Abstract
GLI1 is one of three transcription factors (GLI1, GLI2 and GLI3) that mediate the Hedgehog signal transduction pathway and play important roles in normal development. GLI1 and GLI2 form a positive-feedback loop and function as human oncogenes. The mouse and human GLI1 genes have untranslated 5′ exons and large introns 5′ of the translational start. Here we show that Sonic Hedgehog (SHH) stimulates occupancy in the introns by H3K27ac, H3K4me3 and the histone reader protein BRD4. H3K27ac and H3K4me3 occupancy is not significantly changed by removing BRD4 from the human intron and transcription start site (TSS) region. We identified six GLI binding sites (GBS) in the first intron of the human GLI1 gene that are in regions of high sequence conservation among mammals. GLI1 and GLI2 bind all of the GBS in vitro. Elimination of GBS1 and 4 attenuates transcriptional activation by GLI1. Elimination of GBS1, 2, and 4 attenuates transcriptional activation by GLI2. Eliminating all sites essentially eliminates reporter gene activation. Further, GLI1 binds the histone variant H2A.Z. These results suggest that GLI1 and GLI2 can regulate GLI1 expression through protein-protein interactions involving complexes of transcription factors, histone variants, and reader proteins in the regulatory intron of the GLI1 gene. GLI1 acting in trans on the GLI1 intron provides a mechanism for GLI1 positive feedback and auto-regulation. Understanding the combinatorial protein landscape in this locus will be important to interrupting the GLI positive feedback loop and providing new therapeutic approaches to cancers associated with GLI1 overexpression.
Highlights
The Sonic Hedgehog/Patched/GLI pathway is important for normal development, cancer and congenital anomalies [1,2,3]
Transcription Factor Chromatin immunoprecipitation (ChIP)-seq from ENCODE identified 161 factors in this region and Chromatin State Segmentation by HMM demonstrated the human intron is a regulatory region in nine cell lines
Both GLI2 occupancy of the regulatory region and active epigenetic histone marks are Sonic Hedgehog (SHH) dependent. This SHH effect requires GLI2 and/ or GLI3 as it is absent in GLI2/GLI3 null cells
Summary
The Sonic Hedgehog/Patched/GLI pathway is important for normal development, cancer and congenital anomalies [1,2,3]. SHH signaling, through PTCH/SMO, recruits GLI/SuFu to the ciliary tip where they are rapidly dissociated [4] This promotes nuclear localization of the GLI transcription factors allowing target gene regulation [5,6,7]. Clinical trials have been conducted to establish the efficacy of down-regulating the pathway in patients by interfering with SMO [12,13] This approach may not be effective in many patients because of non-canonical signaling, which directly activates GLI1 and bypasses the block at the level of SMO. Elucidating the combinatorial protein-protein interactions that regulate GLI1 expression will provide new therapeutic approaches to GLI1 induced cancers overcoming the shortfall of upstream inhibitors whose efficacy is limited by non-canonical signaling
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