Genetic and transcriptomic analysis of transcription factor genes in the model halophilic Archaeon: coordinate action of TbpD and TfbA

<p>Abstract</p> <p>Background</p> <p>Archaea are prokaryotic organisms with simplified versions of eukaryotic transcription systems. Genes coding for the general transcription factors TBP and TFB are present in multiple copies in several Archaea, including <it>Halobacterium </it>sp. NRC-1. Multiple TBP and TFBs have been proposed to participate in transcription of genes via recognition and recruitment of RNA polymerase to different classes of promoters.</p> <p>Results</p> <p>We attempted to knock out all six TBP and seven TFB genes in <it>Halobacterium </it>sp. NRC-1 using the <it>ura</it>3-based gene deletion system. Knockouts were obtained for six out of thirteen genes, <it>tbp</it>CDF and <it>tfb</it>ACG, indicating that they are not essential for cell viability under standard conditions. Screening of a population of 1,000 candidate mutants showed that genes which did not yield mutants contained less that 0.1% knockouts, strongly suggesting that they are essential. The transcriptomes of two mutants, Δ<it>tbp</it>D and Δ<it>tfb</it>A, were compared to the parental strain and showed coordinate down regulation of many genes. Over 500 out of 2,677 total genes were regulated in the Δ<it>tbp</it>D and Δ<it>tfb</it>A mutants with 363 regulated in both, indicating that over 10% of genes in both strains require the action of both TbpD and TfbA for normal transcription. Culturing studies on the Δ<it>tbp</it>D and Δ<it>tfb</it>A mutant strains showed them to grow more slowly than the wild-type at an elevated temperature, 49°C, and they showed reduced viability at 56°C, suggesting TbpD and TfbA are involved in the heat shock response. Alignment of TBP and TFB protein sequences suggested the expansion of the TBP gene family, especially in <it>Halobacterium </it>sp. NRC-1, and TFB gene family in representatives of five different genera of haloarchaea in which genome sequences are available.</p> <p>Conclusion</p> <p>Six of thirteen TBP and TFB genes of <it>Halobacterium </it>sp. NRC-1 are non-essential under standard growth conditions. TbpD and TfbA coordinate the expression of over 10% of the genes in the NRC-1 genome. The Δ<it>tbp</it>D and Δ<it>tfb</it>A mutant strains are temperature sensitive, possibly as a result of down regulation of heat shock genes. Sequence alignments suggest the existence of several families of TBP and TFB transcription factors in <it>Halobacterium </it>which may function in transcription of different classes of genes.</p

Experimental determination of the permeability of engineering textiles: Benchmark II

In this second international permeability benchmark, the in-plane permeability values of a carbon fabric were studied by twelve research groups worldwide. One participant also investigated the deformation of the tested carbon fabric. The aim of this work was to obtain comparable results in order to make a step toward standardization of permeability measurements. Unidirectional injections were thus conducted to determine the unsaturated in-plane permeability tensor of the fabric. Procedures used by participants were specified in the guidelines defined for this benchmark. Participants were asked to use the same values for parameters such as fiber volume fraction, injection pressure and fluid viscosity to minimize sources of scatter. The comparison of the results from each participant was encouraging. The scatter between data obtained while respecting the guidelines was below 25%. However, a higher dispersion was observed when some parameters differed from the recommendations of this exercise.The authors are grateful to J.M. Beraud from Hexcel Fabrics for his support that made possible this exercise. The contributions of J.B. Alms, N.C. Correia, S. Advani, E. Ruiz and P.C.T. Goncalves to the preparation of the guidelines document and templates are acknowledged by the participants of this benchmark.Vernet, N.; Ruiz, E.; Advani, S.; Alms, JB.; Aubert, M.; Barburski, M.; Barari, B.... (2014). Experimental determination of the permeability of engineering textiles: Benchmark II. Composites Part A: Applied Science and Manufacturing. 61:172-184. doi:10.1016/j.compositesa.2014.02.010S1721846

Methylation matters: binding of Ets-1 to the demethylated Foxp3 gene contributes to the stabilization of Foxp3 expression in regulatory T cells

