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A placental growth factor is silenced in mouse embryos by the zinc finger protein ZFP568

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Science  19 May 2017:
Vol. 356, Issue 6339, pp. 757-759
DOI: 10.1126/science.aah6895
  • Fig. 1 Acute Zfp568 knockout leads to derepression of Igf2-P0.

    (A) Scatter plots of gene expression in Zfp568 WT and KO ESCs and TSCs, as determined by RNA-seq. (B) Schematic of SNPs in CAST/Eij; C57BL/6 hybrid ESCs used to discriminate Igf2 parental alleles. SNP counts from RNA-seq in Zfp568 WT and KO CAST/Eij; C57BL/6 hybrid ESCs are indicated.

  • Fig. 2 ZFP568 directly targets Igf2-P0 for repression.

    (A) ZFP568 and H3K9me3 ChIP-seq signals at the Igf2 locus in Zfp568 WT and KO ESCs and TSCs. DMR0 is a differentially methylated region overlapping exon 1 of the Igf2-P0 transcript. Igf2as is the Igf2 antisense transcript. (B) Relative luciferase activity in 293T cells overexpressing GFP (G) or ZFP568 (Z) and an SV40 promoter–driven luciferase plasmid containing the ZFP568 consensus (cons) or Igf2 binding site in the forward (F) or reverse (R) orientation. PBSPro is the 18-nucleotide (nt) primer binding site for proline, which is bound by ZFP809 but not ZFP568. t test: Error bars indicate standard deviation; **P < 0.01; n = 3. (C) Fluorescence-polarization binding assay of ZFP568 (ZnF1-11) protein incubated with indicated 6-carboxyfluorescein (FAM)–labeled double-stranded oligonucleotides.

  • Fig. 3 Mutation of the ZFP568-binding site upstream of the Igf2-P0 promoter activates Igf2-P0.

    (A) Sequences of WT and two homozygous mutant ESC lines (with deleted nucleotides indicated in red) relative to the consensus binding site for ZFP568. (B) Igf2-P0 and Zfp568 levels were determined by qRT-PCR in the indicated binding site–mutant ESCs before and after Zfp568 deletion with 4-OHT. t test: Error bars indicate standard deviation; **P < 0.01; n = 3. (C) ZFP568 and H3K9me3 ChIP-seq signals at Igf2 in Zfp568 WT and binding site–mutant ESCs.

  • Fig. 4 Igf2 KO rescues Zfp568 KO–induced lethality.

    (A) Relative Igf2-P0 levels in embryonic and extraembryonic tissues from E8.5 Zfp568-GFPFL/FL and Zfp568KO/KO littermate embryos. t test: Error bars indicate standard deviation; *P < 0.05; n = 3. (B) Bright-field and GFP-fluorescence images of Zfp568-GFPFL/FL and Zfp568KO/KO littermate embryos at E8.5. (C) Images of pups of indicated genotypes from Zfp568 KO and paternal (pat) Igf2 KO crosses at E18.5.

Supplementary Materials

  • A placental growth factor is silenced in mouse embryos by the zinc finger protein ZFP568

    Peng Yang, Yixuan Wang, Don Hoang, Matthew Tinkham, Anamika Patel, Ming-An Sun, Gernot Wolf, Mairead Baker, Huan-Chieh Chien, Kuan-Yu Nick Lai, Xiaodong Cheng, Che-Kun James Shen,* Todd S. Macfarlan

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S9
    • Captions for Tables S1 and S2
    • References
    Table S1
    RNA-seq analysis of Zfp568 WT and KO ESCs, TSCs, F1 Hybrid ESCs and E8 embryos
    Table S2
    Joined tables of ChIP-seq of ZFP568 in ESCs and TSCs and RNA-seq data for Zfp568 WT and KO ESCs and TSCs.

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