Functional Anatomy of Polycomb and Trithorax Chromatin Landscapes in Drosophila Embryos

Polycomb group (PcG) and trithorax group (trxG) proteins are conserved chromatin factors that regulate key developmental genes throughout development. In Drosophila, PcG and trxG factors bind to regulatory DNA elements called PcG and trxG response elements (PREs and TREs). Several DNA binding proteins have been suggested to recruit PcG proteins to PREs, but the DNA sequences necessary and sufficient to define PREs are largely unknown. Here, we used chromatin immunoprecipitation (ChIP) on chip assays to map the chromosomal distribution of Drosophila PcG proteins, the N- and C-terminal fragments of the Trithorax (TRX) protein and four candidate DNA-binding factors for PcG recruitment. In addition, we mapped histone modifications associated with PcG-dependent silencing and TRX-mediated activation. PcG proteins colocalize in large regions that may be defined as polycomb domains and colocalize with recruiters to form several hundreds of putative PREs. Strikingly, the majority of PcG recruiter binding sites are associated with H3K4me3 and not with PcG binding, suggesting that recruiter proteins have a dual function in activation as well as silencing. One major discriminant between activation and silencing is the strong binding of Pleiohomeotic (PHO) to silenced regions, whereas its homolog Pleiohomeotic-like (PHOL) binds preferentially to active promoters. In addition, the C-terminal fragment of TRX (TRX-C) showed high affinity to PcG binding sites, whereas the N-terminal fragment (TRX-N) bound mainly to active promoter regions trimethylated on H3K4. Our results indicate that DNA binding proteins serve as platforms to assist PcG and trxG binding. Furthermore, several DNA sequence features discriminate between PcG- and TRX-N–bound regions, indicating that underlying DNA sequence contains critical information to drive PREs and TREs towards silencing or activation.     

Identification of novel oocyte and granulosa cell markers

Here we present novel gene expression patterns in the ovary as part of an ongoing assessment of published micro-array data from mouse oocytes and embryos. We present the expression patterns of 13 genes that had been determined by micro-array to be expressed in the mature egg, but not during subsequent preimplantation development. In-situ hybridization of sectioned ovaries revealed that these genes were expressed in one of two distinct patterns: (1) oocyte-specific or (2) expressed in both the oocyte and surrounding granulosa cells. Despite the fact that micro-array data demonstrated expression in the egg, several of these genes are expressed at low levels in the oocyte, but strongly expressed in granulosa cells. Eleven of these genes have no reported function or expression during oogenesis, indicating that this approach is a necessary step towards functional annotation of the genome. Also of note is that while some of these gene products have been well characterized in other tissues and cell types, others are relatively unstudied in the literature. Our results provide novel gene expression information that may provide insights into the molecular mechanisms of follicular recruitment, oocyte maturation and ovulation and will direct further experimentation into the role these genes play during oogenesis.     

Crystal Structure of the PIM2 Kinase in Complex with an Organoruthenium Inhibitor

Background

The serine/threonine kinase PIM2 is highly expressed in human leukemia and lymphomas and has been shown to positively regulate survival and proliferation of tumor cells. Its diverse ATP site makes PIM2 a promising target for the development of anticancer agents. To date our knowledge of catalytic domain structures of the PIM kinase family is limited to PIM1 which has been extensively studied and which shares about 50% sequence identity with PIM2.

Principal Findings

Here we determined the crystal structure of PIM2 in complex with an organoruthenium complex (inhibition in sub-nanomolar level). Due to its extraordinary shape complementarity this stable organometallic compound is a highly potent inhibitor of PIM kinases.

Significance

The structure of PIM2 revealed several differences to PIM1 which may be explored further to generate isoform selective inhibitors. It has also demonstrated how an organometallic inhibitor can be adapted to the binding site of protein kinases to generate highly potent inhibitors.

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Genetic differentiation between sun and shade habitats in populations of Lindera benzoin L.

Differences in selection patterns among habitats can alter the distribution of genetic diversity even when this is estimated with neutral markers. For plants, light is an essential resource that can influence both abiotic and biotic components of habitat. We examined genetic differentiation between sun and shade habitats in Lindera benzoin L. (Spicebush), a perennial understory shrub. Genetic diversity of 127 plants from sun and shade habitats in two populations of L. benzoin was determined using 12 polymorphic microsatellite markers. We analyzed patterns of genetic diversity using analysis of molecular variance (AMOVA), and we assessed correlation between genetic and geographic distance using Mantel tests. We found (1) low levels of differentiation among populations (F _ST = 0.028), (2) little evidence of genetic structure within populations due to isolation-by-distance, and (3) some evidence of habitat-based genetic differentiation. Specifically, the AMOVA showed a small (0.5%) but significant portion of overall variation could be explained by differences between habitats. The overall low levels of differentiation we saw were likely a result of extensive gene flow in this dioecious, bird-dispersed species.     