The forkhead-box protein P3 (Foxp3) is a key transcription factor for the development and suppressive activity of regulatory T cells (Tregs), a T cell subset critically involved in the maintenance of self-tolerance and prevention of over-shooting immune responses. However, the transcriptional regulation of Foxp3 expression remains incompletely understood. We have previously shown that epigenetic modifications in the CpG-rich Treg-specific demethylated region (TSDR) in the Foxp3 locus are associated with stable Foxp3 expression. We now demonstrate that the methylation state of the CpG motifs within the TSDR controls its transcriptional activity rather than a Treg-specific transcription factor network. By systematically mutating every CpG motif within the TSDR, we could identify four CpG motifs, which are critically determining the transcriptional activity of the TSDR and which serve as binding sites for essential transcription factors, such as CREB/ATF and NF-κB, which have previously been shown to bind to this element. The transcription factor Ets-1 was here identified as an additional molecular player that specifically binds to the TSDR in a demethylation-dependent manner in vitro. Disruption of the Ets-1 binding sites within the TSDR drastically reduced its transcriptional enhancer activity. In addition, we found Ets-1 bound to the demethylated TSDR in ex vivo isolated Tregs, but not to the methylated TSDR in conventional CD4+ T cells. We therefore propose that Ets-1 is part of a larger protein complex, which binds to the TSDR only in its demethylated state, thereby restricting stable Foxp3 expression to the Treg lineage

The role of epigenetic dysregulation in the epidemic of allergic disease

The epidemic of allergic disease in early life is one of the clearest indicators that the developing immune system is vulnerable to modern environmental changes. A range of environmental exposures epidemiologically associated with allergic disease have been shown to have effects on the foetal immune function in pregnancy, including microbial burden, dietary changes and environmental pollutants. Preliminary studies now suggest that these early effects on immune development may be mediated epigenetically through a variety of processes that collectively modify gene expression and allergic susceptibility and that these effects are potentially heritable across generations. It is also possible that rising rates of maternal allergy, a recognised direct risk factor for infant allergic disease, may be further amplifying the effects of environmental changes. Whilst effective prevention strategies are the ultimate goal in reversing the allergy epidemic, the specific environmental drivers, target genes, and intracellular pathways and mechanisms of early life immune programming are still unclear. It is hoped that identifying genes that are differentially regulated in association with subsequent allergic disease will assist in identifying causal pathways and upstream contributing environmental factors. In this way, epigenetic paradigms are likely to provide valuable insights into how the early environment can be modified to more favourably drive immune development and reverse the allergic epidemic

Distinct regulation of IgE, IgG4 and IgA by T regulatory cells and toll-like receptors

BACKGROUND: Allergic diseases are characterized by the activation of the immune system and formation of immunoglobulin (Ig)E antibodies against normally innocuous environmental antigens, whereas IgG4 and IgA represent noninflammatory and blocking antibody isotypes. The T helper 2 (Th2) cells induce and T regulatory (Treg) cells suppress several features of allergic inflammation. Our aim was to investigate the role of allergen-specific T regulatory type 1 (Tr1) cells and CD4(+)CD25(+) Treg cells and toll-like receptors (TLRs) on IgE, IgG4 and IgA production. METHODS: Germline or productive Ig-transcripts are determined by real-time reverse transcriptase-polymerase chain reaction, secreted Igs are measured by enzyme-linked immunosorbent assay and the frequency of Ig-producing plasma cells is investigated by enzyme-linked immunosorbent spot. Circulating CD4(+)CD25(+) Treg cells and allergen-specific Tr1 cells are used. RESULTS: Both allergen-specific, interleukin-10-secreting Tr1 cells and CD4(+)CD25(+) Treg cells from healthy individuals induced IgG4 and suppressed IgE production in peripheral blood mononuclear cells and purified B-cell cultures. In contrast, induction of IgA production is independent of T-cell help and the role of Tr1 or Treg cells is very limited, whereas it was highly induced by direct B-cell activation via TLR7 and 9. CONCLUSIONS: These data suggest that T regulatory cells may contribute to the suppression of allergic diseases by suppression of IgE and induction of IgG4, whereas IgA production is enhanced by B-cell activation via TLR7 and TLR9