The Schizosaccharomyces pombe JmjC-Protein,Msc1, Prevents H2A.Z Localization in Centromeric and Subtelomeric Chromatin Domains

Eukaryotic genomes are repetitively packaged into chromatin by nucleosomes, however they are regulated by the differences between nucleosomes, which establish various chromatin states. Local chromatin cues direct the inheritance and propagation of chromatin status via self-reinforcing epigenetic mechanisms. Replication-independent histone exchange could potentially perturb chromatin status if histone exchange chaperones, such as Swr1C, loaded histone variants into wrong sites. Here we show that in Schizosaccharomyces pombe, like Saccharomyces cerevisiae, Swr1C is required for loading H2A.Z into specific sites, including the promoters of lowly expressed genes. However S. pombe Swr1C has an extra subunit, Msc1, which is a JumonjiC-domain protein of the Lid/Jarid1 family. Deletion of Msc1 did not disrupt the S. pombe Swr1C or its ability to bind and load H2A.Z into euchromatin, however H2A.Z was ectopically found in the inner centromere and in subtelomeric chromatin. Normally this subtelomeric region not only lacks H2A.Z but also shows uniformly lower levels of H3K4me2, H4K5, and K12 acetylation than euchromatin and disproportionately contains the most lowly expressed genes during vegetative growth, including many meiotic-specific genes. Genes within and adjacent to subtelomeric chromatin become overexpressed in the absence of either Msc1, Swr1, or paradoxically H2A.Z itself. We also show that H2A.Z is N-terminally acetylated before, and lysine acetylated after, loading into chromatin and that it physically associates with the Nap1 histone chaperone. However, we find a negative correlation between the genomic distributions of H2A.Z and Nap1/Hrp1/Hrp3, suggesting that the Nap1 chaperones remove H2A.Z from chromatin. These data describe H2A.Z action in S. pombe and identify a new mode of chromatin surveillance and maintenance based on negative regulation of histone variant misincorporation.     

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites,Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval body wall contains several cell types that are models to study cell and tissue morphogenesis. For example tracheae are used to examine tube morphogenesis¹, and the dendritic arborization (DA) sensory neurons of the Drosophila larva have become a primary system for the elucidation of general and neuron-class-specific mechanisms of dendritic differentiation^2-5 and degeneration⁶.The shape of dendrite branches can vary significantly between neuron classes, and even among different branches of a single neuron^7,8. Genetic studies in DA neurons suggest that differential cytoskeletal organization can underlie morphological differences in dendritic branch shape^4,9-11. We provide a robust immunological labeling method to assay in vivo microtubule organization in DA sensory neuron dendrite arbor (Figures 1, 2, Movie 1). This protocol illustrates the dissection and immunostaining of first instar larva, a stage when active sensory neuron dendrite outgrowth and branching organization is occurring ^12,13.In addition to staining sensory neurons, this method achieves robust labeling of microtubule organization in muscles (Movies 2, 3), trachea (Figure 3, Movie 3), and other body wall tissues. It is valuable for investigators wishing to analyze microtubule organization in situ in the body wall when investigating mechanisms that control tissue and cell shape.Download video file.^{(47M, mov)}     

Inactivation of Bcl-2 through IκB kinase (IKK)-dependent phosphorylation mediates apoptosis upon exposure to 4-hydroxynonenal (HNE)

Apoptosis of macrophage foam cells loaded with modified/oxidized lipids is implicated in destabilization of advanced atherosclerotic plaques in humans. Concentration of HNE, main aldehydic product of plasma LDL peroxidation, elevates in atherosclerotic lesions as well as in cultured cells under oxidative stress. Although this reactive aldehyde has been shown to promote apoptosis with the involvement of p38 MAPK and JNK in various mammalian cell lines, roles of B-cell lymphoma 2 (Bcl-2) family proteins remain to be deciphered. We demonstrated that HNE-induced apoptosis was accompanied by concurrent downregulations of antiapoptotic Bcl-x(L) and Mcl-1 as well as upregulation of proapoptotic Bak. Furthermore, phoshorylation of Bcl-2 at Thr56, Ser70, and probably more phosphorylation sites located on N-terminal loop domain associated with HNE-induced apoptosis in both U937 and HeLa cells while ectopic expression of a phospho-defective Bcl-2 mutant significantly attenuated apoptosis. In parallel to this, HNE treatment caused release of proapoptotic Bax from Bcl-2. Pharmacological inhbition of IKK inhibited HNE-induced Bcl-2 phosphorylation. Similarly, silencing IKKα and -β both ended up with abrogation of Bcl-2 phosphorylation along with attenuation of apoptosis. Moreover, both IKKα and -β coimmunoprecipitated with Bcl-2 and in vitro kinase assay proved the ability of IKK to phosphorylate Bcl-2. In view of these findings and considering HNE inhibits DNA-binding activity of nuclear factor-κB (NF-κB) through prevention of IκB phosphorylation/ubiquitination/proteolysis, IKK appears to directly interfere with Bcl-2 activity through phosphorylation in HNE-mediated apoptosis independent of NF-κB signaling.     

Uroplakin Peptide-Specific Autoimmunity Initiates Interstitial Cystitis/Painful Bladder Syndrome in Mice

The pathophysiology of interstitial cystitis/painful bladder syndrome (IC/PBS) is enigmatic. Autoimmunity and impaired urothelium might lead the underlying pathology. A major shortcoming in IC/PBS research has been the lack of an appropriate animal model. In this study, we show that the bladder specific uroplakin 3A-derived immunogenic peptide UPK3A 65–84, which contains the binding motif for IA^d MHC class II molecules expressed in BALB/c mice, is capable of inducing experimental autoimmune cystitis in female mice of that strain. A highly antigen-specific recall proliferative response of lymph node cells to UPK3A 65–84 was observed, characterized by selectively activated CD4+ T cells with a proinflammatory Th1-like phenotype, including enhanced production of interferon γ and interleukin-2. T cell infiltration of the bladder and bladder-specific increased gene expression of inflammatory cytokines were observed. Either active immunization with UPK3A 65–84 or adoptive transfer of peptide-activated CD4+ T cells induced all of the predominant IC/PBS phenotypic characteristics, including increased micturition frequency, decreased urine output per micturition, and increased pelvic pain responses to stimulation with von Frey filaments. Our study demonstrates the creation of a more specific experimental autoimmune cystitis model that is the first inducible model for IC/PBS that manifests all of the major symptoms of this debilitating condition.