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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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Candidate target genes regulated by Zic3 at 8 hpf and 24 hpf developmental stages.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.
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pgen-1003852-g006: Candidate target genes regulated by Zic3 at 8 hpf and 24 hpf developmental stages.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.

Mentions: To identify potential zic3 targets during late neurogenesis, we performed microarray expression analysis on 24 hpf GFP-positive zic3 expressing cells that were FACS-sorted (Table S8). Comparing expression levels to a control dataset derived from GFP-negative cells (cells negative for zic3 expression), we identified genes enriched in GFP-positive cells (zic3-expressing cells). A total of 689 genes (p-value<0.05; fold change ≥1.5) were enriched in zic3-expressing cells (zic3-coexpressed genes). Among these genes were six members of the Zic family and other genes expressed in the dorsal neural tube. This confirmed the identity of the sorted cells as dorsal neural cells. Among the zic3-coexpressed genes, 167 had at least one peak within 100 kb of their TSS, rendering them putative Zic3 targets (Table S10). Similar to the 8 hpf stage, members of the Wnt pathway were also among the targets. However, Zic3 seems to regulate a different set of Wnt components, including wnt11r and lef1 (Fig. 6, Table S8). qRT-PCR revealed that wnt11r, were down-regulated in Zic3 morphants at 24 hpf (Fig. 4C; Table S7), confirming their positive regulation by Zic3. Two other genes encoding Wnt ligands, wnt10a and wnt10b, were co-expressed with zic3, and regulated upon Zic3 knockdown (Table S7; Fig. 4C) although they were not associated with peaks in ChIP-seq, suggesting that they may be indirect targets of Zic3. A striking difference between 8 hpf and 24 hpf regulatory landscape is apparent from the distinct functions associated with Zic3 target genes at each stage. For example, many genes regulating cell migration and polarity were identified as Zic3 targets at 8 hpf, whereas at 24 hpf neural crest determinants were found. The latter included foxd3, and pax3a which were further confirmed to be responsive to Zic3 knockdown (Fig. 4C, Table S7, S11).


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)

Candidate target genes regulated by Zic3 at 8 hpf and 24 hpf developmental stages.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.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003852-g006: Candidate target genes regulated by Zic3 at 8 hpf and 24 hpf developmental stages.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.
Mentions: To identify potential zic3 targets during late neurogenesis, we performed microarray expression analysis on 24 hpf GFP-positive zic3 expressing cells that were FACS-sorted (Table S8). Comparing expression levels to a control dataset derived from GFP-negative cells (cells negative for zic3 expression), we identified genes enriched in GFP-positive cells (zic3-expressing cells). A total of 689 genes (p-value<0.05; fold change ≥1.5) were enriched in zic3-expressing cells (zic3-coexpressed genes). Among these genes were six members of the Zic family and other genes expressed in the dorsal neural tube. This confirmed the identity of the sorted cells as dorsal neural cells. Among the zic3-coexpressed genes, 167 had at least one peak within 100 kb of their TSS, rendering them putative Zic3 targets (Table S10). Similar to the 8 hpf stage, members of the Wnt pathway were also among the targets. However, Zic3 seems to regulate a different set of Wnt components, including wnt11r and lef1 (Fig. 6, Table S8). qRT-PCR revealed that wnt11r, were down-regulated in Zic3 morphants at 24 hpf (Fig. 4C; Table S7), confirming their positive regulation by Zic3. Two other genes encoding Wnt ligands, wnt10a and wnt10b, were co-expressed with zic3, and regulated upon Zic3 knockdown (Table S7; Fig. 4C) although they were not associated with peaks in ChIP-seq, suggesting that they may be indirect targets of Zic3. A striking difference between 8 hpf and 24 hpf regulatory landscape is apparent from the distinct functions associated with Zic3 target genes at each stage. For example, many genes regulating cell migration and polarity were identified as Zic3 targets at 8 hpf, whereas at 24 hpf neural crest determinants were found. The latter included foxd3, and pax3a which were further confirmed to be responsive to Zic3 knockdown (Fig. 4C, Table S7, S11).

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