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Wael M Elshamy - One of the best experts on this subject based on the ideXlab platform.

  • The molecular underpinning of Geminin-overexpressing triple-negative breast cancer cells homing specifically to lungs
    Cancer Gene Therapy, 2021
    Co-Authors: Eman Sami, Danielle Bogan, Alfredo Molinolo, Jim Koziol, Wael M Elshamy
    Abstract:

    Triple-negative breast cancer (TNBCs) display lung metastasis tropism. However, the mechanisms underlying this organ-specific pattern remains to be elucidated. We sought to evaluate the utility of blocking extravasation to prevent lung metastasis. To identify potential Geminin overexpression-controlled genetic drivers that promote TNBC tumor homing to lungs, we used the differential/suppression subtractive chain (D/SSC) technique. A Geminin overexpression-induced lung metastasis gene signature consists of 24 genes was discovered. We validated overexpression of five of these genes (LGR5, HAS2, CDH11, NCAM2, and DSC2) in worsening lung metastasis-free survival in TNBC patients. Our data demonstrate that LGR5-induced β-catenin signaling and stemness in TNBC cells are Geminin-overexpression dependent. They also demonstrate for the first-time expression of RSPO2 in mouse lung tissue only and exacerbation of its secretion in the circulation of mice that develop Geminin overexpressing/LGR5^+-TNBC lung metastasis. We identified a novel extravasation receptor complex, consists of CDH11, CD44v6, c-Met, and AXL on Geminin overexpressing/LGR5^+-TNBC lung metastatic precursors, inhibition of any of its receptors prevented Geminin overexpressing/LGR5^+-TNBC lung metastasis. Overall, we propose that Geminin overexpression in normal mammary epithelial (HME) cells promotes the generation of TNBC metastatic precursors that home specifically to lungs by upregulating LGR5 expression and promoting stemness, intravasation, and extravasation in these precursors. Circulating levels of RSPO2 and OPN can be diagnostic biomarkers to improve risk stratification of metastatic TNBC to lungs, as well as identifying patients who may benefit from therapy targeting Geminin alone or in combination with any member of the newly discovered extravasation receptor complex to minimize TNBC lung metastasis.

  • targeting axl and rage to prevent Geminin overexpression induced triple negative breast cancer metastasis
    Scientific Reports, 2019
    Co-Authors: Daniel Ryan, James A Koziol, Wael M Elshamy
    Abstract:

    Dissemination of metastatic precursors from primaries is the primary reason for patient death. Dissemination encompasses tumor cells invasion of stroma, followed by intravasation through the endothelium barrier into the bloodstream. Here, we describe how Geminin-overexpressing tumor cells acquire dissemination ability. Acetylated HMGB1 (Ac-HMGB1) secreted by Geminin-overexpressing cells activates RAGE and CXCR4 expression on mesenchymal stem cells (MSCs) located in tumor stroma. Through secreting CXCL12, Geminin-overexpressing cells recruit these CXCR4+-MSCs into the tumor. Within the tumor, MSCs differentiate into S100A4-secreting cancer-associated fibroblasts (CAFs). S100A4, in a reciprocal manner, activates Geminin-overexpressing cells to secrete CCL2 that recruits M0-macrophages from the stroma into the tumor. Within the tumor, CCL2 polarizes M0-macrophages into Gas6-secreting M2-tumor-associated macrophages (M2-TAMs). In concert, Geminin-overexpression, S100A4/RAGE and Gas6/AXL signaling promote the invasive and intravasation abilities in Geminin-overexpressing cells through exacerbating their stemness and epithelial-to-mesenchymal phenotypes and enhancing expression and functional interaction of CD151 and α3β1-integrin in Geminin-overexpressing cells. Tumors formed following injection of Geminin-overexpressing cells admixed with MSCs/CAFs grew faster, metastasized earlier, especially to lungs, and were extremely sensitive to anti-c-Abl, anti-RAGE, and anti-AXL drugs. These data support an intrinsic ability in Geminin-overexpressing tumor cells to promote their metastatic potential through recruitment and bi-directional interactions with MSCs/CAFs and M2-TAMs.

  • Geminin overexpression induces mammary tumors via suppressing cytokinesis
    Oncotarget, 2011
    Co-Authors: Zannel Blanchard, Rohit Malik, Nicole Mullins, Christine Maric, Hugh Luk, David Horio, Brenda Hernandez, Jeffrey Killeen, Wael M Elshamy
    Abstract:

