Cai C et al. (2010), KHDC1B is a novel CPEB binding partner specific...

XB-ART-41817

Mol Biol Cell 2010 Sep 15;2118:3137-48. doi: 10.1091/mbc.E10-03-0255.

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KHDC1B is a novel CPEB binding partner specifically expressed in mouse oocytes and early embryos.

Cai C , Tamai K , Molyneaux K .

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mRNAs required for meiotic maturation and early embryonic development are stored in growing oocytes. These transcripts are translationally repressed until hormonal cues trigger ovulation. Errors in translation underlie some cases of human infertility and are associated with ovarian germ cell tumors. However, it remains unclear how maternal transcripts are kept quiescent in mammals. This study describes a potential translational regulator, KHDC1B. KHDC1B is a member of a small family of KH-domain containing proteins specific to eutherian mammals. Two family members, KHDC1A and 1B, are highly expressed in oocytes. KHDC1A and 1B bind polyU agarose and form oligomers like other KH-domain proteins. The functions of these proteins were tested by expression in Xenopus embryos. KHDC1A caused cell death, whereas KHDC1B caused cleavage arrest. This arrest phenotype was rescued by coexpression of the mouse translational regulator cytoplasmic polyadenylation binding protein 1 (mCPEB1). Coimmunoprecipitation and coimmunostaining experiments confirmed the functional interaction between KHDC1B and mCPEB1. Finally, KHDC1B levels and binding partners were shown to fluctuate with the cell cycle. KHDC1B, via its interaction with mCEPB1, may regulate translation of mRNA targets required for oocyte maturation.

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Species referenced: Xenopus laevis
Genes referenced: cpeb1 eif4e tbx2 tcf7l1 tle1 tle2

