Browsing by Author "Murphy, Susan K"
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Item Open Access ACLY and ACC1 Regulate Hypoxia-Induced Apoptosis by Modulating ETV4 via α-ketoglutarate.(PLoS Genet, 2015-10) Keenan, Melissa M; Liu, Beiyu; Tang, Xiaohu; Wu, Jianli; Cyr, Derek; Stevens, Robert D; Ilkayeva, Olga; Huang, Zhiqing; Tollini, Laura A; Murphy, Susan K; Lucas, Joseph; Muoio, Deborah M; Kim, So Young; Chi, Jen-TsanIn order to propagate a solid tumor, cancer cells must adapt to and survive under various tumor microenvironment (TME) stresses, such as hypoxia or lactic acidosis. To systematically identify genes that modulate cancer cell survival under stresses, we performed genome-wide shRNA screens under hypoxia or lactic acidosis. We discovered that genetic depletion of acetyl-CoA carboxylase (ACACA or ACC1) or ATP citrate lyase (ACLY) protected cancer cells from hypoxia-induced apoptosis. Additionally, the loss of ACLY or ACC1 reduced levels and activities of the oncogenic transcription factor ETV4. Silencing ETV4 also protected cells from hypoxia-induced apoptosis and led to remarkably similar transcriptional responses as with silenced ACLY or ACC1, including an anti-apoptotic program. Metabolomic analysis found that while α-ketoglutarate levels decrease under hypoxia in control cells, α-ketoglutarate is paradoxically increased under hypoxia when ACC1 or ACLY are depleted. Supplementation with α-ketoglutarate rescued the hypoxia-induced apoptosis and recapitulated the decreased expression and activity of ETV4, likely via an epigenetic mechanism. Therefore, ACC1 and ACLY regulate the levels of ETV4 under hypoxia via increased α-ketoglutarate. These results reveal that the ACC1/ACLY-α-ketoglutarate-ETV4 axis is a novel means by which metabolic states regulate transcriptional output for life vs. death decisions under hypoxia. Since many lipogenic inhibitors are under investigation as cancer therapeutics, our findings suggest that the use of these inhibitors will need to be carefully considered with respect to oncogenic drivers, tumor hypoxia, progression and dormancy. More broadly, our screen provides a framework for studying additional tumor cell stress-adaption mechanisms in the future.Item Open Access AHRR Hypomethylation mediates the association between maternal smoking and metabolic profiles in children.(Hepatology communications, 2023-10) Vidal, Adriana C; Chandramouli, Shivram A; Marchesoni, Joddy; Brown, Nia; Liu, Yukun; Murphy, Susan K; Maguire, Rachel; Wang, Yaxu; Abdelmalek, Manal F; Mavis, Alisha M; Bashir, Mustafa R; Jima, Dereje; Skaar, David A; Hoyo, Cathrine; Moylan, Cynthia ABackground
Tobacco smoking during pregnancy is associated with metabolic dysfunction in children, but mechanistic insights remain limited. Hypomethylation of cg05575921 in the aryl hydrocarbon receptor repressor (AHRR) gene is associated with in utero tobacco smoke exposure. In this study, we evaluated whether AHRR hypomethylation mediates the association between maternal smoking and metabolic dysfunction in children.Methods
We assessed metabolic dysfunction using liver fat content (LFC), serum, and clinical data in children aged 7-12 years (n=78) followed since birth. Maternal smoking was self-reported at 12 weeks gestation. Methylation was measured by means of pyrosequencing at 3 sequential CpG sites, including cg05575921, at birth and at ages 7-12. Regression models were used to evaluate whether AHRR methylation mediated the association between maternal smoking and child metabolic dysfunction.Results
Average AHRR methylation at birth was significantly higher among children of nonsmoking mothers compared with children of mothers who smoked (69.8% ± 4.4% vs. 63.5% ± 5.5, p=0.0006). AHRR hypomethylation at birth was associated with higher liver fat content (p=0.01), triglycerides (p=0.01), and alanine aminotransferase levels (p=0.03), and lower HDL cholesterol (p=0.01) in childhood. AHRR hypomethylation significantly mediated associations between maternal smoking and liver fat content (indirect effect=0.213, p=0.018), triglycerides (indirect effect=0.297, p=0.044), and HDL cholesterol (indirect effect = -0.413, p=0.007). AHRR methylation in childhood (n=78) was no longer significantly associated with prenatal smoke exposure or child metabolic parameters (p>0.05).Conclusions
AHRR hypomethylation significantly mediates the association between prenatal tobacco smoke exposure and features of childhood metabolic dysfunction, despite the lack of persistent hypomethylation of AHRR into childhood. Further studies are needed to replicate these findings and to explore their causal and long-term significance.Item Open Access An epigenome-wide association study of child appetitive traits and DNA methylation.