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Mast cells (MCs) have been identified in various tumors; however, the role of these cells in tumorigenesis remains controversial. Here, we quantified MCs in human and murine malignant pleural effusions (MPEs) and evaluated the fate and function of these cells in MPE development. Evaluation of murine MPE-competent lung and colon adenocarcinomas revealed that these tumors actively attract and subsequently degranulate MCs in the pleural space by elaborating CCL2 and osteopontin. MCs were required for effusion development, as MPEs did not form in mice lacking MCs, and pleural infusion of MCs with MPE-incompetent cells promoted MPE formation. Once homed to the pleural space, MCs released tryptase AB1 and IL-1[beta], which in turn induced pleural vasculature leakiness and triggered NF-[kappa]B activation in pleural tumor cells, thereby fostering pleural fluid accumulation and tumor growth. Evaluation of human effusions revealed that MCs are elevated in MPEs compared with benign effusions. Moreover, MC abundance correlated with MPE formation in a human cancer cell-induced effusion model. Treatment of mice with the c-KIT inhibitor imatinib mesylate limited effusion precipitation by mouse and human adenocarcinoma cells. Together, the results of this study indicate that MCs are required for MPE formation and suggest that MC-dependent effusion formation is therapeutically addressable., Introduction Inflammation was recently recognized as an enabling hallmark of cancer that may mediate tumor growth and dissemination instead of tumor eradication (1). Inflammatory signaling networks in the tumor microenvironment [...]

Chromatin licensing and DNA replication factor 1 (CDT1), a protein of the pre‐replicative complex, is essential for loading the minichromosome maintenance complex (MCM) helicases onto the origins of DNA replication. While several studies have shown that dysregulation of CDT1 expression causes re‐replication and DNA damage in cell lines, and CDT1 is highly expressed in several human cancers, whether CDT1 deregulation is sufficient to enhance tumorigenesis in vivo is currently unclear. To delineate its role in vivo, we overexpressed Cdt1 in the mouse colon and induced carcinogenesis using azoxymethane/dextran sodium sulfate (AOM/DSS). Here, we show that mice overexpressing Cdt1 develop a significantly higher number of tumors with increased tumor size, and more severe dysplastic changes (high‐grade dysplasia), compared with control mice under the same treatment. These tumors exhibited an increased growth rate, while cells overexpressing Cdt1 loaded greater amounts of Mcm2 onto chromatin, demonstrating origin overlicensing. Adenomas overexpressing Cdt1 showed activation of the DNA damage response (DDR), apoptosis, formation of micronuclei, and chromosome segregation errors, indicating that aberrant expression of Cdt1 results in increased genomic and chromosomal instability in vivo, favoring cancer development. In line with these results, high‐level expression of CDT1 in human colorectal cancer tissue specimens and colorectal cancer cell lines correlated significantly with increased origin licensing, activation of the DDR, and microsatellite instability (MSI). © 2022 The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

The neural stem cell niche is a key regulator participating in the maintenance, regeneration, and repair of the brain. Within the niche neural stem cells (NSC) generate new neurons throughout life, which is important for tissue homeostasis and brain function. NSCs are regulated by intrinsic and extrinsic factors with cellular metabolism being lately recognized as one of the most important ones, with evidence suggesting that it may serve as a common signal integrator to ensure mammalian brain homeostasis. The aim of this review is to summarize recent insights into how metabolism affects NSC fate decisions in adult neural stem cell niches, with occasional referencing of embryonic neural stem cells when it is deemed necessary. Specifically, we will highlight the implication of mitochondria as crucial regulators of NSC fate decisions and the relationship between metabolism and ependymal cells. The link between primary cilia dysfunction in the region of hypothalamus and metabolic diseases will be examined as well. Lastly, the involvement of metabolic pathways in ependymal cell ciliogenesis and physiology regulation will be discussed. [ABSTRACT FROM AUTHOR]