    Zannel Blanchard * , Rohit Malik * , Nicole Mullins, Christine Maric, Hugh Luk, David Horio, Brenda Hernandez, Jeffrey Killeen and Wael M. ElShamy 1 Cancer Institute and Department of Biochemistry, University of Mississippi Medical Center, 2500 N. State St., G651-6, Jackson, MS 39216 * Denotes equal contribution Received: November 28, 2011; Accepted: December 9, 2011; Published: December 17, 2011; Keywords: Geminin overexpression induces mammary tumors Correspondence: Wael M. ElShamy, email: // // Abstract Aneuploidy plays an important role in the development of cancer. Here, we uncovered an oncogenic role for Geminin in mitotic cells. In addition to chromatin, tyrosine phosphorylated Geminin also localizes to centrosome, spindle, cleavage furrow and midbody during mitosis. Geminin binding to Aurora B prevents its binding to INCENP, and thus activation leading to lack of histone H3-(serine 10) phosphorylation, chromosome condensation failure, aborted cytokinesis and the formation of aneuploid, drug resistance cells. Geminin overexpressing human mammary epithelial cells form aneuploid, aggressive tumors in SCID mice. Geminin is overexpressed in more than half of all breast cancers analyzed. The current study reveals that Geminin is a genuine oncogene that promotes cytokinesis failure and production of aneuploid, aggressive breast tumors when overexpressed and thus a worthy therapeutic target (oncotarget) for aggressive breast cancer.

  • Geminin overexpression prevents the completion of topoisomerase iiα chromosome decatenation leading to aneuploidy in human mammary epithelial cells
    Breast Cancer Research, 2011
    Co-Authors: Lauren Gardner, Nicole Mullins, Rohit Malik, Yoshiko Shimizu, Wael M Elshamy
    Abstract:

    The nuclear enzyme topoisomerase IIα (TopoIIα) is able to cleave DNA in a reversible manner, making it a valuable target for agents such as etoposide that trap the enzyme in a covalent bond with the 5′ DNA end to which it cleaves. This prevents DNA religation and triggers cell death in cancer cells. However, development of resistance to these agents limits their therapeutic use. In this study, we examined the therapeutic targeting of Geminin for improving the therapeutic potential of TopoIIα agents. Human mammary epithelial (HME) cells and several breast cancer cell lines were used in this study. Geminin, TopoIIα and cell division cycle 7 (Cdc7) silencing were done using specific small interfering RNA. Transit or stable inducible overexpression of these proteins and casein kinase Ie (CKIe) were also used, as well as several pharmacological inhibitors that target TopoIIα, Cdc7 or CKIe. We manipulated HME cells that expressed H2B-GFP, or did not, to detect chromosome bridges. Immunoprecipitation and direct Western blot analysis were used to detect interactions between these proteins and their total expression, respectively, whereas interactions on chromosomal arms were detected using a trapped in agarose DNA immunostaining assay. TopoIIα phosphorylation by Cdc7 or CKIe was done using an in vitro kinase assay. The TopoGen decatenation kit was used to measure TopoIIα decatenation activity. Finally, a comet assay and metaphase chromosome spread were used to detect chromosome breakage and changes in chromosome condensation or numbers, respectively. We found that Geminin and TopoIIα interact primarily in G2/M/early G1 cells on chromosomes, that Geminin recruits TopoIIα to chromosomal decatenation sites or vice versa and that Geminin silencing in HME cells triggers the formation of chromosome bridges by suppressing TopoIIα access to chromosomal arms. CKIe kinase phosphorylates and positively regulates TopoIIα chromosome localization and function. CKIe kinase overexpression or Cdc7 kinase silencing, which we show phosphorylates TopoIIα in vitro, restored DNA decatenation and chromosome segregation in Geminin-silenced cells before triggering cell death. In vivo, at normal concentration, Geminin recruits the deSUMOylating sentrin-specific proteases SENP1 and SENP2 enzymes to deSUMOylate chromosome-bound TopoIIα and promote its release from chromosomes following completion of DNA decatenation. In cells overexpressing Geminin, premature departure of TopoIIα from chromosomes is thought to be due to the fact that Geminin recruits more of these deSUMOylating enzymes, or recruits them earlier, to bound TopoIIα. This triggers premature release of TopoIIα from chromosomes, which we propose induces aneuploidy in HME cells, since chromosome breakage generated through this mechanism were not sensed and/or repaired and the cell cycle was not arrested. Expression of mitosis-inducing proteins such as cyclin A and cell division kinase 1 was also increased in these cells because of the overexpression of Geminin. TopoIIα recruitment and its chromosome decatenation function require a normal level of Geminin. Geminin silencing induces a cytokinetic checkpoint in which Cdc7 phosphorylates TopoIIα and inhibits its chromosomal recruitment and decatenation and/or segregation function. Geminin overexpression prematurely deSUMOylates TopoIIα, triggering its premature departure from chromosomes and leading to chromosomal abnormalities and the formation of aneuploid, drug-resistant cancer cells. On the basis of our findings, we propose that therapeutic targeting of Geminin is essential for improving the therapeutic potential of TopoIIα agents.

