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Cited by 152Genetic Syndromes and ImprintingEpigenetics and DNA MethylationGenomics and Chromatin Dynamics

The DOI matches its Crossref record; 69 of the 69 references it cites were found in a registry or an index.

Allele-specific binding of ZFP57 in the epigenetic regulation of imprinted and non-imprinted monoallelic expression

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Strogantsev, Ruslan, Krueger, Felix, Yamazawa, Kazuki, Shi, Hui, Gould, Poppy, Goldman-Roberts, Megan, McEwen, Kirsten, Sun, Bowen, Pedersen, Roger, Ferguson-Smith, Anne C.·2015·Genome Biology
DOI 10.1186/s13059-015-0672-7PMC PMC4491874
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Abstract

Background: Selective maintenance of genomic epigenetic imprints during pre-implantation development is required for parental origin-specific expression of imprinted genes. The Kruppel-like zinc finger protein ZFP57 acts as a factor necessary for maintaining the DNA methylation memory at multiple imprinting control regions in early mouse embryos and embryonic stem (ES) cells. Maternal-zygotic deletion of ZFP57 in mice presents a highly penetrant phenotype with no animals surviving to birth. Additionally, several cases of human transient neonatal diabetes are associated with somatic mutations in the ZFP57 coding sequence. Results: Here, we comprehensively map sequence-specific ZFP57 binding sites in an allele-specific manner using hybrid ES cell lines from reciprocal crosses between C57BL/6J and Cast/EiJ mice, assigning allele specificity to approximately two-thirds of all binding sites. While half of these are biallelic and include endogenous retrovirus (ERV) targets, the rest show monoallelic binding based either on parental origin or on genetic background of the allele. Parental-origin allele-specific binding is methylation-dependent and maps only to imprinting control differentially methylated regions (DMRs) established in the germline. We identify a novel imprinted gene, Fkbp6, which has a critical function in mouse male germ cell development. Genetic background-specific sequence differences also influence ZFP57 binding, as genetic variation that disrupts the consensus binding motif and its methylation is often associated with monoallelic expression of neighboring genes. Conclusions: The work described here uncovers further roles for ZFP57-mediated regulation of genomic imprinting and identifies a novel mechanism for genetically determined monoallelic gene expression.

