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

Amir Ali Abbasi,Greg Elgar,Heather Callaway,Zissis Paparidis,Debbie K Goode,Sajid Malik,Karl-Heinz Grzeschik,Jean-Nicolas Volff

doi:10.1371/journal.pone.0000366

Amir Ali Abbasi, Greg Elgar + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0000366

Copy DOI

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.

Highlights

Mutations in the human transcription factor GLI3 cause a variety of dominant developmental defect syndromes, subsumed under the term ‘‘GLI3 morphopathies’’ [1], including Greig cephalopolysyndactyly syndrome (GCPS) [2,3,4], Pallister-Hall syndrome (PHS) [5], postaxial polydactyly type A (PAPA) [6], and preaxial polydactyly type IV (PPD-IV) [1]]
It is more likely that non-coding sequences conserved between human and from the pufferfish Takifugu (Fugu) and which might be potential enhancers, are restricted to GLI3 introns
Comparison of approximately 1 Mb human genomic DNA sequence encompassing GLI3 and extending up to the flanking genes with the complete assembly of the Takifugu rubripes genome sequence indicates that sequence homology is restricted to the gene region proper (Figure 1)

Summary

Introduction

Mutations in the human transcription factor GLI3 cause a variety of dominant developmental defect syndromes, subsumed under the term ‘‘GLI3 morphopathies’’ [1], including Greig cephalopolysyndactyly syndrome (GCPS) [2,3,4], Pallister-Hall syndrome (PHS) [5], postaxial polydactyly type A (PAPA) [6], and preaxial polydactyly type IV (PPD-IV) [1]]. All GLI3 morphopathies show malformations of the autopod, i.e. polydactylies or syndactylies. Functional haploinsufficiency of GLI3 appears to cause GCPS, since deletions or translocations eliminating one allele as well as missense or nonsense mutations distributed over the entire coding sequence are associated with this phenotype [2,4,12]

Methods

Results

Conclusion