In mammals, DNA is methylated at cytosines within CpG dinucleotides. Properly regulated methylation is crucial for normal development. Inappropriate methylation may contribute to tumorigenesis by silencing tumor-suppressor genes or by activating growth-stimulating genes. Although many genes have been identified that acquire methylation and whose expression is methylation-sensitive, little is known about how DNA methylation is controlled. We have identified a DNA sequence that regulates establishment of DNA methylation in the male germ line at Rasgrf1. In mice, the imprinted Rasgrf1 locus is methylated on the paternal allele within a differentially methylated domain (DMD) 30 kbp 5' of the promoter. Expression is exclusively from the paternal allele in neonatal brain. Methylation is regulated by a repeated sequence, consisting of a 41-mer repeated 40 times, found immediately 3' of the DMD. This sequence is present in organisms in which Rasgrf1 is imprinted. In addition, DMD methylation is required for imprinted Rasgrf1 expression. Together the DMD and repeat element constitute a binary switch that regulates imprinting at the locus.

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...

At the imprinted Rasgrf1 locus in mouse, a cis-acting sequence controls DNA methylation at a differentially methylated domain (DMD). While characterizing epigenetic marks over the DMD, we observed that DNA and H3K27 trimethylation are mutually exclusive, with DNA and H3K27 methylation limited to the paternal and maternal sequences, respectively. The mutual exclusion arises because one mark prevents placement of the other. We demonstrated this in five ways: using 5-azacytidine treatments and mutations at the endogenous locus that disrupt DNA methylation; using a transgenic model in which the maternal DMD inappropriately acquired DNA methylation; and by analyzing materials from cells and embryos lacking SUZ12 and YY1. SUZ12 is part of the PRC2 complex, which is needed for placing H3K27me3, and YY1 recruits PRC2 to sites of action. Results from each experimental system consistently demonstrated antagonism between H3K27me3 and DNA methylation. When DNA methylation was lost, H3K27me3 encroached into sites where it had not been before; inappropriate acquisition of DNA methylation excluded normal placement of H3K27me3, and loss of factors needed for H3K27 methylation enabled DNA methylation to appear where it had been excluded. These data reveal the previously unknown antagonism between H3K27 and DNA methylation and identify a means by which epigenetic states may change during disease and development.

In mammals, imprinted genes have parent-of-origin-specific patterns of DNA methylation that cause allele-specific expression. At Rasgrf1 (encoding RAS protein-specific guanine nucleotide-releasing factor 1), a repeated DNA element is needed to establish methylation and expression of the active paternal allele 1 . At Igf2r (encoding insulin-like growth factor 2 receptor), a sequence called region 2 is needed for methylation of the active maternal allele 2,3 . Here we show that replacing the Rasgrf1 repeats on the paternal allele with region 2 allows both methylation and expression of the paternal copy of Rasgrf1, indicating that sequences that control methylation can function ectopically. Paternal transmission of the mutated allele also induced methylation and expression in trans of the normally unmethylated and silent wild-type maternal allele. Once activated, the wild-type maternal Rasgrf1 allele maintained its activated state in the next generation independently of the paternal allele. These results recapitulate in mice several features in common with paramutation described in plants 4 .Rasgrf1 is methylated on the paternal allele in a differentially methylated domain (DMD) 30 kb 5′ of the promoter. Expression is from the paternal allele in neonatal brain 5 . This imprinting requires a 1.6-kb repeated element located immediately downstream of the DMD consisting of a 41-mer repeated 40 times that regulates establishment of methylation at the DMD 1,6 . The DMD is a methylation-sensitive enhancer-blocking element, which, together with the repeats, functions as a binary switch that regulates imprinting. Sequences regulating DNA methylation have been identified for one other locus, Igf2r. In intron 2 of Igf2r, region 2 controls methylation and allele-specific expression 2,3,7 .We generated mice containing Igf2r region 2 in place of the Rasgrf1 repeats to determine if their activities overlap. Reciprocal crosses were done between mice heterozygous with respect to this allele (Rasgrf1 tm3.1Pds , Fig. 1) and PWK mates to monitor expression from the two alleles in neonatal brain 5 . Similar crosses were done with C57BL/6 mates to evaluate changes in methylation of the Rasgrf1 DMD.Maternal transmission of the Rasgrf1 tm3.1Pds allele (Rasgrf1 −/+ ) had no effect on methylation or expression of the locus, which remained paternal allele-specific and was expressed at levels COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. Fig. 2a and data not shown). Mice with a paternally transmitted repeat deletion lacked both methylation and expression of the paternal allele 1 , but paternal transmission of the Rasgrf1 tm3.1Pds mutation (Rasgrf1 +/− ) permitted expression of the locus, albeit at lower levels than in wild-type mice (Fig. 2b). The paternal Rasgrf1 tm3.1Pds allele also caused derepression of the normally silent wild-type maternal allele. Expression of the mutated paternal allele in Rasgrf1 +/− mice showed that region 2 was able, in part, to replace the function of t...

