BBLN-1 is essential for intermediate filament organization and apical membrane morphology.

Epithelial tubes are essential components of metazoan organ systems that control the flow of fluids and the exchange of materials between body compartments and the outside environment. The size and shape of the central lumen confer important characteristics to tubular organs and need to be carefully controlled. Here, we identify the small coiled-coil protein BBLN-1 as a regulator of lumen morphology in the C. elegans intestine. Loss of BBLN-1 causes the formation of bubble-shaped invaginations of the apical membrane into the cytoplasm of intestinal cells and abnormal aggregation of the subapical intermediate filament (IF) network. BBLN-1 interacts with IF proteins and localizes to the IF network in an IF-dependent manner. The appearance of invaginations is a result of the abnormal IF aggregation, indicating a direct role for the IF network in maintaining lumen homeostasis. Finally, we identify bublin (BBLN) as the mammalian ortholog of BBLN-1. When expressed in the C. elegans intestine, BBLN recapitulates the localization pattern of BBLN-1 and can compensate for the loss of BBLN-1 in early larvae. In mouse intestinal organoids, BBLN localizes subapically, together with the IF protein keratin 8. Our results therefore may have implications for understanding the role of IFs in regulating epithelial tube morphology in mammals.

Host Vesicle Fusion Protein VAPB Contributes to the Nuclear Egress Stage of Herpes Simplex Virus Type-1 (HSV-1) Replication.

The primary envelopment/de-envelopment of Herpes viruses during nuclear exit is poorly understood. In Herpes simplex virus type-1 (HSV-1), proteins pUL31 and pUL34 are critical, while pUS3 and some others contribute; however, efficient membrane fusion may require additional host proteins. We postulated that vesicle fusion proteins present in the nuclear envelope might facilitate primary envelopment and/or de-envelopment fusion with the outer nuclear membrane. Indeed, a subpopulation of vesicle-associated membrane protein-associated protein B (VAPB), a known vesicle trafficking protein, was present in the nuclear membrane co-locating with pUL34. VAPB knockdown significantly reduced both cell-associated and supernatant virus titers. Moreover, VAPB depletion reduced cytoplasmic accumulation of virus particles and increased levels of nuclear encapsidated viral DNA. These results suggest that VAPB is an important player in the exit of primary enveloped HSV-1 virions from the nucleus. Importantly, VAPB knockdown did not alter pUL34, calnexin or GM-130 localization during infection, arguing against an indirect effect of VAPB on cellular vesicles and trafficking. Immunogold-labelling electron microscopy confirmed VAPB presence in nuclear membranes and moreover associated with primary enveloped HSV-1 particles. These data suggest that VAPB could be a cellular component of a complex that facilitates UL31/UL34/US3-mediated HSV-1 nuclear egress.

Modeling oncogenic translocations: distinct roles for double-strand break repair pathways in translocation formation in mammalian cells.

Reciprocal chromosomal translocations are implicated in the etiology of many tumors, including leukemias, lymphomas, and sarcomas. DNA double-strand breaks (DSBs) caused by various cellular processes and exogenous agents are thought to be responsible for the generation of most translocations. Mammalian cells have multiple pathways for repairing DSBs in the chromosomes: non-homologous end-joining (NHEJ), homologous recombination (HR), and single-strand annealing (SSA), which is a specialized pathway involving sequence repeats. In this review, we summarize the various reporters that have been used to examine the potential for each of these DSB repair pathways to mediate translocation formation in mammalian cells. This approach has demonstrated that NHEJ is very proficient at mediating translocation formation, while HR is not because of crossover suppression. Although SSA can efficiently mediate translocations between identical repeats, its contribution to translocation formation is likely very limited because of sequence divergence between repetitive elements in the genome.

Identification of the neurotransmitters involved in modulation of transmitter release from the central terminals of the locust wing hinge stretch receptor.

