Imprinted methylation of the paternal Rasgrf1 allele in mice occurs at a differentially methylated domain (DMD) 30 kbp 5 of the promoter. A repeated sequence 3 of the DMD regulates imprinted methylation, which is required for imprinted expression. Here we identify the mechanism by which methylation controls imprinting. The DMD is an enhancer blocker that binds CTCF in a methylation-sensitive manner. CTCF bound to the unmethylated maternal allele silences expression. CTCF binding to the paternal allele is prevented by repeatmediated methylation, allowing expression. Optimal in vitro enhancer-blocking activity requires CTCF binding sites. The enhancer blocker can be bypassed in vivo and imprinting abolished by placing an extra enhancer proximal to the promoter. Together, the repeats and the DMD constitute a binary switch that regulates Rasgrf1 imprinting.Approximately 70 transcripts undergo genomic imprinting, in which expression is primarily or exclusively from one parental allele while the other allele remains silent. Accompanying allele-specific expression is allele-specific DNA methylation, which is important for imprinted expression (17). In mice, the paternal Rasgrf1 allele is exclusively expressed in neonatal brain, while the maternal allele is silent. The paternal allele is also methylated within a differentially methylated domain (DMD) located 30 kbp 5Ј of the promoter. Immediately 3Ј of the DMD is a repeated sequence element containing 40 copies of a 41-nucleotide (nt) element. DMD methylation requires the repeats. Mice lacking the repeats on the paternal allele fail to establish proper paternal specific DNA methylation during gametogenesis (11,29). Removal of the paternal repeats after fertilization but before implantation causes a loss of previously established methylation (R. Holmes, Y. Chang, and P. D. Soloway, submitted for publication). Collectively, the results show the Rasgrf1 repeats provide a positive signal for establishing and maintaining DNA methylation in mice. In other studies, sequences have been identified that regulate methylation at transgene insertion sites (4, 5, 15) and at the endogenous position of the imprinted H19/Igf2 locus (8,25,27).Paternal allele-specific expression of Rasgrf1 in neonatal brain requires DMD methylation. Mutations that cause inappropriate loss of paternal allele methylation silence paternal allele expression, while mutations that induce maternal allele methylation activate the normally silent maternal allele (11,29; Holmes et al., submitted). The correlation between DMD methylation and expression at Rasgrf1 is reminiscent of the relationship between methylation of the H19 DMD and expression of the tightly linked Igf2 locus. The H19 DMD is a methylation-sensitive enhancer-blocking element that binds CTCF when unmethylated, as on the maternal allele. This enables the enhancer-blocking activity of the DMD to prevent interaction between a downstream enhancer with the upstream Igf2 promoter, thus silencing the maternal Igf2 allele. However, when the DMD is methyla...

No abstract

Epigenetic marks are fundamental to normal development, but little is known about signals that dictate their placement. Insights have been provided by studies of imprinted loci in mammals, where monoallelic expression is epigenetically controlled. Imprinted expression is regulated by DNA methylation programmed during gametogenesis in a sex-specific manner and maintained after fertilization. At Rasgrf1 in mouse, paternal-specific DNA methylation on a differential methylation domain (DMD) requires downstream tandem repeats. The DMD and repeats constitute a binary switch regulating paternal-specific expression. Here, we define sequences sufficient for imprinted methylation using two transgenic mouse lines: One carries the entire Rasgrf1 cluster (RC); the second carries only the DMD and repeats (DR) from Rasgrf1. The RC transgene recapitulated all aspects of imprinting seen at the endogenous locus. DR underwent proper DNA methylation establishment in sperm and erasure in oocytes, indicating the DMD and repeats are sufficient to program imprinted DNA methylation in germlines. Both transgenes produce a DMD-spanning pit-RNA, previously shown to be necessary for imprinted DNA methylation at the endogenous locus. We show that when pit-RNA expression is controlled by the repeats, it regulates DNA methylation in cis only and not in trans. Interestingly, pedigree history dictated whether established DR methylation patterns were maintained after fertilization. When DR was paternally transmitted followed by maternal transmission, the unmethylated state that was properly established in the female germlines could not be maintained. This provides a model for transgenerational epigenetic inheritance in mice.

