The CNS microenvironment promotes leukemia cell survival by disrupting tumor suppression and cell cycle regulation in pediatric T-cell acute lymphoblastic leukemia.

A major obstacle in improving survival in pediatric T-cell acute lymphoblastic leukemia is understanding how to predict and treat leukemia relapse in the CNS. Leukemia cells are capable of infiltrating and residing within the CNS, primarily the leptomeninges, where they interact with the microenvironment and remain sheltered from systemic treatment. These cells can survive in the CNS, by hijacking the microenvironment and disrupting normal functions, thus promoting malignant transformation. While the protective effects of the bone marrow niche have been widely studied, the mechanisms behind leukemia infiltration into the CNS and the role of the CNS niche in leukemia cell survival remain unknown. We identified a dysregulated gene expression profile in CNS infiltrated T-ALL and CNS relapse, promoting cell survival, chemoresistance, and disease progression. Furthermore, we discovered that interactions between leukemia cells and human meningeal cells induced epigenetic alterations, such as changes in histone modifications, including H3K36me3 levels. These findings are a step towards understanding the molecular mechanisms promoting leukemia cell survival in the CNS microenvironment. Our results highlight genetic and epigenetic alterations induced by interactions between leukemia cells and the CNS niche, which could potentially be utilized as biomarkers to predict CNS infiltration and CNS relapse.

Live Detection of Neural Progenitors and Glioblastoma Cells by an Oligothiophene Derivative.

There is an urgent need for simple and non-invasive identification of live neural stem/progenitor cells (NSPCs) in the developing and adult brain as well as in disease, such as in brain tumors, due to the potential clinical importance in prognosis, diagnosis, and treatment of diseases of the nervous system. Here, we report a luminescent conjugated oligothiophene (LCO), named p-HTMI, for non-invasive and non-amplified real-time detection of live human patient-derived glioblastoma (GBM) stem cell-like cells and NSPCs. While p-HTMI stained only a small fraction of other cell types investigated, the mere addition of p-HTMI to the cell culture resulted in efficient detection of NSPCs or GBM cells from rodents and humans within minutes. p-HTMI is functionalized with a methylated imidazole moiety resembling the side chain of histidine/histamine, and non-methylated analogues were not functional. Cell sorting experiments of human GBM cells demonstrated that p-HTMI labeled the same cell population as CD271, a proposed marker for stem cell-like cells and rapidly migrating cells in glioblastoma. Our results suggest that the LCO p-HTMI is a versatile tool for immediate and selective detection of neural and glioma stem and progenitor cells.

Irradiation and lithium treatment alter the global DNA methylation pattern and gene expression underlying a shift from gliogenesis towards neurogenesis in human neural progenitors.

Central nervous system (CNS) tumors account for almost a third of pediatric cancers and are the largest contributor to cancer-related death in children. Cranial radiation therapy (CRT) is, often in combination with chemotherapy and surgery, effective in the treatment of high-grade childhood brain cancers, but it has been associated with late complications in 50-90% of survivors, such as decline in cognition and mood, decreased social competence, and fatigue. A leading hypothesis to explain the decline in cognition, at least partially, is injury to the neural stem and progenitor cells (NSPCs), which leads to apoptosis and altered fate choice, favoring gliogenesis over neurogenesis. Hence, treatments harnessing neurogenesis are of great relevance in this context. Lithium, a well-known mood stabilizer, has neuroprotective and antitumor effects and has been found to reverse irradiation-induced damage in rodents, at least in part by regulating the expression of the glutamate decarboxylase 2 gene (Gad2) via promoter demethylation in rat NSPCs. Additionally, lithium was shown to rescue irradiation-induced cognitive defects in mice. Here, we show that irradiation (IR) alone or in combination with lithium chloride (LiCl) caused major changes in gene expression and global DNA methylation in iPSC-derived human NSPCs (hNSPCs) compared to untreated cells, as well as LiCl-only-treated cells. The pattern of DNA methylation changes after IR-treatment alone was stochastic and observed across many different gene groups, whereas differences in DNA methylation after LiCl-treatment of irradiated cells were more directed to specific promoters of genes, including genes associated with neurogenesis, for example GAD2. Interestingly, IR and IR + LiCl treatment affected the promoter methylation and expression of several genes encoding factors involved in BMP signaling, including the BMP antagonist gremlin1. We propose that lithium in addition to promoting neuronal differentiation, also represses glial differentiation in hNSPCs with DNA methylation regulation being a key mechanism of action.