Toxoplasma gondii protease TgSUB1 is required for cell surface processing of micronemal adhesive complexes and efficient adhesion of tachyzoites

Host cell invasion by Toxoplasma gondii is critically dependent upon adhesive proteins secreted from the micronemes. Proteolytic trimming of microneme contents occurs rapidly after their secretion onto the parasite surface and is proposed to regulate adhesive complex activation to enhance binding to host cell receptors. However, the proteases responsible and their exact function are still unknown. In this report, we show that T. gondii tachyzoites lacking the microneme subtilisin protease TgSUB1 have a profound defect in surface processing of secreted microneme proteins. Notably parasites lack protease activity responsible for proteolytic trimming of MIC2, MIC4 and M2AP after release onto the parasite surface. Although complementation with full‐length TgSUB1 restores processing, complementation of Δsub1 parasites with TgSUB1 lacking the GPI anchor (Δsub1::ΔGPISUB1) only partially restores microneme protein processing. Loss of TgSUB1 decreases cell attachment and in vitro gliding efficiency leading to lower initial rates of invasion. Δsub1 and Δsub1::ΔGPISUB1 parasites are also less virulent in mice. Thus TgSUB1 is involved in micronemal protein processing and regulation of adhesive properties of macromolecular adhesive complexes involved in host cell invasion.     

Evaluation of sediment profile imagery as a tool for assessing water quality in Greenwich Bay,Rhode Island,USA

The Benthic Habitat Quality (BHQ) index was used to assess habitat visible in sediment profile images (SPI) following hypoxia disturbance in a shallow (<10 m) estuarine embayment in Rhode Island, USA. We tested for associations between the BHQ, SPI features and water quality over several assessment windows (1, 3, 7, 14, and 28 days prior to imaging) and at multiple dissolved oxygen (DO) thresholds (2.0 mg l^?1, 2.9 mg l^?1, and 4.8 mg l^?1). Using categorical data analysis, we established empirical relationships between hypoxia prevalence and presence/absence of biogenic features visible in SPI. Fecal pellets, tubes, feeding pits, voids, mounds, and BHQ score were good affirmative features, meaning that their presence (or score greater than 5) indicated a high probability of good water quality. However, low sensitivity to hypoxia precluded their usefulness as indicators, and was attributed to rarity in images and to factors acting on time intervals longer than those examined, e.g. long-term organic enrichment or hypoxia. Burrow structures and the apparent redox potential discontinuity (aRPD), or oxidized layer of surface sediment, were good discriminatory features, with high sensitivity and specificity for both hypoxia and normoxia. Both were strong surrogates for water quality over multiple assessment windows and DO thresholds, and had the highest overall predictive values. We conclude that SPI images can be used to widen the spatial extent of water quality monitoring efforts by utilizing the relationships between aRPD, burrows and hypoxia prevalence.     

Codon usage bias and the evolution of influenza A viruses. Codon Usage Biases of Influenza Virus

Background  

The influenza A virus is an important infectious cause of morbidity and mortality in humans and was responsible for 3 pandemics in the 20^th century. As the replication of the influenza virus is based on its host's machinery, codon usage of its viral genes might be subject to host selection pressures, especially after interspecies transmission. A better understanding of viral evolution and host adaptive responses might help control this disease.     

Real-time monitoring of the membrane-binding and insertion properties of the cholesterol-dependent cytolysin anthrolysin O from Bacillus anthracis

Bacillus anthracis has recently been shown to secrete a potently hemolytic/cytolytic protein that has been designated anthrolysin O (ALO). In this work, we initiated a study of this potential anthrax virulence factor in an effort to understand the membrane-binding properties of this protein. Recombinant anthrolysin O (rALO35-512) and two N-terminally truncated versions of ALO (rALO390-512 and rALO403-512) from B. anthracis were overproduced in Escherichia coli and purified to homogeneity. The role of cholesterol in the cytolytic activity of ALO was probed in cellular cholesterol depletion assays using mouse and human macrophage-like lines, and also Drosophila Schneider 2 cells. Challenging the macrophage cells with rALO35-512, but not rALO390-512 or rALO403-512, resulted in cell death by lysis, with this cytolysis being abolished by depletion of the membrane cholesterol. Drosophila cells, which contain ergosterol as their major membrane sterol, were resistant to rALO-mediated cytolysis. In order to determine the molecular mechanism of this resistance, the interaction of rALO with model membranes comprised of POPC alone, or with a variety of structurally similar sterols including ergosterol, was probed using Biacore. Both rALO35-512 and rALO403-512 demonstrated robust binding to model membranes composed of POPC and cholesterol, with amount of protein bound proportional to the cholesterol content. Ergosterol supported greatly reduced binding of both rALO35-512 and rALO403-512, whereas other sterols tested did not support binding. The rALO403-512--membrane interaction demonstrated an equilibrium dissociation constant (KD) in the low nanomolar range, whereas rALO35-512 exhibited complex kinetics likely due to the multiple events involved in pore formation. These results establish the pivotal role of cholesterol in the action of rALO. The biosensor method developed to measure ALO recognition of cholesterol in a membrane environment could be extended to provide a platform for the screening of inhibitors of other membrane-binding proteins and peptides.