- Email: [email protected]
Regional-specific global cytosine methylation and DNA methyltransferase expression in the adult rat hippocampus
Neuroscience Letters 440 (2008) 49–53
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Regional-specific global cytosine methylation and DNA methyltransferase expression in the adult rat hippocampus Shelley E. Brown a , Ian C.G. Weaver b,1 , Michael J. Meaney b,∗,1 , Moshe Szyf a,∗∗,1 a b
Department of Pharmacology and Therapeutics, McGill University, 3655 Sir William Oslar Promenade, Montr´eal, Qu´ebec H3G 1Y6, Canada Douglas Hospital Research Center, 6875 LaSalle Blvd, Montr´eal, Qu´ebec H4H 1R3, Canada
a r t i c l e
i n f o
Article history: Received 9 December 2007 Received in revised form 30 April 2008 Accepted 7 May 2008 Keywords: DNMT-1 DNMT-3a/b DNA methylation Hippocampus Maternal care
a b s t r a c t Recent observations suggest that DNA methylation plays an important role in memory and long-term potentiation (LTP) in the hippocampus and is involved in programming the offspring epigenome in response to maternal care. Global DNA methylation is believed to be stable postnatally and to be similar across tissues in the adult mammal. It has also been a long held belief that DNA methyltransferases (DNMTs) play a very limited role in postmitotic tissues. Recent data suggests a more dynamic role for DNA methylation in the brain postnatally, therefore we examined the global state of methylation and the expression of the known DNMTs in the different regions of the hippocampus. We observed strikingly different levels of global methylation in the adult rat dentate gyrus (DG) and CA1 region in comparison with the CA2 and CA3 regions. mRNA levels of DNA methyltransferases exhibited similar regional specificity and were correlated with global DNA methylation levels. These regional differences in global methylation and expression of the DNA methylation machinery in the adult brain are consistent with the emerging hypothesis that DNA methylation may play a dynamic physiological role in the adult brain. Crown Copyright © 2008 Published by Elsevier Ireland Ltd. All rights reserved.
Mammalian DNA is modified by addition of a methyl moiety to cytosines, which in most cases reside in cytosine guanosine (CG) dinucleotide sequences [15]. While about 80% of the CG dinucleotide sequences are methylated, the distribution of methylated cytosines in the DNA is cell- and tissue-specific [26]. The DNA methylation pattern is sculpted during development by the action of DNA methyltransferases (DNMTs) and demethylases to generate a tissue-specific pattern of methylation [4]. It was generally believed that although gene-specific differences in methylation are observed between different somatic tissues, the global level of methylation is similar across tissues. In critical regulatory regions DNA methylation silences the expression of genes and was therefore proposed as a mechanism for the differential expression of tissue-specific genes [26], parental imprinting [28], X inactivation [27] and silencing of repetitive and viral parasitic sequences [39]. These functions are stable over the life span, and since such models served as the basis for our understanding of DNA methylation this epigenetic process was considered to be immutable except in cases of pathology. Moreover, DNMTs were believed to maintain the fixed
∗ Corresponding author. Tel: +1 514 761 6131x3938; fax: +1 514 762 3043. ∗∗ Corresponding author. Tel.: +1 514 398 7107; fax: +1 514 398 6690. E-mail addresses: [email protected] (M.J. Meaney), [email protected] (M. Szyf). 1 McGill Program for the Study of Behaviour, Genes and Environment.
