Carina Y. Howell

Genomic Imprinting Disrupted by a Maternal Effect Mutation in the Dnmt1 Gene

Maintenance of genomic methylation patterns in mammalian somatic cells depends on DNA methyltransferase-1 (Dnmt1). Mouse oocytes and preimplantation embryos lack Dnmt1 but express a variant of this protein called Dnmt1o. We eliminated Dnmt1o by deletion of the oocyte-specific promoter and first exon from the Dnmt1 locus. Homozygous animals were normal, but most heterozygous fetuses of homozygous females died during the last third of gestation. Although genomic methylation patterns were established normally in Dnmt1o-deficient oocytes, embryos derived from such oocytes showed a loss of allele-specific expression and methylation at certain imprinted loci. Transient nuclear localization of Dnmt1o in 8-cell embryos suggests that this variant of Dnmt1 provides maintenance methyltransferase activity specifically at imprinted loci during the fourth embryonic S phase.

Neurologic Defects and Selective Disruption of Basement Membranes in Mice Lacking Entactin-1/Nidogen-1

Entactin-1 (nidogen-1) is an ubiquitous component of basement membranes. From in vitro experiments, entactin-1 was assigned a role in maintaining the structural integrity of the basement membrane because of its binding affinity to other components, such as type IV collagen and laminin. Entactin-1 also interacts with integrin receptors on the cell surface to mediate cell adhesion, spreading, and motility. Targeted disruption of the entactin-1 gene in the mouse presented in this study revealed a duplication of the entacin-1 locus. Homozygous mutants for the functional locus lacked entactin-1 mRNA and protein and often displayed seizure-like symptoms and loss of muscle control in the hind legs. The behavior patterns suggested the presence of neurologic deficits in the central nervous system, thus providing genetic evidence linking entactin-1 to proper functions of the neuromuscular system. In homozygous mutants, structural alterations in the basement membranes were found only in selected locations including brain capillaries and the lens capsule. The morphology of the basement membranes in other tissues examined superficially appeared to be normal. These observations suggest that the lost functions of entactin-1 result in pathologic changes that are highly tissue specific.

cis-Acting signal for inheritance of imprinted DNA methylation patterns in the preimplantation mouse embryo.

ABSTRACT The inheritance of gametic methylation patterns is a critical event in the imprinting of genes. In the case of the imprintedRSVIgmyc transgene, the methylation pattern in the unfertilized egg is maintained by the early mouse embryo, whereas the sperm’s methylation pattern is lost in the early embryo. To investigate the cis-acting requirements for this preimplantation stage of genomic imprinting, we examined the fate of different RSVIgmyc methylation patterns, preimposed onRSVIgmyc and introduced into the mouse zygote by pronuclear injection. RSVIgmyc methylation patterns with a low percentage of methylated CpG dinucleotides, generated by using bacterial cytosine methylases with four-base recognition sequences, were lost in the early embryo. In contrast, methylation was maintained when all CpG dinucleotides were methylated with the bacterialSssI (CpG) methylase. This singular maintenance ofRSVIgmyc methylation preimposed with SssI methylase appears to be specific to the early, undifferentiated embryo; differentiated NIH 3T3 fibroblasts transfected with methylated versions of RSVIgmyc maintained all methylation patterns, independent of the level of preimposed methylation. The methylation pattern of the RSVIgmyc allele in adult founder transgenic mice that was produced by pronuclear injection of anSssI-methylated construct could not be distinguished from the maternal RSVIgmyc methylation pattern. Thus, a highly methylated allele in adult mice, normally generated by transmission of RSVIgmyc through the female germ line, was also produced in founder transgenic mice by bypassing gametogenesis and introducing a highly methylated RSVIgmyc into the mouse zygote. These results suggest that RSVIgmyc methylation itself is a cis-acting signal for the preimplantation maintenance of the oocyte’s methylation pattern and, therefore, acis-acting signal for RSVIgmyc imprinting. Furthermore, our inability to identify a sequence element withinRSVIgmyc that was absolutely required for its imprinting suggests that the extent of RSVIgmyc methylation, rather than a particular pattern of methylation, is the principal feature of this imprinting signal.