Lithium treatment reverses irradiation-induced changes in rodent neural progenitors and rescues cognition.

Cranial radiotherapy in children has detrimental effects on cognition, mood, and social competence in young cancer survivors. Treatments harnessing hippocampal neurogenesis are currently of great relevance in this context. Lithium, a well-known mood stabilizer, has both neuroprotective, pro-neurogenic as well as antitumor effects, and in the current study we introduced lithium treatment 4 weeks after irradiation. Female mice received a single 4 Gy whole-brain radiation dose on postnatal day (PND) 21 and were randomized to 0.24% Li2CO3 chow or normal chow from PND 49 to 77. Hippocampal neurogenesis was assessed on PND 77, 91, and 105. We found that lithium treatment had a pro-proliferative effect on neural progenitors, but neuronal integration occurred only after it was discontinued. Also, the treatment ameliorated deficits in spatial learning and memory retention observed in irradiated mice. Gene expression profiling and DNA methylation analysis identified two novel factors related to the observed effects, Tppp, associated with microtubule stabilization, and GAD2/65, associated with neuronal signaling. Our results show that lithium treatment reverses irradiation-induced loss of hippocampal neurogenesis and cognitive impairment even when introduced long after the injury. We propose that lithium treatment should be intermittent in order to first make neural progenitors proliferate and then, upon discontinuation, allow them to differentiate. Our findings suggest that pharmacological treatment of cognitive so-called late effects in childhood cancer survivors is possible.

Nuclear Receptor Binding Protein 2 Is Downregulated in Medulloblastoma, and Reduces Tumor Cell Survival upon Overexpression.

Pseudokinases, comprising 10% of the human kinome, are emerging as regulators of canonical kinases and their functions are starting to be defined. We previously identified the pseudokinase Nuclear Receptor Binding Protein 2 (NRBP2) in a screen for genes regulated during neural differentiation. During mouse brain development, NRBP2 is expressed in the cerebellum, and in the adult brain, mainly confined to specific neuronal populations. To study the role of NRBP2 in brain tumors, we stained a brain tumor tissue array for NRPB2, and find its expression to be low, or absent, in a majority of the tumors. This includes medulloblastoma (MB), a pediatric tumor of the cerebellum. Using database mining of published MB data sets, we also find that NRBP2 is expressed at a lower level in MB than in the normal cerebellum. Recent studies indicate that MB exhibits frequent epigenetic alternations and we therefore treated MB cell lines with drugs inhibiting DNA methylation or histone deacetylation, which leads to an upregulation of NRBP2 mRNA expression, showing that it is under epigenetic regulation in cultured MB cells. Furthermore, forced overexpression of NRBP2 in MB cell lines causes a dramatic decrease in cell numbers, increased cell death, impaired cell migration and inhibited cell invasion in vitro. Taken together, our data indicate that downregulation of NRBP2 may be a feature by which MB cells escape growth regulation.

Stochastic nanoroughness modulates neuron-astrocyte interactions and function via mechanosensing cation channels.

