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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-regulated genes in the Nodal and Wnt signaling pathways.Schematic diagram of the Nodal/TGF-β and Wnt signaling pathways generated by the Ingenuity Pathway Analysis Software. Genes differentially regulated by Zic3 are shown in colour – red for upregulation and green for downregulation of their expression patterns in the microarray data.
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pgen-1003852-g005: Zic3-regulated genes in the Nodal and Wnt signaling pathways.Schematic diagram of the Nodal/TGF-β and Wnt signaling pathways generated by the Ingenuity Pathway Analysis Software. Genes differentially regulated by Zic3 are shown in colour – red for upregulation and green for downregulation of their expression patterns in the microarray data.

Mentions: We identified 1316 genes differentially expressed in MO injected embryos (morphants, fold change >1.2; p≤0.05; Table S3). GO analysis revealed prominent enrichment in functions related to embryonic morphogenesis (Table S4). When the same or higher dose of MO (3.4 ng) was injected, the expression of several representative genes showed similar trend when measured by qPCR. This validated a possibility of their regulation by Zic3 (Fig. 4C; Table S7). We then determined the presence of Zic3 binding peaks within 100 kb of the TSS of these differentially expressed genes, which we defined as a selection criterion for Zic3 target gene. Based on this selection, 454 genes out of the total 1316 were identified as putative targets of Zic3 (Table S5 and Table S6). This set contains genes of the Nodal signaling pathway such as oep, lft1 and pitx2 (Fig. 5). While the presence of Zic3 binding in association with oep suggests direct regulation of Nodal pathway, the association of Zic3 peaks with lft1 and pitx2 suggests that Zic3 could also regulate the pathway through its modulators [42], [43]. These three genes, along with other members of this pathway not associated with Zic3 peaks (foxh1, bon, and gsc), were concurrently upregulated in Zic3 morphants (Fig. 4C; Table S3) suggesting negative regulation of the Nodal pathway by Zic3. Inhibition of Nodal signaling indicates suppression of endodermal fate [15], [44]–[46]. This correlated with broader expression of endodermal marker sox17a in 8 hpf Zic3 morphants (Fig. S4A). The inhibition of endodermal development by Zic3 is in line with previous observation in murine ES cells [38]. Similarly, peaks were associated with three genes of the canonical Wnt signaling pathway: axin1, jun, and vent (Table S5). In support of this association, microarray analysis revealed that the negative regulator of canonical Wnt pathway axin1 was downregulated in Zic3 morphants, while the downstream components jun and vent were upregulated (Fig. 5; Table S3). The expression of some other members of this pathway (axin2 and nlk1) without association with peaks has changed in Zic3 morphants based on microarray data. This implied that such genes could be the indirect targets of Zic3. Such observation provided further support for Zic3 regulation of the canonical Wnt pathway. The inhibition of canonical Wnt signaling by Zic3 was previously reported in frogs as a mechanism for organizer development [16]. Interestingly, Zic3 LOF only affected downstream components of these signaling pathways, and not the ligands, suggesting that at 8 hpf Zic3 is more likely to modulate the response to Wnt signaling in the target cells rather than initiation of signaling.


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-regulated genes in the Nodal and Wnt signaling pathways.Schematic diagram of the Nodal/TGF-β and Wnt signaling pathways generated by the Ingenuity Pathway Analysis Software. Genes differentially regulated by Zic3 are shown in colour – red for upregulation and green for downregulation of their expression patterns in the microarray data.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003852-g005: Zic3-regulated genes in the Nodal and Wnt signaling pathways.Schematic diagram of the Nodal/TGF-β and Wnt signaling pathways generated by the Ingenuity Pathway Analysis Software. Genes differentially regulated by Zic3 are shown in colour – red for upregulation and green for downregulation of their expression patterns in the microarray data.
Mentions: We identified 1316 genes differentially expressed in MO injected embryos (morphants, fold change >1.2; p≤0.05; Table S3). GO analysis revealed prominent enrichment in functions related to embryonic morphogenesis (Table S4). When the same or higher dose of MO (3.4 ng) was injected, the expression of several representative genes showed similar trend when measured by qPCR. This validated a possibility of their regulation by Zic3 (Fig. 4C; Table S7). We then determined the presence of Zic3 binding peaks within 100 kb of the TSS of these differentially expressed genes, which we defined as a selection criterion for Zic3 target gene. Based on this selection, 454 genes out of the total 1316 were identified as putative targets of Zic3 (Table S5 and Table S6). This set contains genes of the Nodal signaling pathway such as oep, lft1 and pitx2 (Fig. 5). While the presence of Zic3 binding in association with oep suggests direct regulation of Nodal pathway, the association of Zic3 peaks with lft1 and pitx2 suggests that Zic3 could also regulate the pathway through its modulators [42], [43]. These three genes, along with other members of this pathway not associated with Zic3 peaks (foxh1, bon, and gsc), were concurrently upregulated in Zic3 morphants (Fig. 4C; Table S3) suggesting negative regulation of the Nodal pathway by Zic3. Inhibition of Nodal signaling indicates suppression of endodermal fate [15], [44]–[46]. This correlated with broader expression of endodermal marker sox17a in 8 hpf Zic3 morphants (Fig. S4A). The inhibition of endodermal development by Zic3 is in line with previous observation in murine ES cells [38]. Similarly, peaks were associated with three genes of the canonical Wnt signaling pathway: axin1, jun, and vent (Table S5). In support of this association, microarray analysis revealed that the negative regulator of canonical Wnt pathway axin1 was downregulated in Zic3 morphants, while the downstream components jun and vent were upregulated (Fig. 5; Table S3). The expression of some other members of this pathway (axin2 and nlk1) without association with peaks has changed in Zic3 morphants based on microarray data. This implied that such genes could be the indirect targets of Zic3. Such observation provided further support for Zic3 regulation of the canonical Wnt pathway. The inhibition of canonical Wnt signaling by Zic3 was previously reported in frogs as a mechanism for organizer development [16]. Interestingly, Zic3 LOF only affected downstream components of these signaling pathways, and not the ligands, suggesting that at 8 hpf Zic3 is more likely to modulate the response to Wnt signaling in the target cells rather than initiation of signaling.

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