Gene therapy is a revolutionary, cutting-edge approach to permanently ameliorate or amend many neuromuscular diseases by targeting their genetic origins. Motor neuron diseases and muscular dystrophies, whose genetic causes are well known, are the frontiers of this research revolution. Several genetic treatments, with diverse mechanisms of action and delivery methods, have been approved during the past decade and have demonstrated remarkable results. However, despite the high number of genetic treatments studied preclinically, those that have been advanced to clinical trials are significantly fewer. The most clinically advanced treatments include adeno-associated virus gene replacement therapy, antisense oligonucleotides, and RNA interference. This review provides a comprehensive overview of the advanced gene therapies for motor neuron diseases (i.e., amyotrophic lateral sclerosis and spinal muscular atrophy) and muscular dystrophies (i.e., Duchenne muscular dystrophy, limb-girdle muscular dystrophy, and myotonic dystrophy) tested in clinical trials. Emphasis has been placed on those methods that are a few steps away from their authoritative approval. [ABSTRACT FROM AUTHOR]

DNA replication initiation requires the loading of MCM2-7 complexes at the origins of replication during G1. Replication licensing renders chromatin competent for DNA replication and its tight regulation is essential to prevent aberrant DNA replication and genomic instability. CDT1 is a critical factor of licensing and its activity is controlled by redundant mechanisms, including Geminin, a protein inhibitor of CDT1. Aberrant CDT1 and Geminin expression have been shown to promote tumorigenesis in vivo and are also evident in multiple human tumors. In this study, we developed an in vitro AlphaScreen™ high-throughput screening (HTS) assay for the identification of small-molecule inhibitors targeting the CDT1/Geminin protein complex. Biochemical characterization of the most potent compound, AF615, provided evidence of specific, dose-dependent inhibition of Geminin binding to CDT1 both in-vitro and in cells. Moreover, compound AF615 induces DNA damage, inhibits DNA synthesis and reduces viability selectively in cancer cell lines, and this effect is CDT1-dependent. Taken together, our data suggest that AF615 may serve as a useful compound to elucidate the role of CDT1/Geminin protein complex in replication licensing and origin firing as well as a scaffold for further medicinal chemistry optimisation. [ABSTRACT FROM AUTHOR]

Cell differentiation is a process that must be precisely regulated for the maintenance of tissue homeostasis. Differentiation towards a multiciliated cell fate is characterized by well‐defined stages, where a transcriptional cascade is activated leading to the formation of multiple centrioles and cilia. Centrioles migrate and dock to the apical cell surface and, acting as basal bodies, give rise to multiple motile cilia. The concerted movement of cilia ensures directional fluid flow across epithelia and defects either in their number or structure can lead to disease phenotypes. Micro‐RNAs (miRNAs; miRs) are small, non‐coding RNA molecules that play an important role in post‐transcriptional regulation of gene expression. miR‐34b/c and miR‐449a/b/c specifically function throughout the differentiation of multiciliated cells, fine‐tuning the expression of many different centriole‐ and cilia‐related genes. They strictly regulate the expression levels of genes that are required both for commitment towards the multiciliated cell fate (e.g. Notch) and for the establishment and maintenance of this fate by regulating the expression of transcription factors and structural components of the pathway. Herein we review miR‐34 and miR‐449 spatiotemporal regulation along with their roles during the different stages of multiciliogenesis. [ABSTRACT FROM AUTHOR]

In eukaryotes, DNA replication progresses through a finely orchestrated temporal and spatial program. The 3D genome structure and nuclear architecture have recently emerged as fundamental determinants of the replication program. Factors with established roles in replication have been recognized as genome organization regulators. Exploiting paradigms from yeasts and mammals, we discuss how DNA replication is regulated in time and space through DNA-associated trans -acting factors, diffusible limiting replication initiation factors, higher-order chromatin folding, dynamic origin localization, and specific nuclear microenvironments. We present an integrated model for the regulation of DNA replication in 3D and highlight the importance of accurate spatio-temporal regulation of DNA replication in physiology and disease. 3D chromatin structure, nuclear architecture, and subnuclear origin localization are critical determinants of the replication program. Opposing activities of the rate limiting Dbf4-dependent kinase and protein phosphatase 1 drive timely origin activation. Bridging proteins recruit positive and negative replication regulators and establish long-range interactions shaping origin positioning and coregulation in 3D. Activity gradients and local enrichments of diffusible initiation factors couple nuclear architecture to the replication program. Subnuclear compartments act as specialized microenvironments providing the appropriate conditions for the execution of timely DNA replication. Aberrations in the DNA replication program affect single nucleotide and copy number alterations along the genome and are linked to cancer and progeria. [ABSTRACT FROM AUTHOR]