  • Geminin is bound to chromatin in g2 m phase to promote proper cytokinesis
    The International Journal of Biochemistry & Cell Biology, 2006
    Co-Authors: Enkeleda Nakuci, Miguel Angel Pujana, Joan Valls, Wael M Elshamy
    Abstract:

    Previous studies suggested that Geminin plays a vital role in both origin assembly and DNA re-replication during S-phase; however, no data to support a role for Geminin in G2/M cells have been described. Here it is shown that in G2/M-phase, Geminin participates in the promotion of proper cytokinesis. This claim can be supported through a series of observations. First, Geminin in G2/M is loaded onto chromatin after it is tyrosine phosphorylated. It is unlike S-phase Geminin that resides in the nuclear soluble fraction, where it is exclusively S/T phosphorylated. Secondly, on chromatin, Geminin gets S/T phosphorylated in late G1; this modification causes the release of Geminin from the chromatin. Cyclins bind and phosphorylate Geminin in a sequential, cell cycle-dependent manner. These modifications correlated well with Geminin departure from the chromatin. This suggests that cyclin functions to either release Geminin from chromatin or at least keep it at bay until late S-phase. Thirdly, depletion of Geminin from a diploid mammary epithelial cell line (HME) causes cells to arrest in late G2/M-phase. Massive serine-10 phosphorylated histone H3 staining and survivin localization to mid-body were observed; this suggests that they could be arrested in either mitosis or at cytokinesis. Finally, while in the absence of Geminin, cyclin B1, chk1 and cdc7 are all over expressed. This paper will demonstrate that only cdc7 is important in maintaining the cytokinesis arrest in the absence of Geminin. Only double depletion of Geminin and cdc7 induce apoptosis. Our results taken together show, for the first time, that phosphorylation-induction activates oscillation of Geminin between both nuclear soluble and chromatin compartments. Chromatin-bound Geminin species functions to initiate or maintain proper cytokineses. In the absence of Geminin, cells arrest in cytokinesis; this defines a novel checkpoint, monitored by cdc7, rather than cyclin B1 or chk1.

Melvin L Depamphilis - One of the best experts on this subject based on the ideXlab platform.

  • Geminin is essential for pluripotent cell viability during teratoma formation but not for differentiated cell viability during teratoma expansion
    Stem Cells and Development, 2017
    Co-Authors: Diane C Adlerwailes, Joshua A Kramer, Melvin L Depamphilis
    Abstract:

    Pluripotent embryonic stem cells (ESCs) are unusual in that Geminin has been reported to be essential either to prevent differentiation by maintaining expression of pluripotency genes or to prevent DNA rereplication-dependent apoptosis. To distinguish between these two incompatible hypotheses, immune-compromised mice were inoculated subcutaneously with ESCs harboring conditional Gmnn alleles alone or together with a tamoxifen-dependent Cre recombinase gene. Mice were then injected with tamoxifen at various times during which the ESCs proliferated and differentiated into a teratoma. For comparison, the same ESCs were cultured in vitro in the presence of monohydroxytamoxifen. The results revealed that Geminin is a haplosufficient gene that is essential for ESC viability before they differentiate into a teratoma, but once a teratoma is established, the differentiated cells can continue to proliferate in the absence of Gmnn alleles, Geminin protein, and pluripotent stem cells. Thus, differentiated cells did not require Geminin for efficient proliferation within the context of a solid tissue, although they did when teratoma cells were cultured in vitro. These results provide proof-of-principle that preventing Geminin function could prevent malignancy in tumors derived from pluripotent cells by selectively eliminating the progenitor cells with little harm to normal cells.

  • Geminin is essential to prevent dna re replication dependent apoptosis in pluripotent cells but not in differentiated cells
    Stem Cells, 2015
    Co-Authors: Yiyuan Huang, Kotaro J Kaneko, Haiyan Pan, Melvin L Depamphilis
    Abstract:

    Geminin is a dual-function protein unique to multicellular animals with roles in modulating gene expression and preventing DNA re-replication. Here, we show that Geminin is essential at the beginning of mammalian development to prevent DNA re-replication in pluripotent cells, exemplified by embryonic stem cells, as they undergo self-renewal and differentiation. Embryonic stem cells, embryonic fibroblasts, and immortalized fibroblasts were characterized before and after Geminin was depleted either by gene ablation or siRNA. Depletion of Geminin under conditions that promote either self-renewal or differentiation rapidly induced DNA re-replication, followed by DNA damage, then a DNA damage response, and finally apoptosis. Once differentiation had occurred, Geminin was no longer essential for viability, although it continued to contribute to preventing DNA re-replication induced DNA damage. No relationship was detected between expression of Geminin and genes associated with either pluripotency or differentiation. Thus, the primary role of Geminin at the beginning of mammalian development is to prevent DNA re-replication-dependent apoptosis, a role previously believed essential only in cancer cells. These results suggest that regulation of gene expression by Geminin occurs only after pluripotent cells differentiate into cells in which Geminin is not essential for viability. Stem Cells 2015;33:3239–3253

  • the dual roles of Geminin during trophoblast proliferation and differentiation
    Developmental Biology, 2014
    Co-Authors: Christelle De Renty, Kotaro J Kaneko, Melvin L Depamphilis
    Abstract:

