase of the expression of a Gli-dependent luciferase reporter, an effect likely attributable to the relief of the Sufu-mediated inhibition of Gli1. Collectively, our data support the involvement of SET in Gli1-mediated transcription and suggest the oncoprotein SET/I2PP2A as a new modulator of Hedgehog signaling.

Highlights

The Hedgehog (Hh) signaling pathway controls cell proliferation and differentiation and is critical for the development of nearly every tissue and organ in vertebrates[1]
This study demonstrated that targeting setb gene expression by injections of setb gRNA with Cas[9] mRNA or seta/b morpholinos into one-cell stage zebrafish embryos resulted in several morphological abnormalities, including defects in spinal cord and eye development[36]
A systematic scoring of the eye defects in setb crispants showed that approximately 80% of the larvae with severe phenotype and 33% of the larvae with moderate phenotype exhibited cyclopia (Fig. 1A), a characteristic embryonic patterning defect reported in the Sonic Hedgehog mutant mouse and zebrafish[37,38]

Summary

Introduction

The Hedgehog (Hh) signaling pathway controls cell proliferation and differentiation and is critical for the development of nearly every tissue and organ in vertebrates[1]. The localization and the activity of Gli transcription factors, control both the amplitude and the outcome of the Hh expression program, especially during development, and are regulated by a complex combination of posttranslational modifications, including phosphorylation8,9, acetylation[10] and ubiquitination/proteasomal t runcation[11]. The oncoprotein SET is mainly a nuclear protein that functions as a histone c haperone[12,13], transcription cofactor[14,15,16], regulator of histone acetylation[17,18], modulator of DNA r epair[19] and as an epigenetic r egulator[20]. SET is an inhibitor of protein phosphatase 2A (PP2A)[21] and has been reported as a signaling amplifier of Rac1-dependent cell m igration[22]. We present several pieces of evidence which support the involvement of SET in Gli1-mediated transcription that plays a crucial role in the Hh signaling cascade

Methods

Results

Conclusion