Maintenance and Acquisition of Genomic DNA Methylation Imprints

    Genomic imprinting is essential for mammalian development. Consistent with this, a majority of cloned embryos die in utero, mainly from the failure to re-establish proper genomic imprints. Cell-based therapies offer great hopes for various degenerative diseases, including Alzheimer’s disease and diabetes. Embryonic stem (ES) cells derived from nuclear transfer experiments and induced pluripotent stem (iPS) cells reprogrammed from adult cells are candidates for this therapeutic approach. However, they all have genomic imprinting defects which can inhibit differentiation and may even cause cancer. Therefore, a requirement for attaining properly differentiated cell lineages for therapeutic applications is a thorough understanding of the underlying mechanisms of genomic imprinting.

     Despite that genomic imprinting was identified in mammals almost three decades ago, many unanswered questions persist about the nature of genomic imprinting memory and about how genomic imprints are established and maintained. Recently, we discovered Zfp57 in a screen for ES cell-specific genes. Zfp57 encodes a KRAB zinc finger protein and exhibits maternal-zygotic embryonic lethality in mouse, the first one identified in mammals. ZFP57 is a key regulator in genomic imprinting at three different phases: establishment, maintenance and re-acquisition of DNA methylation imprints. Consistent with our finding, mutations in human Zfp57 result in hypomethylation at multiple imprinted regions. ZFP57 maintains both paternal and maternal genomic imprints and may target DNA methyltransferases to the imprinting control regions (ICR). Interestingly, these germline-derived differential DNA methylation imprints at the ICRs can be lost in embryos in the absence of both maternal and zygotic Zfp57 and they can be acquired in mouse embryos in the presence of zygotic Zfp57 even though they are not established in the germline. Moreover, acquisition of DNA methylation in mouse embryos occurs in an allele-specific fashion, suggesting the presence of DNA methylation-independent heritable imprinting memory. Therefore, further functional analysis of ZFP57 in genomic imprinting will help us to achieve better understanding of the molecular nature of imprinting memory as well as the temporal and developmental control of DNA methylation imprints.

  To gain insights into the underlying molecular mechanisms of establishment, maintenance and acquisition of DNA methylation genomic imprints, we have identified some ZFP57-associated proteins through co-immunoprecipitation coupled with mass spectrometry. We are in the process of verifying their interactions with ZFP57. Then we will characterize their functions in ES cells as well as in mouse embryos with respect to DNA methylation in genomic imprinting. The approaches we are undertaking are either through gene targeting or RNAi knockdown to reduce or eliminate the functions of a target gene.

   Dysregulation of imprinting genes can lead to cancer, neurological diseases, diabetes and other kinds of human diseases. Indeed, mutations in human ZFP57 are associated with transient neonatal diabetes and congenital heart defects. We have independently discovered that Zfp57 mutant mouse also display defects in the cardiovascular system. Thus, our mutant mouse could serve as a good animal model for analyzing imprinting-related human diseases.

Figure 1.  RNA in-situ hybridization reveals that Zfp57 is expressed specifically in the oocytes (purple stain) within the follicles.

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Figure 2.  Cycle of differential DNA methylation at the imprinting control regions (ICR) in mouse. Differential DNA methylation at the CpG sites of ICRs is reset during gametogenesis. Upon fertilization, the zygote inherits germline-derived genomic DNA methylation imprints as well as non-imprinted methylation. Differential DNA methylation at ICRs are protected from genome-wide demethylation in early embryos whereas non-imprinted methylation is erased during this process. In the absence of ZFP57 (-ZFP57), differential DNA methylation is not protected in early embryos. Without the maternal ZFP57 (-ZFP57), germline DNA methylation is not established at the Snrpn imprinted region. However, methylation could be acquired at the Snrpn ICR in the presence of the zygotic ZFP57 (+ZFP57) in mouse embryos.

© 2008 Mount Sinai Medical Center