Feeding \u3ci\u3eDrosophila\u3c/i\u3e a biotin-deficient diet for multiple generations increases stress resistance and lifespan and alters gene expression and histone biotinylation patterns

Caloric restriction increases stress resistance and lifespan in Drosophila melanogaster and other species. The roles of individual nutrients in stress resistance and longevity are largely unknown. The vitamin biotin is a potential candidate for mediating these effects, given its known roles in stress signaling and gene regulation by epigenetic mechanisms, i.e., biotinylation of histones. Here, we tested the hypothesis that prolonged culture of Drosophila on biotin-deficient medium increases stress resistance and lifespan. Flies were fed a biotin-deficient diet for multiple generations; controls were fed a biotin-normal diet. In some experiments, a third group of flies was fed a biotin-deficient diet for 12 generations and then switched to control diets for two generations to eliminate potential effects of short-term biotin deficiency. Flies fed a biotin-deficient diet exhibited a 30% increase in lifespan. This increase was associated with enhanced resistance to the DNA-damaging agent hydroxyurea and heat stress. Also, fertility increased significantly compared with biotin-normal controls. Biotinylation of histones was barely detectable in biotin-deprived flies, suggesting that epigenetic events might have contributed to effects of biotin deprivation

Histoplasma capsulatum proteome response to decreased iron availability

<p>Abstract</p> <p>Background</p> <p>A fundamental pathogenic feature of the fungus <it>Histoplasma capsulatum </it>is its ability to evade innate and adaptive immune defenses. Once ingested by macrophages the organism is faced with several hostile environmental conditions including iron limitation. <it>H. capsulatum </it>can establish a persistent state within the macrophage. A gap in knowledge exists because the identities and number of proteins regulated by the organism under host conditions has yet to be defined. Lack of such knowledge is an important problem because until these proteins are identified it is unlikely that they can be targeted as new and innovative treatment for histoplasmosis.</p> <p>Results</p> <p>To investigate the proteomic response by <it>H. capsulatum </it>to decreasing iron availability we have created <it>H. capsulatum </it>protein/genomic databases compatible with current mass spectrometric (MS) search engines. Databases were assembled from the <it>H. capsulatum </it>G217B strain genome using gene prediction programs and expressed sequence tag (EST) libraries. Searching these databases with MS data generated from two dimensional (2D) in-gel digestions of proteins resulted in over 50% more proteins identified compared to searching the publicly available fungal databases alone. Using 2D gel electrophoresis combined with statistical analysis we discovered 42 <it>H. capsulatum </it>proteins whose abundance was significantly modulated when iron concentrations were lowered. Altered proteins were identified by mass spectrometry and database searching to be involved in glycolysis, the tricarboxylic acid cycle, lysine metabolism, protein synthesis, and one protein sequence whose function was unknown.</p> <p>Conclusion</p> <p>We have created a bioinformatics platform for <it>H. capsulatum </it>and demonstrated the utility of a proteomic approach by identifying a shift in metabolism the organism utilizes to cope with the hostile conditions provided by the host. We have shown that enzyme transcripts regulated by other fungal pathogens in response to lowering iron availability are also regulated in <it>H. capsulatum </it>at the protein level. We also identified <it>H. capsulatum </it>proteins sensitive to iron level reductions which have yet to be connected to iron availability in other pathogens. These data also indicate the complexity of the response by <it>H. capsulatum </it>to nutritional deprivation. Finally, we demonstrate the importance of a strain specific gene/protein database for <it>H. capsulatum </it>proteomic analysis.</p

Induction of Protective Immunity against Chlamydia muridarum Intravaginal Infection with a Chlamydial Glycogen Phosphorylase

