Project description:The gasotransmitter hydrogen sulfide (H2S), produced by transsulfuration pathway, in recent years has been recognized as a biological mediator playing important role under normal conditions and in various pathologies both in eukaryotes and prokaryotes. The transsulfuration pathway (TSP) includes the conversion of homocysteine to cysteine following the breakdown of methionine. In Drosophila melanogaster and other eukaryotes H2S is produced by cystathionine B-synthase (CBS), cystathionine y-lyase (CSE), and 3-mercaptopyruvate sulftransferase (MST). In the experiments reported here we were able exploring CRISPR technique to obtain single and double deletions of these three major genes responsible for H2S production. In most cases the deletion of one studied gene does not result in the compensatory induction of two other genes responsible for H2S production. Transcriptomic studies demonstrated that the deletions of the above genes alter the genome expression to different degree with more pronounced effect exerted by deletions of CBS gene. Furthermore, the double deletion of both CBS and CSE resulted in cumulative effect on the transcription in the resulted strains. Overall, we found that the obtained deletions affected numerous genes involved in various biological pathways. Characteristically, genes involved in oxidative reduction process, stress-response genes, house-keeping genes, genes participating in olfactory and reproduction were among the most affected. Furthermore, characteristic differences in the response to the deletions of the studied genes were apparently organ specific and have clear-cut gender character. Importantly, the deletions altered global methylation and chromatin state of the experimental strains by increasing the level of inactive chromatin of the genome. Single and double deletions of the three genes responsible for the production of H2S helped to understand new aspects of biological significance of this vital physiological mediator.

Project description:BACKGROUND & AIM: Sessile serrated adenoma/polyps (SSA/Ps) are the likely culprit of ~20% colon cancers but they are molecularly poorly understood. We investigated their epigenetic phenotype using high-throughput analysis of DNA methylation and gene expression. METHODS: 17 SSA/Ps and, as a comparative group, 15 conventional adenomas (all with matched samples of normal mucosa) were prospectively collected during colonoscopy. DNA from the 64 tissues was analyzed, via bisulfite next generation sequencing, for methylation at ~2.7 million CpG sites located prevalently in gene regulatory regions spanning 80.5Mb (~2.5% of the genome). The transcriptome of these samples was also investigated using RNA sequencing. An independent series of 61 archival lesions was used for targeted verification of DNA methylation. RESULTS: Both SSA/Ps and conventional adenomas showed a profound remodelling of their methylome. Cytosine hypermethylation was more pervasive in SSA/Ps than adenomas, in terms of number of hypermethylated regions (22,147 vs 15,965, respectively) and genes (4938 vs 3443, respectively). In addition, the extent of hypermethylation in a given region was higher in SSA/Ps than adenomas. This methylation pattern of SSA/Ps was reminiscent of the CpG island methylator phenotype (CIMP) of their descendant cancers. We have called this phenotype proto-CIMP since most of the hypermethylation occurred in CpG islands and shores. SSA/Ps were also protected from a wave of demethylation that instead occurred in adenomas outside of CpG islands/shores (4288 vs 18,417 hypomethylated regions in SSAPs vs adenomas). Verification studies of six hypermethylated regions demonstrated the high potential of DNA methylation markers for predicting the diagnosis of SSA/Ps and/or adenomas. Proto-CIMP of SSA/Ps was surprisingly associated with a higher number of up- (618) than downregulated (349) genes, while adenomas showed the opposite trend (516 up- vs 712 downregulated genes). CONCLUSIONS: The epigenetic landscape of SSA/Ps differs substantially from that of conventional adenomas. This huge amount of epigenetic variation represents a rich source of promising diagnostic tools, such as novel DNA markers or histologic stainings, for the tailored management of the two most frequent colon cancer precursors.

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Highly recurrent CBS epimutations in gastric cancer CpG island methylator phenotypes and inflammation

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