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	Figure 1. Alignment of KHDC1A and KHDC1B proteins. Overall the proteins are 65% identical (black-boxed amino acids). Identity is highest in the N-terminal region containing the KH domain (arrow).
	Figure 2. Khdc1 family members are expressed in oocytes and early embryos. (A) RT-PCR for Khdc1a and Khdc1b expression in the indicated tissues. Gapdh expression is shown as a loading control. RT- indicates the negative control sample run without reverse transcriptase. (B) RT-PCR for Khdc1a and Khdc1b expression in isolated germinal vesicle (GV) and meiotic M-phase II (MII) oocytes and embryos. The stage of embryo is indicated by the cell number or by embryonic day. (C) RT-PCR for Khdc1a and Khdc1b in fetal (E12.5–16.5) and postnatal (Day 0–16) testes (T) and ovaries (O). E8.5 indicates whole embryonic day 8.5 embryos. (D) In situ hybridization for Khdc1a and Khdc1b in adult ovary sections. Sense probes were used as controls. Solid insets are higher magnification preantral follicles. Preantral follicles contain growing oocytes surrounded by one or a few layers of granulosa cells. Dashed insets are higher-magnification antral follicles. Antral follicles represent the final stage in follicle growth, contain transcriptionally and translationally quiescent oocytes surrounded by multiple layers of granulosa cells, and contain a fluid-filled space termed the antrum. Scale bar = 240 μm.
	Figure 3. Expression profile for KHDC1 proteins. (A) Antibody specificity was tested by Western blotting. 293T cells were transfected with the indicated plasmids. The pan-KHDC1 antibody recognizes both 1A and 1B. The peptide antibody (α-KHDC1A) is specific for 1A. (B) KHDC1A is expressed in multiple tissues as detected using the α-KHDC1A antibody. The predicated size for KHDC1A is 19.6 kDa. In addition to the full-length form, a 15 kDa isoform of 1A is detected in testis. Tubulin is shown as a loading control. (C) The α-KHDC1A antibody detects a 15-kDa isoform in germinal vesicle (GV), germinal vesicle breakdown (GVBD), and meiotic M-phase I (MI) and meiotic M-phase II (MII) stage oocytes. Expression was also detected in early cleavage stage embryos. (D) Immunostaining for pan-KHDC1 in frozen sections taken from adult ovaries. Preantral follicles containing oocytes surrounded by two layers of granulosa cells or antral follicles surrounded by multiple layers of granulosa cells are shown. Staining is either cytoplasmic or nuclear, and arrows indicate the position of the oocyte. The antibody also stains granulosa cells (g) and ovarian stroma (s). An antral follicle processed without primary antibody is shown as a negative control. Scale bar = 82 μm. (E) Immunostaining for pan-KHDC1 during meiotic maturation and early embryonic development. Staining is shown in germinal vesicle (a), germinal vesicle breakdown (b), and meiotic M-phase II (c) stage oocytes and 1-cell (d and e), 2-cell (f), 4-cell (g), 8-cell (h), 16-cell (i), morula (j), and blastocyst (k) stage embryos. A two-cell embryo processed without primary antibody (l) is shown as a negative control. Arrows indicate perinuclear staining. Scale bar = 20 μm.
	Figure 4. KHDC1A and 1B bind polyU and can multimerize like typical KH domain containing proteins. (A) GST-KHDC1A and GST-KHDC1B bind PolyU agarose. GST alone is shown as a control. (B) HA-KHDC1A coimmunoprecipitates with Flag-KHDC1A (lane 1) or Flag-KHDC1B (lane 3) expressed in 293T cells. HA-KHDC1B also coimmunoprecipitates with Flag-KHDC1B (lane 5). This demonstrates that KHDC1 proteins can form homo or hetero multimers.
	Figure 5. Ectopic expression of KHDC1 proteins perturb Xenopus development. (A) 1 ng of Flag-Khdc1a or Flag-Khdc1b RNA was injected into one cell of two-cell stage Xenopus embryos. Phenotypes were scored at stage 6.5 or at stage 8.5. KHDC1A expression caused a small cell phenotype by stage 6.5 (outlines) and cell death by 8.5. KHDC1B expression caused cleavage arrest (outlines) and eventually cell death after stage 8.5 (see D). Scale bar = 500 μm. (B) Ectopic expression of KHDC1B altered microtubule distribution. Frozen sections from control or Khdc1b-injected embryos were stained for β-tubulin. Control cells have normal mitotic spindles. Khdc1b-injected cells have many small foci of β-tubulin. Scale bar = 100 μm. (C) Ectopic expression of KHDC1B altered the distribution of nuclei. The KHDC1B expressing region marked by Flag staining contains very few nuclei. Dotted lines indicate the edge of the tissue section and the Flag expressing region. Scale bar = 250 μm. (d-E) mCPEB1 rescues the KHDC1 phenotypes. 1 ng of the indicated mRNAs was injected into one cell of two-cell stage embryos and embryos were cultured to midblastula transition (stage 9–9.5). (D) Expression of the control KH domain protein ESG1 did not perturb Xenopus development. Expression of KHDC1B by itself caused cell cycle arrest and death by MBT. Coinjection of a control mRNA encoding for luciferase did not rescue the KHDC1B phenotype. However, coinjection of mCpeb1 rescued both the cell cycle arrest phenotype and embryo survival. Expression of mCPEB1 by itself did not perturb Xenopus development. Expression of KHDC1A caused a small cell phenotype and embryonic death. Expression of mCPEB1 partially rescued the KHDC1A phenotype. Scale bar = 500 μm. (E) Percent of control and injected embryos exhibiting normal development to midblastula transition (MBT). n = the total number of embryos counted. Data were collected from three independent experiments, and error bars show SD.
	Figure 6. KHDC1 proteins associate with mCPEB1. (A) Flag-KHDC1A and Flag-KHDC1B coimmunoprecipitate with HA-mCPEB1 expressed in Xenopus embryos or in 293T cells. (B) The interaction between KHDC1B and mCPEB1 is independent of RNA. (C) Full-length mCPEB1 and the deletion constructs used to test for interaction with KHDC1B. The PEST domain is a motif that regulates protein stability. The RRM (RNA recognition motif) and ZF (zinc finger) domains control RNA binding. (D) HA-mCPEB1 and HA-mCPEB1-Cter coimmunoprecipitate with Flag-KHDC1B when coexpressed in 293T cells. HA-CPEB-Nter did not interact with KHDC1B in this assay. * in the input indicates nonspecific signal. For input, 5% of the total lysate was loaded.
	Figure 7. KHDC1 proteins colocalize with mCPEB1 in Xenopus embryos, mammalian cells, and mouse oocytes. 1 ng of HA-mCpeb1 RNA was injected together with (A) 1 ng of Flag-Khdc1a or (B) 1 ng of Flag-Khdc1b mRNA into one cell of two-cell stage Xenopus embryos. Embryos were allowed to develop to stage 6, and protein localization was examined by immunostaining for the Flag (FITC, green) and HA (Cy5, magenta) tags. Images are single optical sections taken via confocal and are representative of the expression pattern seen in the majority of cells. A single blastomere from an injected region is shown. mCPEB1 and KHDC1 proteins accumulated around the spindle in dividing cells. Note the tripolar spindle structure resulting from KHDC1B expression. Scale bar in A = 10 μm. Scale bar in B = 14 μm. (C) Flag-KHDC1A and HA-mCPEB1 were coexpressed in HeLa cells and protein localization was detected as above. Scale bar = 6 μm. (D) Flag-KHDC1B and HA-mCPEB1 were coexpressed in HeLa cells, and their distribution was detected by immunostaining. Scale bar = 10 μm. (E) Immunostaining using the pan-KHDC1 antibody (Cy5, magenta) and a mouse mAb against mCPEB1 (FITC, green) reveal that the endogenous proteins colocalize in GV oocytes. Scale bar = 40 μm. (F) KHDC1 and mCPEB1 also partially colocalize in MI stage oocytes. Note that mCPEB1 is enriched on the spindle, but endogenous KHDC1 proteins are not. Scale bar = 40 μm.
	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.
	Figure 9. A model for the function of KHDC1 proteins during oocyte maturation. KHDC1 proteins accumulate in the cytoplasm of growing oocytes. If 1A activity dominates, 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 translationally quiescent form. When hormonal cues trigger ovulation, KHDC1B activity falls in response to cell-cycle changes. This triggers activation of cytoplasmic polyadenylation and translation of mRNAs required for progression through meiosis.

References [+] :

Barnard, Symplekin and xGLD-2 are required for CPEB-mediated cytoplasmic polyadenylation. 2004, Pubmed, Xenbase