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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.

Colorectal cancer (CRC) is one of the most frequent gastrointestinal cancers worldwide, with high morbidity and mortality rates. Despite numerous attempts to identify prognostic markers for the CRC patients, the significance of the association of cellular proliferation markers with survival is controversial. Here we used immunohistochemistry to detect four markers of cellular proliferation expressed in primary CRC tissue specimens (n = 269) to assess their potential to serve as prognostic factors. CRC cells variably expressed phospho-histone H3 (PHH3) (range, 0-76 per high-powered field (HPF); median, 7 per HPF), cyclin A (CCNA) (range, 11.3-73.7%; median, 32%), geminin (GMNN) (range, 7.8-82.0%; median, 37.1%), and marker of proliferation Ki-67 (MKI67) (range, 4.9-96.6%; median, 49.6%). Among them, patients with PHH3-high (≥7 per HPF) tumors uniquely experienced significantly longer 5-year survival than those with PHH3-low (≤6 per HPF) (81.8% vs. 65.5%; P = 0.0047). Multivariable Cox hazards regression analysis identified PHH3-high (hazard ratio, 0.54; 95% confidence interval, 0.31-0.92; P = 0.025) as potential favorable factors. PHH3 levels inversely associated with pT stage (P < 0.0001) and were significantly and inversely associated with tumor diameter (ρ = -0.314, P < 0.0001). These findings support the use of PHH3 immunohistochemistry for predicting the prognoses of patients with CRC.

Geminin and its binding partner Cdt1 are essential for the regulation of DNA replication. Here we show that the CULLIN3 E3 ubiquitin ligase adaptor protein SPOP binds Geminin at endogenous level and regulates DNA replication. SPOP promotes K27-linked non-degradative poly-ubiquitination of Geminin at lysine residues 100 and 127. This poly-ubiquitination of Geminin prevents DNA replication over-firing by indirectly blocking the association of Cdt1 with the MCM protein complex, an interaction required for DNA unwinding and replication. SPOP is frequently mutated in certain human cancer types and implicated in tumorigenesis. We show that cancer-associated SPOP mutations impair Geminin K27-linked poly-ubiquitination and induce replication origin over-firing and re-replication. The replication stress caused by SPOP mutations triggers replication catastrophe and cell death upon ATR inhibition. Our results reveal a tumor suppressor role of SPOP in preventing DNA replication over-firing and genome instability and suggest that SPOP-mutated tumors may be susceptible to ATR inhibitor therapy., Geminin-Cdt1 plays essential roles in the regulation of DNA replication. Here the authors reveal that the CULLIN3 E3 ubiquitin ligase adaptor protein SPOP prevents DNA replication over-firing and genome instability by affecting Geminin ubiquitination.

The generation of all different type of cells that reside the adult brain, as well as the generation of new born neural and glial cells during adulthood, require the strict balance between self-renewal and differentiation decisions of embryonic and adult neural stem cells. The mechanisms that control these processes are not well understood, however, the members of Geminin superfamily, Geminin, Idas and Lynkeas, were proposed to control cells fate decisions and cell cycle progression making them good candidates for understanding neural stem cells function. Here we show that Geminin coordinates the differentiation of neurons and glial cells of the developing cortex, as upon its conditional inactivation from the central nervous system, alteration of the neuronal output of embryonic neural stem cells and defects in oligodendrogenesis but not in astrogliogenesis were observed. Additionally, Geminin coordinates self-renewal and differentiation decision of adult neural stem cells in the subependymal zone of the lateral ventricles. Thus, Geminin deletion in this population results in the increased self-renewal division of type-B1 slow-dividing neural stem cells, leading to increase in their numbers and in parallel in the decrease in the generation of transit amplifying neural progenitor cells. However transit amplifying neural progenitor cells exhibit increase cell cycle rate resulting in the generation of equal number of neuroblasts and olfactory interneurons as well as in equal number of oligodendrocytes in the corpus callosum. In the present study, extensive expression analysis of Idas and Lynkeas was also performed, in neural stem cells of the developing brain. Our results show that Idas and Lynkeas are expressed in the periventricular zone cells of the lateral ventricle from E16.5 embryonic day in mouse brain and their expression is restricted in radial glial cells committed to the ependymal cells lineage. Upon in vivo overexpression, using in utero electroporation in neural stem cells of the developing cortex, it was shown that Idas and Lynkeas control cell fate decisions of radial glial cells, leading to their premature differentiation towards multiciliated ependymal cells through increase in Foxj1 and c-Myb expression. Summarizing our results, we propose that Geminin controls self-renewal and differentiation decision of embryonic and adult neural stem cells and we also propose a novel function of Idas and Lynkeas, except from their known role in cell cycle progression, in cell fate decisions of radial glial cells towards multiciliated ependymal cell. Our results suggest that Geminin superfamily has a key role in cell fate decisions of embryonic and adult neural stem cells of the mammalian brain.

Background Although hepatocellular regeneration is the cornerstone of liver homeostasis, current techniques for assessing such regeneration are limited. A method for visualizing the regeneration process would provide a means for advanced studies. Therefore, we examined the possibility of using fluorescence ubiquination-based cell cycle indicator (Fucci) mice for direct visualization of hepatocellular regeneration. Materials and methods We performed a two-thirds partial hepatectomy in conventional and Fucci mice. Fucci animals have orange Cdt1 expressed in the G1 phase and green Geminin expressed in S/G2/M phases. Regenerating livers were procured daily for 7 d. Immunohistochemical staining was performed for proliferative Ki67 and mitotic pHH3 serine 10 (pHH3) markers on formalin-fixed, paraffin-embedded tissue sections from conventional mice. The orange Cdt1 and green Geminin fluorescence indicative of the G1 and S/G2/M phases, respectively, were assessed in liver tissues, in vivo and ex vivo, with two-photon laser scanning microscopy. Results Immunostaining with Ki67 and pHH3 revealed a typical profile of hepatocellular regeneration after hepatectomy in conventional mice, although immunostaining required more than a week to process. In contrast, hepatocellular regeneration could be visualized with two-photon microscopy within a few hours in regenerating livers of the Fucci mice. Only orange G1 hepatocytes were seen in the baseline liver specimens; however, multiple bright green and yellow hepatocytes were seen 48 h after hepatectomy, indicating active hepatocytes in the S/G2/M phases of the cell cycle. Conclusions Hepatocellular regeneration is readily visualized in regenerating livers of Fucci mice. The Fucci model is an exciting tool for advanced studies of hepatocellular and liver regeneration.

Background/aim Several links between DNA replication, pluripotency and development have been recently identified. The involvement of miRNA in the regulation of cell cycle events and pluripotency factors has also gained attention. Materials and methods In the present study, we used the g:Profiler platform to analyze transcription factor binding sites, miRNA networks and protein-protein interactions to identify novel links among the aforementioned processes. Results and conclusion A complex circuitry between retinoic acid signaling, SWI/SNF components, pluripotency factors including Oct4, Sox2 and Nanog and cell cycle regulators was identified. It is suggested that the DNA replication inhibitor geminin plays a central role in this circuitry.

Geminin, an inhibitor of the DNA replication licensing factor, chromatin licensing and DNA replication factor (Cdt) 1, is essential for the maintenance of genomic integrity. As a multifunctional protein, geminin is also involved in tumor progression, but the molecular details are largely unknown. Here, we found that lysophosphatidic acid (LPA)–induced upregulation of geminin was specific to gastric cancer cells. LPA acted via LPA receptor (LPAR) 3 and matrix metalloproteinases (MMPs) signaling to transactivate epidermal growth factor receptor (EGFR) (Y1173) and thereby stabilize geminin expression level during the S phase. LPA also induced the expression of deubiquitinating protein (DUB) 3, which prevented geminin degradation. These results reveal a novel mechanism underlying gastric cancer progression that involves the regulation of geminin stability by LPA-induced EGFR transactivation and provide potential targets for the signaling pathway and tumor cell–specific inhibitors.

