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Zfp423 binds autoregulatory sites in p19 cell culture model.

Cho YW, Hong CJ, Hou A, Gent PM, Zhang K, Won KJ, Hamilton BA - PLoS ONE (2013)

Bottom Line: Both sites are significantly enriched in either quantitative PCR or massively parallel sequencing assays.A site in intron 5 acts as a classical enhancer in transient assays, but does not require the consensus motif for activity, suggesting a redundant or modulatory role for Zfp423 binding in this context.We speculate that Zfp423 may repress this enhancer as part of a developmental ratchet.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine and Cellular and Molecular Medicine, Institute for Genomic Medicine and Moores UCSD Cancer Center, University of California San Diego School of Medicine, La Jolla, California, United States of America.

ABSTRACT
Zfp423 is a 30 zinc finger transcription factor that forms regulatory complexes with EBF family members and factors targeted by canonical signaling pathways. Zfp423 mutations produce a range of developmental abnormalities in mice and humans related to the ciliopathies. Surprisingly, computational analysis of clustered Zfp423 and partner motifs in conserved genomic sequences predicts enrichment in Zfp423 and Ebf genes. In cell culture models selected for Zfp423 and EBF expression, we identify strong and reproducible occupancy of two Zfp423 intronic sites using chromatin immunoprecipitation with multiple independent antibodies. Both sites are significantly enriched in either quantitative PCR or massively parallel sequencing assays. A site in intron 5 acts as a classical enhancer in transient assays, but does not require the consensus motif for activity, suggesting a redundant or modulatory role for Zfp423 binding in this context. We speculate that Zfp423 may repress this enhancer as part of a developmental ratchet.

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Conserved non-coding sequences suggest Zfp423 autoregulatory sites.Analysis of vertebrate conserved non-coding sequences for co-occurrence of consensus motifs for Zfp423 (ROAZ), Ebf family members (OLF1), BMP-responsive SMADs and nuclear receptor motifs recognized by retinoic acid receptors (DR1, DR4) shows unexpected enrichment in Zfp423 and Ebf genes. (A) Table shows number of conserved sties for pairwise combinations of consensus binding sequences within 100 bp for two sites, 150 bp for three-site combinations. Alignments based on public assemblies of human (hg18), mouse (mm9), chicken (galGal3) and frog (xenTro2) genomes as of 5/1/2012. Number of sites conserved among the three species used for sorting is further reduced for sites excluding known protein coding sequences (H-M-C excl. cds). (B) Conserved, clustered sites identified in Zfp423 include three positions where Zfp423 and Ebf matrix predictions overlap, are indicated by vertical above a diagram of exon structure and pairwise conservation between mouse and other mammal (M) and non-mammal vertebrate (V) species, showing the locations of predicted binding sites. Adapted from UCSC Genome Browser. Similar schematics for (C) Ebf3 using the same parameters and (D) Ebf1 using larger window sizes. Numbers indicate the frequency of similar predicted sites in the genome for the parameters used to identify each.
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pone-0066514-g001: Conserved non-coding sequences suggest Zfp423 autoregulatory sites.Analysis of vertebrate conserved non-coding sequences for co-occurrence of consensus motifs for Zfp423 (ROAZ), Ebf family members (OLF1), BMP-responsive SMADs and nuclear receptor motifs recognized by retinoic acid receptors (DR1, DR4) shows unexpected enrichment in Zfp423 and Ebf genes. (A) Table shows number of conserved sties for pairwise combinations of consensus binding sequences within 100 bp for two sites, 150 bp for three-site combinations. Alignments based on public assemblies of human (hg18), mouse (mm9), chicken (galGal3) and frog (xenTro2) genomes as of 5/1/2012. Number of sites conserved among the three species used for sorting is further reduced for sites excluding known protein coding sequences (H-M-C excl. cds). (B) Conserved, clustered sites identified in Zfp423 include three positions where Zfp423 and Ebf matrix predictions overlap, are indicated by vertical above a diagram of exon structure and pairwise conservation between mouse and other mammal (M) and non-mammal vertebrate (V) species, showing the locations of predicted binding sites. Adapted from UCSC Genome Browser. Similar schematics for (C) Ebf3 using the same parameters and (D) Ebf1 using larger window sizes. Numbers indicate the frequency of similar predicted sites in the genome for the parameters used to identify each.

