Structural dynamics and epigenetic modifications of Hoxc loci along the anteroposterior body axis in developing mouse embryos

Hox genes are organized as clusters and specify regional identity along the anteroposterior body axis by sequential expression at a specific time and region during embryogenesis. However, the precise mechanisms underlying the sequential spatio-temporal, collinear expression pattern of Hox genes are not fully understood. Since epigenetic modifications such as chromatin architecture and histone modifications have become crucial mechanisms for highly coordinated gene expressions, we examined such modifications. E14.5 mouse embryos were dissected into three parts along the anteroposterior axis: brain, trunk-anterior, and trunk-posterior. Then, structural changes and epigenetic modifications were analyzed along the Hoxc cluster using chromosome conformation capture and chromatin immunoprecipitation-PCR methods. Hox non-expressing brain tissues had more compact, heterochromatin-like structures together with the strong repressive mark H3K27me3 than trunk tissues. In the trunk, however, a more loose euchromatin-like topology with a reduced amount of H3K27me3 modifications were observed along the whole cluster, regardless of their potency in gene activation. The active mark H3K4me3 was rather closely associated with the collinear expression of Hoxc genes; at trunk-anterior tissues, only 3' anterior Hoxc genes were marked by H3K4me3 upon gene activation, whereas whole Hoxc genes were marked by H3K4me3 and showed expression in trunk-posterior tissues. Altogether, these results indicated that loosening of the chromatin architecture and removing H3K27me3 were not sufficient for, but rather the concomitant acquisition of H3K4me3 drove the collinear expression of Hoxc genes.     

Expression of C4.4A, a Structural uPAR Homolog, Reflects Squamous Epithelial Differentiation in the Adult Mouse and during Embryogenesis

The glycosylphosphatidylinositol (GPI)–anchored C4.4A was originally identified as a metastasis-associated protein by differential screening of rat pancreatic carcinoma cell lines. C4.4A is accordingly expressed in various human carcinoma lesions. Although C4.4A is a structural homolog of the urokinase receptor (uPAR), which is implicated in cancer invasion and metastasis, no function has so far been assigned to C4.4A. To assist future studies on its function in both physiological and pathophysiological conditions, the present study provide a global survey on C4.4A expression in the normal mouse by a comprehensive immunohistochemical mapping. This task was accomplished by staining paraffin-embedded tissues with a specific rabbit polyclonal anti-C4.4A antibody. In the adult mouse, C4.4A was predominantly expressed in the suprabasal layers of the squamous epithelia of the oral cavity, esophagus, non-glandular portion of the rodent stomach, anus, vagina, cornea, and skin. This epithelial confinement was particularly evident from the abrupt termination of C4.4A expression at the squamo-columnar transition zones found at the ano-rectal and utero-vaginal junctions, for example. During mouse embryogenesis, C4.4A expression first appears in the developing squamous epithelium at embryonic day 13.5. This anatomical location of C4.4A is thus concordant with a possible functional role in early differentiation of stratified squamous epithelia.     

Real-Time RT-PCR on SAG1 and BAG1 Gene Expression during Stage Conversion in Immunosuppressed Mice Infected with Toxoplasma gondii Tehran Strain

Toxoplasmic encephalitis is caused by reactivation of bradyzoites to rapidly dividing tachyzoites of the apicomplexan parasite Toxoplasma gondii in immunocompromised hosts. Diagnosis of this life-threatening disease is problematic, because it is difficult to discriminate between these 2 stages. Toxoplasma PCR assays using gDNA as a template have been unable to discriminate between an increase or decrease in SAG1 and BAG1 expression between the active tachyzoite stage and the latent bradyzoite stage. In the present study, real-time RT-PCR assay was used to detect the expression of bradyzoite (BAG1)- and tachyzoite-specific genes (SAG1) during bradyzoite/tachyzoite stage conversion in mice infected with T. gondii Tehran strain after dexamethasone sodium phosphate (DXM) administration. The conversion reaction was observed in the lungs and brain tissues of experimental mice, indicated by SAG1 expression at day 6 after DXM administration, and continued until day 14. Bradyzoites were also detected in both organs throughout the study; however, it decreased at day 14 significantly. It is suggested that during the reactivation period, bradyzoites not only escape from the cysts and reinvade neighboring cells as tachyzoites, but also converted to new bradyzoites. In summary, the real-time RT-PCR assay provided a reliable, fast, and quantitative way of detecting T. gondii reactivation in an animal model. Thus, this method may be useful for diagnosing stage conversion in clinical specimens of immunocompromised patients (HIV or transplant patients) for early identification of tachyzoite-bradyzoite stage conversion.     

SuperPartitions: detection and classification of orthologs

The proper detection of orthologs is crucial for evolutionary studies of genes and species. Despite large efforts to solve this problem the methodological situation appears unsettled to a large extent and the “quest for orthologs” is still an ongoing task in large-scale genome comparisons.Here, we introduce a simple operational framework for the detection of orthologs and their classification. The operational framework relies on well-established principles, optimizing their implementation for the considered purposes, and chaining components in coherent procedures: 1) We take advantage of the efficiency and simplicity of the Reciprocal Best Hit (RBH) detections, remedying (by design) the drawback concerning the limitations in terms of 1:1 detections. The procedure is based on the partitioning of Reciprocal Best Hits, with the further merging of partitions including members of the same paralogous classes (“SuperPartition of Orthologs” (SPOs)). 2) We then resort to the conservation profiles of the obtained clusters, allowing simple detection of SPOs containing duplicated members. Based on accepted evolutionary principles, such members can be further tagged as in-paralogs (co-orthologs) or out-paralogs.The method is illustrated and validated by extensive genomic analyses. The performances of the overall approach are characterized in global terms for three sets of species (Chlamydiae, Mycobacteria, Aspergilli), showing that at least 75% of the sets of orthologs contain at most one protein from a given species. The sets including more than one protein from a given species are shown to contain in-paralogs in proportions varying from 28% to 58%. The characterizations also show that the large majority of SPOs are associated with ancestral motifs, and accordingly not prone to chaining effects that might be triggered by multi-domain proteins. Further the SPO formulation is compared to other similarity based ortholog detection methods. Beyond core common results, significant differences are observed between various methods, which can be accounted for to a large extent on conceptual grounds, relative to the different merging schemes involved. Such comparisons highlight a major advantage of the SPO approach concerning the proper clustering of associated paralogs, which appear to be often dispatched spuriously into distinct orthologous classes.Finally the perspectives for future applications and elaborations of SPO-based compositional analyses are discussed.