7 research outputs found
Transcription of the Geminin gene is regulated by a negative-feedback loop
Geminin performs a central function in regulating cellular proliferation and differentiation in development and also in stem cells. Of interest, down-regulation of Geminin induces gene transcription regulated by E2F, indicating that Geminin is involved in regulation of E2F-mediated transcriptional activity. Because transcription of the Geminin gene is reportedly regulated via an E2F-responsive region (E2F-R) located in the first intron, we first used a reporter vector to examine the effect of Geminin on E2F-mediated transcriptional regulation. We found that Geminin transfection suppressed E2F1- and E2F2-mediated transcriptional activation and also mildly suppressed such activity in synergy with E2F5, 6, and 7, suggesting that Geminin constitutes a negative-feedback loop for the Geminin promoter. Of interest, Geminin also suppressed nuclease accessibility, acetylation of histone H3, and trimethylation of histone H3 at lysine 4, which were induced by E2F1 overexpression, and enhanced triÂmethylation of histone H3 at lysine 27 and monoubiquitination of histone H2A at lysine 119 in E2F-R. However, Geminin5EQ, which does not interact with Brahma or Brg1, did not suppress accessibility to nuclease digestion or transcription but had an overall dominant-negative effect. These findings suggest that E2F-mediated activation of Geminin transcription is negatively regulated by Geminin through the inhibition of chromatin remodeling
Manipulation of Cell Cycle and Chromatin Configuration by Means of Cell-Penetrating Geminin
<div><p>Geminin regulates chromatin remodeling and DNA replication licensing which play an important role in regulating cellular proliferation and differentiation. Transcription of the <i>Geminin</i> gene is regulated via an E2F-responsive region, while the protein is being closely regulated by the ubiquitin-proteasome system. Our objective was to directly transduce Geminin protein into cells. Recombinant cell-penetrating Geminin (CP-Geminin) was generated by fusing Geminin with a membrane translocating motif from FGF4 and was efficiently incorporated into NIH 3T3 cells and mouse embryonic fibroblasts. The withdrawal study indicated that incorporated CP-Geminin was quickly reduced after removal from medium. We confirmed CP-Geminin was imported into the nucleus after incorporation and also that the incorporated CP-Geminin directly interacted with Cdt1 or Brahma/Brg1 as the same manner as Geminin. We further demonstrated that incorporated CP-Geminin suppressed S-phase progression of the cell cycle and reduced nuclease accessibility in the chromatin, probably through suppression of chromatin remodeling, indicating that CP-Geminin constitutes a novel tool for controlling chromatin configuration and the cell cycle. Since Geminin has been shown to be involved in regulation of stem cells and cancer cells, CP-Geminin is expected to be useful for elucidating the role of Geminin in stem cells and cancer cells, and for manipulating their activity.</p></div
Molecular interaction of incorporated CP-Geminin with the target substrates.
<p>Expression plasmid vectors for Myc-Cdt1, HA-Brahma-domainII and HA-Brg1-domainII were transfected into NIH 3T3 cells, after which CP-Geminin was added. The cells were then subjected to immunoprecipitation analysis. 1. Control, 2. Flag-Geminin, 3. CP-Geminin including a Flag tag.</p
Generation of recombinant CP-Geminin protein
<p>(A) Structure of CP-Geminin. Amino acid numbers indicate location of the representative domains. The coiled-coil domain is required for the dimerization or multimerization and also for the interaction with Cdt1. MTM: membrane translocating motif from FGF4. (B) Production and affinity-purification of recombinant CP-Geminin. Proteins were separated on SDS-PAGE and subjected to CBB staining. MW: molecular weight.</p
Geminin transduction by means of CP-Geminin
<p>(A) CP-Geminin incorporation into NIH 3T3 cells. FITC-conjugated CP-Geminin and Geminin were added into the medium. One and 12h after the addition, cells were observed under a confocal microscope. The nucleus was stained with Hoechst33342. DIC: differential interference contrast (B) FITC-conjugated CP-Geminin incorporation into leukemic cell lines 24 h after the addition. (C) Dose dependency of CP-Geminin incorporation into NIH 3T3 cells. FITC-conjugated CP-Geminin was added to the medium at the indicated concentration, and 12h after the addition, the cells were examined under a confocal microscope. (D) Withdrawal study of CP-Geminin. FITC-conjugated CP-Geminin was added to the medium and the incorporation was confirmed 12h after the addition. CP-Geminin was then removed by replacing the medium with CP-Geminin with one without CP-Geminin. One and 4h after the removal, the cells were examined under a confocal microscope.</p
Effect of CP-Geminin on chromatin configuration and transcription.
<p>(A) Effect of CP-Geminin on nuclease accessibility of the E2F-R chromatin locus of the <i>Geminin</i> gene. Twenty-four h after transduction of CP-Geminin (1,000 nM), cells were subjected to nuclease accessibility analysis. BSA, control (B) Effect on transcription of the <i>Geminin</i> gene promoter. Twenty four h after transduction of CP-Geminin (1,000 nM), E2F1 (1 ÎĽg) and the luciferase reporter plasmids (1.5 ÎĽg) were co-transfected, and transcription activity of the <i>Geminin</i> gene promoter was examined with the luciferase reporter assay. (C) Effect of CP-Geminin on mRNA expression of the endogenous genes, <i>Geminin</i>, <i>Mcm7</i>, <i>CcnA2</i> and <i>Actb2</i>. Twenty four h after transduction of CP-Geminin (1,000 nM), E2F1 (1 ÎĽg) was transfected, and cells were subjected to TaqMan real-time PCR analysis an additional 24h after the transfection. *: P< 0.01.</p