Diffuse large B-cell lymphoma (DLBCL) harbors defects in the proliferation pathway. We performed multiparameter analysis of proteins expressed during different cell cycle phases and correlated them with clinical parameters of head and neck DLBCLs.Thirty-nine DLBCLs were staged and immunohistochemically stained with MCM2, Ki67, and geminin. The receiver operating characteristic curve and its area under the curve were calculated, and sensitivity vs specificity curve analysis was performed.The highest labeling index was in MCM2, followed by Ki67 and geminin (P.001). All pairs showed significant differences (P.001). The best cutoff points to differentiate limited from advanced disease were 68% and 45% for MCM2 and Ki67, respectively. There was no acceptable cutoff for geminin (area under the curve = 0.667, P = .134). MCM2/Ki67 (P = .293) and geminin/Ki67 (P = .233) ratios did not differ between the stages. The median (interquartile range) of the geminin/Ki67 ratio was 0.57 (0.68), translating to a reduced G1.We suggest a role for cell cycle-related proteins in the biology and behavior of DLBCLs. MCM2 and Ki67 cutoffs can be a potential option to differentiate limited from advanced disease, where imaging and laboratory techniques are unavailable. The G1 decrease and the significantly higher MCM2 expression compared to Ki67 indicate replication disturbances, making factors involved in the G1 phase targets for treatment.

Neurons are postmitotic cells. Reactivation of the cell cycle by neurons has been reported in Alzheimer’s disease (AD) brains and models. This gave rise to the hypothesis that reentering the cell cycle renders neurons vulnerable and thus contributes to AD pathogenesis. Here, we use the fluorescent ubiquitination-based cell cycle indicator (FUCCI) technology to monitor the cell cycle in live neurons. We found transient, self-limited cell cycle reentry activity in naive neurons, suggesting that their postmitotic state is a dynamic process. Furthermore, we observed a diverse response to oligomeric amyloid-β (oAβ) challenge; neurons without cell cycle reentry activity would undergo cell death without activating the FUCCI reporter, while neurons undergoing cell cycle reentry activity at the time of the oAβ challenge could maintain and increase FUCCI reporter signal and evade cell death. Accordingly, we observed marked neuronal FUCCI positivity in the brains of human mutant Aβ precursor protein transgenic (APP23) mice together with increased neuronal expression of the endogenous cell cycle control protein geminin in the brains of 3-mo-old APP23 mice and human AD brains. Taken together, our data challenge the current view on cell cycle in neurons and AD, suggesting that pathways active during early cell cycle reentry in neurons protect from Aβ toxicity.

Multiciliated Ependymal Cells and Adult Neural Stem Cells are components of the adult neurogenic niche, essential for brain homeostasis. These cells share a common glial cell lineage regulated by the Geminin family members Geminin and GemC1/Mcidas. Ependymal precursors require GemC1/Mcidas expression to massively amplify centrioles and become multiciliated cells. Here we show that GemC1-dependent differentiation occurs mostly in cycling Radial Glial Cells, in which a DNA damage response, including replicative stress and dysfunctional telomeres, arrests the cell cycle after the G1/S restriction point due to the activation of the p53-p21 pathway, which contributes to centriole amplification. Telomerase expression in Radial Glial Cells impairs ependymal differentiation and favors the Neural Stem Cell fate.

BackgroundLysophosphatidic acid (LPA) is one of the simplest active phospholipid molecules. Binding to its receptors on the cell surface, LPA initiates various intracellular signal cascades, involving in numerous biological processes, such as cell proliferation, migration, and apoptosis. If abnormalities occur in the processes of LPA production, receptor expression or signal transduction, it may induce certain diseases and even contribute to the occurrence, development and metastasis of cancer. However, whether the initiation of DNA replication is regulated by LPA has not yet been investigated.MethodsFirst, diverse public databases were analyzed to explore the genetic abnormalities affecting geminin. Next, an LPA gradient treatment was performed on gastric cancer cells, followed by detecting geminin expression variation using western blot analysis. Finally, RNAi technology or inhibitors were used to block the biological activity of related factors in the GPCR induced EGFR transactivation signaling pathway to verify whether the effect of LPA evoked gastric cancer DNA replication is dependent on geminin upregulation.ResultsWe found that LPA specifically up-regulated expression of an essential replication negative regulator geminin in early S phase in gastric cancer cell lines, and that the deletion of geminin selectively induced DNA re-replication. Neither of these phenomena has been observed in normal gastric epithelial cells, indicating LPA-induced geminin up-regulation is restricted to tumor cells. Using RNAi or specific inhibitors to block the activity of related factors in the signaling pathway, we found that LPA acts through LPAR3 and downstream coupled MMPs signaling to trans-activate EGFR, increasing the expression level of geminin in S phase. On the other hand, LPA stimulation induced the up-regulation of de-ubiquitinating enzyme 3 (DUB3) in a short time and inhibited the ubiquitination degradation of geminin to enhance geminin stability and positively regulate the DNA replication initiation in gastric cancer cells. Taken together, our results suggested that LPA mediated DNA replication and S-phase cell-cycle progression through a LPAR3/MMPs/EGFR/PI3K/mTOR signaling axis in gastric cancer. ConclusionsOur research is for the first time to study the regulatory effect of LPA-induced EGFR transactivation in DNA replication of tumor cells, and to uncover a novel mechanism for regulating the stability of geminin through LPA and related downstream signaling pathways. All of which will provide potential targets for the development of signaling pathways and tumor cell-specific EGFR transactivation inhibitor for the treatment of gastric cancer.

APC/C-mediated proteolysis of cyclin B and securin promotes anaphase entry, inactivating CDK1 and permitting chromosome segregation, respectively. Reduction of CDK1 activity relieves inhibition of the CDK1-counteracting phosphatases PP1 and PP2A-B55, allowing wide-spread dephosphorylation of substrates. Meanwhile, continued APC/C activity promotes proteolysis of other mitotic regulators. Together, these activities orchestrate a complex series of events during mitotic exit. However, the relative importance of regulated proteolysis and dephosphorylation in dictating the order and timing of these events remains unclear. Using high temporal-resolution proteomics, we compare the relative extent of proteolysis and protein dephosphorylation. This reveals highly-selective rapid proteolysis of cyclin B, securin and geminin at the metaphase-anaphase transition, followed by slow proteolysis of other substrates. Dephosphorylation requires APC/C-dependent destruction of cyclin B and was resolved into PP1-dependent categories with unique sequence motifs. We conclude that dephosphorylation initiated by selective proteolysis of cyclin B drives the bulk of changes observed during mitotic exit., eLife digest New cells are made when a single cell duplicates its DNA and divides into two cells, distributing the DNA equally between them, in a process called mitosis. The splitting of the two copies of DNA happens through a series of controlled events known as mitotic exit. Previous research has suggested that mitotic exit relies on both the destruction of specific proteins and the removal of tags called phosphate groups from other proteins. Phosphate groups modify how proteins behave and their removal can trigger changes in a protein’s activity. Although protein destruction and phosphate group removal were known to be important to mitotic exit, it was not understood how they are coordinated in the cell to ensure the correct order of events. Holder et al. have used a technique called mass spectrometry to monitor the level of thousands of proteins, and any tags attached to them, during mitotic exit in human cells grown in the laboratory. The experiments revealed that the destruction of a single protein, known as cyclin B, plays a major role in triggering subsequent events. The removal of cyclin B activates enzymes known as phosphatases, which remove phosphate groups from proteins. Phosphatases then act on a wide range of proteins in a specific order that depends on the environment surrounding the phosphate group. This ‘chain’ of phosphatase activity determines the order of events during mitotic exit. The findings of Holder et al. contribute to the basic understanding of how mitotic exit works. Errors in the process can affect the stability of a cell’s genome, contributing to diseases such as cancer. In the future, this may help to identify what goes wrong in these cases and potential avenues for developing treatments.