The cullin-RING E3 ubiquitin ligase CRL4Cdt2 has emerged as a master regulator of genome stability, which targets key cell cycle proteins for proteolysis during S phase and after DNA damage. Recent advances shed light on how it couples ubiquitination to DNA synthesis, offering a new paradigm for substrate recognition: Cdt2 binds directly onto proliferating cell nuclear antigen (PCNA) loaded on DNA, which serves as a landing pad for the independent recruitment of the ubiquitin ligase and its substrates. Cyclin-dependent kinases (CDKs) and the ataxia telangiectasia and Rad3-related (ATR) kinase ensure accurate spatiotemporal regulation of CRL4Cdt2 under normal conditions and upon DNA damage. Deregulation of Cdt2 is evident in malignancies and was recently highlighted as a major target of oncogenic viruses, supporting the therapeutic targeting of the ligase as a promising anticancer strategy. CRL4Cdt2 is crucial in preserving the integrity of the genome by ubiquitinating a wide spectrum of factors, directing their proteolysis. CRL4Cdt2 has a unique mode of action: substrates and ubiquitin ligase are independently recruited onto different monomers of trimeric PCNA loaded on DNA. The tight binding of CRL4Cdt2 onto PCNADNA offers a platform for DNA synthesis-coupled ubiquitination of its transiently binding substrates. CRL4Cdt2 targets different substrates in a timely manner, thus enabling cell cycle and DNA repair processes to be completed in an ordered way. The ability of the substrate receptor Cdt2 to recognize its substrates is regulated by multiple post-translational modifications, while Cdt2 is a target of oncogenic viruses. Deregulation of CRL4Cdt2 is implicated in tumorigenesis and novel anticancer therapies are being developed against this ubiquitin ligase. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Lung adenocarcinoma (LADC) is the leading cause of cancer death worldwide. Nevertheless, syngeneic mouse models of the disease are sparse, and cell lines suitable for transplantable and immunocompetent mouse models of LADC remain unmet needs. We established multiple mouse LADC cell lines by repeatedly exposing two mouse strains (FVB, Balb/c) to the tobacco carcinogens urethane or diethylnitrosamine and by culturing out the resulting lung tumours for prolonged periods of time. Characterization of the resulting cell lines (n = 7) showed that they were immortal and phenotypically stable in vitro , and oncogenic, metastatic and lethal in vivo. The primary tumours that gave rise to the cell lines, as well as secondary tumours generated by transplantation of the cell lines, displayed typical LADC features, such as glandular architecture and mucin and thyroid transcription factor 1 expression. Moreover, these cells exhibited marked molecular similarity with human smokers' LADC, including carcinogen-specific Kras point mutations (Kras Q61R in urethane- and Kras Q61H in diethylnitrosamine-triggered cell lines) and Trp53 deletions and displayed stemness features. Interestingly, all cell lines overexpressed proliferin, a murine prolactin orthologue, which functioned as a lung tumour promoter. Furthermore, prolactin was overexpressed and portended poor prognosis in human LADC. In conclusion, we report the first LADC cell lines derived from mice exposed to tobacco carcinogens. These cells closely resemble human LADC and provide a valuable tool for the functional investigation of the pathobiology of the disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Malignant pleural effusion (MPE) is a frequent metastatic manifestation of human cancers. While we previously identified KRAS mutations as molecular culprits of MPE formation, the underlying mechanism remained unknown. Here, we determine that non-canonical IKKα-RelB pathway activation of KRAS-mutant tumor cells mediates MPE development and this is fueled by host-provided interleukin IL-1β. Indeed, IKKα is required for the MPE-competence of KRAS-mutant tumor cells by activating non-canonical NF-κB signaling. IL-1β fuels addiction of mutant KRAS to IKKα resulting in increased CXCL1 secretion that fosters MPE-associated inflammation. Importantly, IL-1β-mediated NF-κB induction in KRAS-mutant tumor cells, as well as their resulting MPE-competence, can only be blocked by co-inhibition of both KRAS and IKKα, a strategy that overcomes drug resistance to individual treatments. Hence we show that mutant KRAS facilitates IKKα-mediated responsiveness of tumor cells to host IL-1β, thereby establishing a host-to-tumor signaling circuit that culminates in inflammatory MPE development and drug resistance. [ABSTRACT FROM AUTHOR]

