Unique ERalpha cistromes control cell type-specific gene regulation

Estrogens play an important role in normal physiology and in a variety of pathological states involving diverse tissues including breast and bone. The mechanism by which estrogens exert cell type- and disease-specific effects, however, remains to be explained. We have compared the gene expression profile of the MCF7 breast cancer cell line with that of the osteoblast-like cell line U2OS-ERalpha by expression microarrays. We find that fewer than 10% of the 17beta-estradiol (E2)-regulated genes are common to both cell types. We have validated this in primary calvarial osteoblasts. To dissect the mechanism underlying the cell type-specific E2 regulation of gene expression in MCF7 and U2OS-ERalpha cells, we compared the ERalpha binding sites on DNA in the two cell types by performing chromatin immunoprecipitation (ChIP) on genomic tiling arrays (ChIP-on-chip). Consistent with the distinct patterns of E2-regulated gene expression in these two cell lines, we find that the vast majority of ERalpha binding sites are also cell type specific and correlate both in position and number with cell type-specific gene regulation. Interestingly, although the forkhead factor FoxA1 plays a critical role in defining the ERalpha cistrome in MCF7 cells, it is not expressed in U2OS-ERalpha cells, and forkhead motifs are not enriched in the ERalpha cistrome in these cells. Finally, the ERalpha cistromes are correlated with cell type-specific epigenetic histone modifications. These results support a model for the cell type-specific action of E2 being driven primarily through specific ERalpha occupancy of epigenetically marked cis-regulatory regions of target genes.     

Rhythms and alternating patterns in plants as emergent properties of a model of interaction between development and functioning

BACKGROUND AND AIMS: To model plasticity of plants in their environment, a new version of the functional-structural model GREENLAB has been developed with full interactions between architecture and functioning. Emergent properties of this model were revealed by simulations, in particular the automatic generation of rhythms in plant development. Such behaviour can be observed in natural phenomena such as the appearance of fruit (cucumber or capsicum plants, for example) or branch formation in trees. METHODS: In the model, a single variable, the source-sink ratio controls different events in plant architecture. In particular, the number of fruits and branch formation are determined as increasing functions of this ratio. For some sets of well-chosen parameters of the model, the dynamical evolution of the ratio during plant growth generates rhythms. KEY RESULTS AND CONCLUSIONS: Cyclic patterns in branch formation or fruit appearance emerge without being forced by the model. The model is based on the theory of discrete dynamical systems. The mathematical formalism helps us to explain rhythm generation and to control the behaviour of the system. Rhythms can appear during both the exponential and stabilized phases of growth, but the causes are different as shown by an analytical study of the system. Simulated plant behaviours are very close to those observed on real plants. With a small number of parameters, the model gives very interesting results from a qualitative point of view. It will soon be subjected to experimental data to estimate the model parameters.     

The artiodactyl <Emphasis Type="Italic">APOBEC3</Emphasis> innate immune repertoire shows evidence for a multi-functional domain organization that existed in the ancestor of placental mammals

Background  

APOBEC3 (A3) proteins deaminate DNA cytosines and block the replication of retroviruses and retrotransposons. Each A3 gene encodes a protein with one or two conserved zinc-coordinating motifs (Z1, Z2 or Z3). The presence of one A3 gene in mice (Z2–Z3) and seven in humans, A3A-H (Z1a, Z2a-Z1b, Z2b, Z2c-Z2d, Z2e-Z2f, Z2g-Z1c, Z3), suggests extraordinary evolutionary flexibility. To gain insights into the mechanism and timing of A3 gene expansion and into the functional modularity of these genes, we analyzed the genomic sequences, expressed cDNAs and activities of the full A3 repertoire of three artiodactyl lineages: sheep, cattle and pigs.     

