Idas, a novel phylogenetically conserved geminin-related protein, binds to geminin and is required for cell cycle progression

Abstract

Development and homeostasis of multicellular organisms relies on an intricate balance between cell proliferation and differentiation. Geminin regulates the cell cycle by directly binding and inhibiting the DNA replication licensing factor Cdt1. Geminin also interacts with transcriptional regulators of differentiation and chromatin remodelling factors, and its balanced interactions are implicated in proliferation-differentiation decisions during development. Here, we describe Idas (Idas being a cousin of the Gemini in Ancient Greek Mythology), a previously uncharacterised coiled-coil protein related to Geminin. We show that human Idas localizes to the nucleus, forms a complex with Geminin both in cells and in vitro through coiled-coil mediated interactions, and can change Geminin subcellular localization. Idas does not associate with Cdt1 and prevents Geminin from binding to Cdt1 in vitro. Idas depletion from cells affects cell cycle progression; cells accumulate in S phase and are unable to efficiently progress to mitosis. Idas protein levels decrease in anaphase, whereas its overexpression causes mitotic defects. During development, we show that Idas exhibits high level expression in the choroid plexus and the cortical hem of the mouse telencephalon. Our data highlight Idas as a novel Geminin binding partner, implicated in cell cycle progression, and a putative regulator of proliferation-differentiation decisions during development.

FIGURE 1.

Idas is a nuclear protein related to Geminin. A, multiple sequence alignment of Geminin in human, mouse, Xenopus, and Drosophila with the predicted Idas sequences in human, mouse, and Xenopus is shown. Black box, conserved coiled-coil region; red box, conserved C-terminal region; gray box, D-box region. Putative D-box (gray) and NLS (black) are underlined. Polar residues in a and d coiled-coil positions are marked by arrowheads, and residues important for Cdt1 binding by dots (black, conserved in Idas; gray, not conserved). B, MCF7 cells were transfected with IdasGFP (a and b), N-IdasGFP (e and f), and C-IdasGFP (g and h). Immunofluorescence of endogenous Geminin is shown for comparison (c and d). DNA was stained with Hoechst.

FIGURE 2.

Idas interacts with Geminin in cell extracts. A, Geminin interacts with Idas through its coiled coil. MCF7 cells were co-transfected with IdasHA and GemininGFP (lanes 1 and 5), Geminin Δ90–120 GFP (lanes 2 and 6), or GFP (lanes 3 and 7). Total extracts (Totals, left) and anti-HA immunoprecipitates (IPs, right) were immunoblotted for GFP (upper) and HA (lower). Lanes 4 and 8, control immunoprecipitation of IdasHA-GemininGFP transfected cells with mouse IgGs. B, endogenous Geminin-Idas interaction is shown. Total cell extracts from HeLa cells were immunoprecipitated with two different purifications of an anti-Geminin mouse monoclonal antibody (lanes 4 and 5). Total lysates and immunoprecipitates were immunoblotted for Idas (upper panel) and Geminin (lower panel). Mouse IgG (mIgG) immunoprecipitates were used as a negative control. C, Idas self-associates. MCF7 cells were co-transfected with IdasHA and GemininGFP (lanes 1 and 5), IdasGFP (lanes 2 and 6), or GFP (lanes 3 and 7). Total cells lysates (left) and anti-HA immunoprecipitates (right) were immunoblotted for GFP and HA. Lanes 4 and 8, control immunoprecipitation with mouse IgGs. D, Geminin competes for Idas-Idas interactions. MCF7 cells were co-transfected with IdasHA, IdasGFP and GFP (lanes 1 and 4) or with IdasHA, IdasGFP, and GemininGFP (lanes 2 and 5). Total cell lysates (left) and anti-HA immunoprecipitates (right) were immunoblotted for GFP (upper) and HA (lower). Open circle, IdasGFP; filled circle, GemininGFP. Lanes 3 and 6, transfection with IdasGFP alone (control). E, Idas does not interact with Cdt1. MCF7 cells were co-transfected with Cdt1-dhcRed and IdasGFP (lane 1), GemininGFP (lane 2), or GFP (lane 3). Anti-GFP immunoprecipitates were immunoblotted for GFP (upper), Cdt1 (middle), and Geminin (lower). Open circle, endogenous Geminin; filled circle, GemininGFP.

FIGURE 3.

Idas directly binds to Geminin in vitro and prevents Geminin from binding Cdt1. A and B, polyacrylamide SDS-PAGE electrophoresis of Geminin, tIdas, dIdas, and Geminin-tIdas (A) and tGeminin-tIdas (B) complexes expressed and purified from E. coli is shown. C, quantification of Geminin and Idas affinities for Cdt1 by surface plasmon resonance is shown. The measured binding affinities of Geminin, dIdas, tIdas, Geminin-dIdas, and Geminin-tIdas complexes to mini-Cdt1 and mini-Cdt1Y170A immobilized on a Biacore T100 chip are shown.

FIGURE 4.

Idas targets Geminin to the nucleus. A, MCF7 cells were co-transfected with GemininGFP and Idas-Cherry (upper) or Cherry vector (lower), and the subcellular localization of GFP (b and e) and Cherry (c and f) was assessed. DNA was stained with DAPI (a and d). B, quantification of GemininGFP subcellular localization after co-transfection with Idas-Cherry (right) or Cherry (left, control) is shown. Nucl, nucleus; cyt, cytoplasm.

