A Systems Biology Approach Reveals the Role of a Novel Methyltransferase in Response to Chemical Stress and Lipid Homeostasis

Using small molecule probes to understand gene function is an attractive approach that allows functional characterization of genes that are dispensable in standard laboratory conditions and provides insight into the mode of action of these compounds. Using chemogenomic assays we previously identified yeast Crg1, an uncharacterized SAM-dependent methyltransferase, as a novel interactor of the protein phosphatase inhibitor cantharidin. In this study we used a combinatorial approach that exploits contemporary high-throughput techniques available in Saccharomyces cerevisiae combined with rigorous biological follow-up to characterize the interaction of Crg1 with cantharidin. Biochemical analysis of this enzyme followed by a systematic analysis of the interactome and lipidome of CRG1 mutants revealed that Crg1, a stress-responsive SAM-dependent methyltransferase, methylates cantharidin in vitro. Chemogenomic assays uncovered that lipid-related processes are essential for cantharidin resistance in cells sensitized by deletion of the CRG1 gene. Lipidome-wide analysis of mutants further showed that cantharidin induces alterations in glycerophospholipid and sphingolipid abundance in a Crg1-dependent manner. We propose that Crg1 is a small molecule methyltransferase important for maintaining lipid homeostasis in response to drug perturbation. This approach demonstrates the value of combining chemical genomics with other systems-based methods for characterizing proteins and elucidating previously unknown mechanisms of action of small molecule inhibitors

Genomic organization and the tissue distribution of alternatively spliced isoforms of the mouse <it>Spatial </it>gene

<p>Abstract</p> <p>Background</p> <p>The stromal component of the thymic microenvironment is critical for T lymphocyte generation. Thymocyte differentiation involves a cascade of coordinated stromal genes controlling thymocyte survival, lineage commitment and selection. The "Stromal Protein Associated with Thymii And Lymph-node" (<it>Spatial</it>) gene encodes a putative transcription factor which may be involved in T-cell development. In the testis, the <it>Spatial </it>gene is also expressed by round spermatids during spermatogenesis.</p> <p>Results</p> <p>The <it>Spatial </it>gene maps to the B3-B4 region of murine chromosome 10 corresponding to the human syntenic region 10q22.1. The mouse <it>Spatial </it>genomic DNA is organised into 10 exons and is alternatively spliced to generate two short isoforms (<it>Spatial</it>-α and -γ) and two other long isoforms (<it>Spatial</it>-δ and -ε) comprising 5 additional exons on the 3' site. Here, we report the cloning of a new short isoform, <it>Spatial</it>-β, which differs from other isoforms by an additional alternative exon of 69 bases. This new exon encodes an interesting proline-rich signature that could confer to the 34 kDa Spatial-β protein a particular function. By quantitative TaqMan RT-PCR, we have shown that the short isoforms are highly expressed in the thymus while the long isoforms are highly expressed in the testis. We further examined the inter-species conservation of <it>Spatial </it>between several mammals and identified that the protein which is rich in proline and positive amino acids, is highly conserved.</p> <p>Conclusions</p> <p>The <it>Spatial </it>gene generates at least five alternative spliced variants: three short isoforms (<it>Spatial</it>-α, -β and -γ) highly expressed in the thymus and two long isoforms (<it>Spatial</it>-δ and -ε) highly expressed in the testis. These alternative spliced variants could have a tissue specific function.</p