Fancm has dual roles in the limiting of meiotic crossovers and germ cell maintenance in mammals.

Meiotic crossovers are required for accurate chromosome segregation and producing new allelic combinations. Meiotic crossover numbers are tightly regulated within a narrow range, despite an excess of initiating DNA double-strand breaks. Here, we reveal the tumor suppressor FANCM as a meiotic anti-crossover factor in mammals. We use unique large-scale crossover analyses with both single-gamete sequencing and pedigree-based bulk-sequencing datasets to identify a genome-wide increase in crossover frequencies in Fancm-deficient mice. Gametogenesis is heavily perturbed in Fancm loss-of-function mice, which is consistent with the reproductive defects reported in humans with biallelic FANCM mutations. A portion of the gametogenesis defects can be attributed to the cGAS-STING pathway after birth. Despite the gametogenesis phenotypes in Fancm mutants, both sexes are capable of producing offspring. We propose that the anti-crossover function and role in gametogenesis of Fancm are separable and will inform diagnostic pathways for human genomic instability disorders.

An epigenetic signature of advanced colorectal cancer metastasis.

Colorectal cancer (CRC) is a leading cause of morbidity and mortality worldwide. The majority of CRC deaths are caused by tumor metastasis, even following treatment. There is strong evidence for epigenetic changes, such as DNA methylation, accompanying CRC metastasis and poorer patient survival. Earlier detection and a better understanding of molecular drivers for CRC metastasis are of critical clinical importance. Here, we identify a signature of advanced CRC metastasis by performing whole genome-scale DNA methylation and full transcriptome analyses of paired primary cancers and liver metastases from CRC patients. We observed striking methylation differences between primary and metastatic pairs. A subset of loci showed coordinated methylation-expression changes, suggesting these are potentially epigenetic drivers that control the expression of critical genes in the metastatic cascade. The identification of CRC epigenomic markers of metastasis has the potential to enable better outcome prediction and lead to the discovery of new therapeutic targets.

A MicroRNA Cluster in the DLK1-DIO3 Imprinted Region on Chromosome 14q32.2 Is Dysregulated in Metastatic Hepatoblastomas.

Hepatoblastoma (HB) is the most common malignant liver neoplasm in children. Despite progress in HB therapy, outcomes for patients with metastatic disease remain poor. Dysregulation of miRNA expression is one of the potential epigenetic mechanisms associated with pathogenesis of HB. However, miRNA profiles related to the different stages of HB tissues and cells, in particular of lung metastatic tumor cells, are unknown. In the present study, using array-based miRNA expression and DNA methylation analysis on formalin-fixed paraffin-embedded tissues, we aimed to identify miRNA changes that can discriminate between lung metastatic tumors, primary tumors (fetal and embryonal subtypes), and nontumorous surrounding livers. Our analysis demonstrated that a large cluster of microRNAs and snoRNAs located within the 14q32.2 DLK1-DIO3 region showed a strikingly upregulated expression pattern in HB tumors, especially metastatic tumors, compared to normal liver tissues. This revealed dysregulation of miRNAs similar to that seen in a malignant stem-like subtype of hepatocellular carcinoma associated with poor prognosis. These findings in HB mirror similar findings made in multiple other cancer types. With further analysis this may in future allow stratification of different stages and types of HB tumors based on their miRNA profiles, which could lead to new approaches to diagnosis and treatment in progressive HB patients.

Reprogramming roadmap reveals route to human induced trophoblast stem cells.

The reprogramming of human somatic cells to primed or naive induced pluripotent stem cells recapitulates the stages of early embryonic development1-6. The molecular mechanism that underpins these reprogramming processes remains largely unexplored, which impedes our understanding and limits rational improvements to reprogramming protocols. Here, to address these issues, we reconstruct molecular reprogramming trajectories of human dermal fibroblasts using single-cell transcriptomics. This revealed that reprogramming into primed and naive pluripotency follows diverging and distinct trajectories. Moreover, genome-wide analyses of accessible chromatin showed key changes in the regulatory elements of core pluripotency genes, and orchestrated global changes in chromatin accessibility over time. Integrated analysis of these datasets revealed a role for transcription factors associated with the trophectoderm lineage, and the existence of a subpopulation of cells that enter a trophectoderm-like state during reprogramming. Furthermore, this trophectoderm-like state could be captured, which enabled the derivation of induced trophoblast stem cells. Induced trophoblast stem cells are molecularly and functionally similar to trophoblast stem cells derived from human blastocysts or first-trimester placentas7. Our results provide a high-resolution roadmap for the transcription-factor-mediated reprogramming of human somatic cells, indicate a role for the trophectoderm-lineage-specific regulatory program during this process, and facilitate the direct reprogramming of somatic cells into induced trophoblast stem cells.

MicroRNA expression patterns and signalling pathways in the development and progression of childhood solid tumours.

