GLI1/GLI2 functional interplay is required to control Hedgehog/GLI targets gene expression

Abstract

The Hedgehog-regulated transcription factors GLI1 and GLI2 play overlapping roles in development and disease, however, the mechanisms underlying their interplay remain elusive. We report for the first time that GLI1 and GLI2 physically and functionally interact in cancer cells. GLI1 and GLI2 were shown to co-immunoprecipitate in PANC1 pancreatic cancer cells and RMS13 rhabdomyosarcoma cells. Mapping analysis demonstrated that the zinc finger domains of both proteins are required for their heteromerization. RNAi knockdown of either GLI1 or GLI2 inhibited expression of a number of well characterized GLI target genes (BCL2, MYCN PTCH2, IL7 and CCND1) in PANC1 cells, whereas PTCH1 expression was only inhibited by GLI1 depletion. qPCR screening of a large set of putative canonical and non-canonical Hedgehog/GLI targets identified further genes (e.g., E2F1, BMP1, CDK2) strongly downregulated by GLI1 and/or GLI2 depletion in PANC1 cells, and demonstrated that ANO1, AQP1 and SOCS1 are upregulated by knockdown of either GLI1 or GLI2. Chromatin immunoprecipitation showed that GLI1 and GLI2 occupied the same regions at the BCL2, MYCN and CCND1 promoters. Further, depletion of GLI1 inhibited GLI2 occupancy at these promoters, suggesting that GLI1/GLI2 interaction is required for the recruitment of GLI2 to these sites. Together, these findings indicate that GLI1 and GLI2 coordinately regulate the transcription of some genes, and provide mechanistic insight into the roles of GLI proteins in carcinogenesis.

Transcriptomic analysis of loss of Gli1 in neural stem cells responding to demyelination in the mouse brain

“…Table 2), consistent with their very distinct responses to demyelination. Many of these genes were downregulated in the nulls in agreement with the role of Gli1 as a transcriptional activator 43,44 .…”

Another twist to the GLI code

“…The proposed specific function of various GLI homo-and heterodimers is indicated by their effects on target genes. GLI1/GLI2 dimers have been identified in [6]. We propose the existence of other homo-and heterodimers (shaded shapes), based on reported biochemical interaction of the individual ZF domains, or unexpected functional outputs.…”

“…The gradient of Hh ligands regulates GLI1 expression and GLI2 and GLI3 PTMs and processing, while other signalling pathways activated by TGF-β and EGFR alter the balance through up-regulation of GLI2 and phosphorylation changes of GLI1 and GLI2. Based on the new findings of Tolosa et al [6] and earlier reports, dimerisation of GLI isoforms through their Zinc Finger (ZF) domain adds another level of transcriptional activity regulation. The proposed specific function of various GLI homo-and heterodimers is indicated by their effects on target genes.…”

“…Ruiz i Altaba's group coined the term 'GLI code' to convey the diverse functions of the three vertebrate GLI isoforms ( Figure 1) [3]. In this model, GLI1 is exclusively a transcriptional activator; however, research in the past decade has shown a darker side of GLI1, uncovering its potential to serve as a transcriptional repressor of selected genes, such as ANO1, CDH1, AQP1 and SOCS1 [4][5][6]. How can GLI1 mechanistically act as a repressor?…”

“…However, the role of GLI1 in maintaining cancer cells phenotype and/or in maintaining a stem-like state suggests a gain of function during tumour formation and progression that cannot totally be replaced by GLI2. A novel study by Tolosa et al [6] sheds new light onto these questions and offers mechanistic explanations for the exceptions to the GLI code.…”