DLK1/DIO3 locus upregulation by a ß-catenin-dependent enhancer drives cell proliferation and liver tumorigenesis.

The CTNNB1 gene, encoding ß-catenin, is frequently mutated in hepatocellular carcinoma (HCC, ∼30%) and in hepatoblastoma (HB, >80%), in which DLK1/DIO3 locus induction is correlated with CTNNB1 mutations. Here, we aim to decipher how sustained ß-catenin activation regulates DLK1/DIO3 locus expression and the role this locus plays in HB and HCC development in mouse models deleted for Apc (ApcΔhep) or Ctnnb1-exon 3 (ß-cateninΔExon3) and in human CTNNB1-mutated hepatic cancer cells. We identified an enhancer site bound by TCF-4/ß-catenin complexes in an open conformation upon sustained ß-catenin activation (DLK1-Wnt responsive element [WRE]) and increasing DLK1/DIO3 locus transcription in ß-catenin-mutated human HB and mouse models. DLK1-WRE editing by CRISPR-Cas9 approach impaired DLK1/DIO3 locus expression and slowed tumor growth in subcutaneous CTNNB1-mutated tumor cell grafts, ApcΔhep HB and ß-cateninΔExon3 HCC. Tumor growth inhibition resulted either from increased FADD expression and subsequent caspase-3 cleavage in the first case or from decreased expression of cell cycle actors regulated by FoxM1 in the others. Therefore, the DLK1/DIO3 locus is an essential determinant of FoxM1-dependent cell proliferation during ß-catenin-driven liver tumorigenesis. Targeting the DLK1-WRE enhancer to silence the DLK1/DIO3 locus might thus represent an interesting therapeutic strategy to restrict tumor growth in primary liver cancers with CTNNB1 mutations.

White Blood Cell BRCA1 Promoter Methylation Status and Ovarian Cancer Risk.

Background: The role of normal tissue gene promoter methylation in cancer risk is poorly understood. Objective: To assess associations between normal tissue BRCA1 methylation and ovarian cancer risk. Design: 2 case-control (initial and validation) studies. Setting: 2 hospitals in Norway (patients) and a population-based study (control participants). Participants: 934 patients and 1698 control participants in the initial study; 607 patients and 1984 control participants in the validation study. Measurements: All patients had their blood sampled before chemotherapy. White blood cell (WBC) BRCA1 promoter methylation was determined by using methylation-specific quantitative polymerase chain reaction, and the percentage of methylation-positive samples was compared between population control participants and patients with ovarian cancer, including the subgroup with high-grade serous ovarian cancer (HGSOC). Results: In the initial study, BRCA1 methylation was more frequent in patients with ovarian cancer than control participants (6.4% vs. 4.2%; age-adjusted odds ratio [OR], 1.83 [95% CI, 1.27 to 2.63]). Elevated methylation, however, was restricted to patients with HGSOC (9.6%; OR, 2.91 [CI, 1.85 to 4.56]), in contrast to 5.1% and 4.0% of patients with nonserous and low-grade serous ovarian cancer (LGSOC), respectively. These findings were replicated in the validation study (methylation-positive status in 9.1% of patients with HGSOC vs. 4.3% of control participants-OR, 2.22 [CI 1.40 to 3.52]-4.1% of patients with nonserous ovarian cancer, and 2.7% of those with LGSOC). The results were not influenced by tumor burden, storage time, or WBC subfractions. In separate analyses of young women and newborns, BRCA1 methylation was detected in 4.1% (CI, 1.8% to 6.4%) and 7.0% (CI, 5.0% to 9.1%), respectively. Limitations: Patients with ovarian cancer were recruited at the time of diagnosis in a hospital setting. Conclusion: Constitutively normal tissue BRCA1 promoter methylation is positively associated with risk for HGSOC. Primary Funding Source: Norwegian Cancer Society.

Comprehensive evaluation of methods to isolate, quantify, and characterize circulating cell-free DNA from small volumes of plasma.

Circulating cell-free DNA (ccfDNA) has great potential for non-invasive diagnostics, and prediction and monitoring of treatment response, but its amount is usually limited. Therefore, the choice of methods to extract and characterize ccfDNA is crucial. In the current study, we performed the most comprehensive comparison of methods for ccfDNA extraction (11 methods), quantification (3 methods), and estimation of the integrity index (2 methods) from small quantities of different kinds of plasma. The QIAamp® Circulating Nucleic Acid Kit and the Norgen Plasma/Serum Circulating DNA Purification Mini Kit showed the best accuracy and reproducibility, but the Norgen kit allowed to extract a higher amount of ccfDNA. This workflow provides a reliable protocol for the multiple applications of ccfDNA in biomedicine.

cBAF generates subnucleosomes that expand OCT4 binding and function beyond DNA motifs at enhancers.

The canonical BRG/BRM-associated factor (cBAF) complex is essential for chromatin opening at enhancers in mammalian cells. However, the nature of the open chromatin remains unclear. Here, we show that, in addition to producing histone-free DNA, cBAF generates stable hemisome-like subnucleosomal particles containing the four core histones associated with 50-80 bp of DNA. Our genome-wide analysis indicates that cBAF makes these particles by targeting and splitting fragile nucleosomes. In mouse embryonic stem cells, these subnucleosomes become an in vivo binding substrate for the master transcription factor OCT4 independently of the presence of OCT4 DNA motifs. At enhancers, the OCT4-subnucleosome interaction increases OCT4 occupancy and amplifies the genomic interval bound by OCT4 by up to one order of magnitude compared to the region occupied on histone-free DNA. We propose that cBAF-dependent subnucleosomes orchestrate a molecular mechanism that projects OCT4 function in chromatin opening beyond its DNA motifs.

Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer's disease.

We identified rare coding variants associated with Alzheimer's disease in a three-stage case-control study of 85,133 subjects. In stage 1, we genotyped 34,174 samples using a whole-exome microarray. In stage 2, we tested associated variants (P < 1 × 10-4) in 35,962 independent samples using de novo genotyping and imputed genotypes. In stage 3, we used an additional 14,997 samples to test the most significant stage 2 associations (P < 5 × 10-8) using imputed genotypes. We observed three new genome-wide significant nonsynonymous variants associated with Alzheimer's disease: a protective variant in PLCG2 (rs72824905: p.Pro522Arg, P = 5.38 × 10-10, odds ratio (OR) = 0.68, minor allele frequency (MAF)cases = 0.0059, MAFcontrols = 0.0093), a risk variant in ABI3 (rs616338: p.Ser209Phe, P = 4.56 × 10-10, OR = 1.43, MAFcases = 0.011, MAFcontrols = 0.008), and a new genome-wide significant variant in TREM2 (rs143332484: p.Arg62His, P = 1.55 × 10-14, OR = 1.67, MAFcases = 0.0143, MAFcontrols = 0.0089), a known susceptibility gene for Alzheimer's disease. These protein-altering changes are in genes highly expressed in microglia and highlight an immune-related protein-protein interaction network enriched for previously identified risk genes in Alzheimer's disease. These genetic findings provide additional evidence that the microglia-mediated innate immune response contributes directly to the development of Alzheimer's disease.