We evaluated 7 C. muridarum ORFs for their ability to induce protection against chlamydial infection in a mouse intravaginal infection model. These antigens, although encoded in C. muridarum genome, are transcriptionally regulated by a cryptic plasmid that is known to contribute to C. muridarum pathogenesis. Of the 7 plasmid-regulated ORFs, the chlamydial glycogen phosphorylase or GlgP, when delivered into mice intramuscularly, induced the most pronounced protective immunity against C. muridarum intravaginal infection. The GlgP-immunized mice displayed a significant reduction in vaginal shedding of live organisms on day 14 after infection. The protection correlated well with a robust C. muridarum-specific antibody and a Th1-dominant T cell responses, which significantly reduced the severity but not overall incidence of hydrosalpinx. The GlgP-induced partial protection against upper genital tract pathology suggests that GlgP may be considered a component for a multi-subunit vaccine. These results have demonstrated that intramuscular immunization of mice with purified proteins can be used to identify vaccine antigens for preventing intravaginal infection with C. trachomatis in humans

Heterochromatin: A Rapidly Evolving Species Barrier

Recent work has shown that changes in the sequence composition of heterochromatin, or in the factors that maintain that heterochromatin, may play an important role in speciation

Heterochromatin Protein 1β (HP1β) has distinct functions and distinct nuclear distribution in pluripotent versus differentiated cells

Background: Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into every cell type and to self-renew. These characteristics correlate with a distinct nuclear architecture, epigenetic signatures enriched for active chromatin marks and hyperdynamic binding of structural chromatin proteins. Recently, several chromatin-related proteins have been shown to regulate ESC pluripotency and/or differentiation, yet the role of the major heterochromatin proteins in pluripotency is unknown. Results: Here we identify Heterochromatin Protein 1β (HP1β) as an essential protein for proper differentiation, and, unexpectedly, for the maintenance of pluripotency in ESCs. In pluripotent and differentiated cells HP1β is differentially localized and differentially associated with chromatin. Deletion of HP1β, but not HP1aα, in ESCs provokes a loss of the morphological and proliferative characteristics of embryonic pluripotent cells, reduces expression of pluripotency factors and causes aberrant differentiation. However, in differentiated cells, loss of HP1β has the opposite effect, perturbing maintenance of the differentiation state and facilitating reprogramming to an induced pluripotent state. Microscopy, biochemical fractionation and chromatin immunoprecipitation reveal a diffuse nucleoplasmic distribution, weak association with chromatin and high expression levels for HP1β in ESCs. The minor fraction of HP1β that is chromatin-bound in ESCs is enriched within exons, unlike the situation in differentiated cells, where it binds heterochromatic satellite repeats and chromocenters. Conclusions: We demonstrate an unexpected duality in the role of HP1β: it is essential in ESCs for maintaining pluripotency, while it is required for proper differentiation in differentiated cells. Thus, HP1β function both depends on, and regulates, the pluripotent state

Heterochromatin Protein 1 (HP1a) Positively Regulates Euchromatic Gene Expression through RNA Transcript Association and Interaction with hnRNPs in Drosophila

Heterochromatin Protein 1 (HP1a) is a well-known conserved protein involved in heterochromatin formation and gene silencing in different species including humans. A general model has been proposed for heterochromatin formation and epigenetic gene silencing in different species that implies an essential role for HP1a. According to the model, histone methyltransferase enzymes (HMTases) methylate the histone H3 at lysine 9 (H3K9me), creating selective binding sites for itself and the chromodomain of HP1a. This complex is thought to form a higher order chromatin state that represses gene activity. It has also been found that HP1a plays a role in telomere capping. Surprisingly, recent studies have shown that HP1a is present at many euchromatic sites along polytene chromosomes of Drosophila melanogaster, including the developmental and heat-shock-induced puffs, and that this protein can be removed from these sites by in vivo RNase treatment, thus suggesting an association of HP1a with the transcripts of many active genes. To test this suggestion, we performed an extensive screening by RIP-chip assay (RNA–immunoprecipitation on microarrays), and we found that HP1a is associated with transcripts of more than one hundred euchromatic genes. An expression analysis in HP1a mutants shows that HP1a is required for positive regulation of these genes. Cytogenetic and molecular assays show that HP1a also interacts with the well known proteins DDP1, HRB87F, and PEP, which belong to different classes of heterogeneous nuclear ribonucleoproteins (hnRNPs) involved in RNA processing. Surprisingly, we found that all these hnRNP proteins also bind heterochromatin and are dominant suppressors of position effect variegation. Together, our data show novel and unexpected functions for HP1a and hnRNPs proteins. All these proteins are in fact involved both in RNA transcript processing and in heterochromatin formation. This suggests that, in general, similar epigenetic mechanisms have a significant role on both RNA and heterochromatin metabolisms

