Dataset Information

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.

SUBMITTER: Abbasi AA

PROVIDER: S-EPMC1838922 | biostudies-literature | 2007 Apr

REPOSITORIES: biostudies-literature

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Human GLI3 intragenic conserved non-coding sequences are tissue-specific enhancers.

Abbasi Amir Ali AA Paparidis Zissis Z Malik Sajid S Goode Debbie K DK Callaway Heather H Elgar Greg G Grzeschik Karl-Heinz KH

PloS one 20070411 4

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 T ...[more]

PMID: 17426814

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Project description:Comparison of polymorphism at synonymous and non-synonymous sites in protein-coding DNA can provide evidence for selective constraint. Non-coding DNA that forms part of the regulatory landscape presents more of a challenge since there is not such a clear-cut distinction between sites under stronger and weaker selective constraint. Here, we consider putative regulatory elements termed Conserved Non-coding Elements (CNEs) defined by their high level of sequence identity across all vertebrates. Some mutations in these regions have been implicated in developmental disorders; we analyse CNE polymorphism data to investigate whether such deleterious effects are widespread in humans. Single nucleotide variants from the HapMap and 1000 Genomes Projects were mapped across nearly 2000 CNEs. In the 1000 Genomes data we find a significant excess of rare derived alleles in CNEs relative to coding sequences; this pattern is absent in HapMap data, apparently obscured by ascertainment bias. The distribution of polymorphism within CNEs is not uniform; we could identify two categories of sites by exploiting deep vertebrate alignments: stretches that are non-variant, and those that have at least one substitution. The conserved category has fewer polymorphic sites and a greater excess of rare derived alleles, which can be explained by a large proportion of sites under strong purifying selection within humans--higher than that for non-synonymous sites in most protein coding regions, and comparable to that at the strongly conserved trans-dev genes. Conversely, the more evolutionarily labile CNE sites have an allele frequency distribution not significantly different from non-synonymous sites. Future studies should exploit genome-wide re-sequencing to obtain better coverage in selected non-coding regions, given the likelihood that mutations in evolutionarily conserved enhancer sequences are deleterious. Discovery pipelines should validate non-coding variants to aid in identifying causal and risk-enhancing variants in complex disorders, in contrast to the current focus on exome sequencing.

Project description:BACKGROUND:Identifying DNA sequences (enhancers) that direct the precise spatial and temporal expression of developmental control genes remains a significant challenge in the annotation of vertebrate genomes. Locating these sequences, which in many cases lie at a great distance from the transcription start site, has been a major obstacle in deciphering gene regulation. Coupling of comparative genomics with functional validation to locate such regulatory elements has been a successful method in locating many such regulatory elements. But most of these studies looked either at a single gene only or the whole genome without focusing on any particular process. The pressing need is to integrate the tools of comparative genomics with knowledge of developmental biology to validate enhancers for developmental transcription factors in greater detail RESULTS:Our results show that near four different genes (nkx3.2, pax9, otx1b and foxa2) in zebrafish, only 20-30% of highly conserved DNA sequences can act as developmental enhancers irrespective of the tissue the gene expresses in. We find that some genes also have multiple conserved enhancers expressing in the same tissue at the same or different time points in development. We also located non-conserved enhancers for two of the genes (pax9 and otx1b). Our modified Bacterial artificial chromosome (BACs) studies for these 4 genes revealed that many of these enhancers work in a synergistic fashion, which cannot be captured by individual DNA constructs and are not conserved at the sequence level. Our detailed biochemical and transgenic analysis revealed Foxa1 binds to the otx1b non-conserved enhancer to direct its activity in forebrain and otic vesicle of zebrafish at 24 hpf. CONCLUSION:Our results clearly indicate that high level of functional conservation of genes is not necessarily associated with sequence conservation of its regulatory elements. Moreover certain non conserved DNA elements might have role in gene regulation. The need is to bring together multiple approaches to bear upon individual genes to decipher all its regulatory elements.

Project description:BackgroundA substantial fraction of non-coding DNA sequences of multicellular eukaryotes is under selective constraint. In particular, approximately 5% of the human genome consists of conserved non-coding sequences (CNSs). CNSs differ from other genomic sequences in their nucleotide composition and must play important functional roles, which mostly remain obscure.ResultsWe investigated relative abundances of short sequence motifs in all human CNSs present in the human/mouse whole-genome alignments vs. three background sets of sequences: (i) weakly conserved or unconserved non-coding sequences (non-CNSs); (ii) near-promoter sequences (located between nucleotides -500 and -1500, relative to a start of transcription); and (iii) random sequences with the same nucleotide composition as that of CNSs. When compared to non-CNSs and near-promoter sequences, CNSs possess an excess of AT-rich motifs, often containing runs of identical nucleotides. In contrast, when compared to random sequences, CNSs contain an excess of GC-rich motifs which, however, lack CpG dinucleotides. Thus, abundance of short sequence motifs in human CNSs, taken as a whole, is mostly determined by their overall compositional properties and not by overrepresentation of any specific short motifs. These properties are: (i) high AT-content of CNSs, (ii) a tendency, probably due to context-dependent mutation, of A's and T's to clump, (iii) presence of short GC-rich regions, and (iv) avoidance of CpG contexts, due to their hypermutability. Only a small number of short motifs, overrepresented in all human CNSs are similar to binding sites of transcription factors from the FOX family.ConclusionHuman CNSs as a whole appear to be too broad a class of sequences to possess strong footprints of any short sequence-specific functions. Such footprints should be studied at the level of functional subclasses of CNSs, such as those which flank genes with a particular pattern of expression. Overall properties of CNSs are affected by patterns in mutation, suggesting that selection which causes their conservation is not always very strong.

Project description:In addition to protein coding sequence, the human genome contains a significant amount of regulatory DNA, the identification of which is proving somewhat recalcitrant to both in silico and functional methods. An approach that has been used with some success is comparative sequence analysis, whereby equivalent genomic regions from different organisms are compared in order to identify both similarities and differences. In general, similarities in sequence between highly divergent organisms imply functional constraint. We have used a whole-genome comparison between humans and the pufferfish, Fugu rubripes, to identify nearly 1,400 highly conserved non-coding sequences. Given the evolutionary divergence between these species, it is likely that these sequences are found in, and furthermore are essential to, all vertebrates. Most, and possibly all, of these sequences are located in and around genes that act as developmental regulators. Some of these sequences are over 90% identical across more than 500 bases, being more highly conserved than coding sequence between these two species. Despite this, we cannot find any similar sequences in invertebrate genomes. In order to begin to functionally test this set of sequences, we have used a rapid in vivo assay system using zebrafish embryos that allows tissue-specific enhancer activity to be identified. Functional data is presented for highly conserved non-coding sequences associated with four unrelated developmental regulators (SOX21, PAX6, HLXB9, and SHH), in order to demonstrate the suitability of this screen to a wide range of genes and expression patterns. Of 25 sequence elements tested around these four genes, 23 show significant enhancer activity in one or more tissues. We have identified a set of non-coding sequences that are highly conserved throughout vertebrates. They are found in clusters across the human genome, principally around genes that are implicated in the regulation of development, including many transcription factors. These highly conserved non-coding sequences are likely to form part of the genomic circuitry that uniquely defines vertebrate development.

Dataset Information

Human GLI3 intragenic conserved non-coding sequences are tissue-specific enhancers.

Publications

Human GLI3 intragenic conserved non-coding sequences are tissue-specific enhancers.

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