CUL-4A Is Critical for Early Embryonic Development

Abstract

Ubiquitin-mediated degradation targets cell cycle regulators for proteolysis. Much of the ubiquitin pathway's substrate specificity is conferred by E3 ubiquitin ligases, and cullins are core components of some E3s. CUL-4A encodes one of six mammalian cullins and is amplified and/or overexpressed in breast cancer, which suggests a role in regulating cell cycle progression. To examine CUL-4A's physiologic function, we generated a CUL-4A deletion mutation in mice. No viable CUL-4A^−/− pups and no homozygous mutant embryos as early as 7.5 days postcoitum (dpc) were recovered. However, CUL-4A^−/− blastocysts are viable, hatch, form an inner cell mass and trophectoderm, and implant (roughly 4.5 dpc), indicating that CUL-4A^−/− embryos die between 4.5 and 7.5 dpc. Despite 87% similarity between the Cul-4A and Cul-4B cullins, the CUL-4A^−/− lethal phenotype indicates that CUL-4A has one or more distinct function(s). Surprisingly, 44% fewer heterozygous pups were recovered than expected by Mendelian genetics, indicating that many heterozygous embryos also die during gestation due to haploinsufficiency. Taken together, our findings indicate that appropriate CUL-4A expression is critical for early embryonic development.

This work was supported by an American Heart Association Midwest Affiliate Scientist Development Grant (9930341Z), an Indiana University Cancer Center Development Grant (NIH P30CA82709), a grant from the Showalter Research Trust Fund, an American Cancer Society Indiana University Institutional Research Grant, and an Indiana University School of Medicine Core Centers of Excellence in Molecular Hematology grant (NIH P30DK49218) awarded to K.T.C. and Riley Memorial Association grants awarded separately to J.C.R. and K.T.C.

We thank Dave Skalnik and Merv Yoder for advice and helpful discussions, Loren Field for advice and for expertly isolating 0.5-dpc embryos, the Indiana University Cancer Center knockout mouse core facility for ES cell transfections, blastocyst injections, and generation of chimeric mice, and Stephany Scruggs, Heather Noffsinger, Candice Horn, and Prianto Moeljadi for diligent technical assistance.