Machine learning enables pan-cancer identification of mutational hotspots at persistent CTCF binding sites.

CCCTC-binding factor (CTCF) is an insulator protein that binds to a highly conserved DNA motif and facilitates regulation of three-dimensional (3D) nuclear architecture and transcription. CTCF binding sites (CTCF-BSs) reside in non-coding DNA and are frequently mutated in cancer. Our previous study identified a small subclass of CTCF-BSs that are resistant to CTCF knock down, termed persistent CTCF binding sites (P-CTCF-BSs). P-CTCF-BSs show high binding conservation and potentially regulate cell-type constitutive 3D chromatin architecture. Here, using ICGC sequencing data we made the striking observation that P-CTCF-BSs display a highly elevated mutation rate in breast and prostate cancer when compared to all CTCF-BSs. To address whether P-CTCF-BS mutations are also enriched in other cell-types, we developed CTCF-INSITE-a tool utilising machine learning to predict persistence based on genetic and epigenetic features of experimentally-determined P-CTCF-BSs. Notably, predicted P-CTCF-BSs also show a significantly elevated mutational burden in all 12 cancer-types tested. Enrichment was even stronger for P-CTCF-BS mutations with predicted functional impact to CTCF binding and chromatin looping. Using in vitro binding assays we validated that P-CTCF-BS cancer mutations, predicted to be disruptive, indeed reduced CTCF binding. Together this study reveals a new subclass of cancer specific CTCF-BS DNA mutations and provides insights into their importance in genome organization in a pan-cancer setting.

Evaluation of diagnostic techniques for early detection of heartworm in experimentally infected dogs: identification of Dirofilaria immitis-derived microRNA in the initial 28 weeks post-inoculation.

BACKGROUND: Dirofilaria immitis, commonly known as heartworm (HW), is a parasitic nematode transmitted by various mosquito species, leading to heartworm disease (HWD) in dogs. Diagnosis of HW typically involves antigen or microfilariae detection, or visualization of adult worms through imaging or post mortem examination. Polymerase chain reaction (PCR) and micro RNA (miRNA) detection have been explored for HW diagnosis. METHODS: Three dogs, previously experimentally infected with HW, underwent blood sampling every 4 weeks for 7 months. Samples were assessed for antigen presence after heat treatment, PCR amplification, and microfilaria examination using Giemsa-stained thick smears. Additionally, whole blood aliquots underwent miRNA deep sequencing and bioinformatic analysis. RESULTS: Heartworm antigen was detectable after heat treatment at 20 weeks post-inoculation and via PCR at 24 weeks, with microfilariae observed in peripheral blood smears at 28 weeks. However, deep miRNA sequencing revealed that the miRNA candidate sequences are not consistently expressed before 28 weeks of infection. CONCLUSIONS: While ancillary molecular methods such as PCR and miRNA sequencing may be less effective than antigen detection for detecting immature larval stages in an early stage of infection, our experimental findings demonstrate that circulating miRNAs can still be detected in 28 weeks post-infection.

Characterisation and reproducibility of the HumanMethylationEPIC v2.0 BeadChip for DNA methylation profiling.

BACKGROUND: The Illumina family of Infinium Methylation BeadChip microarrays has been widely used over the last 15 years for genome-wide DNA methylation profiling, including large-scale and population-based studies, due to their ease of use and cost effectiveness. Succeeding the popular HumanMethylationEPIC BeadChip (EPICv1), the recently released Infinium MethylationEPIC v2.0 BeadChip (EPICv2) claims to extend genomic coverage to more than 935,000 CpG sites. Here, we comprehensively characterise the reproducibility, reliability and annotation of the EPICv2 array, based on bioinformatic analysis of both manifest data and new EPICv2 data from diverse biological samples. RESULTS: We find a high degree of reproducibility with EPICv1, evidenced by comparable sensitivity and precision from empirical cross-platform comparison incorporating whole genome bisulphite sequencing (WGBS), and high correlation between technical sample replicates, including between samples with DNA input levels below the manufacturer's recommendation. We provide a full assessment of probe content, evaluating genomic distribution and changes from previous array versions. We characterise EPICv2's new feature of replicated probes and provide recommendations as to the superior probes. In silico analysis of probe sequences demonstrates that probe cross-hybridisation remains a significant problem in EPICv2. By mapping the off-target sites at single nucleotide resolution and comparing with WGBS we show empirical evidence for preferential off-target binding. CONCLUSIONS: Overall, we find EPICv2 a worthy successor to the previous Infinium methylation microarrays, however some technical issues remain. To support optimal EPICv2 data analysis we provide an expanded version of the EPICv2 manifest to aid researchers in understanding probe design, data processing, choosing appropriate probes for analysis and for integration with methylation datasets from previous versions of the Infinium Methylation BeadChip.