The flight motor system of the locust represents a model preparation for the investigation of neuromodulation. At the wing hinges are stretch receptors important in generating and controlling the flight motor pattern. The forewing stretch receptor (fSR) makes direct cholinergic synapses with depressor motor neurons (MN) controlling that wing, including the first basalar MN (BA1). The fSR/BA1 synapse is modulated by muscarinic cholinergic receptors located on gamma-aminobutyric acid (GABA)-ergic interneurons (Judge and Leitch [1999a] J. Comp. Neurol. 407:103-114; Judge and Leitch [1999b] J. Neurobiol. 40:420-431). However, electrophysiology has shown that fSR/BA is also modulated by biogenic amines (Leitch et al. [2003] J. Comp. Neurol. 462:55-70). We have used electron microscopic immunocytochemistry (ICC) to identify the neurotransmitters in neurons presynaptic to the fSR and to determine the relative proportion of these different classes of modulatory inputs. Approximately 55% of all inputs to the fSR are glutamate-IR, indicating that glutamatergic neurons may also play an important role in presynaptically modulating the fSR terminals. Anti-GABA ICC confirmed that over 40% of inputs to the fSR are GABA-IR (Judge and Leitch [1999a] J. Comp. Neurol. 407:103-114). Labelling sections with an antioctopamine antibody revealed neurons containing distinctive large, electron-dense granules, which could reliably be used to identify them. Aminergic neurons that modulate the synapse may have very few morphologically recognizable synaptic outputs. Although putative octopaminergic processes were found in close contact to horseradish peroxidase-filled fSR profiles, no morphologically recognizable synaptic inputs to the fSR were evident. Collectively, these data suggest that most inputs to the fSR are from either glutamatergic or GABAergic neurons.

Phenotype of cerebellar glutamatergic neurons is altered in stargazer mutant mice lacking brain-derived neurotrophic factor mRNA expression.

Brain-derived neurotrophic factor (BDNF) influences neuronal survival, differentiation, and maturation. More recently, its role in synapse formation and plasticity has also emerged. In the cerebellum of the spontaneous recessive mutant mouse stargazer (stg) there is a specific and pronounced deficit in BDNF mRNA expression. BDNF protein levels in the cerebellum as a whole are reduced by 70%, while in the granule cells (GCs) there is a selective and near total reduction in BDNF mRNA expression. Recently, we published data demonstrating that inhibitory neurons in the cerebella of stgs have significantly reduced levels (approximately 50%) of gamma-aminobutyric acid (GABA) and fewer, smaller inhibitory synapses compared to wildtype (WT) controls. Our current investigations indicate that the stargazer mutation has an even more pronounced effect on the phenotype of glutamatergic neurons in the cerebellum. There is a profound decrease in the levels of glutamate-immunoreactivity (up to 77%) in stg compared to WT controls. The distribution profile of presynaptic vesicles is also markedly different: stgs have proportionally fewer docked vesicles and fewer vesicles located adjacent to the active zone ready to dock than WTs. Furthermore, the thickness of the postsynaptic density (PSD) at mossy fiber-granule cell (MF-GC) and parallel fiber-Purkinje cell (PF-PC) synapses is severely reduced (up to 33% less than WT controls). The number and length of excitatory synapses, however, appear to be relatively unchanged. It is possible that at least some of theses changes in phenotype are directly attributable to the lack of BDNF in the cerebellum of the stg mutant.

Cerebellar Golgi, Purkinje, and basket cells have reduced gamma-aminobutyric acid immunoreactivity in stargazer mutant mice.

The stargazer mutant mouse has characteristic ataxia and head-tossing traits coupled with a severe impairment in the acquisition of classical eye-blink conditioning (Qiao et al. [1996] J. Neurosci. 16:640-648; Qiao et al. [ 1998] J. Neurosci. 18:6990-6999). These phenotypes are thought to be cerebellar mediated and have been attributed to the specific reduction in brain-derived neurotrophic factor (BDNF). The granule cells in the cerebellum of the stargazer mouse exhibit a near-total and exclusive ablation of BDNF mRNA expression and a consequent defect in TrkB receptor signalling. To investigate whether the stargazer mutation and lack of availability of BDNF in the granule cells compromise the phenotype of the cerebellar inhibitory neurons, specifically their immunoreactivity for gamma-aminobutyric acid (GABA); the levels of GABA neurotransmitter expressed in Golgi, Purkinje, and basket cells; and the density of their synaptic contacts were compared in stargazer and wild-type controls using electron microscopy and quantitative immunogold labelling. The data presented in this study clearly show that, in the spontaneous ataxic mutant mouse stargazer, the cerebellar inhibitory neurons have significantly reduced levels of GABA immunoreactivity indicative of a significant decrease in their GABA content compared with wild-type controls. Furthermore, the density of inhibitory synapses between Golgi interneurons and granule cells and also between basket and Purkinje cells in stargazer mutants is reduced to approximately half that in wild-type controls. Whether this reduction in GABA content and inhibitory synapse density is directly attributable to the lack of BDNF in the cerebellum of the stargazer mutant is yet to be proved.