SUMMARYDNA replication initiates with pre-replication complex (pre-RC) formation at replication origins in G1 (replication origin licensing), followed by activation of a pre-RC subset in the S phase. It has been suggested that a checkpoint prevents S phase entry when too few origins are licensed. Yet, we found that in normal cells, complete DNA synthesis inhibition by overexpression of a non-degradable geminin variant, or by CDT1 silencing prevents DNA replication without inducing any checkpoint. Cells continue cycling and enter mitosis, despite the absence of replicated DNA. Most of these unlicensed cells exit mitosis without dividing and enter senescence; however, about 25% of them successfully divide without previous DNA replication, producing daughter cells with half the normal diploid complement of chromosomes (1C). This suggests a potentially attractive strategy to derive haploid cells from any somatic cell type and unveil undescribed aspects of the coordination between DNA replication and cell division in mammals.

SummaryWe cloned and characterized two new coral fluorescent proteins: h2-3 and 1-41. h2-3 formed an obligate dimeric complex and exhibited bright fluorescence. On the other hand, 1-41 formed a highly multimeric complex and exhibited dim red fluorescence. We engineered 1-41 into AzaleaB5, a practically useful red-emitting fluorescent protein for cellular labeling applications. We fused h2-3 and AzaleaB5 to the ubiquitination domains of human Geminin and Cdt1, respectively, to generate a new color variant of Fucci (Fluorescent Ubiquitination-based Cell-Cycle Indicator): Fucci5. We found Fucci5 provided brighter nuclear labeling for monitoring cell cycle progression than the 1st and 2nd generations that used mAG/mKO2 and mVenus/mCherry, respectively.

Multiciliated cells (MCC) project dozens to hundreds of motile cilia from the cell surface to generate fluid flow across epithelial surfaces or turbulence to promote the transport of gametes. The MCC differentiation program is initiated by GEMC1 and MCIDAS, members of the geminin family, that activate key transcription factors, including p73 and FOXJ1, to control the multiciliogenesis program. To support the generation of multiple motile cilia, MCCs must undergo massive centriole amplification to generate a sufficient number of basal bodies (modified centrioles). This transcriptional program involves the generation of deuterosomes, unique structures that act as platforms to regulate centriole amplification, the reactivation of cell cycle programs to control centriole amplification and release, and extensive remodeling of the cytoskeleton. This review will focus on providing an overview of the transcriptional regulation of MCCs and its connection to key processes, in addition to highlighting exciting recent developments and open questions in the field.

Development of the complex structure of the vertebrate limb requires carefully orchestrated interactions between multiple regulatory pathways and proteins. Among these, precise regulation of 5’ Hox transcription factor expression is essential for proper limb bud patterning and elaboration of distinct limb skeletal elements. Here, we identified Geminin (Gmnn) as a novel regulator of this process. A conditional model of Gmnn deficiency resulted in loss or severe reduction of forelimb skeletal elements, while both the forelimb autopod and hindlimb were unaffected. 5’ Hox gene expression expanded into more proximal and anterior regions of the embryonic forelimb buds in this Gmnn-deficient model. A second conditional model of Gmnn deficiency instead caused a similar but less severe reduction of hindlimb skeletal elements and hindlimb polydactyly, while not affecting the forelimb. An ectopic posterior SHH signaling center was evident in the anterior hindlimb bud of Gmnn-deficient embryos in this model. This center ectopically expressed Hoxd13, the HOXD13 target Shh, and the SHH target Ptch1, while these mutant hindlimb buds also had reduced levels of the cleaved, repressor form of GLI3, a SHH pathway antagonist. Together, this work delineates a new role for Gmnn in modulating Hox expression to pattern the vertebrate limb.SummaryThis work identifies a new role for Geminin in mouse limb development. Geminin is a nuclear protein that regulates gene expression to control several other aspects of vertebrate development.

Multiciliated cells (MCCs) project dozens to hundreds of motile cilia from their apical surface to promote the movement of fluids or gametes in the mammalian brain, airway or reproductive organs. Differentiation of MCCs requires the sequential action of the Geminin family transcriptional activators, GEMC1 and MCIDAS, that both interact with E2F4/5-DP1. How these factors activate transcription and the extent to which they play redundant functions remains poorly understood. Here, we demonstrate that the transcriptional targets and proximal proteomes of GEMC1 and MCIDAS are highly similar. However, we identified distinct interactions with SWI/SNF subcomplexes; GEMC1 interacts primarily with the ARID1A containing BAF complex and MCIDAS with the BRD9 containing ncBAF complex. Inhibition of BRD9 impaired MCIDAS-mediated activation of several target genes and compromised MCC differentiation. Our data suggest that the differential engagement of distinct SWI/SNF subcomplexes by GEMC1 and MCIDAS is required for MCC-specific transcriptional regulation.

Endoreplication, known as endocycles or endoreduplication, is a cell cycle variant in which the genomic DNA is re-replicated without mitosis leading to polyploidy. Endoreplication is essential for the development and functioning of the different organs in animals and plants. Deletion of Geminin, a DNA replication licensing inhibitor, causes DNA re-replication or damage. However, the role of Geminin in endoreplication is still unclear. Here, we studied the role of Geminin in the endoreplication of the silk gland cells of silkworms by constructing two transgenic silkworm strains, including BmGeminin1-overexpression and BmGeminin1-RNA interference. Interference of BmGeminin1 led to body weight gain, increased silk gland volume, increased DNA content, and enhanced DNA re-replication activity relative to wild-type Dazao. Meanwhile, overexpression of BmGeminin1 showed an opposite phenotype compared to the BmGem1-RNAi strain. Furthermore, RNA-sequencing of the transgenic strains was carried out to explore how BmGeminin1 regulates DNA re-replication. Our data demonstrated a vital role of Geminin in the regulation of endoreplication in the silk gland of silkworms.

Cell transplantation holds considerable promise for end-stage liver diseases but identifying a suitable, transplantable cell type has been problematic. Here, we describe a novel type of mesenchymal stem cells (MSCs) from human adipose tissue. These cells are different from previously reported MSCs, they are in the euchromatin state with epigenetic multipotency, and express pluripotent markers MYC, KLF4, and GMNN. Most of the genes associated with germ layer specification are modified by H3K4me3 or co-modified by H3K4me3 and H3K27me3. We named this new type of MSCs as adult multipotent adipose-derived stem cells (M-ADSCs). Using a four-step nonviral system, M-ADSCs can be efficiently Induced into hepatocyte like cells with expression of hepatocyte markers, drug metabolizing enzymes and transporters, and the other basic functional properties including albumin (ALB) secretion, glycogen storage, detoxification, low-density lipoprotein intake, and lipids accumulation. In vivo both M-ADSCs-derived hepatoblasts and hepatocytes could form vascularized liver-like tissue, secrete ALB and express metabolic enzymes. Single-cell RNA-seq was used to investigate the important stages in this conversion. M-ADSCs could be converted to a functionally multipotent state during the preinduction stage without undergoing reprogramming process. Our findings provide important insights into mechanisms underlying cell development and conversion.