Histone H3K9 Trimethylase Eggless Controls Germline Stem Cell Maintenance and Differentiation

Epigenetic regulation plays critical roles in the regulation of cell proliferation, fate determination, and survival. It has been shown to control self-renewal and lineage differentiation of embryonic stem cells. However, epigenetic regulation of adult stem cell function remains poorly defined. Drosophila ovarian germline stem cells (GSCs) are a productive adult stem cell system for revealing regulatory mechanisms controlling self-renewal and differentiation. In this study, we show that Eggless (Egg), a H3K9 methyltransferase in Drosophila, is required in GSCs for controlling self-renewal and in escort cells for regulating germ cell differentiation. egg mutant ovaries primarily exhibit germ cell differentiation defects in young females and gradually lose GSCs with time, indicating that Egg regulates both germ cell maintenance and differentiation. Marked mutant egg GSCs lack expression of trimethylated H3K9 (H3k9me3) and are rapidly lost from the niche, but their mutant progeny can still differentiate into 16-cell cysts, indicating that Egg is required intrinsically to control GSC self-renewal but not differentiation. Interestingly, BMP-mediated transcriptional repression of differentiation factor bam in marked egg mutant GSCs remains normal, indicating that Egg is dispensable for BMP signaling in GSCs. Normally, Bam and Bgcn interact with each other to promote GSC differentiation. Interestingly, marked double mutant egg bgcn GSCs are still lost, but their progeny are able to differentiate into 16-cell cysts though bgcn mutant GSCs normally do not differentiate, indicating that Egg intrinsically controls GSC self-renewal through repressing a Bam/Bgcn-independent pathway. Surprisingly, RNAi-mediated egg knockdown in escort cells leads to their gradual loss and a germ cell differentiation defect. The germ cell differentiation defect is at least in part attributed to an increase in BMP signaling in the germ cell differentiation niche. Therefore, this study has revealed the essential roles of histone H3K9 trimethylation in controlling stem cell maintenance and differentiation through distinct mechanisms

Windei, the Drosophila Homolog of mAM/MCAF1, Is an Essential Cofactor of the H3K9 Methyl Transferase dSETDB1/Eggless in Germ Line Development

The epigenetic regulation of gene expression by the covalent modification of histones is a fundamental mechanism required for the proper differentiation of germ line cells during development. Trimethylation of histone 3 lysine 9 (H3K9me3) leads to chromatin silencing and the formation of heterochromatin by recruitment of heterochromatin protein 1 (HP1). dSETDB1/Eggless (Egg), the ortholog of the human methyltransferase SETDB1, is the only essential H3K9 methyltransferase in Drosophila and is required for H3K9 trimethylation in the female germ line. Here we show that Windei (Wde), the Drosophila homolog of mouse mAM and human MCAF1, is an essential cofactor of Egg required for its nuclear localization and function in female germ line cells. By deletion analysis combined with coimmunoprecipitation, we have identified the protein regions in Wde and Egg that are necessary and sufficient for the interaction between the two proteins. We furthermore identified a region of Egg that gets covalently modified by SUMOylation, which may facilitate the formation of higher order chromatin-modifying complexes. Together with Egg, Wde localizes to euchromatin, is enriched on chromosome 4, and binds to the Painting of fourth (POF) protein. Our data provide the first genetic and phenotypic analysis of a mAM/MCAF1 homolog in a model organism and demonstrate its essential function in the survival of germ line cells