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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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Zfp423 intron 5 binding site is an enhancer.(A) Reporter constructs are shown schematically. A pTAL minimal promoter was inserted upstream of the firefly luciferase reporter in pGL4. Fragments of the Zfp423 intron 5 putative enhancer were placed as indicated by the alignment of grey boxes. Position of the Zfp423 consensus match is indicated by XX in the “sites mutated” construct. Horizontal lines indicated constructs tested together, with pGL4-TAL repeated in each group. (B) Reporter gene expression levels, expressed as the ratio of firefly luciferase to co-transfected Renilla luciferase enzymatic activity measured by luminescence. Each measurement was normalized to the average of control vector (pGL4-TAL) samples run on the same day. For each construct, 2–3 DNA preparations were each used in 2–4 independent co-transfections for 9–12 (top eight constructs) or 6 (bottom three constructs) measurements. (C) Sequence changes in the sites mutated construct. Top line shows sequence of the mouse reference clone. The overlapping ROAZ (Zfp423), OLF1 site predicted by SynoR is boxed. Consensus Zfp423 (ROAZ) binding site and an adjoining consensus half-site (grey text) are indicated. Bottom line shows mutated sequence, with altered residues in grey lowercase. (D) As one estimate of the significance level for apparent shifts between constructs, normalized expression values were compared by ANOVA, followed by Tukey's Honest Significant Differences pair-wise comparisons. Calculated p-values are indicated in the intersection between construct designations. Potential scaling differences between sequential experiments on different days (indicated by gaps between cells in the table) may violate some assumptions of the test. (E) ChIP-qPCR performed for transfected plasmids shows a higher enrichment index compared to input and IgG controls (calculated as ΔΔCT for carrying the wild-type site than for the mutated sites shown in (C). The pairwise comparison was significant at p = 0.007 by t-test or 0.03 by the nonparametric equivalent (Wilcoxon signed rank test).
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pone-0066514-g004: Zfp423 intron 5 binding site is an enhancer.(A) Reporter constructs are shown schematically. A pTAL minimal promoter was inserted upstream of the firefly luciferase reporter in pGL4. Fragments of the Zfp423 intron 5 putative enhancer were placed as indicated by the alignment of grey boxes. Position of the Zfp423 consensus match is indicated by XX in the “sites mutated” construct. Horizontal lines indicated constructs tested together, with pGL4-TAL repeated in each group. (B) Reporter gene expression levels, expressed as the ratio of firefly luciferase to co-transfected Renilla luciferase enzymatic activity measured by luminescence. Each measurement was normalized to the average of control vector (pGL4-TAL) samples run on the same day. For each construct, 2–3 DNA preparations were each used in 2–4 independent co-transfections for 9–12 (top eight constructs) or 6 (bottom three constructs) measurements. (C) Sequence changes in the sites mutated construct. Top line shows sequence of the mouse reference clone. The overlapping ROAZ (Zfp423), OLF1 site predicted by SynoR is boxed. Consensus Zfp423 (ROAZ) binding site and an adjoining consensus half-site (grey text) are indicated. Bottom line shows mutated sequence, with altered residues in grey lowercase. (D) As one estimate of the significance level for apparent shifts between constructs, normalized expression values were compared by ANOVA, followed by Tukey's Honest Significant Differences pair-wise comparisons. Calculated p-values are indicated in the intersection between construct designations. Potential scaling differences between sequential experiments on different days (indicated by gaps between cells in the table) may violate some assumptions of the test. (E) ChIP-qPCR performed for transfected plasmids shows a higher enrichment index compared to input and IgG controls (calculated as ΔΔCT for carrying the wild-type site than for the mutated sites shown in (C). The pairwise comparison was significant at p = 0.007 by t-test or 0.03 by the nonparametric equivalent (Wilcoxon signed rank test).

