Abstract
BackgroundGenomic imprinting evolved in a common ancestor to marsupials and eutherian mammals and ensured the transcription of developmentally important genes from defined parental alleles. The regulation of imprinted genes is often mediated by differentially methylated imprinting control regions (ICRs) that are bound by different proteins in an allele-specific manner, thus forming unique chromatin loops regulating enhancer-promoter interactions. Factors that maintain the allele-specific methylation therefore are essential for the proper transcriptional regulation of imprinted genes. Binding of CCCTC-binding factor (CTCF) to the IGF2/H19-ICR1 is thought to be the key regulator of maternal ICR1 function. Disturbances of the allele-specific CTCF binding are causative for imprinting disorders like the Silver-Russell syndrome (SRS) or the Beckwith-Wiedemann syndrome (BWS), the latter one being associated with a dramatically increased risk to develop nephroblastomas.MethodsKaiso binding to the human ICR1 was detected and analyzed by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA). The role of Kaiso-ICR1 binding on DNA methylation was tested by lentiviral Kaiso knockdown and CRISPR/Cas9 mediated editing of a Kaiso binding site.ResultsWe find that another protein, Kaiso (ZBTB33), characterized as binding to methylated CpG repeats as well as to unmethylated consensus sequences, specifically binds to the human ICR1 and its unmethylated Kaiso binding site (KBS) within the ICR1. Depletion of Kaiso transcription as well as deletion of the ICR1-KBS by CRISPR/Cas9 genome editing results in reduced methylation of the paternal ICR1. Additionally, Kaiso affects transcription of the lncRNA H19 and specifies a role for ICR1 in the transcriptional regulation of this imprinted gene.ConclusionsKaiso binding to unmethylated KBS in the human ICR1 is necessary for ICR1 methylation maintenance and affects transcription rates of the lncRNA H19.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0215-4) contains supplementary material, which is available to authorized users.
Highlights
Genomic imprinting evolved in a common ancestor to marsupials and eutherian mammals and ensured the transcription of developmentally important genes from defined parental alleles
These results indicate that Kaiso binds the human imprinting control region 1 (ICR1) including its Kaiso binding site (KBS) in the B4-repeat
While the full-length B-repeat binding is observed on the methylated paternal allele, binding of the B4-KBS can occur on both alleles, since the KBS cannot be methylated
Summary
Genomic imprinting evolved in a common ancestor to marsupials and eutherian mammals and ensured the transcription of developmentally important genes from defined parental alleles. Methylation maintenance in the ICR1 (and Igf2r-DMR) has been described to be independent of ZFP57 binding [17] Another protein that may contribute to the methylated ICR1-mediated effects is ZBTB33 (Kaiso), a member of the pox virus and zinc finger (POZ) family of zinc finger (ZF) transcription factors that can bind methylated CmGCmG motifs via its three Krüppel-like C2H2 ZFs. In the presence of an appropriate consensus sequence around a single CG dinucleotide, it recognizes the CmG motif [18]. Kaiso is the only POZ-ZF protein known to be able to bind methylated CG motifs [18, 19], as well as the specific DNA sequence 5′ TNGCAGGA3′ (Kaiso binding site (KBS)) with even higher affinity [21]. The partially binding of Kaiso to the unmethylated allele [20] and the presence of a KBS, which can potentially be recognized bound by Kaiso on both alleles, led us to investigate whether Kaiso plays a role in the maintenance of human genomic imprinting via binding at the ICR1
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