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Fig. 1 Allele-specific mapping of Zfp57 binding sites. a Schematic diagram of the experimental design. Example polymorphisms are shown for the maternal allele-specific binding of ZFP57 over BL6 (A)–Cast (G) SNP. BL6 allele (blue line) is bound by ZFP57 in BC and Cast (brown line) in the CB line. ASAP allele-specific alignment pipeline devised to map genome-specific reads; ESC embryonic stem cell. b Total number of peaks identified as being biallelic, parental origin-specific (maternal (Mat) and paternal (Pat)) and strain-specific (BL6 and Cast) based on Fisher’s exact test analysis of allele-specific reads under each peak. NI refers to non-informative sites due to lack of SNP spanning reads. c UCSC genome browser views (mm9 assembly) of data showing parental allele-specific binding at the paternally methylated IG-DMR (top left), maternally methylated Snrpn ICR (bottom left), biallelic binding site within Pank4 gene intron on chromosome 4 (top right) and a strain-specific monoallelic binding within the Cmtm4 gene intron on chromosome 8 (bottom right). Tracks in each panel show the following (top-down): histone modification profiles for H3K4me3, H3K9me3 and H4K20me3 [28]; combined read densities for BC and CB ZFP57 ChIP; Bl6/Cast SNPs [24]; density plots for allele-specific reads in BC and CB ChIP; position of known ICRs where found; annotated UCSC genes in the region
Fig. 1 Allele-specific mapping of Zfp57 binding sites. a Schematic diagram of the experimental design. Example polymorphisms are shown for the maternal allele-specific binding of ZFP57 over BL6 (A)–Cast (G) SNP. BL6 allele (blue line) is bound by ZFP57 in BC and Cast (brown line) in the CB line. ASAP allele-specific alignment pipeline devised to map genome-specific reads; ESC embryonic stem cell. b Total number of peaks identified as being biallelic, parental origin-specific (maternal (Mat) and paternal (Pat)) and strain-specific (BL6 and Cast) based on Fisher’s exact test analysis of allele-specific reads under each peak. NI refers to non-informative sites due to lack of SNP spanning reads. c UCSC genome browser views (mm9 assembly) of data showing parental allele-specific binding at the paternally methylated IG-DMR (top left), maternally methylated Snrpn ICR (bottom left), biallelic binding site within Pank4 gene intron on chromosome 4 (top right) and a strain-specific monoallelic binding within the Cmtm4 gene intron on chromosome 8 (bottom right). Tracks in each panel show the following (top-down): histone modification profiles for H3K4me3, H3K9me3 and H4K20me3 [28]; combined read densities for BC and CB ZFP57 ChIP; Bl6/Cast SNPs [24]; density plots for allele-specific reads in BC and CB ChIP; position of known ICRs where found; annotated UCSC genes in the regionCC BY · PubMed Central
Fig. 2 Epigenetic modifications around ZFP57 peaks. a Overlap of ZFP57 peaks with Ensembl annotated gene features (left) and repeat elements (right). Selective enrichment/depletion of ZFP57 peaks are compared against an average of ten independently generated boot-strap sets (Control). b Plot of cumulative read counts for three different histone modifications around ZFP57 peaks found at imprinted germline DMRs and elsewhere. Shown are H3K4me3, H3K9me3 and H4K20me3, and pan-H3 representing overall histone density. c Average DNA methylation at CGIs bound by ZFP57 at ICRs and other non-imprinted loci compared with a randomly selected set of similar size. Data points represent individual CGIs with a horizontal line showing median value for the group. Previously published ES cell reduced representation bisulfite sequencing methylation data were used for this analysis [31]. Non-parametric Kruskall-Wallis tests were performed with significantly different categories denoted (***P < 0.001)
Fig. 2 Epigenetic modifications around ZFP57 peaks. a Overlap of ZFP57 peaks with Ensembl annotated gene features (left) and repeat elements (right). Selective enrichment/depletion of ZFP57 peaks are compared against an average of ten independently generated boot-strap sets (Control). b Plot of cumulative read counts for three different histone modifications around ZFP57 peaks found at imprinted germline DMRs and elsewhere. Shown are H3K4me3, H3K9me3 and H4K20me3, and pan-H3 representing overall histone density. c Average DNA methylation at CGIs bound by ZFP57 at ICRs and other non-imprinted loci compared with a randomly selected set of similar size. Data points represent individual CGIs with a horizontal line showing median value for the group. Previously published ES cell reduced representation bisulfite sequencing methylation data were used for this analysis [31]. Non-parametric Kruskall-Wallis tests were performed with significantly different categories denoted (***P < 0.001)CC BY · PubMed Central
Fig. 3 Identification of Fkbp6 as a novel imprinted gene. a The Fkbp6 gene region with the location of ZFP57 binding within the promoter regions. PSQ1 and PSQ2 denote regions analyzed by bisulphite pyrosequencing shown in (c). b Fkbp6 expression levels and allele-specificity in mouse tissues and ES cells from quantitative RT-PCR and SNP pyrosequencing. The height of each bar represents Fkbp6 mRNA levels normalized to 18 s rRNA and shown relative to liver expression levels. Blue and red stacked bars show relative contribution of BL6 and Cast alleles, respectively, to overall Fkbp6 levels. Upper and lower error bars correspond to standard deviation between biological replicates for PCR and pyrosequencing analysis, respectively (n = 8 replicates per each tissue: 4 BC and 4 CB; n = 4 for ES cell lines (ESCs). c The Fkbp6 promoter is not a somatic DMR. Quantitative bisulphite pyrosequencing analysis of the Fkbp6 promoter region, which assays overall methylation level in the population of cells in a non-allele-specific manner. Relative proportions of black (methylated) and white (unmethylated) filled areas in circles depict average percentage methylation for individual CpGs (n = 4; 2 BC and 2 CB bioreplicates for each tissue type). Numbers adjacent show median percentage methylation values across CpGs covered by PSQ1 and PSQ2 sequencing primers. Grey circles denote CpGs not covered in the assay. Black arrow denotes Fkbp6 transcription start site and direction of expression. Grey arrow underneath indicates position of the Cast-specific CpG (SNP rs13487942), quantified in (d). d Left: Bar chart showing methylation status of the CpGCast when inherited on paternal (BC cross, blue bars) and maternal (CB cross, pink bars) chromosomes. Data from pyrosequencing of adult testis, E16.5 brain and placenta, n = 2 × BC and 2 × CB samples per tissue. Right: Representative bisulphite pyrograms from individual BC and CB placenta tissues. CpGCast region is framed in red, methylation level is calculated from the relative height of ‘T’ (unmethylated, Um) and ‘C’ (methylated, Me) peaks. e Top: Relative enrichment of H3K4 and H3K9 tri-methylation in E16.5 tissues. Data shown relative to input and normalized to a reference non-enriched genomic location. Bottom: Allele ratios from SNP pyrosequencing of ChIP-enriched DNA. Error bars reflect standard deviation between three technical replicates for each sample shown