(Appetite, 2023-10) Harris, Holly A; Friedman, Chloe; Starling, Anne P; Dabelea, Dana; Johnson, Susan L; Fuemmeler, Bernard F; Jima, Dereje; Murphy, Susan K; Hoyo, Cathrine; Jansen, Pauline W; Felix, Janine F; Mulder, Rosa HThe etiology of childhood appetitive traits is poorly understood. Early-life epigenetic processes may be involved in the developmental programming of appetite regulation in childhood. One such process is DNA methylation (DNAm), whereby a methyl group is added to a specific part of DNA, where a cytosine base is next to a guanine base, a CpG site. We meta-analyzed epigenome-wide association studies (EWASs) of cord blood DNAm and early-childhood appetitive traits. Data were from two independent cohorts: the Generation R Study (n = 1,086, Rotterdam, the Netherlands) and the Healthy Start study (n = 236, Colorado, USA). DNAm at autosomal methylation sites in cord blood was measured using the Illumina Infinium HumanMethylation450 BeadChip. Parents reported on their child's food responsiveness, emotional undereating, satiety responsiveness and food fussiness using the Children's Eating Behaviour Questionnaire at age 4-5 years. Multiple regression models were used to examine the association of DNAm (predictor) at the individual site- and regional-level (using DMRff) with each appetitive trait (outcome), adjusting for covariates. Bonferroni-correction was applied to adjust for multiple testing. There were no associations of DNAm and any appetitive trait when examining individual CpG-sites. However, when examining multiple CpGs jointly in so-called differentially methylated regions, we identified 45 associations of DNAm with food responsiveness, 7 associations of DNAm with emotional undereating, 13 associations of DNAm with satiety responsiveness, and 9 associations of DNAm with food fussiness. This study shows that DNAm in the newborn may partially explain variation in appetitive traits expressed in early childhood and provides preliminary support for early programming of child appetitive traits through DNAm. Investigating differential DNAm associated with appetitive traits could be an important first step in identifying biological pathways underlying the development of these behaviors.Item Open Access Analysis of DNA methylation at birth and in childhood reveals changes associated with season of birth and latitude.(Clinical epigenetics, 2023-09) Kadalayil, Latha; Alam, Md Zahangir; White, Cory Haley; Ghantous, Akram; Walton, Esther; Gruzieva, Olena; Merid, Simon Kebede; Kumar, Ashish; Roy, Ritu P; Solomon, Olivia; Huen, Karen; Eskenazi, Brenda; Rzehak, Peter; Grote, Veit; Langhendries, Jean-Paul; Verduci, Elvira; Ferre, Natalia; Gruszfeld, Darek; Gao, Lu; Guan, Weihua; Zeng, Xuehuo; Schisterman, Enrique F; Dou, John F; Bakulski, Kelly M; Feinberg, Jason I; Soomro, Munawar Hussain; Pesce, Giancarlo; Baiz, Nour; Isaevska, Elena; Plusquin, Michelle; Vafeiadi, Marina; Roumeliotaki, Theano; Langie, Sabine AS; Standaert, Arnout; Allard, Catherine; Perron, Patrice; Bouchard, Luigi; van Meel, Evelien R; Felix, Janine F; Jaddoe, Vincent WV; Yousefi, Paul D; Ramlau-Hansen, Cecilia H; Relton, Caroline L; Tobi, Elmar W; Starling, Anne P; Yang, Ivana V; Llambrich, Maria; Santorelli, Gillian; Lepeule, Johanna; Salas, Lucas A; Bustamante, Mariona; Ewart, Susan L; Zhang, Hongmei; Karmaus, Wilfried; Röder, Stefan; Zenclussen, Ana Claudia; Jin, Jianping; Nystad, Wenche; Page, Christian M; Magnus, Maria; Jima, Dereje D; Hoyo, Cathrine; Maguire, Rachel L; Kvist, Tuomas; Czamara, Darina; Räikkönen, Katri; Gong, Tong; Ullemar, Vilhelmina; Rifas-Shiman, Sheryl L; Oken, Emily; Almqvist, Catarina; Karlsson, Robert; Lahti, Jari; Murphy, Susan K; Håberg, Siri E; London, Stephanie; Herberth, Gunda; Arshad, Hasan; Sunyer, Jordi; Grazuleviciene, Regina; Dabelea, Dana; Steegers-Theunissen, Régine PM; Nohr, Ellen A; Sørensen, Thorkild IA; Duijts, Liesbeth; Hivert, Marie-France; Nelen, Vera; Popovic, Maja; Kogevinas, Manolis; Nawrot, Tim S; Herceg, Zdenko; Annesi-Maesano, Isabella; Fallin, M Daniele; Yeung, Edwina; Breton, Carrie V; Koletzko, Berthold; Holland, Nina; Wiemels, Joseph L; Melén, Erik; Sharp, Gemma C; Silver, Matt J; Rezwan, Faisal I; Holloway, John WBackground
Seasonal variations in environmental exposures at birth or during gestation are associated with numerous adult traits and health outcomes later in life. Whether DNA methylation (DNAm) plays a role in the molecular mechanisms underlying the associations between birth season and lifelong phenotypes remains unclear.Methods
We carried out epigenome-wide meta-analyses within the Pregnancy And Childhood Epigenetic Consortium to identify associations of DNAm with birth season, both at differentially methylated probes (DMPs) and regions (DMRs). Associations were examined at two time points: at birth (21 cohorts, N = 9358) and in children aged 1-11 years (12 cohorts, N = 3610). We conducted meta-analyses to assess the impact of latitude on birth season-specific associations at both time points.Results