Mentions: To test whether the strongly-bound site in intron 5 has enhancer activity, we cloned it and several derivatives into a plasmid with a basal promoter driving firefly luciferase as a reporter, co-transfected each construct with a single Renilla luciferase reporter as a transfection control into P19 cells, and measured the ratio of luciferase activities for replicate transfections with each of three independent DNA preparations for each intron 5 construct (Figure 4). The base construct included a 740 bp fragment fully encompassing the vertebrate-conserved sequence, in the same orientation relative to the direction of transcription as it occurs in Zfp423 (Figure 4A). This showed substantial enhancer activity relative to the base vector, pGL4-TAL (Figure 4B). Shorter fragments derived from the 740 bp sequence also showed activity, including a fragment with the overlapping Zfp423/Ebf predicted binding sites mutated (Figure 4C). Having independent DNA preparations with substantial replication measures for each tested construct allowed us to compare relative enhancer strengths compared to the pGL4-TAL base vector transfected on the same day for each experiment with a robust statistical comparison (Figure 4D). Placing the full 740 bp sequence in the reverse orientation showed even higher activity in this assay, while placing it 3′ to the reporter showed essentially the same activity level as the initial construct, satisfying the classical criteria for a transcriptional enhancer. Surprisingly, mutation of several nucleotides in the putative Zfp423 recognition sites to destroy the consensus motifs (location indicated by XX in Figure 4A) did not diminish, but rather slightly increased expression, suggesting that direct binding by Zfp423 may not be self-activating, but perhaps act as negative regulators in some context (the increase was statistically significant by ANOVA; see comparison of ‘sites mut.’ to ‘1–740’ in Figure 4D). As an indicator of direct binding, we quantified relative ChIP/input ratios by qPCR for transfected plasmids (Figure 4E). Six of six paired comparisons (duplicate transfection of three independent preparations of each plasmid) showed greater enrichment index for wild-type than mutated sequence (p = 0.007, paired t-test). A series of deletion constructs suggested the presence of both positive and negative elements within the enhancer and a minimal element of 162 bp that omits the Zfp423 consensus sites had the highest activation level of all tested fragments. In addition to confirming the intron 5 site as an enhancer, these data suggested that Zfp423 binding might be a negative regulator of its own transcription.


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 intron 5 binding site is an enhancer.(A) Reporter constructs are shown schematically. A pTAL minimal promoter was inserted upstream of the firefly luciferase reporter in pGL4. Fragments of the Zfp423 intron 5 putative enhancer were placed as indicated by the alignment of grey boxes. Position of the Zfp423 consensus match is indicated by XX in the “sites mutated” construct. Horizontal lines indicated constructs tested together, with pGL4-TAL repeated in each group. (B) Reporter gene expression levels, expressed as the ratio of firefly luciferase to co-transfected Renilla luciferase enzymatic activity measured by luminescence. Each measurement was normalized to the average of control vector (pGL4-TAL) samples run on the same day. For each construct, 2–3 DNA preparations were each used in 2–4 independent co-transfections for 9–12 (top eight constructs) or 6 (bottom three constructs) measurements. (C) Sequence changes in the sites mutated construct. Top line shows sequence of the mouse reference clone. The overlapping ROAZ (Zfp423), OLF1 site predicted by SynoR is boxed. Consensus Zfp423 (ROAZ) binding site and an adjoining consensus half-site (grey text) are indicated. Bottom line shows mutated sequence, with altered residues in grey lowercase. (D) As one estimate of the significance level for apparent shifts between constructs, normalized expression values were compared by ANOVA, followed by Tukey's Honest Significant Differences pair-wise comparisons. Calculated p-values are indicated in the intersection between construct designations. Potential scaling differences between sequential experiments on different days (indicated by gaps between cells in the table) may violate some assumptions of the test. (E) ChIP-qPCR performed for transfected plasmids shows a higher enrichment index compared to input and IgG controls (calculated as ΔΔCT for carrying the wild-type site than for the mutated sites shown in (C). The pairwise comparison was significant at p = 0.007 by t-test or 0.03 by the nonparametric equivalent (Wilcoxon signed rank test).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675209&req=5