Complex developmental patterns of histone modifications associated with the human beta-globin switch in primary cells.

OBJECTIVE: The regulation of the beta-globin switch remains undetermined, and understanding this mechanism has important benefits for clinical and basic science. Histone modifications regulate gene expression and this study determines the presence of three important histone modifications across the beta-globin locus in erythroblasts with different beta-like globin-expression profiles. Understanding the chromatin associated with weak gamma gene expression in bone marrow cells is an important objective, with the goal of ultimately inducing postnatal expression of weak gamma-globin to cure beta-hemoglobinopathies. MATERIALS AND METHODS: These studies use uncultured primary fetal and bone marrow erythroblasts and human embryonic stem cell-derived primitive-like erythroblasts. Chromatin immunoprecipitation with antibodies against modified histones reveals DNA associated with such histones. Precipitated DNA is quantitated by real-time polymerase chain reaction for 40 sites across the locus. RESULTS: Distribution of histone modifications differs at each developmental stage. The most highly expressed genes at each stage are embedded within large domains of modifications associated with expression (acetylated histone H3 [H3ac] and dimethyl lysine 4 of histone H3 [H3K4me2]). Moderately expressed genes have H3ac and H3K4me2 in the immediate area around the gene. Dimethyl lysine 9 of histone H3 (H3K9me2), a mark associated with gene suppression, is present at the epsilon and gamma genes in bone marrow cells, suggesting active suppression of these genes. CONCLUSION: This study reveals complex patterns of histone modifications associated with highly expressed, moderately expressed, and unexpressed genes. Activation of gamma postnatally will likely require extensive modification of the histones in a large domain around the gamma genes.

Developmentally regulated extended domains of DNA hypomethylation encompass highly transcribed genes of the human beta-globin locus.

OBJECTIVE: DNA methylation has long been implicated in developmental beta-globin gene regulation. However, the mechanism underlying this regulation is unclear, especially because these genes do not contain CpG islands. This has led us to propose and test the hypothesis that, just as for histone modifications, developmentally specific changes in human beta-like globin gene expression are associated with long-range changes in DNA methylation. MATERIALS AND METHODS: Bisulfite sequencing was used to determine the methylation state of individual CpG dinucleotides across the beta-globin locus in uncultured primary human erythroblasts from fetal liver and bone marrow, and in primitive-like erythroid cells derived from human embryonic stem cells. RESULTS: beta-globin locus CpGs are generally highly methylated, but domains of DNA hypomethylation spanning thousands of base pairs are established around the most highly expressed genes during each developmental stage. These large domains of DNA hypomethylation are found within domains of histone modifications associated with gene expression. We also find hypomethylation of a small proportion of gamma-globin promoters in adult erythroid cells, suggesting a mechanism by which adult erythroid cells produce fetal hemoglobin. CONCLUSION: This is one of the first reports to show that changes in DNA methylation patterns across large domains around non-CpG island genes correspond with changes in developmentally regulated histone modifications and gene expression. These data support a new model in which extended domains of DNA hypomethylation and active histone marks are coordinately established to achieve developmentally specific gene expression of non-CpG island genes.

Neither DNA hypomethylation nor changes in the kinetics of erythroid differentiation explain 5-azacytidine's ability to induce human fetal hemoglobin.

5-azacytidine (5-Aza) is a potent inducer of fetal hemoglobin (HbF) in people with beta-thalassemia and sickle cell disease. Two models have been proposed to explain this activity. The first is based on the drug's ability to inhibit global DNA methylation, including the fetal globin genes, resulting in their activation. The second is based on 5-Aza's cytotoxicity and observations that HbF production is enhanced during marrow recovery. We tested these models using human primary cells in an in vitro erythroid differentiation system. We found that doses of 5-Aza that produce near maximal induction of gamma-globin mRNA and HbF do not alter cell growth, differentiation kinetics, or cell cycle, but do cause a localized demethylation of the gamma promoter. However, when we reduced gamma promoter methylation to levels equivalent to those seen with 5-Aza or to the lower levels seen in primary fetal erythroid cells using DNMT1 siRNA and shRNA, we observed no induction of gamma-globin mRNA or HbF. These results suggest that 5-Aza induction of HbF is not the result of global DNA demethylation or of changes in differentiation kinetics, but involves an alternative, previously unrecognized mechanism. Other results suggest that posttranscriptional regulation plays an important role in the 5-Aza response.