The potential of epigenetic therapy to target the 3D epigenome in endocrine-resistant breast cancer.

Three-dimensional (3D) epigenome remodeling is an important mechanism of gene deregulation in cancer. However, its potential as a target to counteract therapy resistance remains largely unaddressed. Here, we show that epigenetic therapy with decitabine (5-Aza-mC) suppresses tumor growth in xenograft models of pre-clinical metastatic estrogen receptor positive (ER+) breast tumor. Decitabine-induced genome-wide DNA hypomethylation results in large-scale 3D epigenome deregulation, including de-compaction of higher-order chromatin structure and loss of boundary insulation of topologically associated domains. Significant DNA hypomethylation associates with ectopic activation of ER-enhancers, gain in ER binding, creation of new 3D enhancer-promoter interactions and concordant up-regulation of ER-mediated transcription pathways. Importantly, long-term withdrawal of epigenetic therapy partially restores methylation at ER-enhancer elements, resulting in a loss of ectopic 3D enhancer-promoter interactions and associated gene repression. Our study illustrates the potential of epigenetic therapy to target ER+ endocrine-resistant breast cancer by DNA methylation-dependent rewiring of 3D chromatin interactions, which are associated with the suppression of tumor growth.

The Domestic Dog as a Laboratory Host for Brugia malayi.

Of the three nematodes responsible for lymphatic filariasis in humans, only Brugia malayi is actively maintained in research settings owing to its viability in small animal hosts, principal among which is the domestic cat. While the microfilaremic feline host is necessary for propagation of parasites on any significant scale, this system is plagued by a number of challenges not as pronounced in canine filarial models. For this reason, we investigated the capacity in which dogs may serve as competent laboratory hosts for B. malayi. We infected a total of 20 dogs by subcutaneous injection of 500 B. malayi third-stage larvae (L3) in either a single (n = 10) or repeated infection events (125 L3 per week for four weeks; n = 10). Within each group, half of the individuals were injected in the inguinal region and half in the dorsum of the hind paw. To track the course of microfilaremia in this host, blood samples were examined by microscopy biweekly for two years following infection. Additionally, to identify cellular responses with potential value as predictors of patency, we measured peripheral blood leukocyte counts for the first year of infection. A total of 10 of 20 dogs developed detectable microfilaremia. Peak microfilaria density varied but attained levels useful for parasite propagation (median = 1933 mL-1; range: 33-9950 mL-1). Nine of these dogs remained patent at 104 weeks. A two-way ANOVA revealed no significant differences between infection groups in lifetime microfilaria production (p = 0.42), nor did regression analysis reveal any likely predictive relationships to leukocyte values. The results of this study demonstrate the competence of the dog as a host for B. malayi and its potential to serve in the laboratory role currently provided by the cat, while also clarifying the potential for zoonosis in filariasis-endemic regions.

DNA methylation is required to maintain both DNA replication timing precision and 3D genome organization integrity.