    Geminin is a protein involved in both DNA replication and cell fate acquisition. Although it is essential for mammalian preimplantation development, its role remains unclear. In one study, ablation of the Geminin gene (Gmnn) in mouse preimplantation embryos resulted in apoptosis, suggesting that Geminin prevents DNA re-replication, whereas in another study it resulted in differentiation of blastomeres into trophoblast giant cells (TGCs), suggesting that Geminin regulates trophoblast specification and differentiation. Other studies concluded that trophoblast differentiation into TGCs is regulated by fibroblast growth factor-4 (FGF4), and that Geminin is required to maintain endocycles. Here we show that ablation of Gmnn in trophoblast stem cells (TSCs) proliferating in the presence of FGF4 closely mimics the events triggered by FGF4 deprivation: arrest of cell proliferation, formation of giant cells, excessive DNA replication in the absence of DNA damage and apoptosis, and changes in gene expression that include loss of Chk1 with up-regulation of p57 and p21. Moreover, FGF4 deprivation of TSCs reduces Geminin to a basal level that is required for maintaining endocycles in TGCs. Thus, Geminin acts both like a component of the FGF4 signal transduction pathway that governs trophoblast proliferation and differentiation, and Geminin is required to maintain endocycles.

  • spotlight on Geminin
    Breast Cancer Research, 2011
    Co-Authors: Melvin L Depamphilis
    Abstract:

    In the previous issue of Breast Cancer Research, Gardner and co-workers describe a novel interaction between Geminin, a protein that prevents reinitiation of DNA replication, and Topoisomerase IIα (TopoIIα), an enzyme essential for removing catenated intertwines between sister chromatids. Geminin facilitates the action of TopoIIα, thereby promoting termination of DNA replication at the same time it inhibits initiation. In this manner, Geminin ensures that cells duplicate their genome once, but only once, each time they divide. Remarkably, either depletion of Geminin or over-expression of Geminin inhibits the action of TopoIIα, thereby making Geminin an excellent target for cancer chemotherapy.

Kristen L Kroll - One of the best experts on this subject based on the ideXlab platform.

  • Geminin deficiency enhances survival in a murine medulloblastoma model by inducing apoptosis of preneoplastic granule neuron precursors
    Genes & Cancer, 2017
    Co-Authors: Savita Sankar, Ethan S Patterson, Laura E Waller, Emily M A Lewis, Caili Tong, Joshua T Dearborn, David F Wozniak, Joshua B Rubin, Kristen L Kroll
    Abstract:

    Medulloblastoma is the most common malignant brain cancer of childhood. Further understanding of tumorigenic mechanisms may define new therapeutic targets. Geminin maintains genome fidelity by controlling re-initiation of DNA replication within a cell cycle. In some contexts, Geminin inhibition induces cancer-selective cell cycle arrest and apoptosis and/or sensitizes cancer cells to Topoisomerase IIα inhibitors such as etoposide, which is used in combination chemotherapies for medulloblastoma. However, Geminin's potential role in medulloblastoma tumorigenesis remained undefined. Here, we found that Geminin is highly expressed in human and mouse medulloblastomas and in murine granule neuron precursor (GNP) cells during cerebellar development. Conditional Geminin loss significantly enhanced survival in the SmoA1 mouse medulloblastoma model. Geminin loss in this model also reduced numbers of preneoplastic GNPs persisting at one postnatal month, while at two postnatal weeks these cells exhibited an elevated DNA damage response and apoptosis. Geminin knockdown likewise impaired human medulloblastoma cell growth, activating G2 checkpoint and DNA damage response pathways, triggering spontaneous apoptosis, and enhancing G2 accumulation of cells in response to etoposide treatment. Together, these data suggest preneoplastic and cancer cell-selective roles for Geminin in medulloblastoma, and suggest that targeting Geminin may impair tumor growth and enhance responsiveness to Topoisomerase IIα-directed chemotherapies.

  • gene regulatory networks in neural cell fate acquisition from genome wide chromatin association of Geminin and zic1
    Scientific Reports, 2016
    Co-Authors: Savita Sankar, Dhananjay Yellajoshyula, Bo Zhang, Bryan Teets, Nicole B Rockweiler, Kristen L Kroll
    Abstract:

    Neural cell fate acquisition is mediated by transcription factors expressed in nascent neuroectoderm, including Geminin and members of the Zic transcription factor family. However, regulatory networks through which this occurs are not well defined. Here, we identified Geminin-associated chromatin locations in embryonic stem cells and Geminin- and Zic1-associated locations during neural fate acquisition at a genome-wide level. We determined how Geminin deficiency affected histone acetylation at gene promoters during this process. We integrated these data to demonstrate that Geminin associates with and promotes histone acetylation at neurodevelopmental genes, while Geminin and Zic1 bind a shared gene subset. Geminin- and Zic1-associated genes exhibit embryonic nervous system-enriched expression and encode other regulators of neural development. Both Geminin and Zic1-associated peaks are enriched for Zic1 consensus binding motifs, while Zic1-bound peaks are also enriched for Sox3 motifs, suggesting co-regulatory potential. Accordingly, we found that Geminin and Zic1 could cooperatively activate the expression of several shared targets encoding transcription factors that control neurogenesis, neural plate patterning, and neuronal differentiation. We used these data to construct gene regulatory networks underlying neural fate acquisition. Establishment of this molecular program in nascent neuroectoderm directly links early neural cell fate acquisition with regulatory control of later neurodevelopment.