A bstract Molecular mechanisms governing maintenance, commitment, and differentiation of stem cells are largely unexploited. Molecules involved in the regulation of multiple cellular processes are of particular importance for stem cell physiology, as they integrate different signals and coordinate cellular decisions related with self-renewal and fate determination. Geminin has emerged as a critical factor in DNA replication and stem cell differentiation in different stem cell populations. Its inhibitory interaction with Cdt1, a member of the prereplicative complex, ensures the controlled timing of DNA replication and, consequently, genomic stability in actively proliferating cells. In embryonic as well as somatic stem cells, Geminin has been shown to interact with transcription factors and epigenetic regulators to drive gene expression programs and ultimately guide cell fate decisions. An ever-growing number of studies suggests that these interactions of Geminin and proteins regulating transcription are conserved among metazoans. Interactions between Geminin and proteins modifying the epigenome, such as members of the repressive Polycomb group and the SWI/SNF proteins of the permissive Trithorax, have long been established. The complexity of these interactions, however, is only just beginning to unravel, revealing key roles on maintaining stem cell self-renewal and fate specification. In this review, we summarize current knowledge and give new perspectives for the role of Geminin on transcriptional and epigenetic regulation, alongside with its regulatory activity in DNA replication and their implication in the regulation of stem and progenitor cell biology. S tem C ells 2017;35:299-310 [ABSTRACT FROM AUTHOR]

Background: In vertebrate organisms, the neural crest (NC) gives rise to multipotential and highly migratory progenitors which are distributed throughout the embryo and generate, among other structures, the peripheral nervous system, including the intrinsic neuroglial networks of the gut, i.e. the enteric nervous system (ENS). The majority of enteric neurons and glia originate from vagal NC-derived progenitors which invade the foregut mesenchyme and migrate rostro-caudally to colonise the entire length of the gut. Although the migratory behaviour of NC cells has been studied extensively, it remains unclear how their properties and response to microenvironment change as they navigate through complex cellular terrains to reach their target embryonic sites. Results: Using conditional gene inactivation in mice we demonstrate here that the cell cycle-dependent protein Geminin (Gem) is critical for the survival of ENS progenitors in a stage-dependent manner. Gem deletion in early ENS progenitors (prior to foregut invasion) resulted in cell-autonomous activation of DNA damage response and p53-dependent apoptosis, leading to severe intestinal aganglionosis. In contrast, ablation of Gem shortly after ENS progenitors had invaded the embryonic gut did not result in discernible survival or migratory deficits. In contrast to other developmental systems, we obtained no evidence for a role of Gem in commitment or differentiation of ENS lineages. The stage-dependent resistance of ENS progenitors to mutation-induced genotoxic stress was further supported by the enhanced survival of post gut invasion ENS lineages to γ-irradiation relative to their predecessors. Conclusions: Our experiments demonstrate that, in mammals, NC-derived ENS lineages are sensitive to genotoxic stress in a stage-specific manner. Following gut invasion, ENS progenitors are distinctly resistant to Gem ablation and irradiation in comparison to their pre-enteric counterparts. These studies suggest that the microenvironment of the embryonic gut protects ENS progenitors and their progeny from genotoxic stress. [ABSTRACT FROM AUTHOR]