pattern of methylation in somatic tissues after birth during cell division. These basic concepts of DNA methylation are challenged by a number of observations. First, DNMTs are expressed in nondividing neuronal cells [14] suggesting that DNMTs are not simply involved in copying the DNA methylation pattern during cell division. Second, the state of DNA methylation of certain genes undergoes rapid and gene-specific changes during long-term potentiation [18] and following contextual fear conditioning in postmitotic neurons [21]. Third, the glucocorticoid receptor (GR) gene in the hippocampus of an adult rat is hypermethylated in response to exogenous infusion of methionine, suggesting that the DNA methylation machinery is active and responsive in the adult postmitotic brain [36]. These findings suggest a role for DNMTs in neuronal plasticity in the adult animal. Three functional DNMTs have been identified. DNMT1 has a 5–30-fold preference for hemi-methylated substrates [38] and has been designated as a maintenance methyltransferase that copies the pattern of methylation from the paternal strand to the nascent strand during cell division. DNMT3A and DNMT3B show equal preference for unmethylated and hemi-methylated DNA in vitro [24], and were proposed as de novo methyltransferases. However, several studies indicate that maintenance of the state of methylation of certain sequences requires the presence of de novo DNMTs [19]. The maintenance of DNA methylation involves most probably a combination of targeting factors [10], chromatin structure [12],
0304-3940/$ – see front matter. Crown Copyright © 2008 Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.05.028
50
S.E. Brown et al. / Neuroscience Letters 440 (2008) 49–53
and a combination of DNMTs [19]. Though DNMT1, DNMT3A and DNMT3B have distinct functions, they are essential for mammalian brain development [23]. In the following studies, we examined the level of global DNA methylation and expression of the DNA methylation machinery in the hippocampus, a brain region that shows considerable plasticity in adulthood. We previously suggested that maternal care in early life influences epigenetic programming and DNA methylation in the adult rat hippocampus [35]. We therefore compared global methylation across hippocampal cell fields between adult rats exposed to high or low licking and grooming (LG) maternal care in the first week of postnatal life, and compared the levels of global DNA methylation to the levels of DNMT expression. The animals used in all studies were derived from Long-Evans hooded rats born in our colony from animals originally obtained ´ from Charles River Canada (St. Constant, Quebec). The animals were mated with males drawn randomly from a breeding stock maintained in our colony. In cases where the offspring of high or low LG mothers were used in studies, no more than two animals per group were drawn from any single litter. At the time of weaning on day 22 of life, pups were housed in same-sex, same litter groups of 3–4 animals per cage until day 45 of life, and two animals per cage from this point until adulthood. All procedures were performed according to guidelines developed by the Canadian Council on Animal Care and protocol approved by the McGill University Animal Care Committee. Maternal behavior was scored using a version of the procedure previously described [5]. The frequency of maternal licking/grooming and arched-back nursing across a large number of mothers is normally and not bi-modally distributed [5]. To define the high and low LG mothers for the current study we observed the maternal behavior in a cohort of 32 mothers and devised the group mean and standard deviation (S.D.) for each behavior over the first 10 days of life. High LG mothers were defined as females for which the frequency of pup LG was greater than 1 standard deviation (S.D.) above the mean. Low LG mothers were defined as females for which the frequency of pup LG was greater than 1 S.D. below the mean. Whole brains were removed from adult (90-day-old) male offspring (n = 6 animals/group) by rapid decapitation less than 1 min following their removal from the home cage. Coronal sections (16 m) corresponding to stereotaxic levels from −2.30 to −3.80 mm from bregma [25] were thaw-mounted onto gelatinsubbed slides and temporarily stored within the cryostat (−20 ◦ C). The slides were vacuum-dried within a desiccator (4 ◦ C, 14 h) and stored at −80 ◦ C. The immunohistochemistry was performed as previously described [36]. Sections were stained with the anti-5mC antibody or the anti-NeuN antibody (Chemicon). Visualization of 5-mC and NeuN labeling was performed using a Zeiss Axioplan 2 Imaging fluorescence microscope, equipped with a high-resolution color digital camera and connected to a computer running Zeiss Axiovision 4.1 Software. The appropriate filter combination and a 63 plan-apochromatic oil-immersion objective was used to capture images over the entire area of the dentate gyrus (DG), and CA1, CA2 and CA3 hippocampal regions of Ammon’s horn. A minimum of three images were taken for each hippocampal region per slide, with four slides examined in total per animal. These images were converted to Tiff format and imported into an MCID Elite image analysis system for quantification. Individual neuronal nuclei were quantified for the intensity of 5-mC staining and the average of the intensity of the cells in each region was averaged. Approximately 20 nuclei were quantified in each field. By averaging the intensity per nucleus we avoid the confounding factor of differences in cell densities in the different brain regions. The analysis was performed by an observer blind to the experimental conditions.