PR-SET7 and SUV4-20H regulate H4 lysine-20 methylation at imprinting control regions in the mouse

Imprinted genes are important in development and their allelic expression is mediated by imprinting control regions (ICRs). On their DNA-methylated allele, ICRs are marked by trimethylation at H3 Lys 9 (H3K9me3) and H4 Lys 20 (H4K20me3), similar to pericentric heterochromatin. Here, we investigate which histone methyltransferases control this methylation of histone at ICRs. We found that inactivation of SUV4-20H leads to the loss of H4K20me3 and increased levels of its substrate, H4K20me1. H4K20me1 is controlled by PR-SET7 and is detected on both parental alleles. The disruption of SUV4-20H or PR-SET7 does not affect methylation of DNA at ICRs but influences precipitation of H3K9me3, which is suggestive of a trans-histone change. Unlike at pericentric heterochromatin, however, H3K9me3 at ICRs does not depend on SUV39H. Our data show not only new similarities but also differences between ICRs and heterochromatin, both of which show constitutive maintenance of methylation of DNA in somatic cells.     

Northwest passages: conservation genetics of Arctic Island wolves

Wolves in the Canadian Arctic Archipelago face several challenges to persistence: a harsh habitat, an unstable prey base, and potentially significant anthropogenic influences. These external factors, if combined with genetic constraints common to island populations, could be particularly difficult to withstand. To determine the genetic status of Arctic Island wolves, we used 14 microsatellite loci to estimate population variation and the extent of inter-island and island-mainland gene flow. All island populations were significantly less variable than mainland wolves; although inbreeding is currently insignificant, the two least variable populations, Banks and the High Arctic (Ellesmere and Devon Islands), showed genetic signatures of recent population declines. Recovery after a bottleneck appears to result, in large part, via recolonization from other islands. These extinction-recolonization dynamics, and the degree of similarity among island wolves revealed by Bayesian clustering, suggest that Arctic Island wolves function as a metapopulation. Persistence of the metapopulation may be supported by periodic migration from mainland populations, occurring primarily through two corridors: Baffin Island in the Eastern Arctic, and Victoria Island in the Western Arctic. This gene flow could be compromised or eliminated by loss—due to climatic warming or increased human activity—of sea ice in the Northwest Passage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Modulation of the phagosome proteome by interferon-gamma

Macrophages are immune cells that function in the clearance of infectious particles. This process involves the engulfment of microbes into phagosomes where these particles are lysed and degraded. In the current study, we used a large scale quantitative proteomics approach to analyze the changes in protein abundance induced on phagosomes by interferon-gamma (IFN-gamma), an inflammatory cytokine that activates macrophages. Our analysis identified 167 IFN-gamma-modulated proteins on phagosomes of which more than 90% were up-regulated. The list of phagosomal proteins regulated by IFN-gamma includes proteins expected to alter phagosome maturation, enhance microbe degradation, trigger the macrophage immune response, and promote antigen loading on major histocompatibility complex (MHC) class I molecules. A dynamic analysis of IFN-gamma-sensitive proteins by Western blot indicated that newly formed phagosomes display a delayed proteolytic activity coupled to an increased recruitment of the MHC class I peptide-loading complex. These phagosomal conditions may favor antigen presentation by MHC class I molecules on IFN-gamma-activated macrophages.     

Family-based association of a low producing lymphotoxin-alpha allele with reduced Plasmodium falciparum parasitemia

Tumor necrosis factor (TNF)-related genes are thought to play a role in human malaria. TNF polymorphisms have been associated with severe malaria, mild malaria, and parasitemia. Lymphotoxin-alpha gene (LTA) that belongs to the TNF family is one such candidate gene. Here we report the family-based association analysis of a cis-regulatory lymphotoxin-alpha polymorphism with parasitemia in two independent populations living in Burkina Faso. Analysis of 199 subjects (34 families) living in an urban endemic area revealed the association of the low producing LTA+80A allele with reduced parasitemia. Furthermore, there was evidence of significant LTA+80-by-age and LTA+80-by-gender interactions. In another set of 318 residents (55 families) in a rural endemic area, we found both the association of the low producing LTA+80A allele with reduced parasitemia and LTA+80-by-age and LTA+80-by-gender interactions. This study suggests that LTA+80 polymorphism influences parasitemia and acts in an age- and gender-dependent manner.     

Prolonged gene silencing in hepatoma cells and primary hepatocytes after small interfering RNA delivery with biodegradable poly(beta-amino esters)

Background

Small interfering (si)RNA mediated inhibition of oncogenes or viral genes may offer great opportunities for the treatment of several diseases such as hepatocellular carcinoma and viral hepatitis. However, the development of siRNAs as therapeutic agents strongly depends on the availability of safe and effective intracellular delivery systems. Poly(β‐amino esters) (PbAEs) are, in contrast to many other cationic polymers evaluated in siRNA delivery, biodegradable into smaller, nontoxic molecules.