Geminin, a DNA replication licensing inhibitor, ensures faithful DNA replication in vertebrates. Several studies have shown that geminin depletion in vitro results in rereplication and DNA damage, whereas increased expression of geminin has been observed in human cancers. However, conditional inactivation of geminin during embryogenesis has not revealed any detectable DNA replication defects. In order to examine its role in vivo, we conditionally inactivated geminin in the murine colon and lung, and assessed chemically induced carcinogenesis. We show here that mice lacking geminin develop a significantly higher number of tumors and bear a larger tumor burden than sham-treated controls in urethane-induced lung and azoxymethane/dextran sodium sulfate-induced colon carcinogenesis. Survival is also significantly reduced in mice lacking geminin during lung carcinogenesis. A significant increase in the total number and grade of lesions (hyperplasias, adenomas, and carcinomas) was also confirmed by hematoxylin and eosin staining. Moreover, increased genomic aberrations, identified by increased ATR and γH2AX expression, was detected with immunohistochemistry analysis. In addition, we analyzed geminin expression in human colon cancer, and found increased expression, as well as a positive correlation with ATM/ATR levels and a non-monotonic association with γH2AX. Taken together, our data demonstrate that geminin acts as a tumor suppressor by safeguarding genome stability, whereas its overexpression is also associated with genomic instability. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Oral squamous cell carcinoma (OSCC) is still an unabated global killer with little advancement in its survival rate. DNA replication licensing proteins and Aurora kinase A are biomarkers that play important roles in genomic stability. The expression profile of minichromosomal maintenance protein 2 (MCM2), Ki67, geminin, and Aurora-A were linked to clinicopathological and outcome parameters, survival, and DNA content in 125 cases of OSCC. Oral fibroepithelial polyps (OFEP) were controls. The OSCC tumour cells were in a rapidly proliferating state, as assessed by the increased expression profile of MCM2, Ki67, geminin, and Aurora-A and of the geminin/Ki67 ratio, and the decrease of the MCM2/Ki67 ratio, in OSCC compared with OFEP (P < 0.000). There was an association between expression of MCM2, Ki67, and geminin and tumour histologic and invasive front grade (P < 0.05). A total of 82% of the OSCC assessed had aneuploid DNA content, which was associated with increased expression intensity of Aurora-A (P = 0.01). Geminin and the geminin/Ki67 ratio were associated with TNM staging (P < 0.05), and weak expression of MCM2, Ki67, geminin, and Aurora-A were predictive of OSCC survival (P < 0.05). Dysregulation of the origin licensing pathway and the mitotic pathway are important events in OSCC, and the combined analysis of these proteins may contribute to improved treatment decisions.

Rapid protein degradation enables cells to quickly modulate protein abundance. Dysregulation of short-lived proteins plays essential roles in disease pathogenesis. A focused map of short-lived proteins remains understudied. Cycloheximide, a translational inhibitor, is widely used in targeted studies to measure degradation kinetics for short-lived proteins. Here, we combined cycloheximide chase assays with advanced quantitative proteomics to map short-lived proteins under translational inhibition in four human cell lines. Among 11,747 quantified proteins, we identified 1,017 short-lived proteins (half-lives ≤ 8hr). These short-lived proteins are less abundant, evolutionarily younger, and less thermally stable than other proteins. We quantified 103 proteins with different stabilities among cell lines. We showed that U2OS and HCT116 cells express truncated forms of ATRX and GMDS, respectively, which have lower stability than their full-length counterparts. This study provides a large-scale resource of human short-lived proteins under translational arrest, leading to untapped avenues of protein regulation for therapeutic interventions.

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.

Viruses commonly create favorable cellular conditions for their survival through multiple mechanisms. MicroRNAs (miRNAs), which function as post-transcriptional regulators, are utilized by human cytomegalovirus (HCMV) in its infection and pathogenesis. In the present study, the DNA replication inhibitor Geminin (GMNN) was identified to be a direct target of hcmv-miR-US5-1. Overexpression of hcmv-miR-US5-1 could block the accumulation of GMNN during HCMV infection, and the decrease of GMNN expression caused by hcmv-miR-US5-1 or GMNN specific siRNA reduced HCMV DNA copies in U373 cells. Meanwhile, ectopic expression of hcmv-miR-US5-1 and consequent lower expression of GMNN influenced host cell cycle and proliferation. These results imply that hcmv-miR-US5-1 may affect viral replication and host cellular environment by regulating expression kinetics of GMNN during HCMV infection. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material is available for this article at 10.1007/s12250-017-4064-x and is accessible for authorized users.

The initiation of DNA replication is strictly regulated by multiple mechanisms to ensure precise duplication of chromosomes. In higher eukaryotes, activity of the Cdt1 protein is temporally regulated during the cell cycle, and deregulation of Cdt1 induces DNA re-replication. In previous studies, we showed that excess Cdt1 inhibits DNA replication by suppressing progression of replication forks in Xenopus egg extracts. Here, we investigated the functional regions of Cdt1 that are required for the inhibition of DNA replication. We constructed a series of N-terminally or C-terminally deleted mutants of Cdt1 and examined their inhibitory effects on DNA replication in Xenopus egg extracts. Our results showed that the region spanning amino acids (a. a.) 255–620 is required for efficient inhibition of DNA replication, and that, within this region, a. a. 255–289 have a critical role in inhibition. Moreover, one of the Cdt1 mutants, Cdt1 R285A, was compromised with respect to the licensing activity but still inhibited DNA replication. This result suggests that Cdt1 has an unforeseen function in the negative regulation of DNA replication, and that this function is located within a molecular region that is distinct from those required for the licensing activity.

Neural stem cells have been considered as a source of stem cells that can be used for cell replacement therapies in neurodegenerative diseases, as they can be isolated and expanded in vitro and can be used for autologous grafting. However, due to low percentages of survival and varying patterns of differentiation, strategies that will enhance the efficacy of transplantation are under scrutiny. In this article, we have examined whether alterations in Geminin's expression, a protein that coordinates the balance between self-renewal and differentiation, can improve the properties of stem cells transplanted in 6-OHDA hemiparkinsonian mouse model. Our results indicate that, in the absence of Geminin, grafted cells differentiating into dopaminergic neurons were decreased, while an increased number of oligodendrocytes were detected. The number of proliferating multipotent cells was not modified by the absence of Geminin. These findings encourage research related to the impact of Geminin on transplantations for neurodegenerative disorders, as an important molecule in influencing differentiation decisions of the cells composing the graft.

Geminin expression is essential for embryonic development and the maintenance of chromosomal integrity. In spite of this protective role, geminin is also frequently overexpressed in human cancers and the molecular mechanisms underlying its role in tumor progression remain unclear. The histone deacetylase HDAC3 modulates transcription factors to activate or suppress transcription. Little is known about how HDAC3 specifies substrates for modulation among highly homologous transcription factor family members. Here, we have demonstrated that geminin selectively couples the transcription factor forkhead box O3 (FoxO3) to HDAC3, thereby specifically facilitating FoxO3 deacetylation. We determined that geminin-associated HDAC3 deacetylates FoxO3 to block its transcriptional activity, leading to downregulation of the downstream FoxO3 target Dicer, an RNase that suppresses metastasis. Breast cancer cells depleted of geminin or HDAC3 exhibited poor metastatic potential that was attributed to reduced suppression of the FoxO3-Dicer axis. Moreover, elevated levels of geminin, HDAC3, or both together with decreased FoxO3 acetylation and reduced Dicer expression were detected in aggressive human breast cancer specimens. These results underscore a prominent role for geminin in promoting breast cancer metastasis via the enzyme-substrate-coupling mechanism in HDAC3-FoxO3 complex formation.

The proliferation-promoting effect of neuropeptide Y (NPY) always functions in low-serum-cultured vascular smooth muscle cells (VSMCs), and the phenotypic switch of VSMCs is regulated by concentrations of serum. Whether the property of the NPY proliferative effect in VSMCs relies on phenotype of VSMCs is unclear. We aimed to explore the role of NPY on proliferation of different VSMC phenotypes in the pathogenesis of atherosclerosis. By stimulating A10 cells with 200 nM NPY in 0.5 or 10% serum, 3H-thymidine and 5-ethynyl-2′-deoxyuridine (EdU) and CCK8 measurements were used to detect VSMC proliferation. RT-PCR and Flow cytometry were performed to detect the factors involved in different properties of the NPY proliferative effect in VSMCs. Instead of facilitating proliferation, NPY had no significant effect on the growth of VSMCs when cultured in 10% serum (VSMCs stayed at synthetic states). The underlying mechanism may be involved in down-regulation of Y1 receptor (P

To ensure correct spatial and temporal patterning, embryos must maintain pluripotent cell populations and control when cells undergo commitment. The newly identified nucleoprotein Akirin has been shown to modulate the innate immune response through epigenetic regulation and to play important roles in other physiological processes, but its role in neural development remains unknown. Here we show that Akirin2 is required for neural development in Xenopus and that knockdown of Akirin2 expands the expression of the neural progenitor marker Sox2 and inhibits expression of the differentiated neuronal marker N-tubulin. Akirin2 acts antagonistically to Geminin, thus regulating Sox2 expression, and maintains the neural precursor state by participating in the Brg1/Brm-associated factor (BAF) complex mediated by BAF53a. Additionally, Akirin2 also modulates N-tubulin expression by acting upstream of neuronal differentiation 1 (NeuroD) and in parallel with neurogenin-related 1 (Ngnr1) during terminal neuronal differentiation. Thus, our results reveal a novel model in which Akirin2 precisely coordinates and temporally controls Xenopus neural development.