Diagnosing systemic lupus erythematosus (SLE) can be challenging as laboratory screening methods, although sensitive, lack specificity. The poor specificity of autoimmune testing produces more false positive results than true positive results. False positive results can cause stress to patients without autoimmune disease and require unnecessary rheumatology consultation to rule out disease. Our objective was to evaluate two screening assays to reduce the number of false positives while maintaining high sensitivity. In this study, we evaluated two immunoassays, the AtheNA Multi-Lyte II ANA System and QUANTA Lite ANA ELISA, to screen patients for SLE. All positive screening results were compared to immunoflourescent ANA testing using theHEp-2000 ANA System. A chart review was performed on all patients tested to determine clinical diagnosis of SLE. The QuantaLite ANA ELISA produced significantly more false positive results than the AtheNA Multi-Lyte II Test System when screening for SLE in our patient population.

Exosomes, extracellular vesicles <150 nm, are vehicles for transporting information (i.e., cargo) allowing tissue to tissue communication. Depending on the cargo, exosomes can have beneficial or detrimental effects. Brown Adipose Tissue (BAT) is a thermogenic organ that modulates metabolism. BAT is also an endocrine organ affecting function of various distant tissue. We have recently shown that BAT is an important modulator of the healthy and diseased heart. Adipose tissue is a large source of circulating exosomes, but the effects of BAT and changing BAT activity on circulating exosome number and cargo are unknown. Identifying the role of BAT in modulating exosome number and cargo is important since the myocardium is highly responsive to exosomes. We used various known approaches that increase BAT activity (cold exposure, BATcold) or decrease BAT activity (BAT removal (BATless), obesity (HFD), aging (old)) and examined the number and content of circulating exosomes. Upon BAT activation via cold exposure, there was a large increase in circulating exosome numbers (see figure). All approaches that results in decreased BAT activity resulted in a decrease in circulating exosome numbers (see figure). We further examined the role of changing BAT activity on the content (i.e., cargo) of the exosomes, specifically focusing on miRNA. Interestingly, changing BAT activity resulted in large changes to the content of the exosomes, with some miRNA increasing levels and other miRNA decreasing levels. Some of these identified miRNA have been shown to exert beneficial effects on the heart and many miRNA having no defined effect on cardiac function. We believe that these BAT activated exosomes have the combination and proportion of circulatory miRNA necessary to enhance and maintain heart function. There is a great need for new strategies and approaches for treatment of cardiovascular disease (CVD). Our data suggest that a novel treatment strategy for CVD can be derived from BAT exosomes.

10045 Background: Approximately 10% of childhood solid tumors occur in individuals with a cancer genetic predisposition. Identifying cancer predisposition permits interventions for prevention and early detection of future cancers in the child and their affected family members and may have other important medical care implications. The American Society of Clinical Oncology (ASCO) guidelines recommend reviewing family history (FHx) to identify high-risk patients who should be offered genetic testing. To our knowledge, adherence to ASCO guidelines by pediatric oncology providers (POPs) has not been studied. Methods: Retrospective chart review of solid tumor patients <18 years of age referred for genetic counseling 1/1/2017-1/31/2019. POP and genetic counselor (GC) FHx documentation was assessed for adherence with the ASCO minimum quality metrics: 2 generation pedigree, patient ethnicity, cancer genetic testing results in relatives (GTRR), and cancer type, age at diagnosis, and lineage (maternal/paternal) for each relative with cancer (RWC). Comparisons of FHx quality between POPs and GCs were evaluated with 1-sided t-tests. Ethnicity was considered assessed if both maternal and paternal ethnicities were documented. GTRR was considered assessed if the results of all relatives who had undergone genetic testing were noted. Cancer type, age at diagnosis, and lineage were considered assessed if they were checked for all RWCs. Whether or not the child underwent genetic testing and their testing results were also documented. Results: Of 129 eligible patients, 102 underwent germline genetic testing. Twelve (12%) had pathogenic alterations in cancer predisposition genes. The POP failed to document FHx in 20 patients (16%), including 2 with pathogenic germline alterations. The POP missed first/second degree RWCs in 32 of 88 patients with RWCs (36%). GCs achieved >99% on each of the 6 ASCO quality metrics, whereas POPs performed significantly more poorly (Table). In patients with RWCs, GCs achieved all 6 metrics in more cases than POPs (99% vs. 3%, p<0.001). Conclusions: POPs performed significantly worse than GCs in documenting family history compliant with ASCO minimum family history guidelines, raising concern for missed genetic cancer predisposition syndrome diagnoses in those not referred to a GC. Routine referral of pediatric solid tumor patients to a GC for family history review and interventions to improve the quality of family history taking by POPs may improve germline cancer predisposition detection, improving care of both patients and their affected family members. [Table: see text]