We identified associations between birth season and DNAm (False Discovery Rate-adjusted p values < 0.05) at two CpGs at birth (winter-born) and four in the childhood (summer-born) analyses when compared to children born in autumn. Furthermore, we identified twenty-six differentially methylated regions (DMR) at birth (winter-born: 8, spring-born: 15, summer-born: 3) and thirty-two in childhood (winter-born: 12, spring and summer: 10 each) meta-analyses with few overlapping DMRs between the birth seasons or the two time points. The DMRs were associated with genes of known functions in tumorigenesis, psychiatric/neurological disorders, inflammation, or immunity, amongst others. Latitude-stratified meta-analyses [higher (≥ 50°N), lower (< 50°N, northern hemisphere only)] revealed differences in associations between birth season and DNAm by birth latitude. DMR analysis implicated genes with previously reported links to schizophrenia (LAX1), skin disorders (PSORS1C, LTB4R), and airway inflammation including asthma (LTB4R), present only at birth in the higher latitudes (≥ 50°N).Conclusions
In this large epigenome-wide meta-analysis study, we provide evidence for (i) associations between DNAm and season of birth that are unique for the seasons of the year (temporal effect) and (ii) latitude-dependent variations in the seasonal associations (spatial effect). DNAm could play a role in the molecular mechanisms underlying the effect of birth season on adult health outcomes.Item Open Access Association between PEG3 DNA methylation and high-grade cervical intraepithelial neoplasia.(Infectious agents and cancer, 2021-06) Bosire, Claire; Vidal, Adriana C; Smith, Jennifer S; Jima, Dereje; Huang, Zhiqing; Skaar, David; Valea, Fidel; Bentley, Rex; Gradison, Margaret; Yarnall, Kimberly SH; Ford, Anne; Overcash, Francine; Murphy, Susan K; Hoyo, CathrineBackground
Epigenetic mechanisms are hypothesized to contribute substantially to the progression of cervical intraepithelial neoplasia (CIN) to cervical cancer, although empirical data are limited.Methods
Women (n = 419) were enrolled at colposcopic evaluation at Duke Medical Center in Durham, North Carolina. Human papillomavirus (HPV) was genotyped by HPV linear array and CIN grade was ascertained by biopsy pathologic review. DNA methylation was measured at differentially methylated regions (DMRs) regulating genomic imprinting of the IGF2/H19, IGF2AS, MESTIT1/MEST, MEG3, PLAGL1/HYMAI, KvDMR and PEG10, PEG3 imprinted domains, using Sequenom-EpiTYPER assays. Logistic regression models were used to evaluate the associations between HPV infection, DMR methylation and CIN risk overall and by race.Results
Of the 419 participants, 20 had CIN3+, 52 had CIN2, and 347 had ≤ CIN1 (CIN1 and negative histology). The median participant age was 28.6 (IQR:11.6) and 40% were African American. Overall, we found no statistically significant association between altered methylation in selected DMRs and CIN2+ compared to ≤CIN1. Similarly, there was no significant association between DMR methylation and CIN3+ compared to ≤CIN2. Restricting the outcome to CIN2+ cases that were HR-HPV positive and p16 staining positive, we found a significant association with PEG3 DMR methylation (OR: 1.56 95% CI: 1.03-2.36).Conclusions
While the small number of high-grade CIN cases limit inferences, our findings suggest an association between altered DNA methylation at regulatory regions of PEG3 and high grade CIN in high-risk HPV positive cases.Item Open Access Association of cord blood methylation fractions at imprinted insulin-like growth factor 2 (IGF2), plasma IGF2, and birth weight.(Cancer causes & control : CCC, 2012-04) Hoyo, Cathrine; Fortner, Kimberly; Murtha, Amy P; Schildkraut, Joellen M; Soubry, Adelheid; Demark-Wahnefried, Wendy; Jirtle, Randy L; Kurtzberg, Joanne; Forman, Michele R; Overcash, Francine; Huang, Zhiqing; Murphy, Susan KPurpose
Altered methylation at Insulin-like Growth Factor 2 (IGF2) regulatory regions has previously been associated with obesity, and several malignancies including colon, esophageal, and prostate adenocarcinomas, presumably via changes in expression and/or loss of imprinting, but the functional significance of these DNA methylation marks have not been demonstrated in humans. We examined associations among DNA methylation at IGF2 differentially methylated regions (DMRs), circulating IGF2 protein concentrations in umbilical cord blood (UCB) and birth weight in newborns.Methods
Questionnaire data were obtained from 300 pregnant women recruited between 2005 and 2009. UCB DNA methylation was measured by bisulfite pyrosequencing. UCB plasma concentrations of soluble IGF2 were measured by ELISA assays. Generalized linear regression models were used to examine the relationship between DMR methylation and IGF2 levels.Results
Lower IGF2 DMR methylation was associated with elevated plasma IGF2 protein concentrations (β = -9.87, p < 0.01); an association that was stronger in infants born to obese women (pre-pregnancy BMI > 30 kg/m(2), β = -20.21, p < 0.0001). Elevated IGF2 concentrations were associated with higher birth weight (p < 0.0001) after adjusting for maternal race/ethnicity, pre-pregnancy BMI, cigarette smoking, gestational diabetes, and infant sex. These patterns of association were not apparent at the H19 DMR.Conclusion
