15626 • The Journal of Neuroscience, October 31, 2012 • 32(44):15626–15642 Behavioral/Systems/Cognitive The Signature of Maternal Rearing in the Methylome in Rhesus Macaque Prefrontal Cortex and T Cells Nadine Provenc¸al,1,2,4* Matthew J. Suderman,1,2,3* Claire Guillemin,1,2,4 Renaud Massart,2 Angela Ruggiero,11 Dongsha Wang,2,4 Allyson J. Bennett,8,10 Peter J. Pierre,9,10 David P. Friedman,10 Sylvana M. Coˆte´,4,5 Michael Hallett,3 Richard E. Tremblay,4,6,7 Stephen J. Suomi,11 and Moshe Szyf1,2 1Sackler Program for Epigenetics and Developmental Psychobiology, 2Department of Pharmacology and Therapeutics, and 3McGill Centre for Bioinformatics, McGill University, Montreal, Quebec, Canada, H3A 0G4, 4Research Unit on Children’s Psycho-Social Maladjustment and St-Justine Hospital Research Centre, 5School of Social and Preventive Medicine, and 6Department of Psychology and Pediatrics, University of Montreal, Montreal, Quebec, Canada, H3C 3J7, 7School of Public Health, Physiotherapy and Population Sciences, University College Dublin, Dublin 4, Ireland, 8Psychology Department and Harlow Center for Biological Psychology and 9Behavioral Management Unit, Wisconsin National Primate Research Center, University of Wisconsin, Madison, Wisconsin 53715, 10Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, and 11Laboratory of Comparative Ethology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-7971 Early-life adversity is associated with a broad scope of life-long health and behavioral disorders. Particularly critical is the role of the mother. A possible mechanism is that these effects are mediated by “epigenetic” mechanisms. Studies in rodents suggest a causal relationship between early-life adversity and changes in DNA methylation in several “candidate genes” in the brain. This study examines whether randomized differential rearing (maternal vs surrogate–peer rearing) of rhesus macaques is associated with differential meth- ylation in early adulthood. The data presented here show that differential rearing leads to differential DNA methylation in both prefrontal cortex and T cells. These differentially methylated promoters tend to cluster by both chromosomal region and gene function. The broad impact of maternal rearing on DNA methylation in both the brain and T cells supports the hypothesis that the response to early-life adversity is system-wide and genome-wide and persists to adulthood. Our data also point to the feasibility of studying the impact of the social environment in peripheral T-cell DNA methylation. Introduction response to early-life adversity. The main question is, what is the Early-life adverse social experiences in humans are associated mechanism that registers the response to early-life adversity and with an increased risk for developing psychiatric disorders, such has a life-long system-wide impact on multiple phenotypes? as anxiety and major depression (Kaufman et al., 2000; McEwen, It is now well established that genes are regulated by epigenetic 2000; Heim and Nemeroff, 2001), as well as increasing vulnera- information, including DNA methylation. There is an associa- bility to developing chronic disease during adulthood (Power et tion of childhood abuse with DNA methylation levels in the pro- al., 2007). The broad range of phenotypes suggests a system-wide moters of ribosomal RNA genes (McGowan et al., 2008) and exon 1f of the glucocorticoid receptor gene promoter (McGowan et al., 2009). Exposure of infant rats to stressed caretakers produced Received March 26, 2012; revised June 4, 2012; accepted June 25, 2012. Authorcontributions:N.P.,M.J.S.,S.J.S.,andM.S.designedresearch;N.P.,M.J.S.,C.G.,R.M.,A.R.,D.W.,andA.J.B. persisting changes in DNA methylation of the brain-derived performed research; A.J.B., P.J.P., D.P.F., S.M.C., M.H., R.E.T., S.J.S., and M.S. contributed unpublished reagents/ nerve growth factor gene promoter in the adult prefrontal cortex analytic tools; N.P., M.J.S., S.J.S., and M.S. analyzed data; N.P., M.J.S., A.J.B., P.J.P., D.P.F., M.H., R.E.T., S.J.S., and (PFC) (Roth et al., 2009). Early-life stress (Gra¨f et al., 2007) in M.S. wrote the paper. mice caused sustained DNA hypomethylation in the arginine va- This work was supported by a grant from the Canadian Institute of Health Research (M.S.), the Sackler Program sopressin gene (Murgatroyd et al., 2009). Although these data in Psychobiology and Epigenetics at McGill University (M.S.), a grant from the European Research Area Neuron Network(M.S.),andbyfundsfromtheDivisionofIntramuralResearch,EuniceKennedyShriverNationalInstitutesof support the hypothesis that DNA methylation is involved in reg- Child Health and Human Development (S.J.S.) and National