Samples were analyzed in triplicate using total hippocampal RNA preparations from 12 different animals (n = 6 animals/ treatment). RNA extraction and reverse-transcription (RT) was performed as previously described [2]. Samples were taken every two cycles from 16 to 25 cycles to ensure the amplification was in the linear range. The primer sequences are: DNMT1 sense, 5 aacggaacactctctctcactca-3 ; anti-sense, 5 -tcactgtccgacttgctcctc-3 . DNMT3A sense, 5 -cagcgtcacacagaagcatatcc-3 ; anti-sense, 5 -ggtcctcactttgctgaacttgg-3 . DNMT3B sense, 5 -gaatttgagcagcccaggttg3 ; anti-sense, 5 -tgaagaagagccttcctgtgcc-3 . Neuronal-specific -III tubulin sense, 5 -tgcgtgtgtacaggtgaatgc-3 ; anti-sense, 5 aggctgcatagtcatttccaag-3 . Quantification of the bands was determined by densitometry using the NIH Image program. To calculate the final signal for each sample, the ROD value of the band for each sample for the gene of interest (dnmt1, dnmt3a, and dnmt3b) was divided by the ROD value the corresponding neuronal-specific -III tubulin band. To examine whether regional differences in DNA methylation exist in the hippocampus and whether these changes are affected by maternal care, dorsal–hippocampal coronal sections from the adult offspring of high and low LG dams were analyzed using antibodies specific for 5-mC to assess genomic methylation levels in neurons. The tissue sections were analyzed thoroughly over the entire area of the dentate gyrus, and CA1, CA2, and CA3 sub-fields of Ammon’s horn (Fig. 1A). We used this method for measuring global DNA methylation since this is the only method that allows assessment of the state of methylation in neurons without contamination by the surrounding glial cells. Representative confocal microscopy images of 5-mC staining within the different regions from each treatment group are shown in Fig. 1A (panels i–viii). Neuronal cells were differentiated from glial cells by using an antibody directed against NeuN, a neuronal marker (Fig. 1B). 5-mC labeling intensity and background were consistent among slides. Individual neuronal nuclei were quantified for their 5-mC staining intensity to account for differences in cell number between the hippocampal regions. ANOVA results indicated a main effect of Region [F(3, 11) = 32.261, p < 0.001], but no significant effect of Group [high versus low LG mother; F(1, 21) = 4.092, p < 0.1] or Group × Region interaction effect [F(1, 3) = 2.094, p < 0.1]. Tukey’s HSD post hoc analysis revealed significantly greater 5-mC staining intensity within the DG (p < 0.009) and CA1 (p < 0.012) hippocampal regions in comparison to the CA2 and CA3 sub-fields of Ammon’s horn. Despite the trend for higher levels of methylation in the CA1 and DG of high LG animals, these differences were not statistically significant (p > 0.05). Although the staining intensity within the different regions was similar regardless of maternal care, differences in staining intensity between the different regions were observed in the adult hippocampus. Notably, the global methylation in the CA2 and CA3 regions was significantly lower than in the CA1 and DG. Our findings reveal a striking difference in global methylation between different regions across the hippocampus, which was not affected by early in life maternal care. The CA1 and DG regions differ in both cell density and cell type. We examined the spatial organization of the DNA methylation within the cells of these two hippocampal regions (Fig. 1C). While DNA methylation staining is distributed throughout the nucleus in the CA1-3 fields (Fig. 1C, upper panel, CA1 region shown), the nucleus of the DG granule cells shows focal 5-mC staining, forming punctate pockets of heterochromatin (Fig. 1C, lower panel). These differences in spatial organization of DNA methylation are surprising and suggest global differences in organization and packaging of the DNA between these two hippocampal regions. Since the changes in DNA methylation observed between CA1, DG, and CA2/3 regions were global, we reasoned that a global mechanism might be responsible for these differences. We therefore tested whether there are regional differences in expression of the
