Interspecific Variation in One-Carbon Metabolism within the Ovarian Follicle, Oocyte, and Preimplantation Embryo: Consequences for Epigenetic Programming of DNA Methylation

One-carbon (1C) metabolism provides methyl groups for the synthesis and/or methylation 20 of purines and pyrimidines, biogenic amines, proteins and phospholipids. Our understanding of 21 how 1C pathways operate, however, pertains mostly to the (rat) liver. Here we report that 22 transcripts for all bar two genes (i.e. BHMT, MAT1A) encoding enzymes in the linked methionine-23 folate cycles are expressed in all cell types within the ovarian follicle, oocyte and blastocyst in the 24 cow, sheep and pig; as well as in rat granulosa cells (GCs) and human KGN cells. BHMT protein 25 was absent in bovine theca and GCs, as was activity of this enzyme in GCs. Mathematical modelling 26 predicted that absence of this enzyme would lead to more volatile S-adenosylmethionine-mediated 27 transmethylation in response to 1C substrate (e.g., methionine) or cofactor provision. We tested the 28 sensitivity of bovine GCs to reduced methionine (from 50 to 10 µM) and observed a diminished flux 29 of 1C units through the methionine cycle. We then used Reduced-Representation Bisulfite 30 Sequencing to demonstrate that this reduction in methionine during bovine embryo culture leads 31 to genome-wide alterations to DNA methylation in >1,600 genes, including a cohort of imprinted 32 genes linked to an abnormal fetal-overgrowth phenotype. Bovine ovarian and embryonic cells are 33 acutely sensitive to methionine, but further experimentation is required to determine the 34 significance of interspecific variation in BHMT expression. 3

Exploiting differential Wnt target gene expression to generate a molecular biomarker for colorectal cancer stratification

OBJECTIVE Pathological Wnt pathway activation is a conserved hallmark of colorectal cancer. Wnt-activating mutations can be divided into: i) ligand-independent (LI) alterations in intracellular signal transduction proteins (, β-catenin), causing constitutive pathway activation and ii) ligand-dependent (LD) mutations affecting the synergistic R-Spondin axis (, -fusions) acting through amplification of endogenous Wnt signal transmembrane transduction. Our aim was to exploit differential Wnt target gene expression to generate a mutation-agnostic biomarker for LD tumours. DESIGN We undertook harmonised multi-omic analysis of discovery (n=684) and validation cohorts (n=578) of colorectal tumours collated from publicly available data and the Stratification in Colorectal Cancer Consortium. We used mutation data to establish molecular ground truth and subdivide lesions into LI/LD tumour subsets. We contrasted transcriptional, methylation, morphological and clinical characteristics between groups. RESULTS Wnt disrupting mutations were mutually exclusive. Desmoplastic stromal upregulation of may compensate for absence of epithelial mutation in a subset of stromal-rich tumours. Key Wnt negative regulator genes were differentially expressed between LD/LI tumours, with targeted hypermethylation of some genes (, ) occurring even in CIMP-negative LD cancers. mRNA expression was used as a discriminatory molecular biomarker to distinguish LD/LI tumours (area under the curve >0.93). CONCLUSIONS Epigenetic suppression of appropriate Wnt negative feedback loops is selectively advantageous in LD tumours and differential expression in LD/LI lesions can be exploited as a molecular biomarker. Distinguishing between LD/LI tumour types is important; patients with LD tumours retain sensitivity to Wnt ligand inhibition and may be stratified at diagnosis to clinical trials of Porcupine inhibitors