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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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Related in: MedlinePlus

Zfp423 binds consensus sites in introns 3 and 5.(A) Semi-quantitative ChIP-PCR assays for the ZNF423 intron 5 site in IMR32 cells, with commercial antibodies against the indicated factors compared with normal serum from same host species and titrated input chromatin. Cycle numbers are indicated to the left. (B) Frequency of observed enrichment for predicted sites tested in replicate experiments in IMR32 cells. Schematic indicates predicted binding sites for Zfp423 (oval), Ebf (circle) and SMAD (diamond). (C–F) Fold enrichment at the orthologous sites in mouse P19 cells, before or after 4 hour treatment with 200 ng/ml BMP2, measured by ChIP-qPCR. Data from Zfp423 antibody E20 are shown. (C) Zfp423 intron 3, (D) Zfp423 intron 5, (E) Ebf1, (F) Ebf3. (G–H) ChIP-qPCR using a custom, affinity-purified antibody against ZNF423 fusion protein shows higher fold discrimination at Zfp423 sites in P19 cells. Experiments in A–B, C–F and G–H were performed independently by different investigators among the authors. * p≥0.05, ** p≥0.01, *** p≥0.001, t-test for comparison to IgG control for same condition and primer pair. (I) Alignment of the predicted binding site in intron 5 and syntenic sites from the indicated species shows strong sequence constraint that fits the overlapping Zfp423 (ROAZ) and EBF (OLF1) consensus motifs. (J) Western blot of mouse forebrain (Brain) and cerebellum (Cbm) extracts from wild-type littermate (+/+) or Zfp423  mutant (nur12) mice. The same blot was stripped and re-probed, using species-specific secondary antibodies coupled to alternate infrared fluors. Reactivity for β-actin and Gapdh are shown as internal loading controls. (K) Western blot of nuclear extracts from P19 cells treated with the indicated shRNA shows high degree of specificity for each Zfp423 antibody. Nxf1 is used as a loading control. Normalized shZfp423 signal <1% of control for each panel. (L) Screen shot from custom UCSC browser tracks showing normalized read density for ChIP-Seq from P19 cells, using custom ZNF423 antibody. Prominent peaks occur over the predicted sites in introns 3 and 5.
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pone-0066514-g003: Zfp423 binds consensus sites in introns 3 and 5.(A) Semi-quantitative ChIP-PCR assays for the ZNF423 intron 5 site in IMR32 cells, with commercial antibodies against the indicated factors compared with normal serum from same host species and titrated input chromatin. Cycle numbers are indicated to the left. (B) Frequency of observed enrichment for predicted sites tested in replicate experiments in IMR32 cells. Schematic indicates predicted binding sites for Zfp423 (oval), Ebf (circle) and SMAD (diamond). (C–F) Fold enrichment at the orthologous sites in mouse P19 cells, before or after 4 hour treatment with 200 ng/ml BMP2, measured by ChIP-qPCR. Data from Zfp423 antibody E20 are shown. (C) Zfp423 intron 3, (D) Zfp423 intron 5, (E) Ebf1, (F) Ebf3. (G–H) ChIP-qPCR using a custom, affinity-purified antibody against ZNF423 fusion protein shows higher fold discrimination at Zfp423 sites in P19 cells. Experiments in A–B, C–F and G–H were performed independently by different investigators among the authors. * p≥0.05, ** p≥0.01, *** p≥0.001, t-test for comparison to IgG control for same condition and primer pair. (I) Alignment of the predicted binding site in intron 5 and syntenic sites from the indicated species shows strong sequence constraint that fits the overlapping Zfp423 (ROAZ) and EBF (OLF1) consensus motifs. (J) Western blot of mouse forebrain (Brain) and cerebellum (Cbm) extracts from wild-type littermate (+/+) or Zfp423 mutant (nur12) mice. The same blot was stripped and re-probed, using species-specific secondary antibodies coupled to alternate infrared fluors. Reactivity for β-actin and Gapdh are shown as internal loading controls. (K) Western blot of nuclear extracts from P19 cells treated with the indicated shRNA shows high degree of specificity for each Zfp423 antibody. Nxf1 is used as a loading control. Normalized shZfp423 signal <1% of control for each panel. (L) Screen shot from custom UCSC browser tracks showing normalized read density for ChIP-Seq from P19 cells, using custom ZNF423 antibody. Prominent peaks occur over the predicted sites in introns 3 and 5.

Mentions: To test whether predicted sites are occupied in cells with relatively high levels of the indicated factors, we performed a series of chromatin immunoprecipitation (ChIP) experiments (Figure 3). Semi-quantitative PCR after ChIP detected ZNF423 binding above background in IMR32 cells at the intron 5 site in two experiments among four replicates (Figure 3A,B). A single experiment further suggested possible binding to a predicted site in EBF3 gene. Interestingly, parallel ChIP-PCR experiments from the same lysates also detected EBF binding to several predicted sites, using an antibody with low discrimination among paralogous EBF members, though ChIP with several SMAD antibodies failed to detect binding under these conditions (Figure 3B).