  • Geminin loss causes neural tube defects through disrupted progenitor specification and neuronal differentiation
    Developmental Biology, 2014
    Co-Authors: Ethan S Patterson, Laura E Waller, Kristen L Kroll
    Abstract:

    Geminin is a nucleoprotein that can directly bind chromatin regulatory complexes to modulate gene expression during development. Geminin knockout mouse embryos are preimplantation lethal by the 32-cell stage, precluding in vivo study of Geminin׳s role in neural development. Therefore, here we used a conditional Geminin allele in combination with several Cre-driver lines to define an essential role for Geminin during mammalian neural tube (NT) formation and patterning. Geminin was required in the NT within a critical developmental time window (embryonic day 8.5–10.5), when NT patterning and closure occurs. Geminin excision at these stages resulted in strongly diminished expression of genes that mark and promote dorsal NT identities and decreased differentiation of ventral motor neurons, resulting in completely penetrant NT defects, while excision after embryonic day 10.5 did not result in NT defects. When Geminin was deleted specifically in the spinal NT, both NT defects and axial skeleton defects were observed, but neither defect occurred when Geminin was excised in paraxial mesenchyme, indicating a tissue autonomous requirement for Geminin in developing neuroectoderm. Despite a potential role for Geminin in cell cycle control, we found no evidence of proliferation defects or altered apoptosis. Comparisons of gene expression in the NT of Geminin mutant versus wild-type siblings at embryonic day 10.5 revealed decreased expression of key regulators of neurogenesis, including neurogenic bHLH transcription factors and dorsal interneuron progenitor markers. Together, these data demonstrate a requirement for Geminin for NT patterning and neuronal differentiation during mammalian neurulation in vivo.

  • Geminin restrains mesendodermal fate acquisition of embryonic stem cells and is associated with antagonism of wnt signaling and enhanced polycomb mediated repression
    Stem Cells, 2013
    Co-Authors: Elizabeth A Caronna, Ethan S Patterson, Pamela M Hummert, Kristen L Kroll
    Abstract:

    Embryonic cells use both growth factor signaling and cell intrinsic transcriptional and epigenetic regulation to acquire early cell fates. Underlying mechanisms that integrate these cues are poorly understood. Here, we investigated the role of Geminin, a nucleoprotein that interacts with both transcription factors and epigenetic regulatory complexes, during fate acquisition of mouse embryonic stem cells. In order to determine Geminin's role in mesendoderm formation, a process which occurs during embryonic gastrulation, we selectively over-expressed or knocked down Geminin in an in vitro model of differentiating mouse embryonic stem cells. We found that Geminin antagonizes mesendodermal fate acquisition, while these cells instead maintain elevated expression of genes associated with pluripotency of embryonic stem cells. During mesendodermal fate acquisition, Geminin knockdown promotes Wnt signaling, while Bmp, Fgf, and Nodal signaling are not affected. Moreover, we showed that Geminin facilitates the repression of mesendodermal genes that are regulated by the Polycomb repressor complex. Geminin directly binds several of these genes, while Geminin knockdown in mesendodermal cells reduces Polycomb repressor complex occupancy at these loci and increases trimethylation of histone H3 lysine 4, which correlates with active gene expression. Together, these results indicate that Geminin is required to restrain mesendodermal fate acquisition of early embryonic cells and that this is associated with both decreased Wnt signaling and enhanced Polycomb repressor complex retention at mesendodermal genes. STEM Cells 2013;31:1477–1487

  • Geminin promotes neural fate acquisition of embryonic stem cells by maintaining chromatin in an accessible and hyperacetylated state
    Proceedings of the National Academy of Sciences of the United States of America, 2011
    Co-Authors: Dhananjay Yellajoshyula, Ethan S Patterson, Matthew S Elitt, Kristen L Kroll
    Abstract:

    Formation of the complex vertebrate nervous system begins when pluripotent cells of the early embryo are directed to acquire a neural fate. Although cell intrinsic controls play an important role in this process, the molecular nature of this regulation is not well defined. Here we assessed the role for Geminin, a nuclear protein expressed in embryonic cells, during neural fate acquisition from mouse embryonic stem (ES) cells. Whereas Geminin knockdown does not affect the ability of ES cells to maintain or exit pluripotency, we found that it significantly impairs their ability to acquire a neural fate. Conversely, Geminin overexpression promotes neural gene expression, even in the presence of growth factor signaling that antagonizes neural transcriptional responses. These data demonstrate that Geminin's activity contributes to mammalian neural cell fate acquisition. We investigated the mechanistic basis of this phenomenon and found that Geminin maintains a hyperacetylated and open chromatin conformation at neural genes. Interestingly, recombinant Geminin protein also rapidly alters chromatin acetylation and accessibility even when Geminin is combined with nuclear extract and chromatin in vitro. Together, these data support a role for Geminin as a cell intrinsic regulator of neural fate acquisition that promotes expression of neural genes by regulating chromatin accessibility and histone acetylation.

Julian J Blow - One of the best experts on this subject based on the ideXlab platform.