Geminin is implicated in regulation of the cell cycle and differentiation. Although loss of Geminin triggers unscheduled DNA rereplication as a result of interruption of its interaction with Cdt1 in some somatic cancer cells, whether such cell cycle regulation also operates in embryonic stem cells (ESCs) has remained unclear. To characterize the Geminin-Cdt1 axis in ESCs and compare it with that in somatic cells, we established conditional knockout (KO) of Geminin in mouse ESCs and mouse embryonic fibroblasts (MEFs). Geminin KO ESCs manifest a large flattened morphology, develop polyploidy accompanied by DNA damage and G2 -M checkpoint activation, and subsequently undergo apoptosis. Rereplication in Geminin KO ESCs was attenuated by inhibition of G2 -M checkpoint signaling or by expression of wild-type Geminin, but not by expression of a Geminin mutant that does not bind to Cdt1, indicating the importance of sequestration of Cdt1 by Geminin in G2 phase. In contrast, Geminin KO MEFs did not manifest disturbance of the cell cycle unless they were treated to force abnormal accumulation of Cdt1. Together, our results indicate that Geminin is a key inhibitor of Cdt1 in mouse ESCs, but that it plays a backup role in MEFs to compensate for accidental up-regulation of Cdt1.

Correct control of DNA replication is crucial to maintain genomic stability in dividing cells. Inappropriate re-licensing of replicated origins is associated with chromosomal instability (CIN), a hallmark of cancer progression that at the same time provides potential opportunities for therapeutic intervention. Geminin is a critical inhibitor of the DNA replication licensing factor Cdt1. To properly achieve its functions, Geminin levels are tightly regulated through the cell cycle by ubiquitin-dependent proteasomal degradation, but the de-ubiquitinating enzymes (DUBs) involved had not been identified. Here we report that DUB3 and USP7 control human Geminin. Overexpression of either DUB3 or USP7 increases Geminin levels through reduced ubiquitination. Conversely, depletion of DUB3 or USP7 reduces Geminin levels, and DUB3 knockdown increases re-replication events, analogous to the effect of Geminin depletion. In exploring potential clinical implications, we found that USP7 and Geminin are strongly correlated in a cohort of invasive breast cancers (P

Geminin plays a critical role in cell cycle regulation by regulating DNA replication and serves as a transcriptional molecular switch that directs cell fate decisions. Spermatogonia lacking Geminin disappear during the initial wave of mitotic proliferation, while geminin is not required for meiotic progression of spermatocytes. It is unclear whether geminin plays a role in pre-meiotic DNA replication in later-stage spermatogonia and their subsequent differentiation. Here, we selectively disrupted Geminin in the male germline using the Stra8-Cre/loxP conditional knockout system. Geminin-deficient mice showed atrophic testes and infertility, concomitant with impaired spermatogenesis and reduced sperm motility. The number of undifferentiated spermatogonia and spermatocytes was significantly reduced; the pachytene stage was impaired most severely. Expression of cell proliferation-associated genes was reduced in Gmnnfl/Δ; Stra8-Cre testes compared to in controls. Increased DNA damage, decreased Cdt1, and increased phosphorylation of Chk1/Chk2 were observed in Geminin-deficient germ cells. These results suggest that geminin plays important roles in pre-meiotic DNA replication and subsequent spermatogenesis.

Background Minichromosome maintenance protein 7 (MCM7) is a downstream of human epidermal growth receptor (HER1) signaling. We examined MCM7, geminin, and HER1 expression in patients with laryngeal squamous cell carcinoma (SCC) treated with radiotherapy and cetuximab. Methods MCM7, geminin, and HER1 were evaluated by immunohistochemistry on 61 patients with laryngeal SCC. The follow-up (median, 32.1 months; range, 2–139 months) went from the beginning of therapy to tumor progression-free survival (PFS) and death (overall survival [OS]). Results MCM7, but not geminin, was associated only with HER1 expression, whereas no association was found with other clinicopathological characteristics. Patients with MCM7 high - geminin high and MCM7 high - geminin low tumor status had a risk of progression 3.1 times and 17.7 times greater, respectively, than patients with MCM7 low – geminin high tumor status. Tumor site, MCM7, and geminin were independent determinants of PFS, whereas MCM7 was an independent prognostic marker of OS. Conclusion MCM7-geminin tumor status may be prognostic for patients with laryngeal SCC treated with cetuximab and radiotherapy. © 2016 Wiley Periodicals, Inc. Head Neck, 2016

The trophoblast cells that take part in placenta formation are characterized by different modes of multiplication of their genome that largely designates their eu- or aneuploidy level. The two main ways of genome multiplication are described in different degree: (a) endoreduplication that involves almost complete shutdown of mitosis and (b) reduced mitosis ('endomitosis') in which, by contrast, entry into mitosis and the passage of its initial stages is a prerequisite of genome multiplication. Endoreduplication observed in the trophoblast giant cells (TGC) in a range of mammalian species implies uncoupling of DNA replication from mitosis achieved by reduction of mitotic Cdk activity. The key role in the regulation of endoreduplication and endomitosis play activity of APC/C complex, geminin and E2F family. A programme of genome multiplication and cell cycle progression may include depolyploidization achieved by specific mitotic or non-mitotic (amitotic) division of the giant nucleus. In some mammalian species (Rodents), this process represents the final step of the giant cell lifespan that coincides with complete cessation of cell or genome reproduction. Meantime, in other species the process may take part in cell reproduction during lengthy pregnancy. The dynamics of fox and human polyploidization is similar by the possibility of a simultaneous increase in the proportion of endopolyploid and low-polyploid cells. Reduced mitoses, endoreduplication and depolyploidization appear to be an evolution strategy allowing to generate the functionally different trophoblast cell populations depending of the lifestyle of life of the animal species. Some placental pathologies may be accounted for disturbance of the programme of the cell/genome reproduction of the giant and low-ploid cell populations.

Introduction: Proliferation markers play a significant role in the biologic behavior of tumors. Geminin is a known inhibitor of the cell cycle and DNA replication and has not been previously reported in cutaneous basal and squamous cell carcinomas of the head and neck. Objectives: We aimed to investigate proliferation markers ki67, MCM2, and geminin in head and neck cutaneous basal and squamous cell carcinomas. Methods: Forty cases of each tumor were immuostained with ki67, MCM2, and geminin followed by assessment of labeling indices (LIs). MCM2/ki67- and geminin/ki67-ratios were also determined; t-test was used for statistical analysis (p

Introduction/Background Homologous recombination deficiency (HRD) is frequent in non-endometrioid endometrial cancer (EC), justifying clinical trials testing PARP-inhibitors. To facilitate this development a pragmatic test to asses HR competency is required. Accumulation of RAD51 protein at sites of DNA double strand breaks, has been put forward as a reliable functional test for HR competency. Here we aimed to test the performance of this pragmatic assay on formalin-fixed paraffin-embedded (FFPE) tumour tissue. Methodology We used a previously described, prospective collected series of 21 EC that underwent primary resection with known HR status based on genomic analyses (next-generation sequencing for HR-core genes and array comparative genomic hybridization/SNP array) as our training cohort. To assure the presence of endogenous DNA damage, a gamma-H2AX staining was performed. Co-immunofluorescence (IF) was performed for both Geminin (marker staining cells in G2/S phase) and RAD51. RAD51 score was calculated as the fraction of Geminin positive cells that showed RAD51 foci (≥ 2) in the nucleus. Final RAD51 scores were correlated to known HR-status. Results All 21 EC showed positivity for gamma-H2AX staining assuring presence of endogenous DNA damage in routine diagnostic FFPE tumour tissue. Among all cases, the RAD51scores varied form 0–72%. The range of RAD51 scores in the five HRD EC was 0–4%, whereas the 16 HRP EC tested showed RAD51 scores ranging from 16–72% (P Conclusion This RAD51 assay can successfully be performed on diagnostic FFPE material and reliably determined HR competency in 100% of our EC cases using cut-off RAD51 score of 10%. We will present the performance of this cut-off on our test-set of HR-annotated EC. This RAD51 assay is a promising cheap, pragmatic assay that can be used in the clinic to select patients that may benefit from platinum compounds and PARP-inhibitor therapy. Disclosure Nothing to disclose.