The development of childhood solid tumours is tied to early developmental processes. These tumours may be complex and heterogeneous, and elucidating the aberrant mechanisms that alter the early embryonic environment and lead to disease is essential to our understanding of how these tumours function. MicroRNAs (miRNAs) are vital regulators of gene expression at all stages of development, and their crosstalk via developmental signalling pathways is essential for orchestrating regulatory control in processes such as proliferation, differentiation and apoptosis of cells. Oncogenesis, from aberrant miRNA expression, can occur through amplification and overexpression of oncogenic miRNAs (oncomiRs), genetic loss of tumour suppressor miRNAs, and global miRNA reduction from genetic and epigenetic alterations in the components regulating miRNA biogenesis. While few driver mutations have been identified in many of these types of tumours, abnormal miRNA expression has been found in a number of childhood solid tumours compared to normal tissue. An exploration of the network of key developmental pathways and interacting miRNAs may provide insight into the development of childhood solid malignancies and how key regulators are affected. Here we present a comprehensive introduction to the roles and implications of miRNAs in normal early development and childhood solid tumours, highlighting several tumours in depth, including embryonal brain tumours, neuroblastoma, osteosarcoma, Wilms tumour, and hepatoblastoma. In light of recent literature describing newer classifications and subtyping of tumours based on miRNA profiling, we discuss commonly identified miRNAs, clusters or families associated with several solid tumours and future directions for improving therapeutic approaches.

Genome-wide methylation sequencing of paired primary and metastatic cell lines identifies common DNA methylation changes and a role for EBF3 as a candidate epigenetic driver of melanoma metastasis.

Epigenetic alterations are increasingly implicated in metastasis, whereas very few genetic mutations have been identified as authentic drivers of cancer metastasis. Yet, to date, few studies have identified metastasis-related epigenetic drivers, in part because a framework for identifying driver epigenetic changes in metastasis has not been established. Using reduced representation bisulfite sequencing (RRBS), we mapped genome-wide DNA methylation patterns in three cutaneous primary and metastatic melanoma cell line pairs to identify metastasis-related epigenetic drivers. Globally, metastatic melanoma cell lines were hypomethylated compared to the matched primary melanoma cell lines. Using whole genome RRBS we identified 75 shared (10 hyper- and 65 hypomethylated) differentially methylated fragments (DMFs), which were associated with 68 genes showing significant methylation differences. One gene, Early B Cell Factor 3 (EBF3), exhibited promoter hypermethylation in metastatic cell lines, and was validated with bisulfite sequencing and in two publicly available independent melanoma cohorts (n = 40 and 458 melanomas, respectively). We found that hypermethylation of the EBF3 promoter was associated with increased EBF3 mRNA levels in metastatic melanomas and subsequent inhibition of DNA methylation reduced EBF3 expression. RNAi-mediated knockdown of EBF3 mRNA levels decreased proliferation, migration and invasion in primary and metastatic melanoma cell lines. Overall, we have identified numerous epigenetic changes characterising metastatic melanoma cell lines, including EBF3-induced aggressive phenotypic behaviour with elevated EBF3 expression in metastatic melanoma, suggesting that EBF3 promoter hypermethylation may be a candidate epigenetic driver of metastasis.

Multi-platform microRNA profiling of hepatoblastoma patients using formalin fixed paraffin embedded archival samples.

BACKGROUND: Formalin fixed paraffin embedded (FFPE) samples are a valuable resource in cancer research and have the potential to be extensively used. However, they are often underused because of degradation and chemical modifications occurring in the RNA that can present obstacles in downstream analysis. In routine medical care, FFPE material is examined and archived, therefore clinical collections of many types of cancers exist. It is beneficial to assess and record the quality of data that can be obtained from this type of material. The current study investigated three independent platforms and their ability to profile microRNAs (miRNAs) within FFPE samples from hepatoblastoma (HB) patients. FINDINGS: Here we present three types of datasets consisting of miRNA profiles for 13 HB patients with different tumour types and molecular variations. The three platforms that were used to generate these data are: next-generation sequencing (Illumina MiSeq), microarray (Affymetrix(®) GeneChip(®) miRNA 3.0 array) and NanoString (nCounter, Human v2 miRNA Assay). The mature miRNAs identified are based on miRBase version 17 and 18. CONCLUSIONS: These datasets provide a global landscape of miRNA expression for a rare childhood cancer that has not previously been well characterised. These data could serve as a resource for future studies aiming to make comparisons of HB miRNA profiles and to document aberrant miRNA expression in this type of cancer.

A cross comparison of technologies for the detection of microRNAs in clinical FFPE samples of hepatoblastoma patients.

Although formalin fixed paraffin embedded (FFPE) tissue is a major biological source in cancer research, it is challenging to work with due to macromolecular fragmentation and nucleic acid crosslinking. Therefore, it is important to characterise the quality of data that can be obtained from FFPE samples. We have compared three independent platforms (next generation sequencing, microarray and NanoString) for profiling microRNAs (miRNAs) using clinical FFPE samples from hepatoblastoma (HB) patients. The number of detected miRNAs ranged from 228 to 345 (median = 294) using the next generation sequencing platform, whereas 79 to 125 (median = 112) miRNAs were identified using microarrays in three HB samples, including technical replicates. NanoString identified 299 to 372 miRNAs in two samples. Between the platforms, we observed high reproducibility and significant levels of shared detection. However, for commonly detected miRNAs, a strong correlation between platforms was not observed. Analysis of 10 additional HB samples with NanoString identified significantly overlapping miRNA expression profiles, and an alternative pattern was identified in a poorly differentiated HB with an aggressive phenotype. This investigation serves as a roadmap for future studies investigating miRNA expression in clinical FFPE samples, and as a guideline for the selection of an appropriate platform.