  • the Geminin and idas coiled coils preferentially form a heterodimer that inhibits Geminin function in dna replication licensing
    Journal of Biological Chemistry, 2013
    Co-Authors: Christophe Caillat, Julian J Blow, Zoi Lygerou, Stavros Taraviras, Dafnieleftheria Pefani, Peter J Gillespie, Anastassis Perrakis
    Abstract:

    Abstract Geminin is an important regulator of proliferation and differentiation in metazoans, which predominantly inhibits the DNA replication licensing factor Cdt1, preventing genome over-replication. We show that Geminin preferentially forms stable coiled-coil heterodimers with its homologue, Idas. In contrast to Idas:Geminin heterodimers, Idas homodimers are thermodynamically unstable and are unlikely to exist as a stable macromolecule under physiological conditions. The crystal structure of the homology regions of Idas in complex with Geminin showed a tight head-to-head heterodimeric coiled-coil. This Idas:Geminin heterodimer binds Cdt1 less strongly than Geminin:Geminin, still with high affinity (~30nM), but with notably different thermodynamic properties. Consistently, in Xenopus egg extracts, Idas:Geminin is less active in licensing inhibition compared to a Geminin:Geminin homodimer. In human cultured cells, ectopic expression of Idas leads to limited over-replication, which is counteracted by Geminin co-expression. The properties of the Idas:Geminin complex suggest it as the functional form of Idas, and provide a possible mechanism to modulate Geminin activity.

  • re replication induced by Geminin depletion occurs from g2 and is enhanced by checkpoint activation
    Journal of Cell Science, 2012
    Co-Authors: Kathleen Klotznoack, Debbie Mcintosh, N J Schurch, Norman Pratt, Julian J Blow
    Abstract:

    To prevent re-replication of DNA in a single cell cycle, the licensing of replication origins by Mcm2-7 is prevented during S and G2 phases. Animal cells achieve this by cell cycle regulated proteolysis of the essential licensing factor Cdt1 and inhibition of Cdt1 by Geminin. Here we investigate the consequences of ablating Geminin in synchronised human U2OS cells. Following Geminin loss, cells complete an apparently normal S phase, but a proportion arrest at the G2/M boundary. When Cdt1 accumulates in these cells, DNA re-replicates, suggesting that the key role of Geminin is to prevent re-licensing in G2. If cell cycle checkpoints are inhibited in cells lacking Geminin, cells progress through mitosis and less re-replication occurs. Checkpoint kinases thereby amplify re-replication into an all-or-nothing response by delaying Geminin-depleted cells in G2. Deep DNA sequencing revealed no preferential re-replication of specific genomic regions after Geminin depletion. This is consistent with the observation that cells in G2 have lost their replication timing information. In contrast, when Cdt1 is overexpressed or is stabilised by the Neddylation inhibitor MLN4924, re-replication can occur throughout S phase.

  • dynamic interactions of high cdt1 and Geminin levels regulate s phase in early xenopus embryos
    Development, 2012
    Co-Authors: J Kisielewska, Julian J Blow
    Abstract:

    Cdt1 plays a key role in licensing DNA for replication. In the somatic cells of metazoans, both Cdt1 and its natural inhibitor Geminin show reciprocal fluctuations in their protein levels owing to cell cycle-dependent proteolysis. Here, we show that the protein levels of Cdt1 and Geminin are persistently high during the rapid cell cycles of the early Xenopus embryo. Immunoprecipitation of Cdt1 and Geminin complexes, together with their cell cycle spatiotemporal dynamics, strongly supports the hypothesis that Cdt1 licensing activity is regulated by periodic interaction with Geminin rather than its proteolysis. Overexpression of ectopic Geminin slows down, but neither arrests early embryonic cell cycles nor affects endogenous Geminin levels; apparent embryonic lethality is observed around 3-4 hours after mid-blastula transition. However, functional knockdown of Geminin by ΔCdt1_193-447, which lacks licensing activity and degradation sequences, causes cell cycle arrest and DNA damage in affected cells. This contributes to subsequent developmental defects in treated embryos. Our results clearly show that rapidly proliferating early Xenopus embryonic cells are able to regulate replication licensing in the persistent presence of high levels of licensing proteins by relying on changing interactions between Cdt1 and Geminin during the cell cycle, but not their degradation.

  • non proteolytic inactivation of Geminin requires cdk dependent ubiquitination
    Nature Cell Biology, 2004
    Co-Authors: Julian J Blow
    Abstract:

    In late mitosis and G1, a complex of the essential initiation proteins Mcm2-7 are assembled onto replication origins to 'license' them for initiation. At other times licensing is inhibited by cyclin-dependent kinases (CDKs) and Geminin, thus ensuring that origins fire only once per cell cycle. Here we show that, paradoxically, CDKs are also required to inactivate Geminin and activate the licensing system. On exit from metaphase in Xenopus laevis egg extracts, CDK-dependent activation of the anaphase-promoting complex (APC/C) results in the transient polyubiquitination of Geminin. This ubiquitination triggers Geminin inactivation without requiring ubiquitin-dependent proteolysis, and is essential for replication origins to become licensed. This reveals an unexpected role for CDKs and ubiquitination in activating chromosomal DNA replication.

