ZFP57 recognizes multiple and closely spaced sequence motif variants to maintain repressive epigenetic marks in mouse embryonic stem cells.
Imprinting Control Regions (ICRs) need to maintain their parental allele-specific DNA methylation during early embryogenesis despite genome-wide demethylation and subsequent de novo methylation. ZFP57 and KAP1 are both required for maintaining the repressive DNA methylation and H3-lysine-9-trimethylation (H3K9me3) at ICRs. In vitro, ZFP57 binds a specific hexanucleotide motif that is enriched at its genomic binding sites. We now demonstrate in mouse embryonic stem cells (ESCs) that SNPs disrupting closely-spaced hexanucleotide motifs are associated with lack of ZFP57 binding and H3K9me3 enrichment. Through a transgenic approach in mouse ESCs, we further demonstrate that an ICR fragment containing three ZFP57 motif sequences recapitulates the original methylated or unmethylated status when integrated into the genome at an ectopic position. Mutation of Zfp57 or the hexanucleotide motifs led to loss of ZFP57 binding and DNA methylation of the transgene. Finally, we identified a sequence variant of the hexanucleotide motif that interacts with ZFP57 both in vivo and in vitro. The presence of multiple and closely located copies of ZFP57 motif variants emerges as a distinct characteristic that is required for the faithful maintenance of repressive epigenetic marks at ICRs and other ZFP57 binding sites.
Institute of Genetics and Biophysics 'A. Buzzati-Traverso', CNR, 80131 Naples, Italy Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, 81100 Caserta, Italy.
Zahra
Z
Anvar
Institute of Genetics and Biophysics 'A. Buzzati-Traverso', CNR, 80131 Naples, Italy Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, 81100 Caserta, Italy.
Marco
M
Cammisa
Institute of Genetics and Biophysics 'A. Buzzati-Traverso', CNR, 80131 Naples, Italy Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, 81100 Caserta, Italy.
Vincenzo
V
Riso
Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, 81100 Caserta, Italy.
Ilaria
I
Baglivo
Institute of Genetics and Biophysics 'A. Buzzati-Traverso', CNR, 80131 Naples, Italy Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, 81100 Caserta, Italy.
Harpreet
H
Kukreja
Institute of Genetics and Biophysics 'A. Buzzati-Traverso', CNR, 80131 Naples, Italy Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, 81100 Caserta, Italy.
Angela
A
Sparago
Institute of Molecular Genetics (IGMM), CNRS UMR5535 and University of Montpellier, 1919 route de Mende, 34293 Montpellier, France.
Michael
M
Girardot
Institute of Genetics and Biophysics 'A. Buzzati-Traverso', CNR, 80131 Naples, Italy.
Shraddha
S
Lad
Istituto per le Applicazioni del Calcolo 'Mauro Picone' (IAC), CNR, 80131 Naples, Italy.
Italia
I
De Feis
Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, 81100 Caserta, Italy.
Flavia
F
Cerrato
Istituto per le Applicazioni del Calcolo 'Mauro Picone' (IAC), CNR, 80131 Naples, Italy.
Claudia
C
Angelini
Institute of Molecular Genetics (IGMM), CNRS UMR5535 and University of Montpellier, 1919 route de Mende, 34293 Montpellier, France.
Robert
R
Feil
Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, 81100 Caserta, Italy.
Paolo V
PV
Pedone
Institute of Genetics and Biophysics 'A. Buzzati-Traverso', CNR, 80131 Naples, Italy Ceinge Biotecnologie Avanzate s.c.a.r.l., 80145 Naples, Italy.
Giovanna
G
Grimaldi
Institute of Genetics and Biophysics 'A. Buzzati-Traverso', CNR, 80131 Naples, Italy Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, 81100 Caserta, Italy andrea.riccio@unina2.it.
Andrea
A
Riccio
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D019175:DNA Methylation; D018392:Genomic Imprinting; D006657:Histones / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D008745:Methylation; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D000066450:Mouse Embryonic Stem Cells / Q000378:metabolism*; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D059372:Nucleotide Motifs / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D000076183:Tripartite Motif-Containing Protein 28
D006657:Histones; D009687:Nuclear Proteins; D012097:Repressor Proteins; C501529:Zfp-57 protein, mouse; C103190:Trim28 protein, mouse; D000076183:Tripartite Motif-Containing Protein 28; D008239:Lysine