Our data suggest that variation in IGF2 DMR methylation is an important mechanism by which circulating IGF2 concentrations, a putative risk factor for obesity and cancers of the colon, esophagus, and prostate, are modulated; associations that may depend on pre-pregnancy obesity.Item Open Access Association of maternal prenatal copper concentration with gestational duration and preterm birth: a multicountry meta-analysis.(The American journal of clinical nutrition, 2024-01) Monangi, Nagendra K; Xu, Huan; Fan, Yue-Mei; Khanam, Rasheeda; Khan, Waqasuddin; Deb, Saikat; Pervin, Jesmin; Price, Joan T; Kaur, Lovejeet; INTERBIO-21st Study Consortium; Al Mahmud, Abdullah; Thanh, Le Quang; Care, Angharad; Landero, Julio A; Combs, Gerald F; Belling, Elizabeth; Chappell, Joanne; Chen, Jing; Kong, Fansheng; Lacher, Craig; Ahmed, Salahuddin; Chowdhury, Nabidul Haque; Rahman, Sayedur; Kabir, Furqan; Nisar, Imran; Hotwani, Aneeta; Mehmood, Usma; Nizar, Ambreen; Khalid, Javairia; Dhingra, Usha; Dutta, Arup; Ali, Said Mohamed; Aftab, Fahad; Juma, Mohammed Hamad; Rahman, Monjur; Ahmed, Tahmeed; Islam, M Munirul; Vwalika, Bellington; Musonda, Patrick; Ashorn, Ulla; Maleta, Kenneth; Hallman, Mikko; Goodfellow, Laura; Gupta, Juhi K; Alfirevic, Ana; Murphy, Susan K; Rand, Larry; Ryckman, Kelli K; Murray, Jeffrey C; Bahl, Rajiv; Litch, James A; Baruch-Gravett, Courtney; Sopory, Shailaja; Chandra Mouli Natchu, Uma; Kumar, Pavitra V; Kumari, Neha; Thiruvengadam, Ramachandran; Singh, Atul Kumar; Kumar, Pankaj; GARBH-Ini study team; Alfirevic, Zarko; Baqui, Abdullah H; Bhatnagar, Shinjini; Hirst, Jane E; Hoyo, Cathrine; Jehan, Fyezah; Jelliffe-Pawlowski, Laura; Rahman, Anisur; Roth, Daniel E; Sazawal, Sunil; Stringer, Jeffrey SA; Ashorn, Per; Zhang, Ge; Muglia, Louis JBackground
Copper (Cu), an essential trace mineral regulating multiple actions of inflammation and oxidative stress, has been implicated in risk for preterm birth (PTB).Objectives
This study aimed to determine the association of maternal Cu concentration during pregnancy with PTB risk and gestational duration in a large multicohort study including diverse populations.Methods
Maternal plasma or serum samples of 10,449 singleton live births were obtained from 18 geographically diverse study cohorts. Maternal Cu concentrations were determined using inductively coupled plasma mass spectrometry. The associations of maternal Cu with PTB and gestational duration were analyzed using logistic and linear regressions for each cohort. The estimates were then combined using meta-analysis. Associations between maternal Cu and acute-phase reactants (APRs) and infection status were analyzed in 1239 samples from the Malawi cohort.Results
The maternal prenatal Cu concentration in our study samples followed normal distribution with mean of 1.92 μg/mL and standard deviation of 0.43 μg/mL, and Cu concentrations increased with gestational age up to 20 wk. The random-effect meta-analysis across 18 cohorts revealed that 1 μg/mL increase in maternal Cu concentration was associated with higher risk of PTB with odds ratio of 1.30 (95% confidence interval [CI]: 1.08, 1.57) and shorter gestational duration of 1.64 d (95% CI: 0.56, 2.73). In the Malawi cohort, higher maternal Cu concentration, concentrations of multiple APRs, and infections (malaria and HIV) were correlated and associated with greater risk of PTB and shorter gestational duration.Conclusions
Our study supports robust negative association between maternal Cu and gestational duration and positive association with risk for PTB. Cu concentration was strongly correlated with APRs and infection status suggesting its potential role in inflammation, a pathway implicated in the mechanisms of PTB. Therefore, maternal Cu could be used as potential marker of integrated inflammatory pathways during pregnancy and risk for PTB.Item Open Access Associations between birth and one year anthropometric measurements and IGF2 and IGF2R genetic variants in African American and Caucasian American infants.(Journal of pediatric genetics, 2013-01) Vidal, Adriana C; Overcash, Francine; Murphy, Susan K; Murtha, Amy P; Schildkraut, Joellen M; Forman, Michele R; Demark-Wahnefried, Wendy; Kurtzberg, Joanne; Skaar, David; Jirtle, Randy L; Hoyo, CathrineInsulin-like growth factor 2 receptor (IGF2R) and insulin-like growth factor 2 (IGF2) genetic variants have been inconsistently associated with low birth weight and birth length in Caucasian and Asian infants, however few studies have included African Americans (AA). Generalized linear models and logistic regression models were used to examine associations between IGF2R single nucleotide polymorphisms (SNP) rs629849 and rs8191754, and IGF2 SNP rs680 and infant anthropometric measurements, in a racially diverse birth cohort in Durham County, North Carolina. Caucasian American (CA) carriers of the IGF2R SNP rs629849 were heavier (P = 0.02) and longer (P = 0.003) at birth, however body size at age 1 yr was similar to that of AA. Birth length significantly differed between carriers and non-carriers of the IGF2 rs680 variant in both AA (P = 0.04) and CA infants (P = 0.03). Both AA and CA carriers were 1 cm shorter at birth compared to non-carriers. We found no evidence for an association between rs8191754 and infant anthropometric measurements. Associations between SNPs andone year weight gain were only observed for rs680; CA infant carriers of rs680 variants weighed less than non-carriers at year one (P = 0.03); however, no associations were found in AA infants at year one. Larger studies using ancestral markers are required to disentangle these associations.Item Open Access Branched chain amino acid transaminase 1 (BCAT1) is overexpressed and hypomethylated in patients with non-alcoholic fatty liver disease who experience adverse clinical events: A pilot study.(PloS one, 2018-01) Wegermann, Kara; Henao, Ricardo; Diehl, Anna Mae; Murphy, Susan K; Abdelmalek, Manal F; Moylan, Cynthia ABackground and objectives