  • Geminin becomes activated as an inhibitor of cdt1 rlf b following nuclear import
    Current Biology, 2002
    Co-Authors: Ben Hodgson, Shusuke Tada, Julian J Blow
    Abstract:

    During late mitosis and early interphase, origins of replication become "licensed" for DNA replication by loading Mcm2-7 complexes [1–5]. Mcm2-7 complexes are removed from origins as replication forks initiate replication, thus preventing rereplication of DNA in a single cell cycle. Premature origin licensing is prevented in metaphase by the action of Geminin, which binds and inhibits Cdt1/RLF-B, a protein that is required for the loading of Mcm2-7 [6–9]. Recombinant Geminin that is added to Xenopus egg extracts is efficiently degraded upon exit from metaphase [10]. Here, we show that recombinant and endogenous forms of Xenopus Geminin behave differently from one another, such that a significant proportion of endogenous Geminin escapes proteolysis upon exit from metaphase. During late mitosis and early G1, the surviving population of endogenous Geminin does not associate with Cdt1/RLF-B and does not inhibit licensing. Following nuclear assembly, Geminin is imported into nuclei and becomes reactivated to bind Cdt1/RLF-B. This reactivated Geminin provides the major nucleoplasmic inhibitor of origin relicensing during late interphase. Since the initiation of replication at licensed origins depends on nuclear assembly [11], our results suggest an elegant and novel mechanism for preventing rereplication of DNA in a single cell cycle.

Thomas J Mcgarry - One of the best experts on this subject based on the ideXlab platform.

  • an inactive Geminin mutant that binds cdt1
    Genes, 2015
    Co-Authors: Marissa Suchyta, Benoit Miotto, Thomas J Mcgarry
    Abstract:

    The initiation of DNA replication is tightly regulated in order to ensure that the genome duplicates only once per cell cycle. In vertebrate cells, the unstable regulatory protein Geminin prevents a second round of DNA replication by inhibiting the essential replication factor Cdt1. Cdt1 recruits mini-chromosome maintenance complex (MCM2-7), the replication helicase, into the pre-replication complex (pre-RC) at origins of DNA replication. The mechanism by which Geminin inhibits MCM2-7 loading by Cdt1 is incompletely understood. The conventional model is that Geminin sterically hinders a direct physical interaction between Cdt1 and MCM2-7. Here, we describe an inactive missense mutant of Geminin, GemininAWA, which binds to Cdt1 with normal affinity yet is completely inactive as a replication inhibitor even when added in vast excess. In fact, GemininAWA can compete with GemininWT for binding to Cdt1 and prevent it from inhibiting DNA replication. GemininAWA does not inhibit the loading of MCM2-7 onto DNA in vivo, and in the presence of GemininAWA, nuclear DNA is massively over-replicated within a single S phase. We conclude that Geminin does not inhibit MCM loading by simple steric interference with a Cdt1-MCM2-7 interaction but instead works by a non-steric mechanism, possibly by inhibiting the histone acetyltransferase HBO1.

  • Geminin is required for mitotic proliferation of spermatogonia
    Developmental Biology, 2012
    Co-Authors: Kelly A Barry, Kathryn M Schultz, Christopher J Payne, Thomas J Mcgarry
    Abstract:

    Spermatogonial stem cells divide throughout life, maintaining their own population and giving rise to differentiated gametes. The unstable regulatory protein Geminin is thought to be one of the factors that determine whether stem cells continue to divide or terminally differentiate. Geminin regulates the extent of DNA replication and is thought to maintain cells in an undifferentiated state by inhibiting various transcription factors and chromatin remodeling proteins. To examine how Geminin might regulate spermatogenesis, we developed two conditional mouse models in which the Geminin gene (Gmnn) is deleted from either spermatogonia or meiotic spermatocytes. Deleting Geminin from spermatogonia causes complete sterility in male mice. Gmnn(-/-) spermatogonia disappear during the initial wave of mitotic proliferation that occurs during the first week of life. Gmnn(-/-) spermatogonia exhibit more double-stranded DNA breaks than control cells, consistent with a defect in DNA replication. They maintain expression of genes associated with the undifferentiated state and do not prematurely express genes characteristic of more differentiated spermatogonia. In contrast, deleting Geminin from spermatocytes does not disrupt meiosis or the differentiation of spermatids into mature sperm. In females, Geminin is not required for meiosis, oocyte differentiation, or fertility after the embryonic period of mitotic proliferation has ceased. We conclude that Geminin is absolutely required for mitotic proliferation of spermatogonia but does not regulate their differentiation. Our results suggest that Geminin maintains replication fidelity during the mitotic phase of spermatogenesis, ensuring the precise duplication of genetic information for transmission to the next generation.

