Limits...
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.

Show MeSH

Related in: MedlinePlus

Distribution of Zic3 peaks as identified in ChIP-seq experiments according to GREAT algorithm.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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3814314&req=5

pgen-1003852-g002: Distribution of Zic3 peaks as identified in ChIP-seq experiments according to GREAT algorithm.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.

Mentions: Sequencing of the 8 hpf ChIP sample generated 23,945,552 reads (11,037,221 or 46% were mapped to the zebrafish genome); the 24 hpf ChIP sample generated 23,083,504 reads (11,797,011 or 51% were mapped). We identified 3209 and 2088 Zic3 binding sites (hereafter referred to as peaks) with high significance value at 8 hpf (Table S13) and 24 hpf (Table S14), respectively. Interestingly, both datasets showed that only a small fraction (8.6% at 8 hpf and 4% at 24 hpf) of the peaks mapped to promoter regions (within 5 kb of transcription start site, TSS), while the rest were aligned to intragenic (26.8% at 8 hpf and 29% at 24 hpf) and intergenic (64.6% at 8 hpf and 67% at 24 hpf) regions (Fig. 2A). This suggested that Zic3 mainly acts via distal regulatory elements. To validate the ChIP-seq performance, we carried out quantitative PCR (qPCR) on randomly selected peaks from the 8 hpf dataset, five within promoter region and sixteen at regions outside of gene promoters. Taking a fold-change of 2 as a cutoff for positive enrichment, the qPCR analysis validated all but one peak tested (Table S1).


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)

Distribution of Zic3 peaks as identified in ChIP-seq experiments according to GREAT algorithm.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.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003852-g002: Distribution of Zic3 peaks as identified in ChIP-seq experiments according to GREAT algorithm.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.
Mentions: Sequencing of the 8 hpf ChIP sample generated 23,945,552 reads (11,037,221 or 46% were mapped to the zebrafish genome); the 24 hpf ChIP sample generated 23,083,504 reads (11,797,011 or 51% were mapped). We identified 3209 and 2088 Zic3 binding sites (hereafter referred to as peaks) with high significance value at 8 hpf (Table S13) and 24 hpf (Table S14), respectively. Interestingly, both datasets showed that only a small fraction (8.6% at 8 hpf and 4% at 24 hpf) of the peaks mapped to promoter regions (within 5 kb of transcription start site, TSS), while the rest were aligned to intragenic (26.8% at 8 hpf and 29% at 24 hpf) and intergenic (64.6% at 8 hpf and 67% at 24 hpf) regions (Fig. 2A). This suggested that Zic3 mainly acts via distal regulatory elements. To validate the ChIP-seq performance, we carried out quantitative PCR (qPCR) on randomly selected peaks from the 8 hpf dataset, five within promoter region and sixteen at regions outside of gene promoters. Taking a fold-change of 2 as a cutoff for positive enrichment, the qPCR analysis validated all but one peak tested (Table S1).

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