S.E. Brown et al. / Neuroscience Letters 440 (2008) 49–53
51
Fig. 1. Region-specific global DNA methylation and DNA methyltransferases mRNA in the rat hippocampus. (A) Confocal photomicrographs of representative 5-mC-positive neurons located within the CA1, CA2, CA3 hippocampal regions and dentate gyrus from adult high and low LG offspring (i–viii). Only large round nuclei corresponding to neuronal nuclei (indicated by arrows pointing downward) were included for analysis, partial or smaller nuclei (indicated by arrows pointing upward) were not included in the quantification. Scale bar, 20 M. Shown beneath is the mean ± S.E.M. number of 5-mC-positive nuclei counted from hippocampal samples (n = 6 animals/group with nine sections/animal). Tukey’s HSD post hoc analysis of CA1 versus CA2/3 (**p < 0.009), DG versus CA2/3 (*p < 0.012). (B) Confocal photomicrographs of representative NeuNpositive neurons located within the CA1, CA2, CA3 hippocampal regions and dentate gyrus from adult high LG offspring. (C) Confocal photomicrographs of representative 5-mC-positive neurons located within the CA1 and DG to illustrate the different organization of DNA methylation in these regions. The representative images are from high LG offspring, however no difference was observed between high and low LG offspring. 5-mC distribution in CA2 and CA3 regions were similar to CA1. (D) Mean ± S.E.M. DNA methyltransferases (DNMT-1, -3A and -3B) mRNA expression within the CA1, CA2–3, and DG. No difference was observed between high and low LG offspring. Student’s t-test analysis of CA1 versus CA2/3 (*p < 0.05), DG versus CA2/3 († p < 0.05), CA1 versus DG (§ p < 0.05), compared within groups.
DNMTs. To determine the levels of DNMTs in the different hippocampal regions, we examined the mRNA levels of dnmt-1, -3a and -3b. The hippocampus was dissected from the rat brain and further sectioned into the CA1, CA2/3, and the DG. Due to difficulty in cleanly separating the CA2 and CA3, and as these regions demonstrated similar levels and spatial organization of DNA methylation, these regions were dissected together. RNA was extracted and reverse transcribed using primers specific for the different isoforms of the dnmts. ANOVA results indicated a primary effect of Region [F(2, 5) = 8.76, p < 0.01] and a significant Region × Enzyme transcript interaction [F(2, 12) = 5.73, p < 0.05]. Thus our study reveals region-specific regulation of dnmt mRNA levels in the adult rat hippocampus. Post hoc analysis revealed that the levels of dnmt1 correlated with the overall levels of genomic DNA methylation, with
the highest levels of mRNA transcription observed in the CA1 and DG which are heavily methylated, while the lowest levels were detected in the CA2/3 (p < 0.05, CA2/3 versus CA1 or DG). Interestingly, the mRNA levels of dnmt3a and 3b displayed a complex relationship with dnmt1 mRNA levels and overall genomic DNA methylation. The mRNA levels of dnmt3a were highest in the DG, and lowest in the CA1 region, while the mRNA levels of dnmt3b displayed the opposite pattern, being highest in the CA1 and lowest in the DG (p < 0.05, CA1 versus DG). Interestingly, the level of dnmt3a in CA2/3 was comparable to the DG, suggesting DNMT3A may play a dominant role in regulating the levels of DNA methylation in this area of the hippocampus. Thus, the expression of dnmt mRNAs in the brain exhibits regional and isotypic specificity. The CA1 and DG regions, which are highly methylated relative to the CA2/3 region,
52
S.E. Brown et al. / Neuroscience Letters 440 (2008) 49–53
