C. Cai et al. Figure 8. KHDC1B protein levels and binding partners are cell cycle regulated. (A) NIH-3T3 cells stably expressing Flag-KHDC1A were synchronized using double thymidine arrest. The Western blot shows Flag-KHDC1A expression at the indicated times after release from arrest. The cell cycle was determined by ﬂow cytometry, and the approximate positions of S and M phases are shown. (B) NIH- 3T3 cells stably expressing Flag-KHDC1B were synchronized, and protein levels were detected as in A. KHDC1B protein levels are elevated in late M and G1 phase. (C) KHDC1B, but not KHDC1A interacted with eIF4E in M-phase cells. In untreated cells, HA-eIF4E associated weakly with Flag-KHDC1B. The precipitation efﬁciency was enhanced if the cells were treated with nocodazole to arrest the cells in M-phase. of SCP160. SCP160 is a KH domain in Saccharomyces cervi- siae, containing seven conserved KH domains with the 3146 GXXG loop and seven diverged KH domains, in which the GXXG loop is interrupted. Mutational analysis revealed that both types of KH domains are essential for SCP160 function; furthermore, the diverged KH domains could functionally replace conserved KH domains (Brykailo et al., 2007). This nucleic acid binding activity of KHDC1 proteins may be modulated by their ability to form oligomers (Figure 4) as proposed for other KH domain– containing proteins (Zorn and Krieg, 1997; Ramos et al., 2002). In fact, recent evidence suggests that the KH domain itself can serve as a protein– protein interaction domain in addition to binding nucleic acids (Git and Standart, 2002). Oocytes are the only cell type where both KHDC1A and KHDC1B are expressed and hence the only tissue type where hetero-oligomers are pos- sible. It will be interesting to test whether this interaction is important for regulating oocyte speciﬁc targets and whether KHDC1A exhibits different localization or activity in tissues lacking KHDC1B. Consistent with previous reports (Rajpal et al., 2003), KHDC1A induced what appeared to be an apoptotic re- sponse when expressed in Xenopus embryos. Additional KH-domain proteins have been proposed to regulate apo- ptosis. In particular, a speciﬁc splice isoform of the QKI protein (QKI-7) can induce apoptosis in cultured cells (Pi- lotte et al., 2001). Curiously, this effect is ameliorated by heterodimerization of QKI-7 with other QKI isoforms, and this regulation appears to be controlled via nuclear localiza- tion of QKI protein complexes. Perhaps similar regulatory interactions exist between the different isoforms of KHDC1A or between KHDC1A and 1B proteins. Unlike KHDC1A, KHDC1B did not induce apoptosis. In- stead, it caused a cell cycle arrest phenotype consistent with a role in translational regulation. Furthermore, we have shown that KHDC1B interacts with mCPEB1, a core compo- nent of the cytoplasmic polyadenylation complex known to regulate oocyte maturation and embryonic cell cycle pro- gression. In Xenopus, the members of the CPEB complex are well characterized and include the poly(A) polymerase GLD2, the deadenylating enzyme PARN, the scaffold pro- tein symplekin, and the eIF4E-binding protein and transla- tional inhibitor Maskin. Known CPEB mRNA targets in Xenopus include the cell cycle regulators c-mos and Cyclin B1. In mice, mCPEB1 appears to regulate follicle growth by controlling the polyadenylation and translation of multiple target genes including Gdf9 (Racki and Richter, 2006), but the molecular details of this regulation are unclear. Based on translational paradigms established in other invertebrate Figure 9. A model for the function of KHDC1 proteins during oocyte maturation. KHDC1 proteins accumulate in the cyto- plasm of growing oocytes. If 1A activity dom- inates, oocytes may undergo cell death. If 1B activity dominates, 1B protein can enter the nucleus where it may assemble into CPEB containing RNP complexes selecting them for repression (Lin et al., 2010). Cell survival is rescued and the oocyte matures into a trans- lationally quiescent form. When hormonal cues trigger ovulation, KHDC1B activity falls in response to cell-cycle changes. This trig- gers activation of cytoplasmic polyadenyla- tion and translation of mRNAs required for progression through meiosis. Molecular Biology of the Cell