Extracellular soluble signals are known to play a critical role in maintaining neuronal function and homeostasis in the CNS. However, the CNS is also composed of extracellular matrix macromolecules and glia support cells, and the contribution of the physical attributes of these components in maintenance and regulation of neuronal function is not well understood. Because these components possess well-defined topography, we theorize a role for topography in neuronal development and we demonstrate that survival and function of hippocampal neurons and differentiation of telencephalic neural stem cells is modulated by nanoroughness. At roughnesses corresponding to that of healthy astrocytes, hippocampal neurons dissociated and survived independent from astrocytes and showed superior functional traits (increased polarity and calcium flux). Furthermore, telencephalic neural stem cells differentiated into neurons even under exogenous signals that favor astrocytic differentiation. The decoupling of neurons from astrocytes seemed to be triggered by changes to astrocyte apical-surface topography in response to nanoroughness. Blocking signaling through mechanosensing cation channels using GsMTx4 negated the ability of neurons to sense the nanoroughness and promoted decoupling of neurons from astrocytes, thus providing direct evidence for the role of nanotopography in neuron-astrocyte interactions. We extrapolate the role of topography to neurodegenerative conditions and show that regions of amyloid plaque buildup in brain tissue of Alzheimer's patients are accompanied by detrimental changes in tissue roughness. These findings suggest a role for astrocyte and ECM-induced topographical changes in neuronal pathologies and provide new insights for developing therapeutic targets and engineering of neural biomaterials.

NCoR controls glioblastoma tumor cell characteristics.

BACKGROUND: We have previously shown that the transcriptional coregulator NCoR represses astrocytic differentiation of neural stem cells, suggesting that NCoR could be a plausible target for differentiation therapy of glioblastoma. METHODS: To study a putative role for NCoR in regulating glioblastoma cell characteristics, we used RNA-mediated knockdown followed by analysis of gene expression, proliferation and cell growth, autophagy, invasiveness in vitro, and tumor formation in vitro and in vivo. We further performed chromatin immunoprecipitation of NCoR followed by genome-wide sequencing in the human glioblastoma cell line U87 in order to reveal NCoR-occupied loci. RESULTS: RNA knockdown of NCoR resulted in a moderate increase in differentiation accompanied by a significant decrease in proliferation in adherent U87 human glioblastoma cells. chromatin immunoprecipitation sequencing approach revealed alternative mechanisms underlying the decrease in proliferation, as NCoR was enriched at promoters of genes associated with autophagy such as ULK3. Indeed, signs of an autophagy response in adherent glioblastoma cells included an increased expression of autophagy genes, such as Beclin1, and increased lipidation and nuclear puncta of LC3. Intriguingly, in parallel to the effects in the adherent cells, NCoR knockdown resulted in a significant increase in anchorage-independent growth, and this glioblastoma cell population showed dramatic increases in invasive properties in vitro and tumor formation capacity in vitro and in vivo along with an increased proliferation rate. CONCLUSION: Our results unveil unexpected aspects of NCoR regulation of tumor characteristics in glioblastoma cells and highlight the need for caution when transposing developmental concepts directly to cancer therapy.

The histone H4 lysine 16 acetyltransferase hMOF regulates the outcome of autophagy.

Autophagy is an evolutionarily conserved catabolic process involved in several physiological and pathological processes. Although primarily cytoprotective, autophagy can also contribute to cell death; it is thus important to understand what distinguishes the life or death decision in autophagic cells. Here we report that induction of autophagy is coupled to reduction of histone H4 lysine 16 acetylation (H4K16ac) through downregulation of the histone acetyltransferase hMOF (also called KAT8 or MYST1), and demonstrate that this histone modification regulates the outcome of autophagy. At a genome-wide level, we find that H4K16 deacetylation is associated predominantly with the downregulation of autophagy-related genes. Antagonizing H4K16ac downregulation upon autophagy induction results in the promotion of cell death. Our findings establish that alteration in a specific histone post-translational modification during autophagy affects the transcriptional regulation of autophagy-related genes and initiates a regulatory feedback loop, which serves as a key determinant of survival versus death responses upon autophagy induction.

Like a rolling histone: epigenetic regulation of neural stem cells and brain development by factors controlling histone acetylation and methylation.