Although the burden of non-alcoholic fatty liver disease (NAFLD) continues to increase worldwide, genetic factors predicting progression to cirrhosis and decompensation in NAFLD remain poorly understood. We sought to determine whether gene expression profiling was associated with clinical decompensation and death in patients with NAFLD, and to assess whether altered DNA methylation contributes to these changes in gene expression.Methods
We performed a retrospective analysis of 86 patients in the Duke NAFLD Clinical Database and Biorepository with biopsy-proven NAFLD whose liver tissue was previously evaluated for gene expression and DNA methylation using array based technologies. We assessed the prospective development of liver and cardiovascular disease related outcomes, including hepatic decompensation as identified by the development of ascites, hepatic encephalopathy, hepatocellular carcinoma, or variceal bleeding as well as stroke and myocardial infarction via medical chart review.Results
Of the 86 patients, 47 had F0-F1 fibrosis and 39 had F3-F4 fibrosis at index liver biopsy. Gene expression probe sets (n = 54,675) were analyzed; 42 genes showed significant differential expression (p<0.05) and a two-fold change in expression between patients with and without any outcome. Two expression probes of the branched chain amino-acid transaminase 1 (BCAT1) gene were upregulated (p = 0.02; fold change 2.1 and 2.2 respectively) in patients with a clinical outcome. Methylation of three of the 34 BCAT1 CpG methylation probes were significantly inversely correlated with BCAT1 expression specific to the probes predictive of clinical deterioration.Conclusion
We found differential gene expression, correlated to changes in DNA methylation, at multiple BCAT1 loci in patients with cardiovascular outcomes and/or hepatic decompensation. BCAT1 catalyzes the transformation of alpha-ketoglutarate to glutamate and has been linked to the presence and severity of NAFLD, possibly through derangements in the balance between glutamate and alpha-ketoglutarate. Given the potential for BCAT1 to identify patients at risk for poor outcomes, and the potential therapeutic implications, these results should be validated in larger prospective studies.Item Embargo Breast cancer cells exhibit a non-linear proliferative dose response to progestins(2023) Dolan, EmmaThe steroid hormone progesterone has complex physiologic effects. In typical development and function, cells respond to progesterone in a dose- and tissue-specific manner. Despite the wide range of physiologic concentrations, canonical effects of progesterone have been characterized in the context of a high physiologic dose (10nM+), relevant during uterine cycling. This narrow focus has produced a gap in knowledge, particularly as it relates to the effects of post-menopausal low concentration progestins (0.1-0.3nM). Given that healthy tissues possess regulatory mechanisms to sense and respond to progesterone in a non-linear dose-specific manner, we hypothesized that breast malignancies would also display discontinuous dose-specific dynamic responses. Our results show that treatment with low dose progestins (0.1-0.3nM) drives proliferation in T47D human breast cancer cells, while high dose progestins (10nM+) inhibit proliferation. Using both unbiased and targeted approaches, we found that low dose progestins facilitate cell cycle entry by enhanced expression of CCND1 (cyclin D1) and SGK1 (serum and glucocorticoid related kinase 1), which are required for initiation of the downstream molecular cascade including phosphorylation of retinoblastoma protein (Rb) and expression E2F1. Expression of CCND1 and SGK1 mRNA are proximal responses to low dose progestin treatment, but transcriptional activation is not mediated by canonical progesterone receptor (PR) activity. Future work is needed to identify previously unexplored mechanisms of PR action in the context of low dose progestin treatments. In summary, these results challenge the assumption of dose response linearity to progestins and show unique functional and molecular effects of low dose progestin treatment. Of potential concern, our findings suggest that certain breast cancers, especially those expressing high levels of PR, may be accelerated by normal post-menopausal circulating concentrations of progestins (0.1-0.3nM). However, these findings also offer a sound rationale for the clinical therapeutic use of high dose progestins for patients with PR+ breast cancer.
