Michaela Schwaiger

Evolutionary conservation of the eumetazoan gene regulatory landscape

Despite considerable differences in morphology and complexity of body plans among animals, a great part of the gene set is shared among Bilateria and their basally branching sister group, the Cnidaria. This suggests that the common ancestor of eumetazoans already had a highly complex gene repertoire. At present it is therefore unclear how morphological diversification is encoded in the genome. Here we address the possibility that differences in gene regulation could contribute to the large morphological divergence between cnidarians and bilaterians. To this end, we generated the first genome-wide map of gene regulatory elements in a nonbilaterian animal, the sea anemone Nematostella vectensis. Using chromatin immunoprecipitation followed by deep sequencing of five chromatin modifications and a transcriptional cofactor, we identified over 5000 enhancers in the Nematostella genome and could validate 75% of the tested enhancers in vivo. We found that in Nematostella, but not in yeast, enhancers are characterized by the same combination of histone modifications as in bilaterians, and these enhancers preferentially target developmental regulatory genes. Surprisingly, the distribution and abundance of gene regulatory elements relative to these genes are shared between Nematostella and bilaterian model organisms. Our results suggest that complex gene regulation originated at least 600 million yr ago, predating the common ancestor of eumetazoans.

Recent advances in genomics and transcriptomics of cnidarians.

The advent of the genomic era has provided important and surprising insights into the deducted genetic composition of the common ancestor of cnidarians and bilaterians. This has changed our view of how genomes of metazoans evolve and when crucial gene families arose and diverged in animal evolution. Sequencing of several cnidarian genomes showed that cnidarians share a great part of their gene repertoire as well as genome synteny with vertebrates, with less gene losses in the anthozoan cnidarian lineage than for example in ecdysozoans like Drosophila melanogaster or Caenorhabditis elegans. The Hydra genome on the other hand has evolved more rapidly indicated by more divergent sequences, more cases of gene losses and many taxonomically restricted genes. Cnidarian genomes also contain a rich repertoire of transcription factors, including those that in bilaterian model organisms regulate the development of key bilaterian traits such as mesoderm, nervous system development and bilaterality. The sea anemone Nematostella vectensis, and possibly cnidarians in general, does not only share its complex gene repertoire with bilaterians, but also the regulation of crucial developmental regulatory genes via distal enhancer elements. In addition, epigenetic modifications on DNA and chromatin are shared among eumetazoans. This suggests that most conserved genes present in our genomes today, as well as the mechanisms guiding their expression, evolved before the divergence of cnidarians and bilaterians about 600 Myr ago.

Altered membrane rigidity via enhanced endogenous cholesterol synthesis drives cancer cell resistance to destruxins

Destruxins, secondary metabolites of entomopathogenic fungi, exert a wide variety of interesting characteristics ranging from antiviral to anticancer effects. Although their mode of action was evaluated previously, the molecular mechanisms of resistance development are unknown. Hence, we have established destruxin-resistant sublines of HCT116 colon carcinoma cells by selection with the most prevalent derivatives, destruxin (dtx)A, dtxB and dtxE. Various cell biological and molecular techniques were applied to elucidate the regulatory mechanisms underlying these acquired and highly stable destruxin resistance phenotypes. Interestingly, well-known chemoresistance-mediating ABC efflux transporters were not the major players. Instead, in dtxA- and dtxB-resistant cells a hyper-activated mevalonate pathway was uncovered resulting in increased de-novo cholesterol synthesis rates and elevated levels of lanosterol, cholesterol as well as several oxysterol metabolites. Accordingly, inhibition of the mevalonate pathway at two different steps, using either statins or zoledronic acid, significantly reduced acquired but also intrinsic destruxin resistance. Vice versa, cholesterol supplementation protected destruxin-sensitive cells against their cytotoxic activity. Additionally, an increased cell membrane adhesiveness of dtxA-resistant as compared to parental cells was detected by atomic force microscopy. This was paralleled by a dramatically reduced ionophoric capacity of dtxA in resistant cells when cultured in absence but not in presence of statins. Summarizing, our results suggest a reduced ionophoric activity of destruxins due to cholesterol-mediated plasma membrane re-organization as molecular mechanism underlying acquired destruxin resistance in human colon cancer cells. Whether this mechanism might be valid also in other cell types and organisms exposed to destruxins e.g. as bio-insecticides needs to be evaluated.

13 C-labelled yeast as internal standard for LC–MS/MS and LC high resolution MS based amino acid quantification in human plasma

HIGHLIGHTSQuantitative methods based on isotope dilution by HRMS and QQQ‐MS were developed for the analysis of underivatised amino acids in human plasma.A fully 13C labelled yeast extract provided internal standards for human plasma samples.For method validation a certified reference material for metabolites in human plasma was used.Reversed phase and hydrophilic interaction liquid chromatography were validated. ABSTRACT Extracts from isotopically labelled organisms can be a versatile source for isotopically labelled chemical compounds providing ideal internal standards in mass spectrometry based assays. In this work, the application of 13C enriched yeast (Pichia pastoris) for accurate absolute metabolite quantification in human samples was investigated. >99% 13C enriched Pichia pastoris was produced via fermentation and extracted employing established protocols. Quantitative assays based on LC‐triple quadrupole mass spectrometry (QQQ‐MS) and LC‐high resolution mass spectrometry (HRMS) were validated using the Standard Reference Material, SRM 1950 – metabolites in frozen human plasma. 14 amino acids (as given in the certificate) were quantified using separations by reversed phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC). The latter chromatographic separation provided retention and selectivity for the amino acid panel, while the studied approaches employing RPLC relied on the selectivity of the MS detection. Cross‐validation using the different MS platforms showed that in all cases the application of in‐vivo labelled standards resulted in a significant improvement of trueness and precision. LODs and LOQs ranged, regardless of the detection system and addition of internal standards, in the same order of magnitude. The linear dynamic range of the employed detection systems was enhanced at least for one order of magnitude for several analytes when the internal standards were applied.