Methods and Results

We show for the first time that PbAE : siRNA complexes, containing 1,4‐butanediol (PbAE1) or 1,6‐hexanediol (PbAE2) diacrylate‐based polymers, induced efficient gene silencing in both hepatoma cells and primary hepatocytes without causing significant cytotoxicity. Furthermore, carriers that slowly release the siRNA into the cytoplasm and hence induce a prolonged gene silencing are of major clinical interest, especially in fast dividing tumour cells. Therefore, we also studied the duration of gene silencing in the hepatoma cells and found that it was maintained for at least 5 days after siRNA delivery with PbAE2, the polymer with the slowest degradation kinetics.

Conclusions

From the time‐dependent cellular distribution of these PbAE : siRNA complexes, we suggest that the slowly degrading PbAE2 causes a sustained endosomal release of siRNA during a much longer period than PbAE1. This may support the hypothesis that the endosomal release mechanism of PbAE : siRNA complexes is based on an increase of osmotic pressure in the endosomal vesicles after polymer hydrolysis. In conclusion, our results show that both PbAEs, and especially PbAE2, open up new perspectives for the development of efficient biodegradable siRNA carriers suitable for clinical applications. Copyright © 2008 John Wiley & Sons, Ltd.     

Reassessing the role of a 3'-UTR-binding translational inhibitor in regulation of circadian bioluminescence rhythm in the dinoflagellate Gonyaulax

The nightly bioluminescence of the dinoflagellate Gonyaulax is a circadian rhythm caused by the presence in cells of specialized bioluminescent organelles, termed scintillons, containing the reaction catalyst luciferase, the substrate luciferin and a luciferin-binding protein (LBP). LBP levels increase at the start of the night phase because of increased protein synthesis rates in vivo, and this regulation has been ascribed to circadian binding of an inhibitory protein factor binding to the 3' untranslated region (UTR) of lbp mRNA at times when LBP is not normally synthesized. To purify and characterize the binding factor, the electrophoretic mobility shift assays and UV crosslinking experiments used to first characterize the factor were repeated. However, neither these protocols nor binding to biotinylated RNA probes confirmed the presence of a specific circadian RNA-binding protein. Furthermore, neither RNA probe screening of a cDNA library expressed in bacteria nor three-hybrid assays in yeast were successful in isolating a cDNA encoding a protein able to bind specifically to the lbp 3'UTR. Taken together, these results suggest that alternative mechanisms for regulating lbp translation should now be examined.     

The exopolysaccharide of Rhizobium sp. YAS34 is not necessary for biofilm formation on Arabidopsis thaliana and Brassica napus roots but contributes to root colonization

Microbial exopolysaccharides (EPSs) play key roles in plant–microbe interactions, such as biofilm formation on plant roots and legume nodulation by rhizobia. Here, we focused on the function of an EPS produced by Rhizobium sp. YAS34 in the colonization and biofilm formation on non-legume plant roots ( Arabidopsis thaliana and Brassica napus ). Using random transposon mutagenesis, we isolated an EPS-deficient mutant of strain YAS34 impaired in a glycosyltransferase gene ( gta ). Wild type and mutant strains were tagged with a plasmid-born GFP and, for the first time, the EPS produced by the wild-type strain was seen in the rhizosphere using selective carbohydrate probing with a fluorescent lectin and confocal laser-scanning microscopy. We show for the fist time that Rhizobium forms biofilms on roots of non-legumes, independently of the EPS synthesis. When produced by strain YAS34 wild type, EPS is targeted at specific parts of the plant root system. Nutrient fluctuations, root exudates and bacterial growth phase can account for such a production pattern. The EPS synthesis in Rhizobium sp. YAS34 is not essential for biofilm formation on roots, but is critical to colonization of the basal part of the root system and increasing the stability of root-adhering soil. Thus, in Rhizobium sp. YAS34 and non-legume interactions, microbial EPS is implicated in root–soil interface, root colonization, but not in biofilm formation.