Item Open Access Cannabinoid exposure and altered DNA methylation in rat and human sperm.(Epigenetics, 2018-01) Murphy, Susan K; Itchon-Ramos, Nilda; Visco, Zachary; Huang, Zhiqing; Grenier, Carole; Schrott, Rose; Acharya, Kelly; Boudreau, Marie-Helene; Price, Thomas M; Raburn, Douglas J; Corcoran, David L; Lucas, Joseph E; Mitchell, John T; McClernon, F Joseph; Cauley, Marty; Hall, Brandon J; Levin, Edward D; Kollins, Scott HLittle is known about the reproductive effects of paternal cannabis exposure. We evaluated associations between cannabis or tetrahydrocannabinol (THC) exposure and altered DNA methylation in sperm from humans and rats, respectively. DNA methylation, measured by reduced representation bisulfite sequencing, differed in the sperm of human users from non-users by at least 10% at 3,979 CpG sites. Pathway analyses indicated Hippo Signaling and Pathways in Cancer as enriched with altered genes (Bonferroni p < 0.02). These same two pathways were also enriched with genes having altered methylation in sperm from THC-exposed versus vehicle-exposed rats (p < 0.01). Data validity is supported by significant correlations between THC exposure levels in humans and methylation for 177 genes, and substantial overlap in THC target genes in rat sperm (this study) and genes previously reported as having altered methylation in the brain of rat offspring born to parents both exposed to THC during adolescence. In humans, cannabis use was also associated with significantly lower sperm concentration. Findings point to possible pre-conception paternal reproductive risks associated with cannabis use.Item Open Access Cannabis use and the sperm epigenome: a budding concern?(Environmental epigenetics, 2020-01) Schrott, Rose; Murphy, Susan KThe United States is swiftly moving toward increased legalization of medical and recreational cannabis. Currently considered the most commonly used illicit psychoactive drug, recreational cannabis is legal in 11 states and Washington, DC, and male use is an important and understudied concern. Questions remain, however, about the potential long-term consequences of this exposure and how cannabis might impact the epigenetic integrity of sperm in such a way that could influence the health and development of offspring. This review summarizes cannabis use and potency in the USA, provides a brief overview of DNA methylation as an epigenetic mechanism that is vulnerable in sperm to environmental exposures including cannabis, and summarizes studies that have examined the effects of parental exposure to cannabis or delta-9 tetrahydrocannabinol (THC, the main psychoactive component of cannabis) on the epigenetic profile of the gametes and behavior of offspring. These studies have demonstrated significant changes to the sperm DNA methylome following cannabis use in humans, and THC exposure in rats. Furthermore, the use of rodent models has shown methylation and behavioral changes in rats born to fathers exposed to THC or synthetic cannabinoids, or to parents who were both exposed to THC. These data substantiate an urgent need for additional studies assessing the effects of cannabis exposure on childhood health and development. This is especially true given the current growing state of cannabis use in the USA.Item Open Access Cannabis use is associated with potentially heritable widespread changes in autism candidate gene DLGAP2 DNA methylation in sperm.(Epigenetics, 2020-01) Schrott, Rose; Acharya, Kelly; Itchon-Ramos, Nilda; Hawkey, Andrew B; Pippen, Erica; Mitchell, John T; Kollins, Scott H; Levin, Edward D; Murphy, Susan KParental cannabis use has been associated with adverse neurodevelopmental outcomes in offspring, but how such phenotypes are transmitted is largely unknown. Using reduced representation bisulphite sequencing (RRBS), we recently demonstrated that cannabis use is associated with widespread DNA methylation changes in human and rat sperm. Discs-Large Associated Protein 2 (DLGAP2), involved in synapse organization, neuronal signaling, and strongly implicated in autism, exhibited significant hypomethylation (p < 0.05) at 17 CpG sites in human sperm. We successfully validated the differential methylation present in DLGAP2 for nine CpG sites located in intron seven (p < 0.05) using quantitative bisulphite pyrosequencing. Intron 7 DNA methylation and DLGAP2 expression in human conceptal brain tissue were inversely correlated (p < 0.01). Adult male rats exposed to delta-9-tetrahydrocannabinol (THC) showed differential DNA methylation at Dlgap2 in sperm (p < 0.03), as did the nucleus accumbens of rats whose fathers were exposed to THC prior to conception (p < 0.05). Altogether, these results warrant further investigation into the effects of preconception cannabis use in males and the potential effects on subsequent generations.Item Open Access Developmental nicotine exposure and masculinization of the rat preoptic area.(Neurotoxicology, 2022-03) Joglekar, Rashmi; Cauley, Marty; Lipsich, Taylor; Corcoran, David L; Patisaul, Heather B; Levin, Edward D; Meyer, Joel N; McCarthy, Margaret M; Murphy, Susan KNicotine is a neuroteratogenic component of tobacco smoke, e-cigarettes, and other products and can exert sex-specific effects in the developing brain, likely mediated through sex hormones. Estradiol modulates expression of nicotinic acetylcholine receptors in rats, and plays critical roles in neurodevelopmental processes, including sexual differentiation of the brain. Here, we examined the effects of developmental nicotine exposure on the sexual differentiation of the preoptic area (POA), a brain region that normally displays robust structural sexual dimorphisms and controls adult mating behavior in rodents. Using a rat model of gestational exposure, developing pups were exposed to nicotine (2 mg/kg/day) via maternal osmotic minipump (subcutaneously, sc) throughout the critical window for brain sexual differentiation. At postnatal day (PND) 4, a subset of offspring was analyzed for epigenetic effects in the POA. At PND40, all offspring were gonadectomized, implanted with a testosterone-releasing capsule (sc), and assessed for male sexual behavior at PND60. Following sexual behavior assessment, the area of the sexually dimorphic nucleus of the POA (SDN-POA) was measured using immunofluorescent staining techniques. In adults, normal sex differences in male sexual behavior and in the SDN-POA area were eliminated in nicotine-treated animals. Using novel analytical approaches to evaluate overall masculinization of the adult POA, we identified significant masculinization of the nicotine-treated female POA. In neonates (PND4), nicotine exposure induced trending alterations in methylation-dependent masculinizing gene expression and DNA methylation levels at sexually-dimorphic differentially methylated regions, suggesting that developmental nicotine exposure is capable of triggering masculinization of the rat POA via epigenetic mechanisms.Item Open Access Direct comparisons of bisulfite pyrosequencing versus targeted bisulfite sequencing.