Geminin (Gmnn) is a coiled-coil nuclear protein originally identified as a DNA replication inhibitor. In addition, nuclear Gmnn regulates gene expression by interacting (through a domain distinct from the Cdt1-interacting domain) with Brg1, the catalytic subunit of the SWI/SNF chromatin remodeling complex. In this context, Gmnn regulates expression of key transcription factors involved in differentiation. The dual role of Gmnn in the cell cycle and differentiation has been viewed as a critical mechanism for linking replication and differentiation. We wanted to test its role in human pancreas development. We found a robust Gmnn expression in the human fetal pancreas but its expression slightly decreased with age. Remarkably the fraction of cells expressing both Gmnn and Ki67 declined much faster with age. We isolated 13WD human fetal pancreas and studied the proportion of replicating Sox9+cells. We compared control human fetal explants with fetal explants transduced with a shRNA lentivirus directed against Gmnn (Gmnnsh) and discovered that Gmnnshexplants display a decrease in proliferation compared to controls after 2 days in culture. RNA sequencing comparing Gmnnshexplants to controls revealed a marked decrease in transcripts associated with endocrine differentiation. To evaluate the effects on endocrine differentiation, human fetal pancreases were isolated at 13WD and treated for 5 days with lentiviruses or casein kinase inhibitors. We detected a significant reduction in the number of insulin+cells after treatment with the casein kinase inhibitor TBB and a significant reduction in the number of glucagon+cells after treatment with TBB or after siCsnk2a1. Overexpression of Gmnn significantly increased the proportion of Neurog3+cells compared to the control explants. Finally, we found that inhibition of Gmnn or casein kinase 2 was capable of significantly reducing expression of Nkx6.1 compared to controls. We conclude that Gmnn may play a critical role pancreas cell replication and endocrine specification in human development. Disclosure L. Baeyens: None. D.W. Scheel: None. K. Yang: None. M. German: Advisory Panel; Self; Encellin. Stock/Shareholder; Self; Encellin, Viacyte, Inc. Funding JDRF

The subventricular zone (SVZ) is one of two main niches where neurogenesis persists during adulthood, as it retains neural stem cells (NSCs) with self-renewal capacity and multi-lineage potency. Another critical cellular component of the niche is the population of postmitotic multiciliated ependymal cells. Both cell types are derived from radial glial cells that become specified to each lineage during embryogenesis. We show here that GemC1, encoding Geminin coiled-coil domain-containing protein 1, is associated with congenital hydrocephalus in humans and mice. Our results show that GemC1 deficiency drives cells toward a NSC phenotype, at the expense of multiciliated ependymal cell generation. The increased number of NSCs is accompanied by increased levels of proliferation and neurogenesis in the postnatal SVZ. Finally, GemC1-knockout cells display altered chromatin organization at multiple loci, further supporting a NSC identity. Together, these findings suggest that GemC1 regulates the balance between NSC generation and ependymal cell differentiation, with implications for the pathogenesis of human congenital hydrocephalus.

Motile cilia on multiciliated cells (MCCs) function in fluid clearance over epithelia. Studies with Xenopus embryos and patients with the congenital respiratory disorder reduced generation of multiple motile cilia, have implicated the nuclear protein MCIDAS (MCI), in the transcriptional regulation of MCC specification and differentiation. Recently, a paralogous protein, GMNC, was also shown to be required for MCC formation. Surprisingly, and in contrast to the presently held view, we find that Mci mutant mice can specify MCC precursors. However, these precursors cannot produce multiple basal bodies, and mature into single ciliated cells. We show that MCI is required specifically to induce deuterosome pathway components for the production of multiple basal bodies. Moreover, GMNC and MCI associate differentially with the cell-cycle regulators E2F4 and E2F5, which enables them to activate distinct sets of target genes (ciliary transcription factor genes versus genes for basal body generation). Our data establish a previously unrecognized two-step model for MCC development: GMNC functions in the initial step for MCC precursor specification. GMNC induces Mci expression, which then drives the second step of basal body production for multiciliation.SUMMARY STATEMENTWe show how two GEMININ family proteins function in mammalian multiciliated cell development: GMNC regulates precursor specification and MCIDAS induces multiple basal body formation for multiciliation.

Background Carcinogenesis occurs when the cell cycle is compromised. Chromatin licensing and DNA replication factor 1, geminin, and γ-H2A histone family member X are expressed in cells in G1 phase, S/G2 /M phases, and apoptosis, respectively, and these three markers may be useful for histological evaluation of malignant lesions. Here, we aimed to identify cell cycle phases and apoptosis using immunohistochemistry in oral epithelial precursor lesions and oral squamous cell carcinoma. Methods Chromatin licensing and DNA replication factor 1, geminin, and γ-H2A histone family member X expression levels were immunohistochemically examined in tissue specimens from 55 patients with oral epithelial precursor lesions and 50 patients with oral squamous cell carcinoma. Associations of clinicopathological variables with marker expression were assessed. Results Chromatin licensing and DNA replication factor 1 was expressed in the prickle cell layer of oral epithelial precursor lesions and many carcinoma cells of oral squamous cell carcinoma. Geminin reactivity was widely distributed in high-grade dysplasia and oral squamous cell carcinoma rather than low-grade or no dysplastic cases. γ-H2A histone family member X was expressed in the superficial layer of oral epithelial precursor lesions and scattered carcinoma cells of oral squamous cell carcinoma. In oral squamous cell carcinoma, lower geminin expression was observed in recurrent cases. Geminin and γ-H2A histone family member X were associated with the degree of differentiation and mode of invasion. Conclusion Chromatin licensing and DNA replication factor 1, geminin, and γ-H2A histone family member X expression levels were correlated with oral carcinogenesis; these markers were associated with clinicopathological behaviors in oral squamous cell carcinoma.

Epigenetic information (epigenome) on chromatin is crucial for the determination of cellular identity and for the expression of cell type-specific biological functions. The cell type-specific epigenome is maintained beyond replication and cell division. Nucleosomes of chromatin just after DNA replication are a mixture of old histones with the parental epigenome and newly synthesized histones without such information. The diluted epigenome is mostly restored within one cell cycle using the epigenome on the parental DNA and nucleosomes as replication templates. However, many important questions about the epigenome replication process remain to be clarified. In this study, we investigated the model system comprising of dimethylated histone H3 lysine 9 (H3K9me2) and its regulation by the lysine methyltransferase G9a. Using this epigenome model system, we addressed whether H3K9me2 can be induced in specific cell cycle stages, especially G1. Using cell cycle-specific degrons, we achieved G1 or late G1-to M phases specific accumulation of exogenous G9a in G9a deficient cells. Importantly, global levels of H3K9me2 were significantly recovered by both cell types. These data indicate that H3K9me2 may be plastic and inducible, even in the long-living, terminally-differentiated, post-mitotic, G0-G1 cell population in vivo. This knowledge is valuable in designing epigenome-manipulation-based treatments for diseases.