GemC1, together with Idas and Geminin, an important regulator of DNA-replication licensing and differentiation decisions, constitute a superfamily sharing a homologous central coiled-coil domain. To better understand this family of proteins, the crystal structure of a GemC1 coiled-coil domain variant engineered for better solubility was determined to 2.2 Å resolution. GemC1 shows a less typical coiled coil compared with the Geminin homodimer and the Geminin-Idas heterodimer structures. It is also shown that both in vitro and in cells GemC1 interacts with Geminin through its coiled-coil domain, forming a heterodimer that is more stable that the GemC1 homodimer. Comparative analysis of the thermal stability of all of the possible superfamily complexes, using circular dichroism to follow the unfolding of the entire helix of the coiled coil, or intrinsic tryptophan fluorescence of a unique conserved N-terminal tryptophan, shows that the unfolding of the coiled coil is likely to take place from the C-terminus towards the N-terminus. It is also shown that homodimers show a single-state unfolding, while heterodimers show a two-state unfolding, suggesting that the dimer first falls apart and the helices then unfold according to the stability of each protein. The findings argue that Geminin-family members form homodimers and heterodimers between them, and this ability is likely to be important for modulating their function in cycling and differentiating cells. [ABSTRACT FROM AUTHOR]

Multiciliated cells are abundant in the epithelial surface of different tissues, including cells lining thewalls of the lateral ventricles in the brain and the airway epithelium. Their main role is to control fluid flow and defects in their differentiation are implicated in many human disorders, such as hydrocephalus, accompanied by defects in adult neurogenesis and mucociliary disorder in the airway system. Here we show that Mcidas, which is mutated in human mucociliary clearance disorder, and GemC1 (Gmnc or Lynkeas), previously implicated in cell cycle progression, are key regulators ofmulticiliated ependymal cell generation in the mouse brain. Overexpression and knockdown experiments show that Mcidas and GemC1 are sufficient and necessary for cell fate commitment and differentiation of radial glial cells to multiciliated ependymal cells. Furthermore, we show that GemC1 and Mcidas operate in hierarchical order, upstream of Foxj1 and c-Myb transcription factors, which are known regulators of ependymal cell generation, and that Notch signaling inhibits GemC1 and Mcidas function. Our results suggest that Mcidas and GemC1 are key players in the generation of multiciliated ependymal cells of the adult neurogenic niche. [ABSTRACT FROM AUTHOR]

Balancing stem cell self-renewal and initiation of lineage specification programs is essential for the development and homeostasis of the hematopoietic system. We have specifically ablated geminin in the developing murine hematopoietic system and observed profound defects in the generation of mature blood cells, leading to embryonic lethality. Hematopoietic stem cells (HSCs) accumulated in the fetal liver following geminin ablation, while committed progenitors were reduced. Genome-wide transcriptome analysis identified key HSC transcription factors as being up-regulated upon geminin deletion, revealing a gene network linked with geminin that controls fetal hematopoiesis. In order to obtain mechanistic insight into the ability of geminin to regulate transcription, we examined Hoxa9 as an example of a key gene in definitive hematopoiesis. We demonstrate that in human K562 cells geminin is associated with HOXA9 regulatory elements and its absence increases HOXA9 transcription similarly to that observed in vivo. Moreover, silencing geminin reduced recruitment of the PRC2 component SUZ12 to the HOXA9 locus and resulted in an increase in RNA polymerase II recruitment and H3K4 trimethylation (H3K4me3), whereas the repressive marks H3K9me3 and H3K27me3 were reduced. The chromatin landscape was also modified at the regulatory regions of HOXA10 and GATA1. K562 cells showed a reduced ability to differentiate to erythrocytes and megakaryocytes upon geminin silencing. Our data suggest that geminin is indispensable for fetal hematopoiesis and regulates the generation of a physiological pool of stem and progenitor cells in the fetal hematopoietic system. [ABSTRACT FROM AUTHOR]

