TOR complex 2 in fission yeast is required for chromatin-mediated gene silencing and assembly of heterochromatic domains at subtelomeres.

The conserved serine/threonine protein kinase target of rapamycin (TOR) is a major regulator of eukaryotic cellular and organismal growth and a valuable target for drug therapy. TOR forms the core of two evolutionary conserved complexes, TOR complex 1 (TORC1) and TORC2. In the fission yeast Schizosaccharomyces pombe, TORC2 responds to glucose levels and, by activating the protein kinase Gad8 (an orthologue of human AKT), is required for well-regulated cell cycle progression, starvation responses, and cell survival. Here, we report that TORC2-Gad8 is also required for gene silencing and the formation of heterochromatin at the S. pombe mating-type locus and at subtelomeric regions. Deletion of TORC2-Gad8 resulted in loss of the heterochromatic modification of histone 3 lysine 9 dimethylation (H3K9me2) and an increase in euchromatic modifications, including histone 3 lysine 4 trimethylation (H3K4me3) and histone 4 lysine 16 acetylation (H4K16Ac). Accumulation of RNA polymerase II (Pol II) at subtelomeric genes in TORC2-Gad8 mutant cells indicated a defect in silencing at the transcriptional level. Moreover, a concurrent decrease in histone 4 lysine 20 dimethylation (H4K20me2) suggested elevated histone turnover. Loss of gene silencing in cells lacking TORC2-Gad8 is partially suppressed by loss of the anti-silencer Epe1 and fully suppressed by loss of the Pol II-associated Paf1 complex, two chromatin regulators that have been implicated in heterochromatin stability and spreading. Taken together, our findings suggest that TORC2-Gad8 signaling contributes to epigenetic stability at subtelomeric regions and the mating-type locus in S. pombe.

Heterozygous APC germline mutations impart predisposition to colorectal cancer.

Familial adenomatous polyposis (FAP) is an inherited syndrome caused by a heterozygous adenomatous polyposis coli (APC) germline mutation, associated with a profound lifetime risk for colorectal cancer. While it is well accepted that tumorigenic transformation is initiated following acquisition of a second mutation and loss of function of the APC gene, the role of heterozygous APC mutation in this process is yet to be discovered. This work aimed to explore whether a heterozygous APC mutation induces molecular defects underlying tumorigenic transformation and how different APC germline mutations predict disease severity. Three FAP-human embryonic stem cell lines (FAP1/2/3-hESC lines) carrying germline mutations at different locations of the APC gene, and two control hESC lines free of the APC mutation, were differentiated into colon organoids and analyzed by immunohistochemistry and RNA sequencing. In addition, data regarding the genotype and clinical phenotype of the embryo donor parents were collected from medical records. FAP-hESCs carrying a complete loss-of-function of a single APC allele (FAP3) generated complex and molecularly mature colon organoids, which were similar to controls. In contrast, FAP-hESCs carrying APC truncation mutations (FAP1 and FAP2) generated only few cyst-like structures and cell aggregates of various shape, occasionally with luminal parts, which aligned with their failure to upregulate critical differentiation genes early in the process, as shown by RNA sequencing. Abnormal disease phenotype was shown also in non-pathological colon of FAP patients by the randomly distribution of proliferating cells throughout the crypts, compared to their focused localization in the lower part of the crypt in healthy/non-FAP patients. Genotype/phenotype analysis revealed correlations between the colon organoid maturation potential and FAP severity in the carrier parents. In conclusion, this study suggest that a single truncated APC allele is sufficient to initiate early molecular tumorigenic activity. In addition, the results hint that patient-specific hESC-derived colon organoids can probably predict disease severity among FAP patients.