express two isotypes at higher levels than the CA2/3 region; dnmt1 and dnmt3b for CA1 and dnmt1 and dnmt3a for DG. Our study reveals region-specific variations in levels of global genomic DNA methylation and in expression of dnmt mRNAs across the dorsal hippocampus. The overall levels of DNA methylation were highest in the CA1 and dentate gyrus, and lowest in the CA2 and CA3 regions (Fig. 1A). However, the differences were not limited merely to the absolute levels of DNA methylation, but rather there were detectable differences in the spatial organization of the methylated DNA in the dentate gyrus as compared to cells in the Ammon’s horn (Fig. 1C). The idea that DNA methylation plays a role in nuclear organization and higher order chromatin architecture has been previously proposed based on data from cells with a knockout of DNA methylation enzymes [11]. Our data suggests that similar processes might be defining physiological and functional differences in the brain. Although we have previously shown that gene expression is significantly altered in the hippocampus of adult rats as a function of maternal care early in life [37], here we report the 5-mC staining intensity within the different sub-fields remained the same, regardless of maternal care. These findings remain consistent with our observations that suggest that alterations of cytosine methylation in the adult brain through maternal behavior are highly gene-specific and do not emerge as a function of a widespread alteration in the epigenome [36]. Additionally, most methylated CGs in the genome are found within repetititve sequences and not in promoter of genes, thus changes in methylation in the promoters of genes affected by maternal care may not affect the overall level of CG methylation. The differences in overall levels and spatial organization of methylated DNA reflect differences in cell type and physiological function. The DG is comprised of small tightly packed granule cells, whereas the CA1–3 contain larger and more widely separated pyramidal neurons and interneurons. Additionally, the dentate gyrus is a site of neurogenesis, even into adulthood [17]. Therefore the differences in spatial organization of DNA methylation between these different regions of the hippocampus might relate to differences in cell type and function. The CA1 differs from the CA2 and CA3 of the Ammon’s horn in their neuronal inputs, their role in learning and memory consolidation, and in their susceptibility to cytotoxicity. While the CA3 receives input from the dentate gyrus and layer II of the entorhinal cortex [30], the CA1 receives input not only from the CA3 through the trisynaptic circuit, but also from the subiculum. These differences in input may be associated with the differences in levels of DNA methylation. Synaptic transmission may cause changes in the epigenome; it was previously shown that MeCp2 binding to specific promoters may be affected through synaptic transmission [7], and changes in MeCp2 levels can affect the levels of global DNA methylation [1]. The CA1 and CA3 regions differ in their roles in contextual memory. The CA3 region is important for formation of the context while the CA1 is involved in memory consolidation [8]. Furthermore, recent studies have found that increased DNA methylation is crucial for memory consolidation [21]. Together the differences in DNA methylation levels between the CA1 and CA3 could be related to the differences in function. Previous studies have found that the levels of DNA methylation are coordinated with the levels of DNA methyltransferases [20,31]. Indeed, we found that mRNA levels of the DNA methyltransferases dnmt1, dnmt3a, and dnmt3b correlated with the levels of DNA methylation (Fig. 1D). Levels of dnmt1 correlated most closely with the overall levels of DNA methylation, with highest expression in the dentate gyrus and CA1 cell fields. Levels of dnmt3a and 3b expression were highest in the dentate gyrus and the CA1, respectively. Interestingly, the pattern of expression of dnmt3a is similar to