BACKGROUND: The development of the nervous system is a highly organized process involving the precise and coordinated timing of many complex events. These events require proper expression of genes promoting survival, differentiation, and maturation, but also repression of alternative cell fates and restriction of cell-type-specific gene expression. SCOPE OF THE REVIEW: As the enzymes mediating post-translational histone acetylation and methylation are regulating higher order chromatin structure and controlling gene transcription, knowledge of the roles for these enzymes becomes crucial for understanding neural development and disease. The widespread expression and general biological roles for chromatin-modifying factors have hampered the studies of such enzymes in neural development, but in recent years, in vivo and in vitro studies have started to shed light on the various processes these enzymes regulate. In this review we summarize the implications of chromatin-modifying enzymes in neural development, with particular emphasis on enzymes regulating histone acetylation and methylation. MAJOR CONCLUSIONS: Enzymes controlling histone acetylation and methylation are involved in the whole process of neural development, from controlling proliferation and undifferentiated, "poised", state of stem cells to promoting and inhibiting neurogenic and gliogenic pathways and neuronal survival as well as neurite outgrowth. GENERAL SIGNIFICANCE: Aberrant enzymatic activities of histone acetyl transferases, deacetylases, and demethylases have been chemically and genetically associated with neural developmental disorders and cancer. Future studies may aim at linking the genetic and developmental studies to more in-depth biochemical characterization to provide a clearer picture of how to improve the diagnosis, prognosis, and treatment of such disorders. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

Targeted intra-arterial transplantation of stem cells to the injured CNS is more effective than intravenous administration: engraftment is dependent on cell type and adhesion molecule expression.

Stem cell transplantation procedures using intraparenchymal injections cause tissue injury in addition to associated surgical risks. Intravenous cell administration give engraftment in parenchymal lesions although the method has low efficacy and specificity. In pathological conditions with inflammation, such as traumatic brain injury, there is a transient up-regulation of ICAM-1 and VCAM-1 which might provide environmental cues for migration of stem cells from blood to parenchyma. The aim of this study was to i) analyze the effect of intra-arterial administration on cellular engraftment, ii) compare engraftment and side effects between three different stem cell systems, and iii) analyze gene expression in these three systems. We performed specific intra-arterial transplantations with human mesenchymal stem cells (hMSCs), human neural progenitor cells (hNPCs), and rat neural progenitor cells (rNPCs) in a rat model of traumatic brain injury. These results were compared to the intravenous route for each cell type, respectively. Analysis of engraftment and recipient characterization was performed by immunohistochemistry. We further characterized the different types of cells by microarray and RT-qPCR analysis. Specific intra-arterial transplantations produced significantly higher engraftment compared to intravenous transplantation with hMSCs and rNPCs. No engraftment was detected after intra-arterial or intravenous administration of hNPCs. Characterization of integrin expression indicated that CD49dVCAM-1 and possibly ICAM-1 interactions through CD18 and CD11a, respectively, are important for engraftment after intravascular cell administration. No side effects, such as thromboembolic complications, were detected. When translating stem cell therapies to clinical practice, the route of transplantation and the properties of the cell lines (homing, diapedesis, and migration) become important. This study supports the use of selective intra-arterial transplantation for improving engraftment after traumatic brain injury. In addition, we conclude that careful analysis of cells intended for local, intra-arterial transplantation with respect to integrin expression is important.

Tracheobronchial transplantation with a stem-cell-seeded bioartificial nanocomposite: a proof-of-concept study.

