Defining the optimal animal model for translational research using gene set enrichment analysis

The mouse is the main model organism used to study the functions of human genes because most biological processes in the mouse are highly conserved in humans. Recent reports that compared identical transcriptomic datasets of human inflammatory diseases with datasets from mouse models using traditional gene‐to‐gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. To reduce susceptibility to biased interpretation, all genes of interest for the biological question under investigation should be considered. Thus, standardized approaches for systematic data analysis are needed. We analyzed the same datasets using gene set enrichment analysis focusing on pathways assigned to inflammatory processes in either humans or mice. The analyses revealed a moderate overlap between all human and mouse datasets, with average positive and negative predictive values of 48 and 57% significant correlations. Subgroups of the septic mouse models (i.e., Staphylococcus aureus injection) correlated very well with most human studies. These findings support the applicability of targeted strategies to identify the optimal animal model and protocol to improve the success of translational research

The initiator core promoter element antagonizes repression of TATA-directed transcription by negative cofactor NC2.

International audienceCore promoter regions of protein-coding genes in metazoan genomes are structurally highly diverse and can contain several distinct core promoter elements, which direct accurate transcription initiation and determine basal promoter strength. Diversity in core promoter structure is an important aspect of transcription regulation in metazoans as it provides a basis for gene-selective function of activators and repressors. The basal activity of TATA box-containing promoters is dramatically enhanced by the initiator element (INR), which can function in concert with the TATA box in a synergistic manner. Here we report that a functional INR provides resistance to NC2 (Dr1/DRAP1), a general repressor of TATA promoters. INR-mediated resistance to NC2 is established during transcription initiation complex assembly and requires TBP-associated factors (TAFs) and TAF- and INR-dependent cofactor activity. Remarkably, the INR appears to stimulate TATA-dependent transcription similar to activators by strongly enhancing recruitment of TFIIA and TFIIB and, at the same time, by compromising NC2 binding

MicroRNA Profiling as Tool for <i>In Vitro</i> Developmental Neurotoxicity Testing: The Case of Sodium Valproate

<div><p>Studying chemical disturbances during neural differentiation of murine embryonic stem cells (mESCs) has been established as an alternative <i>in vitro</i> testing approach for the identification of developmental neurotoxicants. miRNAs represent a class of small non-coding RNA molecules involved in the regulation of neural development and ESC differentiation and specification. Thus, neural differentiation of mESCs <i>in vitro</i> allows investigating the role of miRNAs in chemical-mediated developmental toxicity. We analyzed changes in miRNome and transcriptome during neural differentiation of mESCs exposed to the developmental neurotoxicant sodium valproate (VPA). A total of 110 miRNAs and 377 mRNAs were identified differently expressed in neurally differentiating mESCs upon VPA treatment. Based on miRNA profiling we observed that VPA shifts the lineage specification from neural to myogenic differentiation (upregulation of muscle-abundant miRNAs, <i>mir-206, mir-133a</i> and <i>mir-10a</i>, and downregulation of neural-specific <i>mir-124a, mir-128</i> and <i>mir-137</i>). These findings were confirmed on the mRNA level and via immunochemistry. Particularly, the expression of myogenic regulatory factors (MRFs) as well as muscle-specific genes (<i>Actc1, calponin</i>, <i>myosin light chain, asporin, decorin</i>) were found elevated, while genes involved in neurogenesis (e.g. <i>Otx1</i>, <i>2, and Zic3, 4, 5</i>) were repressed. These results were specific for valproate treatment and―based on the following two observations―most likely due to the inhibition of histone deacetylase (HDAC) activity: (i) we did not observe any induction of muscle-specific miRNAs in neurally differentiating mESCs exposed to the unrelated developmental neurotoxicant sodium arsenite; and (ii) the expression of muscle-abundant <i>mir-206</i> and <i>mir-10a</i> was similarly increased in cells exposed to the structurally different HDAC inhibitor trichostatin A (TSA). Based on our results we conclude that miRNA expression profiling is a suitable molecular endpoint for developmental neurotoxicity. The observed lineage shift into myogenesis, where miRNAs may play an important role, could be one of the developmental neurotoxic mechanisms of VPA.</p></div

Valproate effects on viability and expression of β-III-Tubulin.

<p>The cells were induced to differentiate into neurons for 16 days under continuous substance exposure. Cell viability was estimated using CellTiterBlue assay and is shown as a percentage of solvent control (A), expression of β-III-tubulin was analyzed by flow cytometry and is shown as a percentage of solvent control for each concentration tested (B). Results represent a mean of three independent differentiation experiments ± SEM.</p

Gene expression under VPA treatment.

<p>A. RT-PCR verification of Affymetrix whole genome array data. The graph demonstrates mean of log₂ fold change in three independent differentiation processes (VPA vs. solvent control) ± SEM for nine upregulated and four downregulated mRNAs. (n = 3 independent biological replicates, t-test, *p<0.05, **p<0.01, ***p<0.001). B. Induction of expression of myogenic regulation factors (MRFs) by VPA in neural-differentiated ES cells. The graph demonstrates mean of log₂ fold change (VPA vs. solvent control) ± SEM. (n = 3 independent biological replicates, t-test, *p<0.05, **p<0.01, ***p<0.001).</p