Institute on Alcohol Abuse and Alcoholism (D.P.F.), istering early-life adversity in the DNA, they do not explain the National Institutes of Health. M.S. and S.J.S. are fellows of the Canadian Institute for Advanced Research. N.P. was range of phenotypes affected by child adversity. supported by a grant to R.E.T. from the Canadian Institute of Health Research. C.G. and D.W. were supported by There are critical challenges in expanding the initial findings grants to S.M.C. from FRSQ. *N.P. and M.J.S. contributed equally to this work. in rodents, in which causality could be tested, to humans. It is Correspondence should be addressed to either of the following: Stephen Suomi, Laboratory of Comparative impossible to randomize early-life stress in humans, and there- Ethology,EuniceKennedyShriverNationalInstituteofChildHealthandHumanDevelopment,NationalInstitutesof fore it is hard to tell whether the differences observed are driven Health, Department of Health and Human Services, Bethesda, MD 20892-7971, E-mail: [email protected]; by innate genetic differences or whether they are environmentally orMosheSzyf,DepartmentofPharmacologyandTherapeutics,McGillUniversity,3655PromonadeSirWilliamOsler, Montreal, Quebec, H3G 1Y6, Canada, E-mail: [email protected]. driven. Nonhuman primates offer several advantages in address- DOI:10.1523/JNEUROSCI.1470-12.2012 ing these challenges, including a close evolutionary and pheno- Copyright © 2012 the authors 0270-6474/12/3215626-17$15.00/0 typical relationship with humans. In the model examined here, Provenc¸al, Suderman et al. • Maternal Rearing in the Methylome of Rhesus Monkey J. Neurosci., October 31, 2012 • 32(44):15626–15642 • 15627 rhesus macaque monkeys are randomly assigned at birth to dif- effect. A craniotomy was performed to expose the cerebral cortex and ferential rearing conditions by either their mother or an inani- cerebellum, followed by a thoracotomy and perfusion through the left mate, cloth-covered surrogate. The surrogate rearing serves as a ventricle of the heart for 1.5 min with a chilled, oxygenated buffer solu- controlled and relevant early-life stressor. In nonhuman primate tion. The rest of the brainstem was then exposed, and the brain was models, maternal deprivation with some form of social contact removed within 5 min. Prefrontal tissue was flash frozen in isopentane at Ϫ55°C within 15 min of death and kept at Ϫ80°C until DNA/RNA disrupts the mother–infant relationship, leading to important extraction. The tissue consisted mostly of dorsolateral and ventrolateral emotional and social disturbances and behavioral abnormalities, PFC rostral to the caudal end of the arcuate sulcus, dorsal to the cingulate such as motor stereotypes (Suomi, 1991; Champoux et al., 2002; sulcus and lateral to the lateral orbital sulcus. PFC DNA was extracted Barr et al., 2003). Peer-reared macaques develop inadequate so- using the Qiagen DNeasy kit and RNA extraction using Trysol (Invitro- cial skills, are highly reactive and aggressive, and, as adults, show gen), both following the protocol of the manufacturer. increased voluntary alcohol consumption and typically rank at the bottom of the social dominance hierarchy (Suomi, 1991; Barr MeDIP and hybridization to microarrays et al., 2003). We examined here in parallel genome-wide pro- The MeDIP analysis was adapted from Keshet et al. (2006). Briefly, 2 ␮g moter methylation profiles from isolated T cells and from the of each of the T cells and PFC DNA were sonicated, and methylated DNA ␮ PFC of adult rhesus males subjected to maternal and surrogate was immunoprecipitated with 10 g of anti-5-methyl-cytosine (Calbio- rearing conditions after birth using the method of methylated chem). For the infant samples, the amount of T-cell DNA collected from the original 3 ml of blood was insufficient to perform one MeDIP per DNA immunoprecipitation (MeDIP) with comprehensive sample. Therefore, equal amounts of DNA from the six MR infants genome-wide promoter microarray hybridization, statistical (187.5 ng each) and the five SPR infants (300 ng each) were pooled to analysis, and false discovery rate (FDR) correction. We provide perform the MeDIP with 1.5 ␮g DNA of starting materials in each group. evidence that the impact of the social environment on DNA Before sonication, two control plasmids were added to the DNA (6 pg methylation is genome-wide and can be seen in brain as well as each): an unmethylated GFP plasmid and an in vitro methylated Lu- peripheral T cells. ciferase plasmid. The DNA–antibody complex was immunoprecipitated with 5 mg of protein A, and the methylated DNA was eluted with 150 ␮l of TE at 1.5% SDS. The input and bound fractions were then purified,