BACKGROUND: Tracheal tumours can be surgically resected but most are an inoperable size at the time of diagnosis; therefore, new therapeutic options are needed. We report the clinical transplantation of the tracheobronchial airway with a stem-cell-seeded bioartificial nanocomposite. METHODS: A 36-year-old male patient, previously treated with debulking surgery and radiation therapy, presented with recurrent primary cancer of the distal trachea and main bronchi. After complete tumour resection, the airway was replaced with a tailored bioartificial nanocomposite previously seeded with autologous bone-marrow mononuclear cells via a bioreactor for 36 h. Postoperative granulocyte colony-stimulating factor filgrastim (10 µg/kg) and epoetin beta (40,000 UI) were given over 14 days. We undertook flow cytometry, scanning electron microscopy, confocal microscopy epigenetics, multiplex, miRNA, and gene expression analyses. FINDINGS: We noted an extracellular matrix-like coating and proliferating cells including a CD105+ subpopulation in the scaffold after the reseeding and bioreactor process. There were no major complications, and the patient was asymptomatic and tumour free 5 months after transplantation. The bioartificial nanocomposite has patent anastomoses, lined with a vascularised neomucosa, and was partly covered by nearly healthy epithelium. Postoperatively, we detected a mobilisation of peripheral cells displaying increased mesenchymal stromal cell phenotype, and upregulation of epoetin receptors, antiapoptotic genes, and miR-34 and miR-449 biomarkers. These findings, together with increased levels of regenerative-associated plasma factors, strongly suggest stem-cell homing and cell-mediated wound repair, extracellular matrix remodelling, and neovascularisation of the graft. INTERPRETATION: Tailor-made bioartificial scaffolds can be used to replace complex airway defects. The bioreactor reseeding process and pharmacological-induced site-specific and graft-specific regeneration and tissue protection are key factors for successful clinical outcome. FUNDING: European Commission, Knut and Alice Wallenberg Foundation, Swedish Research Council, StratRegen, Vinnova Foundation, Radiumhemmet, Clinigene EU Network of Excellence, Swedish Cancer Society, Centre for Biosciences (The Live Cell imaging Unit), and UCL Business.

Notch induces cyclin-D1-dependent proliferation during a specific temporal window of neural differentiation in ES cells.

The Notch signaling pathway controls cell fate choices at multiple steps during cell lineage progression. To produce the cell fate choice appropriate for a particular stage in the cell lineage, Notch signaling needs to interpret the cell context information for each stage and convert it into the appropriate cell fate instruction. The molecular basis for this temporal context-dependent Notch signaling output is poorly understood, and to study this, we have engineered a mouse embryonic stem (ES) cell line, in which short pulses of activated Notch can be produced at different stages of in vitro neural differentiation. Activation of Notch signaling for 6h specifically at day 3 during neural induction in the ES cells led to significantly enhanced cell proliferation, accompanied by Notch-mediated activation of cyclin D1 expression. A reduction of cyclin-D1-expressing cells in the developing CNS of Notch signaling-deficient mouse embryos was also observed. Expression of a dominant negative form of cyclin D1 in the ES cells abrogated the Notch-induced proliferative response, and, conversely, a constitutively active form of cyclin D1 mimicked the effect of Notch on cell proliferation. In conclusion, the data define a novel temporal context-dependent function of Notch and a critical role for cyclin D1 in the Notch-induced proliferation in ES cells.

Red wine triggers cell death and thioredoxin reductase inhibition: effects beyond resveratrol and SIRT1.

Red wine contains antioxidants and is at moderate amounts believed to exert certain positive health effects. Resveratrol is one of the most studied antioxidants in red wine and has been suggested to activate the longevity- and metabolism-associated histone deacetylase SIRT1. Here we show that relatively low concentrations of resveratrol (0.5-3 microM) specifically inhibited neuronal differentiation of neural stem cells in a SIRT1-dependent manner whereas higher concentrations of resveratrol (> or =10 microM) induced a SIRT1-independent cell death. Surprisingly, using a cell based assay, we found that small amounts of red wine (1-5% v/v)--but not white wine--induced a massive and rapid cell death of various cell types, including neural stem cells and several cancer cell lines. This red wine-induced cell death was ethanol-, SIRT1- and resveratrol-independent but associated with increased oxidative stress and inhibition of thioredoxin reductase (TrxR) activity. The TrxR inhibition correlated with the red color (absorbance at 520 nm) of the wines demonstrating that pigment components of red wine can exert profound cellular effects. Our results unveil important roles for SIRT1 and TrxR in resveratrol and red wine-mediated effects on progenitor and cancer cells, and demonstrate that cellular responses to red wine may be more complex than generally appreciated.

Protein kinase C-dependent phosphorylation regulates the cell cycle-inhibitory function of the p73 carboxy terminus transactivation domain.

