G2E3 Is Required for Early Embryonic Development.

Abstract

G2E3 is a putative ubiquitin ligase (E3) identified in a microarray screen for genes encoding mitotic regulatory proteins and DNA damage responsive genes. This protein was predicted to function as an ubiquitin ligase based on the presence of a c-terminal HECT domain, a common E3 catalytic domain. Three n-terminal PHD domains likely function as protein-protein interaction domains. We have demonstrated that G2E3 localizes to the nucleus and nucleolus of HeLa cells with dynamic localization responsive to external stimuli. For example, G2E3 rapidly delocalized from the nucleolus after treatment with DNA damaging agents, a property shared with several other proteins involved in DNA damage responses. The protein is extremely labile as a result of proteasome-mediated degradation, accumulating in nucleoli and aggresomes after proteasome inhibition. In this study, we determined the effect of G2E3 gene disruption. Mice were generated with a germline mutation inactivating the G2E3 gene. Animals with a heterozygous inactivation of the G2E3 gene were phenotypically normal with no overt change in development, growth, longevity, or fertility. In contrast, mice with disruption of both alleles did not survive to birth. We genotyped mice from G2E3+/− intercrosses at several embryonic stages and found that no homozygous mutant animals were identified as early as E8.5. To determine whether G2E3−/− animals survive through the earliest stages of development, blastocysts were isolated and genotyped by nested PCR. At E3.5, G2E3 gene disruption does not appear to affect viability since the numbers of wild-type, heterozygous knockout, and homozygous knockout blastocysts are similar to the expected 1:2:1 ratio. Therefore, G2E3 gene disruption does not appear to impair viability in the earliest stages of development but causes embryonic lethality prior to or at about the time of implantation. We further show by β-galactosidase staining that G2E3 is predominantly expressed within the central nervous system of the developing embryo and in the early stages of limb bud formation. These studies show that G2E3 plays an essential role in early mammalian development. The development of conditional knockout animals may be very valuable in determining the precise function of G2E3 in development and the DNA damage response.