ZFP57 / imprinting
ZFP57 is a KRAB zinc-finger protein that reads a six-base sequence, TGCCGC, but only when the CpG inside it is methylated. That combination of a short site and a methylation requirement lets it mark the methylated parental allele at imprinting control regions, the germline-methylated switches that restrict about two dozen loci to expression from one parent's chromosome. ZFP57 binds together with the corepressor TRIM28/KAP1 and DNA methyltransferases, shielding those regions from the wave of demethylation that follows fertilization. The larger zinc finger ZNF445 reads the same element and covers most of the same regions, which is why loss of ZFP57 alone produces imprinting defects with incomplete penetrance. In humans, recessive ZFP57 mutations cause transient neonatal diabetes with hypomethylation at multiple imprinted loci.
The factors
ZFP57 (6p22.1, formerly C6orf40) is a KRAB zinc-finger protein with a short tandem array of C2H2 fingers, of which only the first two are needed for sequence recognition. Like other KZFPs it carries an N-terminal KRAB domain that binds the corepressor TRIM28/KAP1 28273063, and in mouse embryonic stem cells ZFP57 and KAP1 occupy imprinting control regions (ICRs) together with DNA methyltransferases and the hemimethylated-DNA-binding protein NP95/UHRF1 (Quenneville et al. 2011) 22055183. ZFP57 is a maternal-zygotic effect gene: protein and transcript are supplied by the oocyte and the gene is also expressed in the embryo (Li et al. 2008) 18854139. ZNF445 (3p21.31, also catalogued as ZKSCAN15/ZSCAN47) is a much larger zinc finger that binds the same regions, can be traced back to the origins of imprinting, and is expressed earlier in the embryo than ZNF/ZFP57 (Takahashi et al. 2019) 30602440. This entry holds five matrices for the two proteins; a sixth label, the Vierstra archetype AC0364, is annotated only as "ZFP".
The motif
The literature consensus is the hexamer TGCCGC carrying a methylated CpG 22055183. A 1.0 Angstrom crystal structure of the two relevant Zfp57 fingers on fully methylated DNA shows finger 1 contacting the 5' half (TGC) and finger 2 the 3' half (CGC). The two 5-methylcytosines are read asymmetrically: one packs against Arg178, which simultaneously contacts the neighbouring 3' guanine in a 5mC-Arg-G arrangement, while the other is read through a layer of ordered water. Affinity falls for unmodified DNA and for the oxidation products of 5mC, and two point mutations found in patients abolish binding (Liu et al. 2012) 23059534. The atlas matrices agree with this. JASPAR MA1583.2 is TGCCGCA; HOCOMOCO, CIS-BP and the Vierstra archetype all give GCGGCA, which is the reverse complement of TGCCGC, so the apparent disagreement with the literature consensus is only a choice of strand 37962376,37971293. Family cohesion is 0.76, the highest value among the zinc-finger entries in this group and the level expected of a genuinely single motif. One member does not fit: the CIS-BP model for ZNF445 (M07674) is a 29 bp, 31-bit matrix unrelated to the hexamer, and nothing else in the atlas resembles it.
Where it acts
ICRs, also called germline differentially methylated regions, number on the order of 20 to 25 in human and mouse and include the regions controlling SNRPN, KCNQ1OT1, H19/IGF2, GNAS and PLAGL1. ZFP57 and KAP1 bind only the H3K9me3-bearing, DNA-methylated allele, so occupancy is monoallelic by construction (Quenneville et al. 2011) 22055183. Binding is not confined to ICRs: several tens of additional methylated loci carry the hexamer and lose methylation when ZFP57 is deleted in ES cells 22055183, and allele-specific ZFP57 binding also occurs at non-imprinted monoallelically expressed regions (Strogantsev et al. 2015) 26025256. Real ICRs usually contain a cluster of hexamers rather than one copy, and clustering discriminates far better than any single match.