Developmental- and differentiation-specific patterns of human gamma- and beta-globin promoter DNA methylation.

The mechanisms underlying the human fetal-to-adult beta-globin gene switch remain to be determined. While there is substantial experimental evidence to suggest that promoter DNA methylation is involved in this process, most data come from studies in nonhuman systems. We have evaluated human gamma- and beta-globin promoter methylation in primary human fetal liver (FL) and adult bone marrow (ABM) erythroid cells. Our results show that, in general, promoter methylation and gene expression are inversely related. However, CpGs at -162 of the gamma promoter and -126 of the beta promoter are hypomethylated in ABM and FL, respectively. We also studied gamma-globin promoter methylation during in vitro differentiation of erythroid cells. The gamma promoters are initially hypermethylated in CD34(+) cells. The upstream gamma promoter CpGs become hypomethylated during the preerythroid phase of differentiation and are then remethylated later, during erythropoiesis. The period of promoter hypomethylation correlates with transient gamma-globin gene expression and may explain the previously observed fetal hemoglobin production that occurs during early adult erythropoiesis. These results provide the first comprehensive survey of developmental changes in human gamma- and beta-globin promoter methylation and support the hypothesis that promoter methylation plays a role in human beta-globin locus gene switching.

Ikaros increases normal apoptosis in adult erythroid cells.

Ikaros is a critical transcriptional regulator of hematopoietic cell differentiation. In addition to its effects on the lymphoid system and hematopoietic stem-cell compartment, we have previously shown that Ikaros is also required for normal erythroid development. In this report, we compare Ikaros-dependent gene expression in erythroid cells of mice lacking the Ikaros protein with that of normal mice in purified adult bone-marrow erythroid cells (BMRC). Gene expression, measured by Affymetrix microarray analysis, indicates that in the BMRC of Ikaros-null mice, there is significant up-regulation of SMADs 6 and 7, serine protease inhibitor 3, and immediate-early protein 3 (IER3), all proteins that play a modulating role in apoptosis. We investigate the role of Ikaros in oxidative stress-induced apoptosis using Annexin-V staining and FACS analysis. We find a decrease in apoptosis in the BMRC of Ikaros-null mice compared to normal mice. This effect is also seen in nonerythroid cells but is stronger in BMRC. We conclude that normal Ikaros function increases normal apoptosis in erythroid cells. The data also suggest that Ikaros plays a role in apoptosis-mediated events in other normal hematopoietic cell lineages.

Up-regulation of AMP-activated kinase by dysfunctional cystic fibrosis transmembrane conductance regulator in cystic fibrosis airway epithelial cells mitigates excessive inflammation.

AMP-activated kinase (AMPK) is a ubiquitous metabolic sensor that inhibits the cystic fibrosis (CF) transmembrane conductance regulator (CFTR). To determine whether CFTR reciprocally regulates AMPK function in airway epithelia and whether such regulation is involved in lung inflammation, AMPK localization, expression, and activity and cellular metabolic profiles were compared as a function of CFTR status in CF and non-CF primary human bronchial epithelial (HBE) cells. As compared with non-CF HBE cells, CF cells had greater and more diffuse AMPK staining and had greater AMPK activity than their morphologically matched non-CF counterparts. The cellular [AMP]/[ATP] ratio was higher in undifferentiated than in differentiated non-CF cells, which correlated with AMPK activity under these conditions. However, this nucleotide ratio did not predict AMPK activity in differentiating CF cells. Inhibiting channel activity in non-CF cells did not affect AMPK activity or metabolic status, but expressing functional CFTR in CF cells reduced AMPK activity without affecting cellular [AMP]/[ATP]. Therefore, lack of functional CFTR expression and not loss of channel activity in CF cells appears to up-regulate AMPK activity in CF HBE cells, presumably through non-metabolic effects on upstream regulatory pathways. Compared with wild-type CFTR-expressing immortalized CF bronchial epithelial (CFBE) cells, DeltaF508-CFTR-expressing CFBE cells had greater AMPK activity and greater secretion of tumor necrosis factor-alpha and the interleukins IL-6 and IL-8. Further pharmacologic AMPK activation inhibited inflammatory mediator secretion in both wild type- and DeltaF508-expressing cells, suggesting that AMPK activation in CF airway cells is an adaptive response that reduces inflammation. We propose that therapies to activate AMPK in the CF airway may be beneficial in reducing excessive airway inflammation, a major cause of CF morbidity.