DNA replication timing and three-dimensional (3D) genome organization are associated with distinct epigenome patterns across large domains. However, whether alterations in the epigenome, in particular cancer-related DNA hypomethylation, affects higher-order levels of genome architecture is still unclear. Here, using Repli-Seq, single-cell Repli-Seq, and Hi-C, we show that genome-wide methylation loss is associated with both concordant loss of replication timing precision and deregulation of 3D genome organization. Notably, we find distinct disruption in 3D genome compartmentalization, striking gains in cell-to-cell replication timing heterogeneity and loss of allelic replication timing in cancer hypomethylation models, potentially through the gene deregulation of DNA replication and genome organization pathways. Finally, we identify ectopic H3K4me3-H3K9me3 domains from across large hypomethylated domains, where late replication is maintained, which we purport serves to protect against catastrophic genome reorganization and aberrant gene transcription. Our results highlight a potential role for the methylome in the maintenance of 3D genome regulation.

Prenatal exposure to bisphenols affects pregnancy outcomes and offspring development in rats.

The objective of this study was to evaluate the effects of gestational exposure to low doses of bisphenol A (BPA), bisphenol S (BPS), and bisphenol F (BPF) on pregnancy outcomes and offspring development. Pregnant Sprague-Dawley rats were orally dosed with vehicle, 5 µg/kg body weight (BW)/day of BPA, BPS and BPF, or 1 µg/kg BW/day of BPF on gestational days 6-21. Pregnancy and gestational outcomes, including number of abortions and stillbirths, were monitored. Male and female offspring were subjected to morphometry at birth, followed by pre- and post-weaning body weights, post-weaning food and water intakes, and adult organ weights. Ovarian follicular counts were also obtained from adult female offspring. We observed spontaneous abortions in over 80% of dams exposed to 5 µg/kg of BPF. BPA exposure increased Graafian follicles in female offspring, while BPS and BPF exposure decreased the number of corpora lutea, suggesting reduced ovulation rates. Moreover, BPA exposure increased male kidney and prostate gland weights, BPF decreased epididymal adipose tissue weights, and BPS had modest effects on male abdominal adipose tissue weights. Prenatal BPS exposure reduced anogenital distance (AGD) in male offspring, suggesting possible feminization, whereas both BPS and BPA induced oxidative stress in the testes. These results indicate that prenatal exposure to BPF affects pregnancy outcomes, BPS alters male AGD, and all three bisphenols alter certain organ weights in male offspring and ovarian function in female offspring. Altogether, it appears that prenatal exposure to BPA or its analogues can induce reproductive toxicity even at low doses.

Independent and combined effects of Bisphenol A and Diethylhexyl Phthalate on gestational outcomes and offspring development in Sprague-Dawley rats.

Bisphenol A (BPA) and Diethylhexyl Phthalate (DEHP) are well-studied endocrine disrupting chemicals (EDCs), however, the effects of mixtures of these EDCs are not. To assess the consequences of prenatal exposure to a mixture of these EDCs, dams were orally administered either saline (control), BPA (5 µg/kg BW/day), high dose DEHP (HD-D; 7.5 mg/kg BW/day), or a combination of BPA with HD-D in experiment 1; saline, BPA (5 µg/kg BW/day), low-dose DEHP (LD-D; 5 µg/kg BW/day) or a combination of BPA with LD-D in experiment 2. Gestational weights, number of abortions, litter size and weights, number of live births and stillbirths were recorded. Morphometric measures were obtained at birth and body weight, food and water intake were monitored weekly from postnatal weeks 3-12. Offspring were sacrificed at 16-24 weeks of age and organ weights were measured. The abortion rate of dams exposed to HD-D and the mixtures, BPA + LD-D and BPA + HD-D were higher at 9, 14 and 27% respectively. Prenatal exposure to BPA or HD-D significantly decreased relative thymus weights in male but not female offspring. Apoptotic cells were detected in thymus sections of both male and female offspring prenatally exposed to DEHP. Relative heart weights increased in BPA + HD-D exposed male offspring compared to the other groups. The results indicate that a mixture of BPA and DEHP, produced a pronounced effect on pregnancy outcomes. Male offspring appear to be more susceptible to the programming effects of these EDCs or their mixture suggesting a need to reconsider the possible additive, antagonistic or synergistic effects of EDC mixtures.