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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 overexpression represses intron 5 enhancer activity in P19 cells.(A) pGL4 reporter with the intron 5 enhancer was similarly active when co-transfected with shRNA directed against Zfp423 or a control. A similar plasmid with a region encompassing the intron 3 binding site had no activity above the pTAL minimal promoter. (B) Co-transfection with a plasmid expressing FLAG-tagged human ZNF423 reduced expression of the intron 5 reporter relative to a pcDNA vector control. This effect did not occur between paired samples with the Zfp423 consensus motifs mutated (intron5m). (C) An independent series of co-transfection assays indicates Ebf1-dependence of the intron 5 enhancer in P19 cells. ZNF423 overexpression and Ebf1 knockdown shows comparable reductions in enhancer activity (p<10−7, Tukey HSD pair-wise comparisons to control after ANOVA). Combining ZNF423 overexpression and Ebf1 knockdown showed further reduction in reporter activity (p = 0.03–0.0000001), while shRNA against endogenous Zfp423 showed no additional effect on activity in Ebf1 knockdown cells. Each construct was assayed in duplicate for each of three independent DNA preparations. (D–E) Immunofluorescent detection of Zfp423 by either E20 (D) or our custom affinity purified antibody (E) in P19 cells transfected with plasmid expressing EGFP and either scrambled control or Zfp423-targeted shRNA shows specific reduction or loss of immunoreactivity in nuclei of Zfp423 depleted cells (outline in middle panels). (F) Western blotting with either commercial (left panel) or custom (right panel) antibody showed effective reduction of P19 Zfp423 levels after transfection with plasmid expressing Zfp423-directed shRNA compared with internal loading controls. Repeated experiments showed 60–99% reduction. (G) Western blotting with a pan-EBF antibody showed moderate reduction (44–68% of control levels) after transfection with plasmid expressing Ebf1-directed shRNA. (H) Western blotting showed overexpression of ZNF423 in pcDNA-FLAG-ZNF423 transfected cells. The transfected product showed 1.4× endogenous level, indicating 2.4-fold expression level for endogenous and transfected products combined.
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pone-0066514-g005: Zfp423 overexpression represses intron 5 enhancer activity in P19 cells.(A) pGL4 reporter with the intron 5 enhancer was similarly active when co-transfected with shRNA directed against Zfp423 or a control. A similar plasmid with a region encompassing the intron 3 binding site had no activity above the pTAL minimal promoter. (B) Co-transfection with a plasmid expressing FLAG-tagged human ZNF423 reduced expression of the intron 5 reporter relative to a pcDNA vector control. This effect did not occur between paired samples with the Zfp423 consensus motifs mutated (intron5m). (C) An independent series of co-transfection assays indicates Ebf1-dependence of the intron 5 enhancer in P19 cells. ZNF423 overexpression and Ebf1 knockdown shows comparable reductions in enhancer activity (p<10−7, Tukey HSD pair-wise comparisons to control after ANOVA). Combining ZNF423 overexpression and Ebf1 knockdown showed further reduction in reporter activity (p = 0.03–0.0000001), while shRNA against endogenous Zfp423 showed no additional effect on activity in Ebf1 knockdown cells. Each construct was assayed in duplicate for each of three independent DNA preparations. (D–E) Immunofluorescent detection of Zfp423 by either E20 (D) or our custom affinity purified antibody (E) in P19 cells transfected with plasmid expressing EGFP and either scrambled control or Zfp423-targeted shRNA shows specific reduction or loss of immunoreactivity in nuclei of Zfp423 depleted cells (outline in middle panels). (F) Western blotting with either commercial (left panel) or custom (right panel) antibody showed effective reduction of P19 Zfp423 levels after transfection with plasmid expressing Zfp423-directed shRNA compared with internal loading controls. Repeated experiments showed 60–99% reduction. (G) Western blotting with a pan-EBF antibody showed moderate reduction (44–68% of control levels) after transfection with plasmid expressing Ebf1-directed shRNA. (H) Western blotting showed overexpression of ZNF423 in pcDNA-FLAG-ZNF423 transfected cells. The transfected product showed 1.4× endogenous level, indicating 2.4-fold expression level for endogenous and transfected products combined.

