Human GLI3 Intragenic Conserved Non-Coding Sequences Are Tissue-Specific Enhancers

Abstract

The zinc-finger transcription factor GLI3 is a key regulator of development, acting as a primary transducer of Sonic hedgehog (SHH) signaling in a combinatorial context dependent fashion controlling multiple patterning steps in different tissues/organs. A tight temporal and spatial control of gene expression is indispensable, however, cis-acting sequence elements regulating GLI3 expression have not yet been reported. We show that 11 ancient genomic DNA signatures, conserved from the pufferfish Takifugu (Fugu) rubripes to man, are distributed throughout the introns of human GLI3. They map within larger conserved non-coding elements (CNEs) that are found in the tetrapod lineage. Full length CNEs transiently transfected into human cell cultures acted as cell type specific enhancers of gene transcription. The regulatory potential of these elements is conserved and was exploited to direct tissue specific expression of a reporter gene in zebrafish embryos. Assays of deletion constructs revealed that the human-Fugu conserved sequences within the GLI3 intronic CNEs were essential but not sufficient for full-scale transcriptional activation. The enhancer activity of the CNEs is determined by a combinatorial effect of a core sequence conserved between human and teleosts (Fugu) and flanking tetrapod-specific sequences, suggesting that successive clustering of sequences with regulatory potential around an ancient, highly conserved nucleus might be a possible mechanism for the evolution of cis-acting regulatory elements.

Wnt canonical pathway restricts graded Shh/Gli patterning activity through the regulation of Gli3 expression

“…Gain and loss of ␤-catenin/Tcf function in chick embryos also directly regulates Gli3 expression. Furthermore we characterized 245 RESEARCH ARTICLE Wnt activity in neural tube patterning four enhancer modules within the human GLI3 locus (Abbasi et al, 2007) in which core-consensus Tcf-binding sites are highly conserved throughout vertebrate species. We showed that two of these enhancer modules (HCNR2 and HCNR3) contain sufficient information to direct expression of Gli3 to the dorsal spinal cord, and that activity of these two modules is dependent on ␤-Catenin/Tcf transcriptional activity.…”

“…A total of 13 (R1-13) highly conserved non-coding DNA regions (HCNR) were found on a global alignment of ~300 kb, among widely divergent vertebrate species [including human, mouse, chick, Xenopus and Fugu (see Fig. S3 in the supplementary material) (Abbasi et al, 2007)]. These HCNR are distributed across almost the entire GLI3 locus interval, with at least one element on each intron (Fig.…”

Phylogenetic study and barcoding of the blood cockle, Tegillarca granosa, found on the west coast of peninsular Malaysia using the COI gene

“…The presence of conserved regions in the mitochondrial genome is often utilized, particularly in phylogenetic studies ranging from parasites (Gibson et al, 2005) to fish (Abbasi et al, 2007). According to Remigio and Hebert (2003), shellfish identification using the conserved regions of a sequence is remarkable especially with the COI gene because it enables the recovery of shallow divergences and more robust estimates of shellfish relationships.…”

Antagonism between Hedgehog and Wnt signaling pathways regulates tumorigenicity (Review)

“…Furthermore, previous studies characterized four highly conserved non-coding DNA regions (HCNRs) within the human GLI3 locus that work as potential enhancer modules; it was demonstrated that HCNR2 and HCNR3 contain sufficient information to direct the expression of GLI3 in the dorsal spinal cord, and that the activity of these two modules is dependent on β-catenin/Tcf transcriptional activity (90)(91)(92). Collectively, these data demonstrate that the Wnt/β-catenin pathway downregulates GLI3 expression, indicating an indirect mechanism initiated by Wnt signaling to repress Shh activity in the dorsal neural tube (90)(91)(92).…”