Background: Maintenance of genome integrity is crucial for the propagation of the genetic information. Cdt1 is a major component of the pre-replicative complex, which controls once per cell cycle DNA replication. Upon DNA damage, Cdt1 is rapidly targeted for degradation. This targeting has been suggested to safeguard genomic integrity and prevent rereplication while DNA repair is in progress. Cdt1 is deregulated in tumor specimens, while its aberrant expression is linked with aneuploidy and promotes tumorigenesis in animal models. The induction of lesions in DNA is a common mechanism by which many cytotoxic anticancer agents operate, leading to cell cycle arrest and apoptosis. Methodology/Principal Finding: In the present study we examine the ability of several anticancer drugs to target Cdt1 for degradation. We show that treatment of HeLa and HepG2 cells with MMS, Cisplatin and Doxorubicin lead to rapid proteolysis of Cdt1, whereas treatment with 5-Fluorouracil and Tamoxifen leave Cdt1 expression unaffected. Etoposide affects Cdt1 stability in HepG2 cells and not in HeLa cells. RNAi experiments suggest that Cdt1 proteolysis in response to MMS depends on the presence of the sliding clamp PCNA. Conclusion/Significance: Our data suggest that treatment of tumor cells with commonly used chemotherapeutic agents induces differential responses with respect to Cdt1 proteolysis. Information on specific cellular targets in response to distinct anticancer chemotherapeutic drugs in different cancer cell types may contribute to the optimization of the efficacy of chemotherapy. [ABSTRACT FROM AUTHOR]

During cortical development, coordination of proliferation and differentiation ensures the timely generation of different neural progenitor lineages that will give rise to mature neurons and glia. Geminin is an inhibitor of DNA replication and it has been proposed to regulate cell proliferation and fate determination during neurogenesis via interactions with transcription factors and chromatin remodeling complexes. To investigate the in vivo role of Geminin in the maintenance and differentiation of cortical neural progenitors, we have generated mice that lack Geminin expression in the developing cortex. Our results show that loss of Geminin leads to the expansion of neural progenitor cells located at the ventricular and subventricular zones of the developing cortex. Early cortical progenitors lacking Geminin exhibit a longer S-phase and a reduced ability to generate early born neurons, consistent with a preference on self-renewing divisions. Overexpression of Geminin in progenitor cells of the cortex reduces the number of neural progenitor cells, promotes cell cycle exit and subsequent neuronal differentiation. Our study suggests that Geminin has an important role during cortical development in regulating progenitor number and ultimately neuron generation. S TEM C ELLS 2011;29:1269-1282 [ABSTRACT FROM AUTHOR]

Hepatocyte nuclear factor 4 (HNF-4) is a member of the nuclear-receptor gene superfamily. HNF-4 binds to response elements of several liver-enriched genes and exhibits a restricted pattern of expression, suggesting an important role for HNF-4 in tissue-specific gene regulation. Here, we report the generation of three mutated forms of the HNF-4 protein, their effects on the ability of the protein to transactivate through HNF-4—response elements, and their ability to suppress transactivation by the wild-type protein. Two mutated forms of the HNF-4 protein, one in which the DNA-binding domain has been deleted and another in which the HNF-4 proximal box has been replaced by that of the glucocorticoid receptor, behaved as inhibitors of the wild-type protein. The properties of a carboxy-terminal-deletion mutant allow us to propose a region of HNF-4 involved in transactivation. [ABSTRACT FROM AUTHOR]

The rRNA genes [ribosomal DNA (rDNA)] are organized in a prominent nuclear compartment, the nucleolus. It is now well established that the nucleolus functions beyond ribosome biosynthesis, regulating several physiological cellular responses. The nucleoli constitute dynamic genomic/nuclear hubs and demonstrate unique inherent characteristics, rendering them ideal to sense, signal, and respond to various intrinsic and environmental insults. Here, we discuss emerging findings supporting direct links between rDNA/nucleolar instability and cellular senescence/organismal aging from yeast to mammals. Moreover, we highlight evidence that nucleolar functionality and rDNA architecture impact on meiotic/transgenerational rejuvenation, thus revealing causality underlying connections between rDNA/nucleolar instability and aging. The ribosomal DNA (rDNA) consists of multiple tandem arrayed rRNA-coding genes and organizes the nucleolus, a dynamic, phase-separated subnuclear compartment where rRNA synthesis, processing, and ribosome assembly takes place. rDNA is the most intensely transcribed region of the eukaryotic genome and constitutes a phylogenetically conserved fragile site, which is inherently unstable and capable of sensing and disseminating stress signals. Recent data directly implicate rDNA/nucleolar instability with senescence onset and organismal aging from yeast to mammals. The nucleolus undergoes characteristic morphological and functional alterations early upon senescence/aging establishment, driving the senescence phenotype. Reversal of rDNA/nucleolar instability was shown to take place upon cellular/organismal rejuvenescence, highlighting causality linking rDNA/nucleolar instability with aging. [ABSTRACT FROM AUTHOR]