Biology and disease
Mice lacking only zygotic Zfp57 show partial neonatal lethality; removing maternal and zygotic function together is close to fully embryonic lethal, and maternal methylation imprints fail to be established at the Snrpn region (Li et al. 2008) 18854139. In humans, recessive ZFP57 mutations cause transient neonatal diabetes mellitus with mosaic hypomethylation at multiple imprinted loci, the first heritable multi-locus imprinting disorder compatible with life (Mackay et al. 2008) 18622393, with long-term clinical follow-up reported later (Boonen et al. 2013) 23150280. Penetrance is incomplete, which is what motivated the search that found ZNF445: in mice Zfp445 and Zfp57 together maintain all but one ICR, and in humans ZNF445 is intolerant of loss-of-function variation, suggesting it carries more of the load than ZFP57 (Takahashi et al. 2019) 30602440.
Reading a hit
A six-base site occurs by chance roughly once every few kilobases per strand, so an isolated TGCCGC match licenses no conclusion. Three things make a hit interesting: the CpG is methylated in your sample, several copies cluster within a few hundred bases, and the interval overlaps an annotated germline DMR. Position-weight-matrix scanning cannot see methylation, so a scan returns candidate sites only. ZFP57 and ZNF445 cannot be separated from sequence. Neither can every GCGGC-containing zinc-finger motif: the HOCOMOCO model for ZNF729, a KRAB zinc finger with no known imprinting role, is TGCGGCAG, and its nearest neighbour anywhere in the atlas is the ZFP57 matrix. The HOCOMOCO ZFP57 model itself is ChIP-derived (evidence code P, grade B), so confidence that this protein binds this sequence rests on the structure and biochemistry in the primary papers rather than on the motif model.
How this was worked out
Knocking out Zfp57 in mouse showed it is a maternal-zygotic effect gene required to maintain both maternal and paternal methylation imprints: zygotic loss gives partial neonatal lethality, loss of both maternal and zygotic function is highly penetrant embryonic lethality, and maternal imprints at Snrpn fail to be established.
Still open then: What DNA sequence ZFP57 recognized, and whether it bound imprinting control regions directly, were unknown.
Mutations in ZFP57 were found in seven pedigrees with transient neonatal diabetes and mosaic hypomethylation at several imprinted loci, the first heritable multi-locus imprinting disorder compatible with life.
Still open then: The incomplete and mosaic pattern of hypomethylation had no mechanistic explanation.
- 2011highChIP-seq and ChIP-chip in mouse ES cells, deletion of Zfp57, Kap1 and DNMTs, motif discovery22055183
ZFP57 and KAP1 were shown to bind the H3K9me3-bearing, DNA-methylated allele of imprinting control regions in embryonic stem cells, and the recognized element was identified as the methylated hexanucleotide TGCCGC, present in all imprinting control regions and in several tens of other methylated loci.
Still open then: How a zinc finger could require methylation of its own recognition site was not explained structurally.
A 1.0 Angstrom crystal structure of the two relevant Zfp57 fingers on fully methylated DNA showed the hexamer read across two fingers with the two 5-methylcytosines contacted asymmetrically, one through a 5mC-Arg-G interaction with Arg178 and the other through ordered water, with reduced affinity for unmodified DNA and for 5mC oxidation products.
Allele-resolved mapping in hybrid ES cells assigned allele specificity to about two-thirds of ZFP57 sites and showed that parental-origin-specific, methylation-dependent binding occurs only at germline differentially methylated regions, while other sites are biallelic or vary with genetic background, including endogenous retrovirus targets and a newly imprinted gene, Fkbp6.
ZNF445 was shown to bind imprinting control regions in mouse and human and to act with ZFP57 in maintaining all but one mouse ICR; because ZNF445 is expressed earlier and is intolerant of loss-of-function variation in humans, the ZFP57-centered account of imprint maintenance was revised toward a two-factor model in which ZNF445 may carry more of the human load.
Still open then: No human with biallelic ZNF445 loss has been described, so its clinical importance is inferred rather than observed.
Still unresolved
- The division of labour between ZFP57 and ZNF445 in humans is unsettled: mouse genetics assigns imprint maintenance to the two factors jointly, while human expression timing and constraint data suggest ZNF445 carries more of it, and no patient with biallelic ZNF445 loss has been reported to test this.
- Why ZFP57 loss produces mosaic, incompletely penetrant hypomethylation that varies between loci and between patients has no mechanistic explanation.
- The CIS-BP model for ZNF445 is a 29 bp matrix unrelated to the methylated hexamer, which contradicts the literature claim that ZNF445 reads the same element as ZFP57; neither the matrix nor a hexamer-based ZNF445 model has been checked against in vitro binding data.