Actively proliferating cancer cells require sufficient amount of NADH and NADPH for biogenesis and to protect cells from the detrimental effect of reactive oxygen species. As both normal and cancer cells share the same NAD biosynthetic and metabolic pathways, selectively lowering levels of NAD(H) and NADPH would be a promising strategy for cancer treatment. Targeting nicotinamide phosphoribosyltransferase (NAMPT), a rate limiting enzyme of the NAD salvage pathway, affects the NAD and NADPH pool. Similarly, lowering NADPH by mutant isocitrate dehydrogenase 1/2 (IDH1/2) which produces D-2-hydroxyglutarate (D-2HG), an oncometabolite that downregulates nicotinate phosphoribosyltransferase (NAPRT) via hypermethylation on the promoter region, results in epigenetic regulation. NADPH is used to generate D-2HG, and is also needed to protect dihydrofolate reductase, the target for methotrexate, from degradation. NAD and NADPH pools in various cancer types are regulated by several metabolic enzymes, including methylenetetrahydrofolate dehydrogenase, serine hydroxymethyltransferase, and aldehyde dehydrogenase. Thus, targeting NAD and NADPH synthesis under special circumstances is a novel approach to treat some cancers. This article provides the rationale for targeting the key enzymes that maintain the NAD/NADPH pool, and reviews preclinical studies of targeting these enzymes in cancers.

ICU bedside nurses see their involvement in discussions of prognosis, goals of care, and palliative care as a key element of overall quality of patient care. Based on the barriers participants identified regarding their engagement, interventions are needed to ensure that nurses have the education, opportunities, and support to actively participate in these discussions.

Background Integrating palliative care into intensive care units (ICUs) requires involvement of bedside nurses, who report inadequate education in palliative care. Objective To implement and evaluate a palliative care professional development program for ICU bedside nurses. Methods From May 2013 to January 2015, palliative care advanced practice nurses and nurse educators in 5 academic medical centers completed a 3-day train-the-trainer program followed by 2 years of mentoring to implement the initiative. The program consisted of 8-hour communication workshops for bedside nurses and structured rounds in ICUs, where nurse leaders coached bedside nurses in identifying and addressing palliative care needs. Primary outcomes were nurses' ratings of their palliative care communication skills in surveys, and nurses' identification of palliative care needs during coaching rounds. Results Each center held at least 6 workshops, training 428 bedside nurses. Nurses rated their skill level higher after the workshop for 15 tasks (eg, responding to family distress, ensuring families understand information in family meetings, all P < .01 vs preworkshop). Coaching rounds in each ICU took a mean of 3 hours per month. For 82% of 1110 patients discussed in rounds, bedside nurses identified palliative care needs and created plans to address them. Conclusions Communication skills training workshops increased nurses' ratings of their palliative care communication skills. Coaching rounds supported nurses in identifying and addressing palliative care needs. (American Journal of Critical Care. 2017; 26:361-371) by AACN on May 12, 2018 http://ajcc.aacnjournals.org/ Downloaded from P alliative care is a specialty and focus of care that aims to improve quality of care for patients who have serious and complex illnesses and their families. [1][2][3] Patients in intensive care units (ICUs) and their families have palliative care needs, including emotional support, management of pain and symptoms, and clinician-family communication to ensure that patients receive treatments that are consistent with their goals. [3][4][5][6][7][8][9][10][11][12][13] AJCC AMERICAN JOURNAL OF CRITICAL CARE, September 2017, Volume 26, No. 5 www.ajcconline.orgIn the ICU, palliative care is provided along with life-sustaining therapies and may be delivered by the ICU team (primary palliative care), a palliative care consult service (specialty palliative care), or both. 2,3,14 A number of barriers to integrating palliative care into the ICU have been identified, including inadequate training of clinicians and misperceptions that such treatment is the same as hospice, comfortfocused care, or end-of-life care. 3 Families, physicians, and nurses identify involvement of bedside nurses as a key factor in the quality of ICU palliative care. [15][16][17][18][19][20] Nurses' training and constancy at the bedside position them to identify palliative care needs, coordinate communication among families and an array of clinicians, and support and educate families. 15,16,19...

IntroductionAn estimated 25% of the 1.2 million individuals living with human immunodeficiency virus (HIV) in the U.S. are co-infected with hepatitis C (HCV). The Centers for Disease Control and Prevention recommends HCV testing for high-risk groups. Our goal was to measure the impact of bundled HIV and HCV testing vs. HIV testing alone on test acceptance and identification of HCV and HIV.MethodsWe conducted a two-armed, randomized controlled trial on a convenience sample of 478 adult patients in the Jacobi Medical Center emergency department from December 2012 to May 2013. Participants were randomized to receive either an offer of bundled HIV/HCV testing or HIV testing alone. We compared the primary outcome, HIV test acceptance, between the two groups. Secondary outcomes included HIV and HCV prevalence, and HCV test acceptance, refusal, risk, and knowledge.ResultsWe found no significant difference in HIV test acceptance between the bundled HCV/HIV (91.8%) and HIV-only (90.6%) groups (p=0.642). There were also no significant differences in test acceptance based on gender, race, or ethnicity. A majority of participants (76.6%) reported at least one HCV risk factor. No participants tested positive for HIV, and one (0.5%) tested positive for HCV.ConclusionIntegrating bundled, rapid HCV/HIV testing into an established HIV testing program did not significantly impact HIV test acceptance. Future screening efforts for HCV could be integrated into current HIV testing models to target high-risk cohorts.