The transcription factor p73, a member of the p53 family of proteins, is involved in the regulation of cell cycle progression and apoptosis. However, the regulatory mechanisms controlling the distinct roles for p73 in these two processes have remained unclear. Here, we report that p73 is able to induce cell cycle arrest independently of its amino-terminal transactivation domain, whereas this domain is crucial for p73 proapoptotic functions. We also characterized a second transactivation domain in the carboxy terminus of p73 within amino acid residues 381 to 399. This carboxy terminus transactivation domain was found to preferentially regulate genes involved in cell cycle progression. Moreover, its activity is regulated throughout the cell cycle and modified by protein kinase C-dependent phosphorylation at serine residue 388. Our results suggest that this novel posttranslational modification within the p73 carboxy terminus transactivation domain is involved in the context-specific guidance of p73 toward the selective induction of cell cycle arrest.

Subcellular receptor redistribution and enhanced microspike formation by a Ret receptor preferentially recruiting Dok.

Ret is a receptor tyrosine kinase for the GDNF family of ligands and plays important roles during nervous system development for cell proliferation, cell migration and neurite growth. Signaling initiated from intracellular tyrosine 1062, by recruitment of several different phosphotyrosine binding (PTB) proteins (i.e. Shc, Frs2 and Dok), is important for these biological effects. By a single amino acid substitution in the PTB domain binding sequence of Ret, we have rewired the receptor such that it preferentially recruits Dok (Ret(Dok+)) with little or no remaining interactions with Shc and Frs2. Ret(Dok+) displays a sustained MAP kinase activation and a loss of Akt signaling compared to Ret(WT). We show that early events after ligand stimulation of Ret(Dok+) include massive formation of fine microspikes that are believed to be priming structures for neurite growth from the cell soma. The Ret(Dok+) receptors relocated in the membrane compartment into focal clusters at the tip of the microspikes, which was associated with Cdc42 activation. These results suggest that engagement of different adaptor proteins by Ret results in very different downstream signaling and functions within neurons and that Dok recruitment leads to a rapid receptor relocation and formation of microspikes.

SMRT-mediated repression of an H3K27 demethylase in progression from neural stem cell to neuron.

A series of transcription factors critical for maintenance of the neural stem cell state have been identified, but the role of functionally important corepressors in maintenance of the neural stem cell state and early neurogenesis remains unclear. Previous studies have characterized the expression of both SMRT (also known as NCoR2, nuclear receptor co-repressor 2) and NCoR in a variety of developmental systems; however, the specific role of the SMRT corepressor in neurogenesis is still to be determined. Here we report a critical role for SMRT in forebrain development and in maintenance of the neural stem cell state. Analysis of a series of markers in SMRT-gene-deleted mice revealed the functional requirement of SMRT in the actions of both retinoic-acid-dependent and Notch-dependent forebrain development. In isolated cortical progenitor cells, SMRT was critical for preventing retinoic-acid-receptor-dependent induction of differentiation along a neuronal pathway in the absence of any ligand. Our data reveal that SMRT represses expression of the jumonji-domain containing gene JMJD3, a direct retinoic-acid-receptor target that functions as a histone H3 trimethyl K27 demethylase and which is capable of activating specific components of the neurogenic program.

Getting the right stuff: controlling neural stem cell state and fate in vivo and in vitro with biomaterials.

Stem cell therapy holds great promises in medical treatment by, e.g., replacing lost cells, re-constitute healthy cell populations and also in the use of stem cells as vehicles for factor and gene delivery. Embryonic stem cells have rightfully attracted a large interest due to their proven capacity of differentiating into any cell type in the embryo in vivo. Tissue-specific stem cells are however already in use in medical practice, and recently the first systematic medical trials involving human neural stem cell (NSC) therapy have been launched. There are yet many obstacles to overcome and procedures to improve. To ensure progress in the medical use of stem cells increased basic knowledge of the molecular mechanisms that govern stem cell characteristics is necessary. Here we provide a review of the literature on NSCs in various aspects of cell therapy, with the main focus on the potential of using biomaterials to control NSC characteristics, differentiation, and delivery. We summarize results from studies on the characteristics of endogenous and transplanted NSCs in rodent models of neurological and cancer diseases, and highlight recent advancements in polymer compatibility and applicability in regulating NSC state and fate. We suggest that the development of specially designed polymers, such as hydrogels, is a crucial issue to improve the outcome of stem cell therapy in the central nervous system.