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)

Zfp423 binds consensus sites in introns 3 and 5.(A) Semi-quantitative ChIP-PCR assays for the ZNF423 intron 5 site in IMR32 cells, with commercial antibodies against the indicated factors compared with normal serum from same host species and titrated input chromatin. Cycle numbers are indicated to the left. (B) Frequency of observed enrichment for predicted sites tested in replicate experiments in IMR32 cells. Schematic indicates predicted binding sites for Zfp423 (oval), Ebf (circle) and SMAD (diamond). (C–F) Fold enrichment at the orthologous sites in mouse P19 cells, before or after 4 hour treatment with 200 ng/ml BMP2, measured by ChIP-qPCR. Data from Zfp423 antibody E20 are shown. (C) Zfp423 intron 3, (D) Zfp423 intron 5, (E) Ebf1, (F) Ebf3. (G–H) ChIP-qPCR using a custom, affinity-purified antibody against ZNF423 fusion protein shows higher fold discrimination at Zfp423 sites in P19 cells. Experiments in A–B, C–F and G–H were performed independently by different investigators among the authors. * p≥0.05, ** p≥0.01, *** p≥0.001, t-test for comparison to IgG control for same condition and primer pair. (I) Alignment of the predicted binding site in intron 5 and syntenic sites from the indicated species shows strong sequence constraint that fits the overlapping Zfp423 (ROAZ) and EBF (OLF1) consensus motifs. (J) Western blot of mouse forebrain (Brain) and cerebellum (Cbm) extracts from wild-type littermate (+/+) or Zfp423  mutant (nur12) mice. The same blot was stripped and re-probed, using species-specific secondary antibodies coupled to alternate infrared fluors. Reactivity for β-actin and Gapdh are shown as internal loading controls. (K) Western blot of nuclear extracts from P19 cells treated with the indicated shRNA shows high degree of specificity for each Zfp423 antibody. Nxf1 is used as a loading control. Normalized shZfp423 signal <1% of control for each panel. (L) Screen shot from custom UCSC browser tracks showing normalized read density for ChIP-Seq from P19 cells, using custom ZNF423 antibody. Prominent peaks occur over the predicted sites in introns 3 and 5.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675209&req=5

pone-0066514-g003: Zfp423 binds consensus sites in introns 3 and 5.(A) Semi-quantitative ChIP-PCR assays for the ZNF423 intron 5 site in IMR32 cells, with commercial antibodies against the indicated factors compared with normal serum from same host species and titrated input chromatin. Cycle numbers are indicated to the left. (B) Frequency of observed enrichment for predicted sites tested in replicate experiments in IMR32 cells. Schematic indicates predicted binding sites for Zfp423 (oval), Ebf (circle) and SMAD (diamond). (C–F) Fold enrichment at the orthologous sites in mouse P19 cells, before or after 4 hour treatment with 200 ng/ml BMP2, measured by ChIP-qPCR. Data from Zfp423 antibody E20 are shown. (C) Zfp423 intron 3, (D) Zfp423 intron 5, (E) Ebf1, (F) Ebf3. (G–H) ChIP-qPCR using a custom, affinity-purified antibody against ZNF423 fusion protein shows higher fold discrimination at Zfp423 sites in P19 cells. Experiments in A–B, C–F and G–H were performed independently by different investigators among the authors. * p≥0.05, ** p≥0.01, *** p≥0.001, t-test for comparison to IgG control for same condition and primer pair. (I) Alignment of the predicted binding site in intron 5 and syntenic sites from the indicated species shows strong sequence constraint that fits the overlapping Zfp423 (ROAZ) and EBF (OLF1) consensus motifs. (J) Western blot of mouse forebrain (Brain) and cerebellum (Cbm) extracts from wild-type littermate (+/+) or Zfp423 mutant (nur12) mice. The same blot was stripped and re-probed, using species-specific secondary antibodies coupled to alternate infrared fluors. Reactivity for β-actin and Gapdh are shown as internal loading controls. (K) Western blot of nuclear extracts from P19 cells treated with the indicated shRNA shows high degree of specificity for each Zfp423 antibody. Nxf1 is used as a loading control. Normalized shZfp423 signal <1% of control for each panel. (L) Screen shot from custom UCSC browser tracks showing normalized read density for ChIP-Seq from P19 cells, using custom ZNF423 antibody. Prominent peaks occur over the predicted sites in introns 3 and 5.
Mentions: To test whether predicted sites are occupied in cells with relatively high levels of the indicated factors, we performed a series of chromatin immunoprecipitation (ChIP) experiments (Figure 3). Semi-quantitative PCR after ChIP detected ZNF423 binding above background in IMR32 cells at the intron 5 site in two experiments among four replicates (Figure 3A,B). A single experiment further suggested possible binding to a predicted site in EBF3 gene. Interestingly, parallel ChIP-PCR experiments from the same lysates also detected EBF binding to several predicted sites, using an antibody with low discrimination among paralogous EBF members, though ChIP with several SMAD antibodies failed to detect binding under these conditions (Figure 3B).

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