the previously published pattern of expression of the methylatedDNA binding domain protein MeCp2, which shows high levels of expression in the DG in the adult hippocampus as compared to the Ammon’s horn [3]. No regional differences in expression have been found for the other MBD proteins, with the exception of MBD3, which shows higher levels of expression in the CA1–4 as compared to the dentate gyrus during embryonic development, but these differences are lost in adulthood [16]. A number of studies have examined differential mRNA expression between the CA1 and CA3 regions of the hippocampus [13,32]. Several genes were found to have higher levels of expression in the CA3 as compared to the CA1, including AMPA receptors GluR1 and 2, and cytoskeletal genes ACTB and ␥-actin [13]. However, without further analysis of the methylation pattern of the specific promoters of these genes it cannot be determined whether their expression is correlated to the global DNA methylation levels observed in this study. It was thought that the DNA methylation pattern was established during development and maintained through cell divisions by maintenance methyltransferases. According to this model there would be no need for DNA methyltransferases in non-dividing cells such as neurons. However it is apparent in this study as well as others that DNA methyltransferases are present in postmitotic neurons [9,14]. This raises the possibility that the DNA methylation pattern is less static and more dynamic than previously believed. Recent studies have found that DNA methyltransferases require targeting through other enzymes to methylate target promoters [29,34]. As well, the DNA methylation pattern in the brain is modulated through environmental signals [35]. Therefore it is possible that the DNA methylation pattern is undergoing constant remodeling and requires the presence of DNA methyltransferases to allow adaptation at the level of the DNA methylation pattern in response to environmental cues. In dividing cells, the expression of DNMT1 is crucial for cell survival, as in its absence cells undergo arrest in G1/S [22], a DNA damage response is triggered [33], and the cells undergo mitotic catastrophe [6]. Although the changes observed with the lack of DNMT1 pertain to dividing cells, there could be an as yet unidentified role for DNA methyltransferases independent of its catalytic activity in non-dividing cells. Acknowledgements The authors thank Alain Niveleau for providing the anti-5methylcytosine antibody, and Jacynthe Laliberte´ for her assistance with the confocal microscopy. SEB and ICGW are supported by fellowships from the CIHR. This study was funded by grants from the NCIC awarded to MS and from the HFSP, CIHR and NICHD awarded to MS and MJM. References [1] A. Brero, H.P. Easwaran, D. Nowak, I. Grunewald, T. Cremer, H. Leonhardt, M.C. Cardoso, Methyl CpG-binding proteins induce large-scale chromatin reorganization during terminal differentiation, J. Cell Biol. 169 (2005) 733– 743. [2] S.E. Brown, M. Szyf, Epigenetic programming of the ribosomal RNA promoter by MBD3, Mol. Cell Biol. (2007). [3] S. Cassel, M.O. Revel, C. Kelche, J. Zwiller, Expression of the methyl-CpG-binding protein MeCP2 in rat brain. An ontogenetic study, Neurobiol. Dis. 15 (2004) 206–211. [4] H. Cedar, A. Razin, DNA methylation and development, Biochim. Biophys. Acta 1049 (1990) 1–8. [5] F.A. Champagne, D.D. Francis, A. Mar, M.J. Meaney, Variations in maternal care in the rat as a mediating influence for the effects of environment on development, Physiol. Behav. 79 (2003) 359–371. [6] T. Chen, S. Hevi, F. Gay, N. Tsujimoto, T. He, B. Zhang, Y. Ueda, E. Li, Complete inactivation of DNMT1 leads to mitotic catastrophe in human cancer cells, Nat. Genet. 39 (2007) 391–396.
