Genome Wide Analysis Reveals Zic3 Interaction with Distal Regulatory Elements of Stage Specific Developmental Genes in Zebrafish

<div><p>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 <i>in vivo</i>, 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 <i>zic3</i>-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 <i>in vivo</i> 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.</p></div

Candidate target genes regulated by Zic3 at 8 hpf and 24 hpf developmental stages.

<p>Target genes are grouped based on their signaling pathway or functions. Changes in expression in microarray are represented by red and green backgrounds for up- and down-regulation respectively.</p

Distribution of Zic3 peaks as identified in ChIP-seq experiments according to GREAT algorithm.

<p>A, distribution of peaks located in promoter (within 5 kb upstream of TSS), intragenic, and intergenic regions. B, gene association rule of ‘basal plus 100 kb’ according to GREAT algorithm. C, percentage of peaks associated with none, one, or two genes based on the gene association rule in B. D, number of peaks present in each distance categories along the x-axis, with regards to TSS of associated gene. E, region map showing overlap between genomic locations of peaks in 8 hpf and 24 hpf datasets. F–I, list of biological processes and tissue specific expression terms enriched among Zic3-associated genes at 8 hpf (F, G) and 24 hpf (H, I) according to DAVID GO terms. Light and dark grey bars represent the expected and observed enrichments of functional categories indicated along the y-axis.</p

Zic3 regulates neural-specific expression through CNEs.

<p>A, number of CNEs found among the Zic3 peaks. A large subset was conserved between zebrafish and <i>Tetraodon</i>, 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 <i>gfp</i> (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.</p

Zic3 binding sites associated with genes from the Nodal and Wnt pathway genes.

<p>UCSC browser image depicting genomic locations of Zic3 peaks identified near Nodal (A) and Wnt (B) pathway genes at 8 hpf. Single black vertical bars below histogram - peaks called by QuEST algorithm, blue horizontal bars - annotated exons (tall boxes), UTRs (half-sized boxes), introns (lines, arrowheads denote transcript orientation); Zic3 binding sites with negative (red arrows) and positive (green arrows) enhancer activity. Scale bars are indicated by black horizontal line at the top of each panel. C, list of tested fragments associated with Nodal and Wnt pathway genes. Enhancer-driven expression was assayed by qRT-PCR of <i>gfp</i> at 8 hpf and through microscopic observation of GFP expression pattern at 24 hpf. Between 50 to 100 embryos were assayed in each experimental time point. D–G, representative figure of <i>gfp</i> expression driven by selected fragments of Zic3 binding sites in F₀ embryos at 24 hpf, immunostained with anti-GFP antibody. D*, F*, dorsal view; E*, G*, lateral view.</p