(microPublication biology, 2021-01) King, Dillon E; Sparling, A Clare; Joglekar, Rashmi; Meyer, Joel N; Murphy, Susan KDNA methylation is an important epigenetic mechanism involved in proper genome function. Bisulfite pyrosequencing (PSQ) is a commonly used technique to quantify DNA methylation. Although very accurate, bisulfite pyrosequencing can be expensive and time consuming for large-scale quantitative DNA methylation analysis at the single nucleotide level. High throughput DNA methylation sequencing has the potential to address these limitations, but its comparability to other methylation detection methods has not been well studied. We compared QIAseq Targeted Methyl Panel technologies (QMS) and PSQ by analyzing four CpG sites within four genes involved in neurodevelopment. QMS and PSQ had an average 5.6% difference in the detected level of DNA methylation for the same four CpG sites. However, we observed a strong correlation in the levels of methylation across all four CpG sites between the two technologies. These findings demonstrate the comparability of QMS relative to PSQ in the ability to accurately quantify DNA methylation at specific CpG sites.Item Open Access Distribution of HPV genotypes in cervical intraepithelial lesions and cervical cancer in Tanzanian women.(Infect Agent Cancer, 2011-11-14) Vidal, Adriana C; Murphy, Susan K; Hernandez, Brenda Y; Vasquez, Brandi; Bartlett, John A; Oneko, Olola; Mlay, Pendo; Obure, Joseph; Overcash, Francine; Smith, Jennifer S; van der Kolk, Mike; Hoyo, CathrineBACKGROUND: Infection with human papillomavirus (HPV) is associated with uterine cervical intraepithelial neoplasia (CIN) and invasive cancers (ICC). Approximately 80% of ICC cases are diagnosed in under-developed countries. Vaccine development relies on knowledge of HPV genotypes characteristic of LSIL, HSIL and cancer; however, these genotypes remain poorly characterized in many African countries. To contribute to the characterization of HPV genotypes in Northeastern Tanzania, we recruited 215 women from the Reproductive Health Clinic at Kilimanjaro Christian Medical Centre. Cervical scrapes and biopsies were obtained for cytology and HPV DNA detection. RESULTS: 79 out of 215 (36.7%) enrolled participants tested positive for HPV DNA, with a large proportion being multiple infections (74%). The prevalence of HPV infection increased with lesion grade (14% in controls, 67% in CIN1 cases and 88% in CIN2-3). Among ICC cases, 89% had detectable HPV. Overall, 31 HPV genotypes were detected; the three most common HPV genotypes among ICC were HPV16, 35 and 45. In addition to these genotypes, co-infection with HPV18, 31, 33, 52, 58, 68 and 82 was found in 91% of ICC. Among women with CIN2-3, HPV53, 58 and 84/83 were the most common. HPV35, 45, 53/58/59 were the most common among CIN1 cases. CONCLUSIONS: In women with no evidence of cytological abnormalities, the most prevalent genotypes were HPV58 with HPV16, 35, 52, 66 and 73 occurring equally. Although numerical constraints limit inference, findings that 91% of ICC harbor only a small number of HPV genotypes suggests that prevention efforts including vaccine development or adjuvant screening should focus on these genotypes.Item Open Access DNA Methylation in Babies Born to Nonsmoking Mothers Exposed to Secondhand Smoke during Pregnancy: An Epigenome-Wide Association Study.(Environmental health perspectives, 2021-05-19) Fuemmeler, Bernard F; Dozmorov, Mikhail G; Do, Elizabeth K; Zhang, Junfeng Jim; Grenier, Carole; Huang, Zhiqing; Maguire, Rachel L; Kollins, Scott H; Hoyo, Cathrine; Murphy, Susan KBackground
Maternal smoking during pregnancy is related to altered DNA methylation in infant umbilical cord blood. The extent to which low levels of smoke exposure among nonsmoking pregnant women relates to offspring DNA methylation is unknown.Objective
This study sought to evaluate relationships between maternal prenatal plasma cotinine levels and DNA methylation in umbilical cord blood in newborns using the Infinium HumanMethylation 450K BeadChip.Methods
Participants from the Newborn Epigenetics Study cohort who reported not smoking during pregnancy had verified low levels of cotinine from maternal prenatal plasma (0 ng/mL to <4 ng/mL), and offspring epigenetic data from umbilical cord blood were included in this study (n=79). Multivariable linear regression models were fit to the data, controlling for cell proportions, age, race, education, and parity. Estimates represent changes in response to any 1-ng/mL unit increase in exposure.Results
Multivariable linear regression models yielded 29,049 CpGs that were differentially methylated in relation to increases in cotinine at a 5% false discovery rate. Top CpGs were within or near genes involved in neuronal functioning (PRKG1, DLGAP2, BSG), carcinogenesis (FHIT, HSPC157) and inflammation (AGER). Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses suggest cotinine was related to methylation of gene pathways controlling neuronal signaling, metabolic regulation, cell signaling and regulation, and cancer. Further, enhancers associated with transcription start sites were enriched in altered CpGs. Using an independent sample from the same study population (n=115), bisulfite pyrosequencing was performed with infant cord blood DNA for two genes within our top 20 hits (AGER and PRKG1). Results from pyrosequencing replicated epigenome results for PRKG1 (cg17079497, estimate=-1.09, standard error (SE)=0.45, p=0.018) but not for AGER (cg09199225; estimate=-0.16, SE=0.21, p=0.44).Discussion