Strict regulation of DNA replication is of fundamental significance for the maintenance of genome stability. Licensing of origins of DNA replication is a critical event for timely genome duplication. Errors in replication licensing control lead to genomic instability across evolution. Here, we present accumulating evidence that aberrant replication licensing is linked to oncogene-induced replication stress and poses a major threat to genome stability, promoting tumorigenesis. Oncogene activation can lead to defects in where along the genome and when during the cell cycle licensing takes place, resulting in replication stress. We also discuss the potential of replication licensing as a specific target for novel anticancer therapies. Oncogene-induced replication stress is a major driver of tumorigenesis. Aberrant replication licensing is an important mediator of oncogene-induced replication stress, leading to genomic instability and malignant transformation. Novel anticancer approaches are being developed that target origin licensing to selectively eliminate cancer cells. [ABSTRACT FROM AUTHOR]

For genomic integrity to be maintained, the cell cycle and DNA damage responses must be linked. Cdt1, a G1-specific cell-cycle factor, is targeted for proteolysis by the Cul4-Ddb1Cdt2 ubiquitin ligase following DNA damage. Using a laser nanosurgery microscope to generate spatially restricted DNA damage within the living cell nucleus, we show that Cdt1 is recruited onto damaged sites in G1 phase cells, within seconds of DNA damage induction. PCNA, Cdt2, Cul4, DDB1 and p21Cip1 also accumulate rapidly to damaged sites. Cdt1 recruitment is PCNA-dependent, whereas PCNA and Cdt2 recruitment are independent of Cdt1. Fitting of fluorescence recovery after photobleaching profiles to an analytic reaction-diffusion model shows that Cdt1 and p21Cip1 exhibit highly dynamic binding at the site of damage, whereas PCNA appears immobile. Cdt2 exhibits both a rapidly exchanging and an apparently immobile subpopulation. Our data suggest that PCNA provides an immobile binding interface for dynamic Cdt1 interactions at the site of damage, which leads to rapid Cdt1 recruitment to damaged DNA, preceding Cdt1 degradation. [ABSTRACT FROM AUTHOR]

Wound healing response plays a central part in chronic inflammation, affecting millions of people worldwide. It is a dynamic process that can lead to fibrosis, if tissue damage is irreversible and wound resolution is not attained. It is clear that there is a tight interconnection among wound healing, fibrosis and a variety of chronic disease conditions, demonstrating the heterogeneity of this pathology. Based on our further understanding of the cellular and molecular mechanisms underpinning tissue repair, new therapeutic approaches have recently been developed that target different aspects of the wound healing process and fibrosis. Nevertheless, several issues still need to be taken into consideration when designing modern wound healing drug delivery formulations. In this review, we highlight novel pharmacological agents that hold promise for targeting wound repair and fibrosis. We also focus on drug-delivery systems that may enhance current and future therapies. [ABSTRACT FROM AUTHOR]

Neural crest cells comprise a multipotent, migratory cell population that generates a diverse array of cell and tissue types, during vertebrate development. Enteric Nervous System controls the function of the gastrointestinal tract and is mainly derived from the vagal and sacral neural crest cells. Deregulation on self-renewal and differentiation of the enteric neural crest cells is evident in enteric nervous system disorders, such as Hirschsprung disease, characterized by the absence of ganglia in a variable length of the distal bowel. Here we show that Geminin is essential for Enteric Nervous System generation as mice that lacked Geminin expression specifically in neural crest cells revealed decreased generation of vagal neural crest cells, and enteric neural crest cells (ENCCs). Geminin-deficient ENCCs showed increased apoptosis and decreased cell proliferation during the early stages of gut colonization. Furthermore, decreased number of committed ENCCs in vivo and the decreased self-renewal capacity of enteric progenitor cells in vitro, resulted in almost total aganglionosis resembling a severe case of Hirschsprung disease. Our results suggest that Geminin is an important regulator of self-renewal and survival of enteric nervous system progenitor cells. [ABSTRACT FROM AUTHOR]