Mentions: Since the predicted Zfp423 binding sites were not required for enhancer activity on a heterologous promoter and deletion of these binding sites appears to increase enhancer strength, we next tested whether ZNF423 expression had any effect on reporter gene expression (Figure 5). Co-transfection of reporter constructs with a short hairpin RNA (shRNA) targeting Zfp423 mRNA that reduces Zfp423 to ∼20% normal levels in P19 cells did not reproducibly alter reporter activity in triplicate measures from a single DNA preparation per construct (Figure 5A). In parallel, a construct including the conserved segment at the intron 3 site showed no enhancer activity either before or after knockdown of Zfp423 RNA, in comparison to pGL4-TAL. However, overexpression of human ZNF423 substantially attenuated expression of the reporter, compared to pcDNA expression vector control (Figure 5B), providing further evidence for a negative effect of Zfp423 at high expression levels. This effect was not seen in co-transfection when the Zfp423 binding sites were mutated, suggesting a direct effect of binding. Since the Zfp423 binding site overlaps a predicted binding site for EBF family members, we then tested whether reducing Ebf expression in P19 cells affects reporter activity (Figure 5C). From duplicate measurements for each of three independent DNA preparations per construct, we again saw no effect of shRNA targeting Zfp423, but a highly significant decrease on overexpression of human ZNF423 (p<10−7, Tukey HSD pair-wise test after one-factor ANOVA). Targeting Ebf1 with shRNA resulted in a similar loss of reporter expression (p<10−7), suggesting that Ebf1 contributes to activation of this enhancer. Targeting Ebf2, which appeared to be expressed at lower levels based on qRT-PCR data (Figure 2C) had only a modest effect. Simultaneously targeting both Ebf1 and Zfp423 by co-transfection of shRNA constructs was not significantly different from targeting Ebf1 alone. However, overexpression of ZNF423 together with Ebf1 shRNA further reduced reporter expression (p = 0.029) below the level achieved with Ebf1 shRNA alone. Expression of the GFP-marked shRNA construct dramatically reduced Zfp423 immunofluorescence in P19 nuclei using either the E20 (Figure 5D) or custom made (Figure 5E) antibody. Western blots confirmed effective reduction of protein expression after shRNA transfections (Figure 5F,G) and overexpression after transfection with human ZNF423 expression plasmid (Figure 5H).


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 overexpression represses intron 5 enhancer activity in P19 cells.(A) pGL4 reporter with the intron 5 enhancer was similarly active when co-transfected with shRNA directed against Zfp423 or a control. A similar plasmid with a region encompassing the intron 3 binding site had no activity above the pTAL minimal promoter. (B) Co-transfection with a plasmid expressing FLAG-tagged human ZNF423 reduced expression of the intron 5 reporter relative to a pcDNA vector control. This effect did not occur between paired samples with the Zfp423 consensus motifs mutated (intron5m). (C) An independent series of co-transfection assays indicates Ebf1-dependence of the intron 5 enhancer in P19 cells. ZNF423 overexpression and Ebf1 knockdown shows comparable reductions in enhancer activity (p<10−7, Tukey HSD pair-wise comparisons to control after ANOVA). Combining ZNF423 overexpression and Ebf1 knockdown showed further reduction in reporter activity (p = 0.03–0.0000001), while shRNA against endogenous Zfp423 showed no additional effect on activity in Ebf1 knockdown cells. Each construct was assayed in duplicate for each of three independent DNA preparations. (D–E) Immunofluorescent detection of Zfp423 by either E20 (D) or our custom affinity purified antibody (E) in P19 cells transfected with plasmid expressing EGFP and either scrambled control or Zfp423-targeted shRNA shows specific reduction or loss of immunoreactivity in nuclei of Zfp423 depleted cells (outline in middle panels). (F) Western blotting with either commercial (left panel) or custom (right panel) antibody showed effective reduction of P19 Zfp423 levels after transfection with plasmid expressing Zfp423-directed shRNA compared with internal loading controls. Repeated experiments showed 60–99% reduction. (G) Western blotting with a pan-EBF antibody showed moderate reduction (44–68% of control levels) after transfection with plasmid expressing Ebf1-directed shRNA. (H) Western blotting showed overexpression of ZNF423 in pcDNA-FLAG-ZNF423 transfected cells. The transfected product showed 1.4× endogenous level, indicating 2.4-fold expression level for endogenous and transfected products combined.
