Intronic regulation of human GLI1 DNA by cis DNA elements and epigenetic marks

Abstract

In humans, a large cancer burden is associated with dysregulation of the Hedgehog/Patched/GLI (HH/PCTH/GLI) pathway. The canonical Hedgehog signal (including Sonic Hedgehog [SHH]) is mediated by the GLI family of transcription factors (GLI1, GLI2 and GLI3). GLI1 plays an important role in normal development and differentiation and is a human oncogene that is believed to be active in up to one‐third of all cancers. GLI1 gene targets sustain proliferation, inhibit apoptosis, and promote angiogenesis and tumor cell migration. Wild‐type p53 competes with GLI1 for the co‐activator TAF9, inhibiting GLI1's oncogenic activity. GLI1 and GLI2 form a positive‐feedback loop that drives their expression. Mechanisms that regulate this positive‐feedback loop remain unknown. We identified 6 GLI binding sites (GBS) in the large first intron of the human GLI1 gene that are highly conserved among mammals and are associated with histone H3K27Ac marks and DNase hypersensitivity clusters. GLI1 binds all of the GBS and GLI2 binds 2 of them, shown using gel mobility shift assays. Elimination of some of the GBS attenuates transcriptional activation. Removal of all GBS eliminates reporter gene activation. Preliminary evidence suggests GLI1 multimerizes and can bind the pioneer protein CBP. ChIP qPCR shows SHH signal dependent (Smoothened agonist) occupancy of the 1st intron near the GBS by GLI2, and increased occupancy by the histones H3K4me3 and H3K27Ac and the histone bromodomain reader protein BRD4. BRD4 can be driven off the intron and transcription start site region with a small inhibitory molecule, I‐BET151, providing a possible route to disrupting GLI1 expression. Further, GLI1 binds the histone variant H2A.Z. These results suggest that GLI1 and GLI2 could auto‐regulate GLI1 expression at GBS in the 1st intron of GLI1 through protein‐protein interactions involving complexes of the transcription factors, histone variants, and reader proteins. Given the widespread involvement of GLI1 and GLI2 in human cancers including medulloblastoma and rhabdomyosarcoma, it is important to try and understand key elements of the regulation of their expression.Support or Funding InformationSupported in part by PHS grant ES1054, Illinois Excellence in Academic Medicine award, Illinois Regenerative Medicine Institute and the Eisenberg Foundation for Charities.