and fig. S9A). Despite this lack of gross morphological phenotype at late fetal stages, only two
Zfp568KO/KO; Igf2KO/KO pups have been recovered after birth, both of which were found dead
(fig. S9, B and C). Nonetheless, these data demonstrate a genetic interaction between Zfp568
Our results provide biochemical and genetic evidence that the KRAB-zinc finger protein ZFP568 is
a specific and direct repressor of Igf2-P0 in mice.
Recent studies have revealed that the majority of
KRAB-ZFPs interact with and likely restrict the
expression of specific retrotransposon families, potentially facilitating the domestication of retrotransposons and the evolution of gene regulatory networks
(15–19). Whether the binding site for ZFP568 at Igf2-
P0 was derived from an ancient and since decayed
retrotransposon or was generated by genetic drift is
unknown, but our findings nonetheless demonstrate that KRAB-ZFPs can evolve essential roles in
precision developmental gene silencing. Notably,
ZFP568 orthologs have been detected in all eutherian mammals examined and contain a conserved
zinc “fingerprint” (16), suggesting that their DNA-binding specificity is conserved. Intriguingly, Zfp57,
another KRAB-ZFP that emerged in mammals, is a
critical factor that maintains genomic imprints at
Igf2-P0 (20, 21), a process linked to viviparity (22).
Furthermore, the human Zfp568 ortholog, Znf568,
is among the most-rapidly evolving human genes,
with three common allele variants found within
human populations that have been linked to relative
head size at birth (12). Thus, KRAB-ZFP/KAP1–
dependent regulation of Igf2-P0 has shaped, and
likely continues to shape, the evolution of mammals.
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We thank K. Pfeifer and C. Gebert for Igf2 KO mice. We thank
S. Coon, J. Iben, and T. Li for Next Generation Sequencing
(NGS) support. This work was supported by NIH grants
1ZIAHD008933 (T.S.M.) and GM049245-23 (A.P. and X.C.),
Ministry of Science and Technology (MOST) Frontier of Science
Award, Academia Sinica Senior Investigator Award (C.-K.J.S.),
National Natural Science Foundation of China (NSFC) 31471392,
and Future Scientists Exchange Program of the China Scholarship
Council (CSC) (Y. W.). NGS data have been deposited in the
Gene Expression Omnibus (GEO) database (GSE84832).
Zfp568-GFPFL/FL mice are available from C.-K.J.S. under a
material transfer agreement with the Academia Sinica, Taipei,
Taiwan, Republic of China.
Materials and Methods
Figs. S1 to S9
Tables S1 and S2
1 August 2016; accepted 20 April 2017
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.