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pone-0066514-g005: Zfp423 overexpression represses intron 5 enhancer activity in P19 cells.(A) pGL4 reporter with the intron 5 enhancer was similarly active when co-transfected with shRNA directed against Zfp423 or a control. A similar plasmid with a region encompassing the intron 3 binding site had no activity above the pTAL minimal promoter. (B) Co-transfection with a plasmid expressing FLAG-tagged human ZNF423 reduced expression of the intron 5 reporter relative to a pcDNA vector control. This effect did not occur between paired samples with the Zfp423 consensus motifs mutated (intron5m). (C) An independent series of co-transfection assays indicates Ebf1-dependence of the intron 5 enhancer in P19 cells. ZNF423 overexpression and Ebf1 knockdown shows comparable reductions in enhancer activity (p<10−7, Tukey HSD pair-wise comparisons to control after ANOVA). Combining ZNF423 overexpression and Ebf1 knockdown showed further reduction in reporter activity (p = 0.03–0.0000001), while shRNA against endogenous Zfp423 showed no additional effect on activity in Ebf1 knockdown cells. Each construct was assayed in duplicate for each of three independent DNA preparations. (D–E) Immunofluorescent detection of Zfp423 by either E20 (D) or our custom affinity purified antibody (E) in P19 cells transfected with plasmid expressing EGFP and either scrambled control or Zfp423-targeted shRNA shows specific reduction or loss of immunoreactivity in nuclei of Zfp423 depleted cells (outline in middle panels). (F) Western blotting with either commercial (left panel) or custom (right panel) antibody showed effective reduction of P19 Zfp423 levels after transfection with plasmid expressing Zfp423-directed shRNA compared with internal loading controls. Repeated experiments showed 60–99% reduction. (G) Western blotting with a pan-EBF antibody showed moderate reduction (44–68% of control levels) after transfection with plasmid expressing Ebf1-directed shRNA. (H) Western blotting showed overexpression of ZNF423 in pcDNA-FLAG-ZNF423 transfected cells. The transfected product showed 1.4× endogenous level, indicating 2.4-fold expression level for endogenous and transfected products combined.
Mentions: Since the predicted Zfp423 binding sites were not required for enhancer activity on a heterologous promoter and deletion of these binding sites appears to increase enhancer strength, we next tested whether ZNF423 expression had any effect on reporter gene expression (Figure 5). Co-transfection of reporter constructs with a short hairpin RNA (shRNA) targeting Zfp423 mRNA that reduces Zfp423 to ∼20% normal levels in P19 cells did not reproducibly alter reporter activity in triplicate measures from a single DNA preparation per construct (Figure 5A). In parallel, a construct including the conserved segment at the intron 3 site showed no enhancer activity either before or after knockdown of Zfp423 RNA, in comparison to pGL4-TAL. However, overexpression of human ZNF423 substantially attenuated expression of the reporter, compared to pcDNA expression vector control (Figure 5B), providing further evidence for a negative effect of Zfp423 at high expression levels. This effect was not seen in co-transfection when the Zfp423 binding sites were mutated, suggesting a direct effect of binding. Since the Zfp423 binding site overlaps a predicted binding site for EBF family members, we then tested whether reducing Ebf expression in P19 cells affects reporter activity (Figure 5C). From duplicate measurements for each of three independent DNA preparations per construct, we again saw no effect of shRNA targeting Zfp423, but a highly significant decrease on overexpression of human ZNF423 (p<10−7, Tukey HSD pair-wise test after one-factor ANOVA). Targeting Ebf1 with shRNA resulted in a similar loss of reporter expression (p<10−7), suggesting that Ebf1 contributes to activation of this enhancer. Targeting Ebf2, which appeared to be expressed at lower levels based on qRT-PCR data (Figure 2C) had only a modest effect. Simultaneously targeting both Ebf1 and Zfp423 by co-transfection of shRNA constructs was not significantly different from targeting Ebf1 alone. However, overexpression of ZNF423 together with Ebf1 shRNA further reduced reporter expression (p = 0.029) below the level achieved with Ebf1 shRNA alone. Expression of the GFP-marked shRNA construct dramatically reduced Zfp423 immunofluorescence in P19 nuclei using either the E20 (Figure 5D) or custom made (Figure 5E) antibody. Western blots confirmed effective reduction of protein expression after shRNA transfections (Figure 5F,G) and overexpression after transfection with human ZNF423 expression plasmid (Figure 5H).

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