Mentions: To identify candidate target genes for Zfp423, we first looked for consensus binding sites in regions conserved among vertebrate genomes (Figure 1). Using the web-based SynoR software tool, which uses a matrix representation to account for degeneracy in binding sites [16], we separately examined paired or clustered sites for Zfp423 [11] and its best-characterized binding partners, Ebf (including Olf1, represented by a distinct sequence matrix [17]), SMAD, and Retinoic acid receptor in sequences conserved across vertebrate species pairs (human-chick, mouse-chick, mouse-frog), across a range of parameters for number (1–4) of sites and maximum distance (100–400 bp) between sites for at least two component factors. Multiple binding matrices were used for Ebf (EBF_Q6 and OLF1_01) and SMAD (SMAD_Q6, SMAD_Q6_01, and SMAD4_Q6) family members. This analysis resulted in a surprisingly small number of sites genome wide; distributions of such sites for 100 bp windows with 2 sites or 150 bp windows for three sites are tabulated (Figure 1A). We identified 60 conserved non-coding sites containing a Zfp423 consensus site within 100 bp of either a consensus motif for one of its known binding partners or a second Zfp423 site, with syntenic site predictions in human, mouse and chicken. Surprisingly, four of these 60 robustly predicted clusters occur in the Zfp423 and Ebf3 genes (Figure 1B,C). Broadening the criteria to allow up to 200 bp between sites and to allow Ebf-only or SMAD-only clusters finds three additional sites in or adjacent to Ebf1 (Figure 1D). This enrichment of clustered sites for known interacting factors in genes encoding those factors represents a dramatic enrichment above genome-wide expectation and led us to test whether these sites might be functional.


Zfp423 binds autoregulatory sites in p19 cell culture model.

Cho YW, Hong CJ, Hou A, Gent PM, Zhang K, Won KJ, Hamilton BA - PLoS ONE (2013)

Conserved non-coding sequences suggest Zfp423 autoregulatory sites.Analysis of vertebrate conserved non-coding sequences for co-occurrence of consensus motifs for Zfp423 (ROAZ), Ebf family members (OLF1), BMP-responsive SMADs and nuclear receptor motifs recognized by retinoic acid receptors (DR1, DR4) shows unexpected enrichment in Zfp423 and Ebf genes. (A) Table shows number of conserved sties for pairwise combinations of consensus binding sequences within 100 bp for two sites, 150 bp for three-site combinations. Alignments based on public assemblies of human (hg18), mouse (mm9), chicken (galGal3) and frog (xenTro2) genomes as of 5/1/2012. Number of sites conserved among the three species used for sorting is further reduced for sites excluding known protein coding sequences (H-M-C excl. cds). (B) Conserved, clustered sites identified in Zfp423 include three positions where Zfp423 and Ebf matrix predictions overlap, are indicated by vertical above a diagram of exon structure and pairwise conservation between mouse and other mammal (M) and non-mammal vertebrate (V) species, showing the locations of predicted binding sites. Adapted from UCSC Genome Browser. Similar schematics for (C) Ebf3 using the same parameters and (D) Ebf1 using larger window sizes. Numbers indicate the frequency of similar predicted sites in the genome for the parameters used to identify each.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675209&req=5

