Polycomb-group complex 1 acts as an E3 ubiquitin ligase for Geminin to sustain hematopoietic stem cell activity

Abstract

Polycomb-group (PcG) genes encode multimeric nuclear protein complexes, PcG complex 1 and 2. PcG complex 2 was proved to induce transcription repression and to further methylate histone H3 at lysine-27 (H3K27). Subsequently PcG complex 1 is recruited through recognition of methylated H3K27 and maintains the transcription silencing by mediating monoubiquitination of histone H2A at lysine-119. Genetic evidence demonstrated a crucial role for PcG complex 1 in stem cells, and Bmi1, a member of PcG complex 1, was shown to sustain adult stem cells through direct repression of the INK4a locus encoding cyclin-dependent kinase inhibitor, p16CKI, and p19ARF. The molecular functions of PcG complex 1, however, remain insufficiently understood. In our study, deficiency of Rae28, a member of PcG complex 1, was found to impair ubiquitin-proteasome-mediated degradation of Geminin, an inhibitor of DNA replication licensing factor Cdt1, and to increase protein stability. The resultant accumulation of Geminin, based on evidence from retroviral transduction experiments, presumably eliminated hematopoietic stem cell activity in Rae28-deficient mice. Rae28 mediates recruiting Scmh1, which provides PcG complex 1 an interaction domain for Geminin. Moreover, PcG complex 1 acts as the E3 ubiquitin ligase for Geminin, as we demonstrated in vivo as well as in vitro by using purified recombinant PcG complex 1 reconstituted in insect cells. Our findings suggest that PcG complex 1 supports the activity of hematopoietic stem cells, in which high-level Geminin expression induces quiescence securing genome stability, by enhancing cycling capability and hematopoietic activity through direct regulation of Geminin.

Fig. 1.

Expression of Geminin, Cdt1, and PcG members. (A) CD34⁻KSL, CD34⁺KSL, progenitors, and their progeny subpopulations were isolated from murine BM cells, and mRNA expression was examined by quantitative RT-PCR. The mRNA expression levels are shown as ratios to the level in GAPDH. Lane 1, CD34⁻KSL (LT-HSCs); lane 2, CD34⁺KSL (MPPs); lane 3, progenitors; lane 4, Ter119⁺ cells (erythroid cells); lane 5, Gr1⁺ cells (granulocytes); lane 6, CD3⁺ cells (T cells); lane 7, B220⁺ cells (B cells). (B) Geminin expression in G₀/G₁-phase CD34⁻KSL, CD34⁺KSL, and progenitors was further examined at the protein level by cell sorting. Average intensity of Geminin expression in three independent experiments is shown with SEMs.

Fig. 2.

Geminin expression and its effect on LTR activity. (A) Geminin protein expression in FL. FL were examined by immunoblot analysis with an anti-Geminin antibody. β-Actin detection confirmed equal amounts of protein on the filter. Wild type: Rae^+/+. (B) Stability of Geminin. FL were pulse-labeled with [³⁵S]methionine in FL. Geminin was immunoprecipitated and was detected by autoradiography. (C) Cdt1 and Mcm2in chromatin fraction of 15.5-days-postcoitum FL. S, soluble fraction; C, chromatin fraction. Histone H2A was detected as a control. (D) Effect of exogeneous Geminin on LTR activity. Retrovirally transduced FL were injected into lethally irradiated syngeneic mice, and the repopulation was examined 1 and 3 months after injection. The number of recipient mice is indicated above each bar. d.p.c., days postcoitum.

Fig. 3.

Involvement of UPS in the regulation of Geminin and Scmh1. (A) Sensitivity to MG132. The lanes of Geminin and Scmh1 show the effect of MG132 on the endogenous and exogeneous expression in U-2 OS cells, respectively. α-Tubulin detection confirmed equal amounts of protein on the filter. (B) In vivo ubiquitination of Geminin. Either Geminin or Geminin-DBD was cotransfected with ubiquitin into HEK-293 cells and was detected by immunoblot analysis. Cellular synchronization was done by thymidine treatment. DNA content is shown below the blots to confirm cellular synchronization.

Fig. 4.

Reconstitution of PcG complex 1 in Sf9 cells and the E3 ubiquitin ligase activity for Geminin. (A) Pull-down assay of the recombinant PcG complex 1. Schematic representation of the complex is shown below the blots. Note that Rae28 and Scmh1 interact with GST-Ring1B even in the absence of Bmi1. Affinity-purified recombinant complex was subjected to in vitro ubiquitination reaction (myc-Geminin+E1+E2+ubiquitin) with indicated modifications. B–E shows E3 ubiquitin ligase activity for Geminin of the recombinant complexes. (B) E1 and E2 dependency. (C) Reaction with biotin-tagged ubiquitin (Biotin-ubiquitin). The reaction products were immunoblotted with an anti-myc antibody (Right) and were also immunoprecipitated with an anti-myc antibody, and molecules modified with biotin-ubiquitin were detected with the VECTASTAIN ABC kit (Left). (D) Effect of methyl-ubiquitin. Mobility-shifted bands other than the lower two may represent polyubiquitinated Geminin. Mono- and polyubiquitinated molecules are indicated by Ub1 and Ubn, respectively. (E) Comparison of E3 ubiquitin ligase activity among PC1–4, PC1–3(-Bmi1), PC1–3(-Rae28), PC1–3(-Scmh1), and GST-Ring1B. *, no reaction. One microgram of PC1–4 was subjected to the reaction, and the amount of GST-Ring1B in the other complexes was adjusted to that in 1 μg of PC1–4. E3 ubiquitin ligase activity was reduced in PC1–3 and GST-Ring1B. Note that polyubiquitinated Geminin was hardly detectable in PC1–3(-Rae28), PC1–3(-Scmh1), and GST-Ring1B.

Fig. 5.

PcG complex 1 directly regulates Geminin by acting as the E3 ubiquitin ligase. Cdt1 is loaded on chromatin and licenses the chromatin for DNA replication, whereas Geminin inhibits the licensing through the direct interaction with Cdt1. PcG complex 1 induces polyubiquitination of Geminin by acting as the E3 ubiquitin ligase, and the polyubiquitin chain may serve as a recognition signal for the proteasome to regulate stability of Geminin. Ub, ubiquitin.