A distinct combination of transcription factors elicits the acquisition of a specific fate and the initiation of a differentiation program. Multiciliated cells (MCCs) are a specialized type of epithelial cells that possess dozens of motile cilia on their apical surface. Defects in cilia function have been associated with ciliopathies that affect many organs, including brain and airway epithelium. Here we show that the geminin coiled-coil domain-containing protein 1 GemC1 (also known as Lynkeas) regulates the transcriptional activation of p73, a transcription factor central to multiciliogenesis. Moreover, we show that GemC1 acts in a trimeric complex with transcription factor E2F5 and tumor protein p73 (officially known as TP73), and that this complex is important for the activation of the p73 promoter. We also provide in vivo evidence that GemC1 is necessary for p73 expression in different multiciliated epithelia. We further show that GemC1 regulates multiciliogenesis through the control of chromatin organization, and the epigenetic marks/tags of p73 and Foxj1. Our results highlight novel signaling cues involved in the commitment program of MCCs across species and tissues. This article has an associated First Person interview with the first author of the paper.

Geminin is an important regulator of DNA replication and cell differentiation, but its role in female reproduction remains uncertain. Maternal geminin does not regulate oocyte meiotic maturation but does control accurate DNA replication. Geminin deletion in oocytes results in impaired embryo development and reduced fertility., Geminin controls proper centrosome duplication, cell division, and differentiation. We investigated the function of geminin in oogenesis, fertilization, and early embryo development by deleting the geminin gene in oocytes from the primordial follicle stage. Oocyte-specific disruption of geminin results in low fertility in mice. Even though there was no evident anomaly of oogenesis, oocyte meiotic maturation, natural ovulation, or fertilization, early embryo development and implantation were impaired. The fertilized eggs derived from mutant mice showed developmental delay, and many were blocked at the late zygote stage. Cdt1 protein was decreased, whereas Chk1 and H2AX phosphorylation was increased, in fertilized eggs after geminin depletion. Our results suggest that disruption of maternal geminin may decrease Cdt1 expression and cause DNA rereplication, which then activates the cell cycle checkpoint and DNA damage repair and thus impairs early embryo development.

Background: Homologous recombination deficiency (HRD) in cancer cells can occur due to mutations (germline or sporadic), methylation or other epigenetic causes. HRD leads to a defect in the conservative, error-free DNA repair mechanism and is associated with enhanced susceptibility to DNA targeting chemotherapy. Currently functional HRD assays are not broadly available for clinical use. Many of the HRD assays used in the experimental setting require fresh frozen tissue for optimal results, or require specialized expertise to interpret the results. We evaluated an immunohistochemical (IHC) assay using formalin fixed paraffin embedded (FFPE) tissue to measure protein expression of Rad51 and geminin, a cell proliferation marker, to assess HRD in breast cancer cell line models and clinical breast cancer samples. We hypothesize that Rad51, which is involved in the later stages of HR, can serve as a functional marker of HRD. Methods: The MCF-7 human breast cancer cell line was used as a model with intact HR. Western blotting of total cell lysates from cells grown in culture was performed to confirm HR response following treatment with DNA damaging chemotherapeutic agents, cisplatin and doxorubicin. Paclitaxel, a microtubule targeting agent, was used as a negative control. Mice with MCF-7 xenograft tumors were also treated with cisplatin, or doxorubicin at two dose levels (low and high) and various time points post treatment to assess the dose and time response to HR markers. Tumors from mice treated with paclitaxel were used as a negative control. Xenograft tumors were fixed and analyzed by IHC using an antibody specific for total Rad51 and geminin expression. DNA damage was also assessed in a portion of the tumor using a pulse gel electrophoresis assay. We also analyzed FFPE breast cancer clinical samples from patients with BRCA1 mutations for Rad51 and geminin expression. Results: In MCF-7 grown in vitro, total Rad51 was elevated as soon as 4 hours following exposure to doxorubicin and cisplatin, but not in response to paclitaxel treatment. In xenograft tumors, baseline Rad51 and geminin expression were relatively high illustrating proficient HR in an actively proliferating tumor model. Rad51 expression increased post treatment with cisplatin and doxorubicin as early as 6hrs and peaked at 16-24hrs. Geminin expression correlated well with expression of Rad51 at baseline and in time response to treatment. Pulse gel electrophoresis in paired tumor samples confirmed DNA damage was occurring compared to vehicle control treated tumors. However, this technique did not show a strong dose or time response. Five breast tumors from patients with known BRCA1 mutations were stained for Rad51 and geminin expression. High geminin expression and low Rad51 expression was noted in the majority of these tumors. Conclusions: An IHC assay using FFPE tissue to measure Rad51/geminin is a promising method to assess HRD in breast cancer. Further analytical and clinical validation of this approach is ongoing. Citation Format: Chalasani P, Nagy D, Livingston RB, Weterings E, Nagle R, Singh S, Barnes M, Grogan T, Ridder R, Baker AF, Kandavel S. Evaluating Rad51/geminin protein expression as an indicator of homologous recombination deficiency in breast cancer models. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-07-07.

Achieving complete and precise genome duplication requires that each genomic segment be replicated only once per cell division cycle. Protecting large eukaryotic genomes from re-replication requires an overlapping set of molecular mechanisms that prevent the first DNA replication step, the DNA loading of MCM helicase complexes to license replication origins, after S phase begins. Previous reports have defined many such origin licensing inhibition mechanisms, but the temporal relationships among them are not clear, particularly with respect to preventing re-replication in G2 and M phases. Using a combination of mutagenesis, biochemistry, and single cell analyses in human cells, we define a new mechanism that prevents re-replication through hyperphosphorylation of the essential MCM loading protein, Cdt1. We demonstrate that Cyclin A/CDK1 can hyperphosphorylate Cdt1 to inhibit MCM re-loading in G2 phase. The mechanism of inhibition is to block Cdt1 binding to MCM independently of other known Cdt1 inactivation mechanisms such as Cdt1 degradation during S phase or Geminin binding. Moreover, our findings suggest that Cdt1 dephosphorylation at the mitosis-to-G1 phase transition re-activates Cdt1. We propose that multiple distinct, non-redundant licensing inhibition mechanisms act in a series of sequential relays through each cell cycle phase to ensure precise genome duplication., Author summary The initial step of DNA replication is loading the DNA helicase, MCM, onto DNA during the first phase of the cell division cycle. If MCM loading occurs inappropriately onto DNA that has already been replicated, then cells risk DNA re-replication, a source of endogenous DNA damage and genome instability. How mammalian cells prevent any sections of their very large genomes from re-replicating is still not fully understood. We found that the Cdt1 protein, one of the critical MCM loading factors, is inhibited specifically in late cell cycle stages through a mechanism involving protein phosphorylation. This phosphorylation prevents Cdt1 from binding MCM; when Cdt1 cannot be phosphorylated MCM is inappropriately re-loaded onto DNA and cells are prone to re-replication. When cells divide and transition into G1 phase, Cdt1 is then dephosphorylated to re-activate it for MCM loading. Based on these findings we assert that the different mechanisms that cooperate to avoid re-replication are not redundant. Instead, different cell cycle phases are dominated by different re-replication control mechanisms. These findings have implications for understanding how genomes are duplicated precisely once per cell cycle and shed light on how that process is perturbed by changes in Cdt1 levels or phosphorylation activity.