pone-0066514-g001: Conserved non-coding sequences suggest Zfp423 autoregulatory sites.Analysis of vertebrate conserved non-coding sequences for co-occurrence of consensus motifs for Zfp423 (ROAZ), Ebf family members (OLF1), BMP-responsive SMADs and nuclear receptor motifs recognized by retinoic acid receptors (DR1, DR4) shows unexpected enrichment in Zfp423 and Ebf genes. (A) Table shows number of conserved sties for pairwise combinations of consensus binding sequences within 100 bp for two sites, 150 bp for three-site combinations. Alignments based on public assemblies of human (hg18), mouse (mm9), chicken (galGal3) and frog (xenTro2) genomes as of 5/1/2012. Number of sites conserved among the three species used for sorting is further reduced for sites excluding known protein coding sequences (H-M-C excl. cds). (B) Conserved, clustered sites identified in Zfp423 include three positions where Zfp423 and Ebf matrix predictions overlap, are indicated by vertical above a diagram of exon structure and pairwise conservation between mouse and other mammal (M) and non-mammal vertebrate (V) species, showing the locations of predicted binding sites. Adapted from UCSC Genome Browser. Similar schematics for (C) Ebf3 using the same parameters and (D) Ebf1 using larger window sizes. Numbers indicate the frequency of similar predicted sites in the genome for the parameters used to identify each.
Mentions: To identify candidate target genes for Zfp423, we first looked for consensus binding sites in regions conserved among vertebrate genomes (Figure 1). Using the web-based SynoR software tool, which uses a matrix representation to account for degeneracy in binding sites [16], we separately examined paired or clustered sites for Zfp423 [11] and its best-characterized binding partners, Ebf (including Olf1, represented by a distinct sequence matrix [17]), SMAD, and Retinoic acid receptor in sequences conserved across vertebrate species pairs (human-chick, mouse-chick, mouse-frog), across a range of parameters for number (1–4) of sites and maximum distance (100–400 bp) between sites for at least two component factors. Multiple binding matrices were used for Ebf (EBF_Q6 and OLF1_01) and SMAD (SMAD_Q6, SMAD_Q6_01, and SMAD4_Q6) family members. This analysis resulted in a surprisingly small number of sites genome wide; distributions of such sites for 100 bp windows with 2 sites or 150 bp windows for three sites are tabulated (Figure 1A). We identified 60 conserved non-coding sites containing a Zfp423 consensus site within 100 bp of either a consensus motif for one of its known binding partners or a second Zfp423 site, with syntenic site predictions in human, mouse and chicken. Surprisingly, four of these 60 robustly predicted clusters occur in the Zfp423 and Ebf3 genes (Figure 1B,C). Broadening the criteria to allow up to 200 bp between sites and to allow Ebf-only or SMAD-only clusters finds three additional sites in or adjacent to Ebf1 (Figure 1D). This enrichment of clustered sites for known interacting factors in genes encoding those factors represents a dramatic enrichment above genome-wide expectation and led us to test whether these sites might be functional.

Bottom Line: Both sites are significantly enriched in either quantitative PCR or massively parallel sequencing assays.A site in intron 5 acts as a classical enhancer in transient assays, but does not require the consensus motif for activity, suggesting a redundant or modulatory role for Zfp423 binding in this context.We speculate that Zfp423 may repress this enhancer as part of a developmental ratchet.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine and Cellular and Molecular Medicine, Institute for Genomic Medicine and Moores UCSD Cancer Center, University of California San Diego School of Medicine, La Jolla, California, United States of America.

ABSTRACT
Zfp423 is a 30 zinc finger transcription factor that forms regulatory complexes with EBF family members and factors targeted by canonical signaling pathways. Zfp423 mutations produce a range of developmental abnormalities in mice and humans related to the ciliopathies. Surprisingly, computational analysis of clustered Zfp423 and partner motifs in conserved genomic sequences predicts enrichment in Zfp423 and Ebf genes. In cell culture models selected for Zfp423 and EBF expression, we identify strong and reproducible occupancy of two Zfp423 intronic sites using chromatin immunoprecipitation with multiple independent antibodies. Both sites are significantly enriched in either quantitative PCR or massively parallel sequencing assays. A site in intron 5 acts as a classical enhancer in transient assays, but does not require the consensus motif for activity, suggesting a redundant or modulatory role for Zfp423 binding in this context. We speculate that Zfp423 may repress this enhancer as part of a developmental ratchet.

Show MeSH
Related in: MedlinePlus