Secondhand smoke exposure among nonsmoking women may alter DNA methylation in regions involved in development, carcinogenesis, and neuronal functioning. These novel findings suggest that even low levels of smoke exposure during pregnancy may be sufficient to alter DNA methylation in distinct sites of mixed umbilical cord blood leukocytes in pathways that are known to be altered in cord blood from pregnant active smokers. https://doi.org/10.1289/EHP8099.Item Open Access Effect of Prenatal Smoke Exposure on Birth Weight: The Moderating Role of Maternal Depressive Symptoms.(Nicotine & tobacco research : official journal of the Society for Research on Nicotine and Tobacco, 2018-12-24) Schechter, Julia; Do, Elizabeth K; Zhang, Junfeng Jim; Hoyo, Cathrine; Murphy, Susan K; Kollins, Scott H; Fuemmeler, BernardIntroduction:Both prenatal smoke exposure and depression have been linked to lower birth weight, a risk factor for morbidity and mortality. Few studies have looked at the interaction between these risk factors and none have used a biomarker to objectively measure prenatal smoke exposure. The current study sought to examine independent and interactive effects of cotinine and depression on birth weight. The effect of race was also explored. Method:Data were drawn from a prospective study of pregnant women (N=568) in the southeastern U.S. Maternal demographic, health information, depressive symptoms, and birth data were collected via self-report and medical record abstraction. Prenatal blood samples were assayed for cotinine. Results:Controlling for covariates, multiple regression analyses indicated that both cotinine and depressive symptoms independently predicted lower birth weight and a significant interaction was also observed. Upon probing the interaction, a negative association between cotinine levels and birth weight was found in the context of higher depression but not lower depression scores. Similarly, logistic regression analyses revealed a significant interaction between cotinine and depression, such that cotinine predicted having a baby < 2500 g among women who fell above the indicated cut-off score. African American women had the highest levels of cotinine and lowest weight babies; however, race was not a significant moderator. Conclusions:Results suggest prenatal smoke exposure has a greater negative effect on birth weight for women endorsing co-occurring depressive symptoms. Findings can inform targeted interventions and assist medical providers with identifying women at increased risk for poor perinatal outcomes. Implications:Despite the common occurrence of smoking during pregnancy and prenatal depression, the interaction between these risk factors on birth weight has rarely been examined. Further, the extant results have been mixed, likely due in part to difficulties in measurement. The current study was the first to use prenatal cotinine to assess bias-free, continuous levels of prenatal smoke exposure. Results indicate that prenatal cotinine was a significant predictor of birth weight only in the context of maternal depressive symptoms. These findings have important implications for mitigating negative perinatal outcomes for pregnant women and their children.Item Open Access Epigenetic alterations in cytochrome P450 oxidoreductase (Por) in sperm of rats exposed to tetrahydrocannabinol (THC).(Scientific reports, 2020-07-23) Acharya, Kelly S; Schrott, Rose; Grenier, Carole; Huang, Zhiqing; Holloway, Zade; Hawkey, Andrew; Levin, Edward D; Murphy, Susan KAs marijuana legalization is increasing, research regarding possible long-term risks for users and their offspring is needed. Little data exists on effects of paternal tetrahydrocannabinol (THC) exposure prior to reproduction. This study determined if chronic THC exposure alters sperm DNA methylation (DNAm) and if such effects are intergenerationally transmitted. Adult male rats underwent oral gavage with THC or vehicle control. Differentially methylated (DM) loci in motile sperm were identified using reduced representation bisulfite sequencing (RRBS). Another cohort was injected with vehicle or THC, and sperm DNAm was analyzed. Finally, THC-exposed and control adult male rats were mated with THC-naïve females. DNAm levels of target genes in brain tissues of the offspring were determined by pyrosequencing. RRBS identified 2,940 DM CpGs mapping to 627 genes. Significant hypermethylation was confirmed (p < 0.05) following oral THC administration for cytochrome P450 oxidoreductase (Por), involved in toxin processing and disorders of sexual development. Por hypermethylation was not observed after THC injection or in the subsequent generation. These results support that THC alters DNAm in sperm and that route of exposure can have differential effects. Although we did not observe evidence of intergenerational transmission of the DNAm change, larger studies are required to definitively exclude this possibility.Item Open Access Epigenetic Regulation of Claudin-1 in the Development of Ovarian Cancer Recurrence and Drug Resistance.(Frontiers in oncology, 2021-01) Visco, Zachary R; Sfakianos, Gregory; Grenier, Carole; Boudreau, Marie-Helene; Simpson, Sabrina; Rodriguez, Isabel; Whitaker, Regina; Yao, Derek Y; Berchuck, Andrew; Murphy, Susan K; Huang, ZhiqingOver 21,000 women are diagnosed with ovarian cancer (OC) in the United States each year and over half that number succumb to this disease annually, often due to recurrent disease. A deeper understanding of the molecular events associated with recurrent disease is needed to identify potential targets. Using genome-scale DNA methylation and gene expression data for 16 matched primary-recurrent advanced stage serous epithelial OCs, we discovered that Claudin-1 (CLDN1), a tight junction protein, shows a stronger correlation between expression and methylation in recurrent versus primary OC at multiple CpG sites (R= -0.47 to -0.64 versus R= -0.32 to -0.57, respectively). An independent dataset showed that this correlation is stronger in tumors from short-term (<3y) survivors than in tumors from long-term (>7y) survivors (R= -0.41 to -0.46 versus R= 0.06 to -0.19, respectively). The presence of this inverse correlation in short-term survivors and recurrent tumors suggests an important role for this relationship and potential predictive value for disease prognosis. CLDN1 expression increased following pharmacologic inhibition of DNA methyltransferase activity (p< 0.001), thus validating the role of methylation in CLDN1 gene inhibition. CLDN1 knockdown enhanced chemosensitivity and suppressed cell proliferation, migration, and wound healing (p< 0.05). Stable CLDN1 knockdown in vivo resulted in reduced xenograft tumor growth but did not reach significance. Our results indicate that the relationship between CLDN1 methylation and expression plays an important role in OC aggressiveness and recurrence.
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