Apoptotic cell death can be initiated through the extrinsic and intrinsic signaling pathways. While cell cycle progression promotes the responsiveness to intrinsic apoptosis induced by genotoxic stress or spindle poisons, this has not yet been studied conclusively for extrinsic apoptosis. Here, we combined fluorescence-based time-lapse monitoring of cell cycle progression and cell death execution by long-term time-lapse microscopy with sampling-based mathematical modeling to study cell cycle dependency of TRAIL-induced extrinsic apoptosis in NCI-H460/geminin cells. In particular, we investigated the interaction of cell death timing and progression of cell cycle states. We not only found that TRAIL prolongs cycle progression, but in reverse also that cell cycle progression affects the kinetics of TRAIL-induced apoptosis: Cells exposed to TRAIL in G1 died significantly faster than cells stimulated in S/G2/M. The connection between cell cycle state and apoptosis progression was captured by developing a mathematical model, for which parameter estimation revealed that apoptosis progression decelerates in the second half of the cell cycle. Similar results were also obtained when studying HCT-116 cells. Our results therefore reject the null hypothesis of independence between cell cycle progression and extrinsic apoptosis and, supported by simulations and experiments of synchronized cell populations, suggest that unwanted escape from TRAIL-induced apoptosis can be reduced by enriching the fraction of cells in G1 phase. Besides novel insight into the interrelation of cell cycle progression and extrinsic apoptosis signaling kinetics, our findings are therefore also relevant for optimizing future TRAIL-based treatment strategies., Author summary TRAIL (TNF-related apoptosis-inducing ligand) induces cell death preferentially in cancer cells. Whether cell cycle progression notably affects extrinsic apoptosis has remained unclear, since systematic experimental and mathematical studies to quantitatively understand such interdependencies remained challenging. Here, we applied statistical and mechanistic modeling, linked with experimental analyses, to determine if cell cycle and apoptosis progression are interconnected. Using sample-based modeling, we demonstrate that times required to commit apoptotic cell death depend on the cell cycle position at the time of TRAIL exposure, with delays manifesting during S phase, around a time that can be defined as a point of apoptosis deceleration (PAD). Overall, cells receiving TRAIL in the G1 phase were more likely to die, providing scope to optimize TRAIL-based treatment strategies with respect to cell cycle dynamics.

Neuroblastoma derived from primitive sympathetic neural precursors is a common type of solid tumor in infants. MYCN proto-oncogene bHLH transcription factor (MYCN) amplification and 1p36 deletion are important factors associated with the poor prognosis of neuroblastoma. Expression levels of MYCN and c-MYB proto-oncogene transcription factor (c-myb) decline during the differentiation of neuroblastoma cells; E2F transcription factor 1 (E2F1) activates the MYCN promoter. However, the underlying mechanism of MYCN overexpression and amplification requires further investigation. In the present study, potential c-Myb target genes, and the effect of c-myb RNA interference (RNAi) on MYCN expression and amplification were investigated in MYCN-amplified neuroblastoma cell lines. The mRNA expression levels and MYCN gene copy number in five neuroblastoma cell lines were determined by quantitative polymerase chain reaction. In addition, variations in potential target gene expression and MYCN gene copy number between pre- and post-c-myb RNAi treatment groups in MYCN-amplified Kelly, IMR32, SIMA and MHH-NB-11 cell lines, normalized to those of non-MYCN-amplified SH-SY5Y, were examined. To determine the associations between gene expression levels and chromosomal aberrations, MYCN amplification and 1p36 alterations in interphases/metaphases were analyzed using fluorescence in situ hybridization. Statistical analyses revealed correlations between 1p36 alterations and the expression of c-myb, MYB proto-oncogene like 2 (B-myb) and cyclin dependent kinase inhibitor 1A (p21). Additionally, the results of the present study also demonstrated that c-myb may be associated with E2F1 and L3MBTL1 histone methyl-lysine binding protein (L3MBTL1) expression, and that E2F1 may contribute to MYCN, B-myb, p21 and chromatin licensing and DNA replication factor 1 (hCdt1) expression, but to the repression of geminin (GMNN). On c-myb RNAi treatment, L3MBTL1 expression was silenced, while GMNN was upregulated, indicating G(2)/M arrest. In addition, MYCN gene copy number increased following treatment with c-myb RNAi. Notably, the present study also reported a 43.545% sequence identity between upstream of MYCN and Drosophila melanogaster amplification control element 3, suggesting that expression and/or amplification mechanisms of developmentally-regulated genes may be evolutionarily conserved. In conclusion, c-myb may be associated with regulating MYCN expression and amplification. c-myb, B-myb and p21 may also serve a role against chromosome 1p aberrations. Together, it was concluded that MYCN gene is amplified during S phase, potentially via a replication-based mechanism.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus, which maintains the persistent infection of the host by intermittently reactivating from latently infected cells to produce viral progenies. While it is established that the replication and transcription activator (RTA) viral transcription factor is required for the induction of lytic viral genes for KSHV lytic reactivation, it is still unknown to what extent RTA alters the host transcriptome to promote KSHV lytic cycle and viral pathogenesis. To address this question, we performed a comprehensive time course transcriptome analysis during KSHV reactivation in B-cell lymphoma cells and determined RTA-binding sites on both the viral and host genomes, which resulted in the identification of the core RTA-induced host genes (core RIGs). We found that the majority of RTA-binding sites at core RIGs contained the canonical RBP-Jκ-binding DNA motif. Subsequently, we demonstrated the vital role of the Notch signaling transcription factor RBP-Jκ for RTA-driven rapid host gene induction, which is consistent with RBP-Jκ being essential for KSHV lytic reactivation. Importantly, many of the core RIGs encode plasma membrane proteins and key regulators of signaling pathways and cell death; however, their contribution to the lytic cycle is largely unknown. We show that the cell cycle and chromatin regulator geminin and the plasma membrane protein gamma-glutamyltransferase 6, two of the core RIGs, are required for efficient KSHV reactivation and virus production. Our results indicate that host genes that RTA rapidly and directly induces can be pivotal for driving the KSHV lytic cycle. IMPORTANCE The lytic cycle of KSHV is involved not only in the dissemination of the virus but also viral oncogenesis, in which the effect of RTA on the host transcriptome is still unclear. Using genomics approaches, we identified a core set of host genes which are rapidly and directly induced by RTA in the early phase of KSHV lytic reactivation. We found that RTA does not need viral cofactors but requires its host cofactor RBP-Jκ for inducing many of its core RIGs. Importantly, we show a critical role for two of the core RIGs in efficient lytic reactivation and replication, highlighting their significance in the KSHV lytic cycle. We propose that the unbiased identification of RTA-induced host genes can uncover potential therapeutic targets for inhibiting KSHV replication and viral pathogenesis.

The histoarchitecture and function of eye and forebrain depend on a well-controlled balance between cell proliferation and differentiation. For example, the binding of the cell cycle regulator GEMININ to CDT1, which is a part of the pre-replication complex, promotes cell differentiation. Homeodomain transcription factors SIX3 and SIX6 also interact with GEMININ of which SIX3-GEMININ interaction promotes cell proliferation, whereas the nature of SIX6-GEMININ interaction has not been studied to date. We investigated SIX3/SIX6 and GEMININ interactions using bimolecular fluorescence complementation, surface plasmon resonance and isothermal titration calorimetry. Interactions between SIX3/SIX6 and GEMININ were detected in mammalian cells in culture. The presence of the C-terminal regions of SIX3 and SIX6 proteins, but not their SIX domains or homeodomains as previously thought, were required for interaction with GEMININ. Interestingly, the disordered C- and N- terminal regions of GEMININ were involved in binding to SIX3/SIX6. The coiled-coil region of GEMININ, which is the known protein-binding domain and also interacts with CDT1, was not involved in GEMININ-SIX3/SIX6 interaction. Using SPR and ITC, SIX3 bound GEMININ with a micromolar affinity and the binding stoichiometry was 1:2 (SIX3 - GEMININ). The present study gives new insights into the binding properties of SIX proteins, especially the role of their variable and disordered C-terminal regions., Highlights • C-terminal regions of SIX3/SIX6 bind GEMININ. • GEMININ coiled-coil region is not involved in SIX3/SIX6 interaction. • C- and N-terminal regions of GEMININ bind SIX3/SIX6. • SIX3 binds GEMININ with a binding stoichiometry of 1:2.