Fig. 3 Identification of Fkbp6 as a novel imprinted gene. a The Fkbp6 gene region with the location of ZFP57 binding within the promoter regions. PSQ1 and PSQ2 denote regions analyzed by bisulphite pyrosequencing shown in (c). b Fkbp6 expression levels and allele-specificity in mouse tissues and ES cells from quantitative RT-PCR and SNP pyrosequencing. The height of each bar represents Fkbp6 mRNA levels normalized to 18 s rRNA and shown relative to liver expression levels. Blue and red stacked bars show relative contribution of BL6 and Cast alleles, respectively, to overall Fkbp6 levels. Upper and lower error bars correspond to standard deviation between biological replicates for PCR and pyrosequencing analysis, respectively (n = 8 replicates per each tissue: 4 BC and 4 CB; n = 4 for ES cell lines (ESCs). c The Fkbp6 promoter is not a somatic DMR. Quantitative bisulphite pyrosequencing analysis of the Fkbp6 promoter region, which assays overall methylation level in the population of cells in a non-allele-specific manner. Relative proportions of black (methylated) and white (unmethylated) filled areas in circles depict average percentage methylation for individual CpGs (n = 4; 2 BC and 2 CB bioreplicates for each tissue type). Numbers adjacent show median percentage methylation values across CpGs covered by PSQ1 and PSQ2 sequencing primers. Grey circles denote CpGs not covered in the assay. Black arrow denotes Fkbp6 transcription start site and direction of expression. Grey arrow underneath indicates position of the Cast-specific CpG (SNP rs13487942), quantified in (d). d Left: Bar chart showing methylation status of the CpGCast when inherited on paternal (BC cross, blue bars) and maternal (CB cross, pink bars) chromosomes. Data from pyrosequencing of adult testis, E16.5 brain and placenta, n = 2 × BC and 2 × CB samples per tissue. Right: Representative bisulphite pyrograms from individual BC and CB placenta tissues. CpGCast region is framed in red, methylation level is calculated from the relative height of ‘T’ (unmethylated, Um) and ‘C’ (methylated, Me) peaks. e Top: Relative enrichment of H3K4 and H3K9 tri-methylation in E16.5 tissues. Data shown relative to input and normalized to a reference non-enriched genomic location. Bottom: Allele ratios from SNP pyrosequencing of ChIP-enriched DNA. Error bars reflect standard deviation between three technical replicates for each sample shownCC BY · PubMed Central
Fig. 4 Genetic analysis of ZFP57 consensus binding motif. a Consensus ZFP57 binding motif identified using MEME algorithm analysis of DNA sequences underlying ChIP-seq peaks. b Distribution of genetically determined monoallelic Zfp57 peaks based on motif integrity on the unbound vs. bound alleles: SNPs disrupting all of the motifs (five sites, red), some of the motifs with at least one remaining intact (eight sites, orange) and none of the motifs (seven sites, green). No motifs could be found on either genetic alleles in two cases (grey). c Diagram indicating which bases within the consensus are varied between BL6 and Cast genomes in strain-specific peaks. Long bars, short bars and triangle indicate deletion, SNP and insertion events, respectively. d Putative mechanisms for ZFP57 allele-specific binding. Top: ZFP57 bound, motif intact and methylated. Bottom left: ZFP57 does not bind because motif is mutated. Bottom right: ZFP57 does not bind because motif is unmethylated in a given genetic background
Fig. 4 Genetic analysis of ZFP57 consensus binding motif. a Consensus ZFP57 binding motif identified using MEME algorithm analysis of DNA sequences underlying ChIP-seq peaks. b Distribution of genetically determined monoallelic Zfp57 peaks based on motif integrity on the unbound vs. bound alleles: SNPs disrupting all of the motifs (five sites, red), some of the motifs with at least one remaining intact (eight sites, orange) and none of the motifs (seven sites, green). No motifs could be found on either genetic alleles in two cases (grey). c Diagram indicating which bases within the consensus are varied between BL6 and Cast genomes in strain-specific peaks. Long bars, short bars and triangle indicate deletion, SNP and insertion events, respectively. d Putative mechanisms for ZFP57 allele-specific binding. Top: ZFP57 bound, motif intact and methylated. Bottom left: ZFP57 does not bind because motif is mutated. Bottom right: ZFP57 does not bind because motif is unmethylated in a given genetic backgroundCC BY · PubMed Central
Fig. 5 Validation of strain-specific sites. a Independent ChIP-quantitative PCR validation of Zfp57 binding to non-imprinted monoallelic peaks. Overall enrichment levels are shown relative to negative control region (REF) and normalized to non-specific IgG pull-down. b Allele-specific analysis using SNP pyrosequencing of ChIP and 5mC-DIP enriched DNA showing Zfp57 binding is directed by both underlying DNA sequence and methylation status of the allele. Error bars represent standard deviation between three technical replicates
Fig. 5 Validation of strain-specific sites. a Independent ChIP-quantitative PCR validation of Zfp57 binding to non-imprinted monoallelic peaks. Overall enrichment levels are shown relative to negative control region (REF) and normalized to non-specific IgG pull-down. b Allele-specific analysis using SNP pyrosequencing of ChIP and 5mC-DIP enriched DNA showing Zfp57 binding is directed by both underlying DNA sequence and methylation status of the allele. Error bars represent standard deviation between three technical replicatesCC BY · PubMed Central
Fig. 6 Strain-specific expression bias at genes around non-imprinted Zfp57 peaks. Allelic activity and expression level for eight genes found to have significant allelic bias in hybrid ES cells (ES, clear dots) and their neural stem cell derivatives (NS, filled dots); see also Fig. S10 in Additional file 1 and Table 3. The corresponding genomic DNA standard (expected 50 % allele composition) used to control for PCR bias and pyrosequencing artifacts is shown as a fitted linear regression (red dotted line). Allelic composition found by SNP pyrosequencing is plotted along the y-axes as percentage of BL6 expression (0 % = pure Cast, 100 % = pure BL6 and 50 % = perfect biallelic) and overall expression level by quantitative RT-PCR is plotted along the x-axes as absolute levels of the control hybrid genomic DNA (log2 nanograms/PCR). The title in each graph shows gene name and closest associated ZFP57 peak along with its relative position with respect to the gene. Note that for Sdk2 a dilution series of neural stem cell cDNA (high level biallelic expression) was used as a control since the relevant exon boundary panning primers could not amplify genomic DNA
Fig. 6 Strain-specific expression bias at genes around non-imprinted Zfp57 peaks. Allelic activity and expression level for eight genes found to have significant allelic bias in hybrid ES cells (ES, clear dots) and their neural stem cell derivatives (NS, filled dots); see also Fig. S10 in Additional file 1 and Table 3. The corresponding genomic DNA standard (expected 50 % allele composition) used to control for PCR bias and pyrosequencing artifacts is shown as a fitted linear regression (red dotted line). Allelic composition found by SNP pyrosequencing is plotted along the y-axes as percentage of BL6 expression (0 % = pure Cast, 100 % = pure BL6 and 50 % = perfect biallelic) and overall expression level by quantitative RT-PCR is plotted along the x-axes as absolute levels of the control hybrid genomic DNA (log2 nanograms/PCR). The title in each graph shows gene name and closest associated ZFP57 peak along with its relative position with respect to the gene. Note that for Sdk2 a dilution series of neural stem cell cDNA (high level biallelic expression) was used as a control since the relevant exon boundary panning primers could not amplify genomic DNACC BY · PubMed Central