High susceptibility of neural stem cells to methylmercury toxicity: effects on cell survival and neuronal differentiation.

Neural stem cells (NSCs) play an essential role in both the developing embryonic nervous system through to adulthood where the capacity for self-renewal may be important for normal function of the CNS, such as in learning, memory and response to injury. There has been much excitement about the possibility of transplantation of NSCs to replace damaged or lost neurones, or by recruitment of endogenous precursors. However, before the full potential of NSCs can be realized, it is essential to understand the physiological pathways that control their proliferation and differentiation, as well as the influence of extrinsic factors on these processes. In the present study we used the NSC line C17.2 and primary embryonic cortical NSCs (cNSCs) to investigate the effects of the environmental contaminant methylmercury (MeHg) on survival and differentiation of NSCs. The results show that NSCs, in particular cNSCs, are highly sensitive to MeHg. MeHg induced apoptosis in both models via Bax activation, cytochrome c translocation, and caspase and calpain activation. Remarkably, exposure to MeHg at concentrations comparable to the current developmental exposure (via cord blood) of the general population in many countries inhibited spontaneous neuronal differentiation of NSCs. Our studies also identified the intracellular pathway leading to MeHg-induced apoptosis, and indicate that NSCs are more sensitive than differentiated neurones or glia to MeHg-induced cytotoxicity. The observed effects of MeHg on NSC differentiation offer new perspectives for evaluating the biological significance of MeHg exposure at low levels.

A basic peptide within the juxtamembrane region is required for EGF receptor dimerization.

The epidermal growth factor receptor (EGFR) is fundamental for normal cell growth and organ development, but has also been implicated in various pathologies, notably tumors of epithelial origin. We have previously shown that the initial 13 amino acids (P13) within the intracellular juxtamembrane region (R645-R657) are involved in the interaction with calmodulin, thus indicating an important role for this region in EGFR function. Here we show that P13 is required for proper dimerization of the receptor. We expressed either the intracellular domain of EGFR (TKJM) or the intracellular domain lacking P13 (DeltaTKJM) in COS-7 cells that express endogenous EGFR. Only TKJM was immunoprecipitated with an antibody directed against the extracellular part of EGFR, and only TKJM was tyrosine phosphorylated by endogenous EGFR. Using SK-N-MC cells, which do not express endogenous EGFR, that were stably transfected with either wild-type EGFR or recombinant full-length EGFR lacking P13 demonstrated that P13 is required for appropriate receptor dimerization. Furthermore, mutant EGFR lacking P13 failed to be autophosphorylated. P13 is rich in basic amino acids and in silico modeling of the EGFR in conjunction with our results suggests a novel role for the juxtamembrane domain (JM) of EGFR in mediating intracellular dimerization and thus receptor kinase activation and function.

Novel isoforms of the TFIID subunit TAF4 modulate nuclear receptor-mediated transcriptional activity.

The transcription factor TFIID consists of TATA-binding protein (TBP) and TBP-associated factors (TAFs). TAFs are essential for modulation of transcriptional activity but the regulation of TAFs is complex and many important aspects remain unclear. In this study, we have identified and characterized five novel truncated forms of the TFIID subunit TAF4 (TAF(II)135). Analysis of the mouse gene structure revealed that all truncations were the results of alternative splicing and resulted in the loss of domains or parts of domains implicated in TAF4 functional interactions. Results from transcriptional assays showed that several of the TAF4 isoforms exerted dominant negative effects on TAF4 activity in nuclear receptor-mediated transcriptional activation. In addition, alternative TAF4 isoforms could be detected in specific cell types. Our results indicate an additional level of complexity in TAF4-mediated regulation of transcription and suggest context-specific roles for these new TAF4 isoforms in transcriptional regulation in vivo.