FIGURE 5.

Idas RNAi prevents normal cell cycle progression. A, quantification of Idas mRNA and protein levels in HeLa cells after Idas RNAi, relative to control RNAi, by real time PCR (graph) and Western blot analysis using an Idas-specific antibody (inset, hIdasAb1) 48 h post-transfection. B and C, HeLa cells were transfected with Idas siRNA or control siRNA and 48 h post-transfection pulsed with BrdU for 30 min, fixed, and immunolabeled with anti-BrdU and anti-Geminin or anti-Cdt1 antibodies. DNA was stained with Hoechst. Quantification of cells positively stained for BrdU, Geminin, and Cdt1 in Idas siRNA-treated normalized to control-treated HeLa cells is shown in B. For each experiment, the % positive cells in control-treated cells was set to 1. -Fold increase/decrease is shown as a mean of three independent experiments, with S.D. More than 400 cells were measured for each experiment. Representative images are shown in C, with % positive cells ±S.D. indicated in each image. D, HeLa cells were treated with Idas or control siRNA and 48 h post-transfection were treated with BrdU for a total of 24 h. Time points were collected at 1, 2, 4, 6, 8, 10, 12, 16, 18, and 24 h after BrdU addition. Cells were fixed and stained with anti-BrdU antibody. DNA was stained with Hoechst. The BrdU labeling index was plotted against the BrdU pulse time. E, HeLa cells were treated with Idas or control siRNA, and 48 h post-transfection cells were treated with BrdU for 30 min, then BrdU was washed out, and cells were left in a BrdU-free medium. After BrdU removal, time points were collected at 30 min and 4, 8, and 12 h. Cells were fixed and immunolabeled with anti-BrdU and anti-Cdt1 antibodies. DNA was stained with Hoechst. The percentage of all cells scoring positive for BrdU (upper) and the percentage of BrdU-positive cells that show mitotic nuclear morphology (middle) or are positive for Cdt1 (bottom) are plotted at each time point to determine progression into Mitosis and G₁ phase, respectively.

FIGURE 6.

A, Idas is degraded during anaphase. HeLa cells stably expressing IdasGFP were synchronized in mitosis by nocodazole, released, and GFP (middle) and Geminin (right) were detected by immunofluorescence. Cells are arranged by mitotic stage based on Hoechst staining. B, quantification of IdasGFP-, Geminin-, Cyclin A-, and Cyclin B1-expressing cells by mitotic stage is shown. See supplemental Fig. 3 for images. C, 293 T cells transfected with IdasGFP, N-IdasGFP, C-IdasGFP, or GFP were arrested in mitosis by nocodazole and released, and the % of cells expressing GFP in different mitotic stages was assessed. See supplemental Fig. 3 for images. D, Idas overexpression causes mitotic defects. 293 T cells were transfected with IdasGFP or GFP as control and treated with nocodazole for 16 h. After release, double immunofluorescence for α-tubulin and GFP was carried out. Examples of cells with abnormal spindles observed after IdasGFP transfection are shown. E, different passages of HeLa cells stably expressing Idas-GFP were stained for GFP and α-tubulin. Multinuclear giant cells were observed (E) and quantified (F). Parental HeLa cells were used as control.

FIGURE 7.

Expression of Idas and Geminin in the developing mouse brain. A, in situ hybridization for Idas (a) and Geminin (b) on an E12,5 dpc mouse embryo is shown. B, double immunofluorescence experiments for Idas (green, a, c, and e) and Geminin (green, b, d, and f) together with the radial glial marker Pax6 (red) on an E12.5 dpc mouse embryo are shown. Boxed areas in a and b are depicted in higher magnification in c and e and in d and f, respectively. Note the nuclear localization of Idas. Ch, cortical hem; Cp, choroid plexus; LV, lateral ventricle; Th, thalamus. C, shown is Western blot analysis of whole cell extracts from HeLa cells non-transfected (lane 1), transfected with IdasGFP (lane 2), or IdasHA (lane 3) and the choroid plexus derived Z310 cells (lane 4) using the anti-Idas specific antibody hIdasAb2. Anti-tubulin serves as a loading control. Star, nonspecific band. Endogenous Idas is not detectable in HeLa cells in this experiment due to its lower expression levels. D, Z310 cells were fixed and processed for immunofluorescence using the anti-hIdas hIdasAb2 antibody. Endogenous Idas appears in the nucleus excluded from the nucleoli.

FIGURE 8.

The Geminin superfamily is implicated in the regulation of DNA replication. Graphic representation of Geminin, Idas, and GEMC1 proteins share the Geminin like coiled-coil domain (shown in purple). Idas and GEMC1 also share a C-terminal conserved box (shown in gray) not present on Geminin. Idas interacts with Geminin and inhibits Geminin interactions with Cdt1. GEMC1 was shown to interact with TopBP1 and to regulate Cdc45 loading to chromatin (43). MCM, Mini-Chromosome Maintenance complex (MCM2–7); ORC, Origin Recognition Complex; polA, polymerase A.