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Metadata

Authors
Strogantsev, R., Krueger, F., Yamazawa, K., Shi, H., Gould, P., Goldman-Roberts, M., McEwen, K., Sun, B., Pedersen, R., & Ferguson-Smith, A. C.
Affiliations
  • Babraham Institute (GB)
  • University of Cambridge (GB)
ORCID
Venue
Genome Biology
Publisher
Springer Science and Business Media LLC
Published
2015-05-30
Volume
16
Issue
1
Pages
112-112
ISSN
1465-6906 (ISSN-L)
DOI
10.1186/s13059-015-0672-7
PMC
PMC4491874
OpenAlex
W2098398752
Cited by
152
References
69
Citations by year
2026: 11 · 2025: 8 · 2024: 4 · 2023: 11 · 2022: 24 · 2021: 8 · 2020: 15 · 2019: 22 · 2018: 12 · 2017: 15 · 2016: 15 · 2015: 7
FWCI
11.05
Type
journal article
Language
en
Open access
Licence
Locations
  • Genome biology (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Apollo (University of Cambridge) (submittedVersion, cc-by)
Funders
  • Wellcome Trust (095606, 095606/Z/11/Z)
  • European Commission (317146, 290123)
  • Biotechnology and Biological Sciences Research Council (BB/G020930/1)
  • Japan Society for the Promotion of Science (15K15478, 25713040)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic Syndromes and Imprinting
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
Keywords
ZFP57, genomic imprinting, monoallelic expression, allele-specific binding, DNA methylation, imprinting control regions, differentially methylated regions, FKBP6, endogenous retroviruses, embryonic stem cells, genetic background effects, male germ cell development, transient neonatal diabetes, Krüppel-like zinc finger protein
MeSH terms
Alleles, Animals, Binding Sites, Cell Line, DNA-Binding Proteins, Mice, Inbred C57BL, Repressor Proteins, Genetic Variation, Genomic Imprinting, DNA Methylation, Histone Code, Mice, Kruppel-Like Transcription Factors

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