pone-0066514-g004: Zfp423 intron 5 binding site is an enhancer.(A) Reporter constructs are shown schematically. A pTAL minimal promoter was inserted upstream of the firefly luciferase reporter in pGL4. Fragments of the Zfp423 intron 5 putative enhancer were placed as indicated by the alignment of grey boxes. Position of the Zfp423 consensus match is indicated by XX in the “sites mutated” construct. Horizontal lines indicated constructs tested together, with pGL4-TAL repeated in each group. (B) Reporter gene expression levels, expressed as the ratio of firefly luciferase to co-transfected Renilla luciferase enzymatic activity measured by luminescence. Each measurement was normalized to the average of control vector (pGL4-TAL) samples run on the same day. For each construct, 2–3 DNA preparations were each used in 2–4 independent co-transfections for 9–12 (top eight constructs) or 6 (bottom three constructs) measurements. (C) Sequence changes in the sites mutated construct. Top line shows sequence of the mouse reference clone. The overlapping ROAZ (Zfp423), OLF1 site predicted by SynoR is boxed. Consensus Zfp423 (ROAZ) binding site and an adjoining consensus half-site (grey text) are indicated. Bottom line shows mutated sequence, with altered residues in grey lowercase. (D) As one estimate of the significance level for apparent shifts between constructs, normalized expression values were compared by ANOVA, followed by Tukey's Honest Significant Differences pair-wise comparisons. Calculated p-values are indicated in the intersection between construct designations. Potential scaling differences between sequential experiments on different days (indicated by gaps between cells in the table) may violate some assumptions of the test. (E) ChIP-qPCR performed for transfected plasmids shows a higher enrichment index compared to input and IgG controls (calculated as ΔΔCT for carrying the wild-type site than for the mutated sites shown in (C). The pairwise comparison was significant at p = 0.007 by t-test or 0.03 by the nonparametric equivalent (Wilcoxon signed rank test).
Mentions: To test whether the strongly-bound site in intron 5 has enhancer activity, we cloned it and several derivatives into a plasmid with a basal promoter driving firefly luciferase as a reporter, co-transfected each construct with a single Renilla luciferase reporter as a transfection control into P19 cells, and measured the ratio of luciferase activities for replicate transfections with each of three independent DNA preparations for each intron 5 construct (Figure 4). The base construct included a 740 bp fragment fully encompassing the vertebrate-conserved sequence, in the same orientation relative to the direction of transcription as it occurs in Zfp423 (Figure 4A). This showed substantial enhancer activity relative to the base vector, pGL4-TAL (Figure 4B). Shorter fragments derived from the 740 bp sequence also showed activity, including a fragment with the overlapping Zfp423/Ebf predicted binding sites mutated (Figure 4C). Having independent DNA preparations with substantial replication measures for each tested construct allowed us to compare relative enhancer strengths compared to the pGL4-TAL base vector transfected on the same day for each experiment with a robust statistical comparison (Figure 4D). Placing the full 740 bp sequence in the reverse orientation showed even higher activity in this assay, while placing it 3′ to the reporter showed essentially the same activity level as the initial construct, satisfying the classical criteria for a transcriptional enhancer. Surprisingly, mutation of several nucleotides in the putative Zfp423 recognition sites to destroy the consensus motifs (location indicated by XX in Figure 4A) did not diminish, but rather slightly increased expression, suggesting that direct binding by Zfp423 may not be self-activating, but perhaps act as negative regulators in some context (the increase was statistically significant by ANOVA; see comparison of ‘sites mut.’ to ‘1–740’ in Figure 4D). As an indicator of direct binding, we quantified relative ChIP/input ratios by qPCR for transfected plasmids (Figure 4E). Six of six paired comparisons (duplicate transfection of three independent preparations of each plasmid) showed greater enrichment index for wild-type than mutated sequence (p = 0.007, paired t-test). A series of deletion constructs suggested the presence of both positive and negative elements within the enhancer and a minimal element of 162 bp that omits the Zfp423 consensus sites had the highest activation level of all tested fragments. In addition to confirming the intron 5 site as an enhancer, these data suggested that Zfp423 binding might be a negative regulator of its own transcription.

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