- Whether ZFP57 binding outside germline differentially methylated regions, at biallelic endogenous retrovirus copies and at background-dependent monoallelic sites, has any regulatory consequence is untested.
A TGCCGC match is a candidate, not a site. The hexamer is short enough to appear roughly a million times in the genome, and the property that makes it functional, CpG methylation, is invisible to motif scanning. Treat a hit as meaningful only with methylation data, clustered copies, or germline-DMR annotation. The motif does not distinguish ZFP57 from ZNF445, and short GCGGC-containing motifs of unrelated KRAB zinc fingers such as ZNF729 score almost as well. The ZFP57 models in HOCOMOCO and the Vierstra set are ChIP-derived or unnamed; the in vitro and structural support comes from the primary literature, not from these matrices.
References
- Quenneville S, Verde G, Corsinotti A, et al. In embryonic stem cells, ZFP57/KAP1 recognize a methylated hexanucleotide to affect chromatin and DNA methylation of imprinting control regions. Mol Cell 2011;44:361-72. PMID 22055183
- Liu Y, Toh H, Sasaki H, et al. An atomic model of Zfp57 recognition of CpG methylation within a specific DNA sequence. Genes Dev 2012;26:2374-9. PMID 23059534
- Li X, Ito M, Zhou F, et al. A maternal-zygotic effect gene, Zfp57, maintains both maternal and paternal imprints. Dev Cell 2008;15:547-57. PMID 18854139
- Mackay DJ, Callaway JL, Marks SM, et al. Hypomethylation of multiple imprinted loci in individuals with transient neonatal diabetes is associated with mutations in ZFP57. Nat Genet 2008;40:949-51. PMID 18622393
- Takahashi N, Coluccio A, Thorball CW, et al. ZNF445 is a primary regulator of genomic imprinting. Genes Dev 2019;33:49-54. PMID 30602440
- Strogantsev R, Krueger F, Yamazawa K, et al. Allele-specific binding of ZFP57 in the epigenetic regulation of imprinted and non-imprinted monoallelic expression. Genome Biol 2015;16:112. PMID 26025256
- Boonen SE, Mackay DJ, Hahnemann JM, et al. Transient neonatal diabetes, ZFP57, and hypomethylation of multiple imprinted loci: a detailed follow-up. Diabetes Care 2013;36:505-12. PMID 23150280
- Imbeault M, Helleboid PY, Trono D. KRAB zinc-finger proteins contribute to the evolution of gene regulatory networks. Nature 2017;543:550-554. PMID 28273063
- Vorontsov IE, Eliseeva IA, Zinkevich A, et al. HOCOMOCO in 2024: a rebuild of the curated collection of binding models for human and mouse transcription factors. Nucleic Acids Res 2024;52:D154-D163. PMID 37971293
- Rauluseviciute I, Riudavets-Puig R, Blanc-Mathieu R, et al. JASPAR 2024: 20th anniversary of the open-access database of transcription factor binding profiles. Nucleic Acids Res 2024;52:D174-D182. PMID 37962376
5 motifs in this family — JASPAR 1 · HOCOMOCO 1 · CIS-BP 2 · Vierstra 1
| motif | database | consensus | bits |
|---|---|---|---|
| M07674_2.00 | CIS-BP | AAATCCttGACaAtCaAGGgTTCcctCaC | 30.9 |
| ZFP57.H14CORE.0.P.BPB | HOCOMOCO | GCGGCA | 12.3 |
| M07756_2.00 | CIS-BP | GCGGCA | 12 |
| ZFP57 (MA1583.2) | JASPAR | TGCCGCA | 11.3 |
| AC0364:ZFP:C2H2_ZF | Vierstra | GCGGCA | 11 |
HOCOMOCO tags read as assay + quality grade: P ChIP-seq, S HT-SELEX, M methyl-HT-SELEX, G GHT-SELEX, I SMiLE-seq, B PBM; grade A means two or more assay types agreed. A P-only matrix is a record of where the protein was crosslinked, which need not be a sequence it binds itself.
The factors, elsewhere
These entries are written from the primary literature and every citation is checked against PubMed, but this one can still be out of date, too confident, or simply wrong — especially where a motif’s published site and its database matrices disagree. Corrections and additions are welcome, and they are the fastest way to make this page better for everyone.