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Genome wide analysis reveals Zic3 interaction with distal regulatory elements of stage specific developmental genes in zebrafish.

Winata CL, Kondrychyn I, Kumar V, Srinivasan KG, Orlov Y, Ravishankar A, Prabhakar S, Stanton LW, Korzh V, Mathavan S - PLoS Genet. (2013)

Bottom Line: Here we use the zebrafish as a model to study the developmental role of Zic3 in vivo, by applying a combination of two powerful genomics approaches--ChIP-seq and microarray.Analysis of Zic3 binding sites revealed a distribution biased towards distal intergenic regions, indicative of a long distance regulatory mechanism; some of these binding sites are highly conserved during evolution and act as functional enhancers.This demonstrated that Zic3 regulation of developmental genes is achieved predominantly through long distance regulatory mechanism and revealed that developmental transitions could be accompanied by dramatic changes in regulatory landscape.

View Article: PubMed Central - PubMed

Affiliation: Human Genetics, Genome Institute of Singapore, Singapore, Singapore.

ABSTRACT
Zic3 regulates early embryonic patterning in vertebrates. Loss of Zic3 function is known to disrupt gastrulation, left-right patterning, and neurogenesis. However, molecular events downstream of this transcription factor are poorly characterized. Here we use the zebrafish as a model to study the developmental role of Zic3 in vivo, by applying a combination of two powerful genomics approaches--ChIP-seq and microarray. Besides confirming direct regulation of previously implicated Zic3 targets of the Nodal and canonical Wnt pathways, analysis of gastrula stage embryos uncovered a number of novel candidate target genes, among which were members of the non-canonical Wnt pathway and the neural pre-pattern genes. A similar analysis in zic3-expressing cells obtained by FACS at segmentation stage revealed a dramatic shift in Zic3 binding site locations and identified an entirely distinct set of target genes associated with later developmental functions such as neural development. We demonstrate cis-regulation of several of these target genes by Zic3 using in vivo enhancer assay. Analysis of Zic3 binding sites revealed a distribution biased towards distal intergenic regions, indicative of a long distance regulatory mechanism; some of these binding sites are highly conserved during evolution and act as functional enhancers. This demonstrated that Zic3 regulation of developmental genes is achieved predominantly through long distance regulatory mechanism and revealed that developmental transitions could be accompanied by dramatic changes in regulatory landscape.

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Zic3 regulates neural-specific expression through CNEs.A, number of CNEs found among the Zic3 peaks. A large subset was conserved between zebrafish and Tetraodon, while a smaller subset was conserved between zebrafish and human. B, distribution of CNE peaks with regards to their distance from TSS of genes. Enrichment of biological process (C) and tissue-specific expression (D) terms among genes associated with CNE peaks. Light and dark grey bars represent expected and observed enrichments of functional categories according to DAVID GO terms. E–H, representative figure of 24 hpf F0 embryos expressing gfp (left panel) driven by Zic3 CNE peaks shown in UCSC browser image (right panel, green arrows); black horizontal line at the top of each panel represents 100 kb. E*, H*, dorsal view; F*, G*, lateral view.
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pgen-1003852-g008: Zic3 regulates neural-specific expression through CNEs.A, number of CNEs found among the Zic3 peaks. A large subset was conserved between zebrafish and Tetraodon, while a smaller subset was conserved between zebrafish and human. B, distribution of CNE peaks with regards to their distance from TSS of genes. Enrichment of biological process (C) and tissue-specific expression (D) terms among genes associated with CNE peaks. Light and dark grey bars represent expected and observed enrichments of functional categories according to DAVID GO terms. E–H, representative figure of 24 hpf F0 embryos expressing gfp (left panel) driven by Zic3 CNE peaks shown in UCSC browser image (right panel, green arrows); black horizontal line at the top of each panel represents 100 kb. E*, H*, dorsal view; F*, G*, lateral view.

Mentions: To study whether Zic3 binding sites were evolutionarily conserved, we overlapped the 8 hpf dataset with a list of known conserved non-coding elements (CNEs; ANCORA database) [64].We identified 228 peaks as CNEs conserved between zebrafish and Tetraodon, and 56 as CNEs conserved between zebrafish and humans (Fig. 8A), with 31 in common between the two groups. Similar to the distribution profile of the full set of peaks, these CNE peaks were mostly located outside of the basal promoter region (Fig. 8B). Genes associated with these CNEs were enriched for developmental functions and neural tissue-specific expression (Fig. 8C,D; Table S2).


