Abstract
G2E3 is a putative ubiquitin ligase (E3) identified in a microarray screen for mitotic regulatory proteins. It shuttles between the cytoplasm and nucleus, concentrating in nucleoli and relocalizing to the nucleoplasm in response to DNA damage. In this study, we demonstrate that G2E3 is an unusual ubiquitin ligase that is essential in early embryonic development to prevent apoptotic death. This protein has a catalytically inactive HECT domain and two distinct RING-like ubiquitin ligase domains that catalyze lysine 48-linked polyubiquitination. To address in vivo function, we generated a knock-out mouse model of G2E3 deficiency that incorporates a beta-galactosidase reporter gene under control of the endogenous promoter. Animals heterozygous for G2E3 inactivation are phenotypically normal with no overt change in development, growth, longevity, or fertility, whereas G2E3 null embryos die prior to implantation. Although normal numbers of G2E3(-/-) blastocysts are present at embryonic day 3.5, these blastocysts involute in culture as a result of massive apoptosis. Using beta-galactosidase staining as a marker for protein expression, we demonstrate that G2E3 is predominantly expressed within the central nervous system and the early stages of limb bud formation of the developing embryo. In adult animals, the most intense staining is found in Purkinje cell bodies and cells lining the ductus deferens. In summary, G2E3 is a dual function ubiquitin ligase essential for prevention of apoptosis in early embryogenesis.
Highlights
Many proteins involved in cell cycle regulation and DNA damage signaling are known, we and others have conducted studies to identify other molecules involved in these processes (4 –7)
G2E3 Is a Dual Function Ubiquitin Ligase—G2E3 has four domains that might function in ubiquitin ligation including three RING-like domains in the N-terminal half of the protein and one HECT domain in the C terminus, shown schematically in Fig. 1A and in a previous publication [19]
We found that the insertion lies ϳ570 base pairs including those from retinoblastoma-related protein 2 (Rbr2) downstream from the end of exon 13, disrupting the HECT
Summary
Preparation of Recombinant G2E3 and E6-AP—Cysteine to alanine mutations of G2E3 were generated in PHD/RING1 (C84A), PHD/RING2 (C147A), PHD/RING3 (C258A/C261A), and HECT (C666A) domains using standard PCR mutagenesis. For pre-implantation blastocyst genotyping, lysis was performed in 2 l of genomic lysis buffer (20 mM Tris, pH 8.0, 100 mM KCl, 4 mM MgCl2, 0.9% Nonidet P-40, 0.9% Triton X-100, and 300 ng/ml proteinase K) followed by heat inactivation of the proteinase K. Blastocysts were subjected to microscopic analysis prior to PCR genotyping In these cases, the blastocysts were washed with PBS and air dried prior to lysis in 15 l of genomic lysis buffer. To perform Annexin-V staining, blastocysts were harvested at E3.5 and cultured for 3 days. -Galactosidase Staining of Mouse Embryos—Embryos were harvested from timed pregnant female mice at the indicated day following detection of a vaginal plug and washed in PBS.
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