  • Geminin is required for zygotic gene expression at the xenopus mid blastula transition
    PLOS ONE, 2012
    Co-Authors: Sarah L Kerns, Kelly A Barry, Kathryn M Schultz, Tina Thorne, Thomas J Mcgarry
    Abstract:

    In many organisms early development is under control of the maternal genome and zygotic gene expression is delayed until the mid-blastula transition (MBT). As zygotic transcription initiates, cell cycle checkpoints become activated and the tempo of cell division slows. The mechanisms that activate zygotic transcription at the MBT are incompletely understood, but they are of interest because they may resemble mechanisms that cause stem cells to stop dividing and terminally differentiate. The unstable regulatory protein Geminin is thought to coordinate cell division with cell differentiation. Geminin is a bi-functional protein. It prevents a second round of DNA replication during S and G2 phase by binding and inhibiting the essential replication factor Cdt1. Geminin also binds and inhibits a number of transcription factors and chromatin remodeling proteins and is thought to keep dividing cells in an undifferentiated state. We previously found that the cells of Geminin-deficient Xenopus embryos arrest in G2 phase just after the MBT then disintegrate at the onset of gastrulation. Here we report that they also fail to express most zygotic genes. The gene expression defect is cell-autonomous and is reproduced by over-expressing Cdt1 or by incubating the embryos in hydroxyurea. Geminin deficient and hydroxyurea-treated blastomeres accumulate DNA damage in the form of double stranded breaks. Bypassing the Chk1 pathway overcomes the cell cycle arrest caused by Geminin depletion but does not restore zygotic gene expression. In fact, bypassing the Chk1 pathway by itself induces double stranded breaks and abolishes zygotic transcription. We did not find evidence that Geminin has a replication-independent effect on transcription. We conclude that Geminin is required to maintain genome integrity during the rapid cleavage divisions, and that DNA damage disrupts zygotic gene transcription at the MBT, probably through activation of DNA damage checkpoint pathways.

  • Geminin deficient neural stem cells exhibit normal cell division and normal neurogenesis
    PLOS ONE, 2011
    Co-Authors: Kathryn M Schultz, Ghazal Banisadr, Ruben Omar Lastra, Tammy L Mcguire, John A Kessler, Richard J Miller, Thomas J Mcgarry
    Abstract:

    Neural stem cells (NSCs) are the progenitors of neurons and glial cells during both embryonic development and adult life. The unstable regulatory protein Geminin (Gmnn) is thought to maintain neural stem cells in an undifferentiated state while they proliferate. Geminin inhibits neuronal differentiation in cultured cells by antagonizing interactions between the chromatin remodeling protein Brg1 and the neural-specific transcription factors Neurogenin and NeuroD. Geminin is widely expressed in the CNS during throughout embryonic development, and Geminin expression is down-regulated when neuronal precursor cells undergo terminal differentiation. Over-expression of Geminin in gastrula-stage Xenopus embryos can expand the size of the neural plate. The role of Geminin in regulating vertebrate neurogenesis in vivo has not been rigorously examined. To address this question, we created a strain of Nestin-Cre/Gmnnfl/fl mice in which the Geminin gene was specifically deleted from NSCs. Interestingly, we found no major defects in the development or function of the central nervous system. Neural-specific GmnnΔ/Δ mice are viable and fertile and display no obvious neurological or neuroanatomical abnormalities. They have normal numbers of BrdU+ NSCs in the subgranular zone of the dentate gyrus, and GmnnΔ/Δ NSCs give rise to normal numbers of mature neurons in pulse-chase experiments. GmnnΔ/Δ neurosphere cells differentiate normally into both neurons and glial cells when grown in growth factor-deficient medium. Both the growth rate and the cell cycle distribution of cultured GmnnΔ/Δ neurosphere cells are indistinguishable from controls. We conclude that Geminin is largely dispensable for most of embryonic and adult mammalian neurogenesis.

  • Geminin prevents rereplication during xenopus development
    Journal of Biological Chemistry, 2007
    Co-Authors: Sarah L Kerns, Susanna J Torke, Jacqueline M Benjamin, Thomas J Mcgarry
    Abstract:

    To maintain a stable genome, it is essential that replication origins fire only once per cell cycle. The protein Geminin is thought to prevent a second round of DNA replication by inhibiting the essential replication factor Cdt1. Geminin also affects the development of several different organs by binding and inhibiting transcription factors and chromatin-remodeling proteins. It is not known if the defects in Geminin-deficient organisms are due to overreplication or to effects on cell differentiation. We previously reported that Geminin depletion in Xenopus causes early embryonic lethality due to a Chk1-dependent G(2) cell cycle arrest just after the midblastula transition. Here we report that expressing a non-Geminin-binding Cdt1 mutant in Xenopus embryos exactly reproduces the phenotype of Geminin depletion. Expressing the same mutant in replication extracts induces a partial second round of DNA replication within a single S phase. We conclude that Geminin is required to suppress a second round of DNA replication in vivo and that the phenotype of Geminin-depleted Xenopus embryos is caused by abnormal Cdt1 regulation. Expressing a nondegradable Cdt1 mutant in embryos also reproduces the Geminin-deficient phenotype. In cell extracts, the nondegradable mutant has no effect by itself but augments the amount of rereplication observed when Geminin is depleted. We conclude that Cdt1 is regulated both by Geminin binding and by degradation.