Quantification of in vivo progenitor mutation accrual with ultra-low error rate and minimal input DNA using SIP-HAVA-seq.

Assaying in vivo accrual of DNA damage and DNA mutations by stem cells and pinpointing sources of damage and mutations would further our understanding of aging and carcinogenesis. Two main hurdles must be overcome. First, in vivo mutation rates are orders of magnitude lower than raw sequencing error rates. Second, stem cells are vastly outnumbered by differentiated cells, which have a higher mutation rate-quantification of stem cell DNA damage and DNA mutations is thus best performed from small, well-defined cell populations. Here we report a mutation detection technique, based on the "duplex sequencing" principle, with an error rate below ∼10-10 and that can start from as little as 50 pg DNA. We validate this technique, which we call SIP-HAVA-seq, by characterizing Caenorhabditis elegans germline stem cell mutation accrual and asking how mating affects that accrual. We find that a moderate mating-induced increase in cell cycling correlates with a dramatic increase in accrual of mutations. Intriguingly, these mutations consist chiefly of deletions in nonexpressed genes. This contrasts with results derived from mutation accumulation lines and suggests that mutation spectrum and genome distribution change with replicative age, chronological age, cell differentiation state, and/or overall worm physiological state. We also identify single-stranded gaps as plausible deletion precursors, providing a starting point to identify the molecular mechanisms of mutagenesis that are most active. SIP-HAVA-seq provides the first direct, genome-wide measurements of in vivo mutation accrual in stem cells and will enable further characterization of underlying mechanisms and their dependence on age and cell state.

Reactivation of death receptor 4 (DR4) expression sensitizes medulloblastoma cell lines to TRAIL.

OBJECT: Apoptosis, a key cellular response to therapeutic agents is often inactivated in tumor cells. In this study, we evaluated the expression of the tumor necrosis family of death receptors, DR4 and DR5, in medulloblastoma tumor samples and cell lines to determine if epigenetic modulation of gene expression could sensitize tumor cell lines to TRAIL-mediated apoptosis. METHODS: Human medulloblastoma samples and cell lines were analyzed for DR4 and DR5 expression by quantitative PCR and immunofluorescence assays. Cell lines with downregulated expression of one or both genes were treated with the histone deacetylase inhibitor, MS-275, and the expression of DR4 and DR5 measured by quantitative PCR, Western blotting, flow cytometry and chromatin immunoprecipitation assays. Induction of apoptosis in the presence of MS-275 was evaluated by TUNEL assay and its ability to augment TRAIL-mediated cytotoxicity was determined by MTT assays, Western blotting and flow cytometry. RESULTS: Compared to normal cerebellum, DR4, but not DR5 expression was consistently downregulated in medulloblastoma tumor samples and in Daoy and D283 cell lines. Interestingly, MS-275 decreased cell growth and induced apoptosis in Daoy and D283 cells. In Daoy cells, this coincided with increased histone H3 and H4 acetylation at the DR4 promoter and enhanced DR4 gene and protein expression as well as elevated Caspase-8 activity. The involvement of DR4 in the cellular response to MS-275 was further confirmed by the observation that knockdown of DR4 and FADD abrogated apoptosis. Further, addition of TRAIL to MS-275 treated cells resulted in an enhancement of apoptosis, suggesting that the upregulated death receptors were functional. CONCLUSION: Our study provides an understanding of the role of DR4 in apoptosis of medulloblastoma cell lines and suggests a potential contribution of aberrant histone deacetylation to the resistance of medulloblastoma cells to therapeutic death.

Regulation of USP37 Expression by REST-Associated G9a-Dependent Histone Methylation.

The deubiquitylase (DUB) USP37 is a component of the ubiquitin system and controls cell proliferation by regulating the stability of the cyclin-dependent kinase inhibitor 1B, (CDKN1B/p27Kip1). The expression of USP37 is downregulated in human medulloblastoma tumor specimens. In the current study, we show that USP37 prevents medulloblastoma growth in mouse orthotopic models, suggesting that it has tumor-suppressive properties in this neural cancer. Here, we also report on the mechanism underlying USP37 loss in medulloblastoma. Previously, we observed that the expression of USP37 is transcriptionally repressed by the RE1 silencing transcription factor (REST), which requires chromatin remodeling factors for its activity. Genetic and pharmacologic approaches were employed to identify a specific role for G9a, a histone methyltransferase (HMT), in promoting methylation of histone H3 lysine-9 (H3K9) mono- and dimethylation, and surprisingly trimethylation, at the USP37 promoter to repress its gene expression. G9a inhibition also blocked the tumorigenic potential of medulloblastoma cells in vivo Using isogenic low- and high-REST medulloblastoma cells, we further showed a REST-dependent elevation in G9a activity, which further increased mono- and trimethylation of histone H3K9, accompanied by downregulation of USP37 expression. Together, these findings reveal a role for REST-associated G9a and histone H3K9 methylation in the repression of USP37 expression in medulloblastoma.Implications: Reactivation of USP37 by G9a inhibition has the potential for therapeutic applications in REST-expressing medulloblastomas. Mol Cancer Res; 15(8); 1073-84. ©2017 AACR.

Semi-permeable Diffusion Barriers Enhance Patterning Robustness in the C. elegans Germline.

Positional information derived from local morphogen concentration plays an important role in patterning. A key question is how morphogen diffusion and gene expression regulation shape positional information into an appropriate profile with suitably low noise. We address this question using a model system--the C. elegans germline--whose regulatory network has been well characterized genetically but whose spatiotemporal dynamics are poorly understood. We show that diffusion within the germline syncytium is a critical control of stem cell differentiation and that semi-permeable diffusion barriers present at key locations make it possible--in combination with a feedback loop in the germline regulatory network--for mitotic zone size to be robust against spatial noise in Notch signaling. Spatial averaging within compartments defined by diffusion barriers is an advantageous patterning strategy, which attenuates noise while still allowing for sharp transitions between compartments. This strategy could apply to other organs.