Genome wide analysis reveals Zic3 interaction with distal regulatory elements of stage specific developmental genes in zebrafish.

Winata CL, Kondrychyn I, Kumar V, Srinivasan KG, Orlov Y, Ravishankar A, Prabhakar S, Stanton LW, Korzh V, Mathavan S - PLoS Genet. (2013)

Zic3 regulates neural-specific expression through CNEs.A, number of CNEs found among the Zic3 peaks. A large subset was conserved between zebrafish and Tetraodon, while a smaller subset was conserved between zebrafish and human. B, distribution of CNE peaks with regards to their distance from TSS of genes. Enrichment of biological process (C) and tissue-specific expression (D) terms among genes associated with CNE peaks. Light and dark grey bars represent expected and observed enrichments of functional categories according to DAVID GO terms. E–H, representative figure of 24 hpf F0 embryos expressing gfp (left panel) driven by Zic3 CNE peaks shown in UCSC browser image (right panel, green arrows); black horizontal line at the top of each panel represents 100 kb. E*, H*, dorsal view; F*, G*, lateral view.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814314&req=5

pgen-1003852-g008: Zic3 regulates neural-specific expression through CNEs.A, number of CNEs found among the Zic3 peaks. A large subset was conserved between zebrafish and Tetraodon, while a smaller subset was conserved between zebrafish and human. B, distribution of CNE peaks with regards to their distance from TSS of genes. Enrichment of biological process (C) and tissue-specific expression (D) terms among genes associated with CNE peaks. Light and dark grey bars represent expected and observed enrichments of functional categories according to DAVID GO terms. E–H, representative figure of 24 hpf F0 embryos expressing gfp (left panel) driven by Zic3 CNE peaks shown in UCSC browser image (right panel, green arrows); black horizontal line at the top of each panel represents 100 kb. E*, H*, dorsal view; F*, G*, lateral view.
Mentions: To study whether Zic3 binding sites were evolutionarily conserved, we overlapped the 8 hpf dataset with a list of known conserved non-coding elements (CNEs; ANCORA database) [64].We identified 228 peaks as CNEs conserved between zebrafish and Tetraodon, and 56 as CNEs conserved between zebrafish and humans (Fig. 8A), with 31 in common between the two groups. Similar to the distribution profile of the full set of peaks, these CNE peaks were mostly located outside of the basal promoter region (Fig. 8B). Genes associated with these CNEs were enriched for developmental functions and neural tissue-specific expression (Fig. 8C,D; Table S2).

Bottom Line: Here we use the zebrafish as a model to study the developmental role of Zic3 in vivo, by applying a combination of two powerful genomics approaches--ChIP-seq and microarray.Analysis of Zic3 binding sites revealed a distribution biased towards distal intergenic regions, indicative of a long distance regulatory mechanism; some of these binding sites are highly conserved during evolution and act as functional enhancers.This demonstrated that Zic3 regulation of developmental genes is achieved predominantly through long distance regulatory mechanism and revealed that developmental transitions could be accompanied by dramatic changes in regulatory landscape.

View Article: PubMed Central - PubMed

Affiliation: Human Genetics, Genome Institute of Singapore, Singapore, Singapore.

ABSTRACT
Zic3 regulates early embryonic patterning in vertebrates. Loss of Zic3 function is known to disrupt gastrulation, left-right patterning, and neurogenesis. However, molecular events downstream of this transcription factor are poorly characterized. Here we use the zebrafish as a model to study the developmental role of Zic3 in vivo, by applying a combination of two powerful genomics approaches--ChIP-seq and microarray. Besides confirming direct regulation of previously implicated Zic3 targets of the Nodal and canonical Wnt pathways, analysis of gastrula stage embryos uncovered a number of novel candidate target genes, among which were members of the non-canonical Wnt pathway and the neural pre-pattern genes. A similar analysis in zic3-expressing cells obtained by FACS at segmentation stage revealed a dramatic shift in Zic3 binding site locations and identified an entirely distinct set of target genes associated with later developmental functions such as neural development. We demonstrate cis-regulation of several of these target genes by Zic3 using in vivo enhancer assay. Analysis of Zic3 binding sites revealed a distribution biased towards distal intergenic regions, indicative of a long distance regulatory mechanism; some of these binding sites are highly conserved during evolution and act as functional enhancers. This demonstrated that Zic3 regulation of developmental genes is achieved predominantly through long distance regulatory mechanism and revealed that developmental transitions could be accompanied by dramatic changes in regulatory landscape.

Show MeSH
Related in: MedlinePlus