S.E. Brown et al. / Neuroscience Letters 440 (2008) 49–53 [7] W.G. Chen, Q. Chang, Y. Lin, A. Meissner, A.E. West, E.C. Griffith, R. Jaenisch, M.E. Greenberg, Derepression of BDNF transcription involves calcium-dependent phosphorylation of MeCP2, Science 302 (2003) 885–889. [8] S. Daumas, H. Halley, B. Frances, J.M. Lassalle, Encoding, consolidation, and retrieval of contextual memory: differential involvement of dorsal CA3 and CA1 hippocampal subregions, Learn. Mem. 12 (2005) 375–382. [9] J. Deng, M. Szyf, Downregulation of DNA (cytosine-5-)methyltransferase is a late event in NGF-induced PC12 cell differentiation, Brain Res. Mol. Brain Res. 71 (1999) 23–31. [10] L. Di Croce, V.A. Raker, M. Corsaro, F. Fazi, M. Fanelli, M. Faretta, F. Fuks, F. Lo Coco, T. Kouzarides, C. Nervi, S. Minucci, P.G. Pelicci, Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor, Science 295 (2002) 1079–1082. [11] J. Espada, E. Ballestar, R. Santoro, M.F. Fraga, A. Villar-Garea, A. Nemeth, L. LopezSerra, S. Ropero, A. Aranda, H. Orozco, V. Moreno, A. Juarranz, J.C. Stockert, G. Langst, I. Grummt, W. Bickmore, M. Esteller, Epigenetic disruption of ribosomal RNA genes and nucleolar architecture in DNA methyltransferase 1 (Dnmt1) deficient cells, Nucleic Acids Res. 35 (2007) 2191–2198. [12] R.J. Gibbons, T.L. McDowell, S. Raman, D.M. O’Rourke, D. Garrick, H. Ayyub, D.R. Higgs, Mutations in ATRX, encoding a SWI/SNF-like protein, cause diverse changes in the pattern of DNA methylation, Nat. Genet. 24 (2000) 368–371. [13] S.D. Ginsberg, S. Che, Expression profile analysis within the human hippocampus: comparison of CA1 and CA3 pyramidal neurons, J. Comp. Neurol. 487 (2005) 107–118. [14] K. Goto, M. Numata, J.I. Komura, T. Ono, T.H. Bestor, H. Kondo, Expression of DNA methyltransferase gene in mature and immature neurons as well as proliferating cells in mice, Differentiation 56 (1994) 39–44. [15] Y. Gruenbaum, R. Stein, H. Cedar, A. Razin, Methylation of CpG sequences in eukaryotic DNA, FEBS Lett. 124 (1981) 67–71. [16] B.P. Jung, G. Zhang, R. Nitsch, J. Trogadis, S. Nag, J.H. Eubanks, Differential expression of methyl CpG-binding domain containing factor MBD3 in the developing and adult rat brain, J. Neurobiol. 55 (2003) 220–232. [17] M.S. Kaplan, J.W. Hinds, Neurogenesis in the adult rat: electron microscopic analysis of light radioautographs, Science 197 (1977) 1092–1094. [18] J.M. Levenson, T.L. Roth, F.D. Lubin, C.A. Miller, I.C. Huang, P. Desai, L.M. Malone, J.D. Sweatt, Evidence that DNA (cytosine-5) methyltransferase regulates synaptic plasticity in the hippocampus, J. Biol. Chem. 281 (2006) 15763–15773. [19] G. Liang, M.F. Chan, Y. Tomigahara, Y.C. Tsai, F.A. Gonzales, E. Li, P.W. Laird, P.A. Jones, Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements, Mol. Cell Biol. 22 (2002) 480–491. [20] D. Lucifero, S. La Salle, D. Bourc’his, J. Martel, T.H. Bestor, J.M. Trasler, Coordinate regulation of DNA methyltransferase expression during oogenesis, BMC Dev. Biol. 7 (2007) 36. [21] C.A. Miller, J.D. Sweatt, Covalent modification of DNA regulates memory formation, Neuron 53 (2007) 857–869. [22] S. Milutinovic, Q. Zhuang, A. Niveleau, M. Szyf, Epigenomic stress response. Knockdown of DNA methyltransferase 1 triggers an intra-S-phase arrest of DNA replication and induction of stress response genes, J. Biol. Chem. 278 (2003) 14985–14995.
53
[23] M. Okano, D.W. Bell, D.A. Haber, E. Li, DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development, Cell 99 (1999) 247–257. [24] M. Okano, S. Xie, E. Li, Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases, Nat. Genet. 19 (1998) 219– 220. [25] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, San Diego, 1996. [26] A. Razin, A.D. Riggs, DNA methylation and gene function, Science 210 (1980) 604–610. [27] A.D. Riggs, X inactivation, differentiation, and DNA methylation, Cytogenet. Cell Genet. 14 (1975) 9–25. [28] C. Sapienza, Parental imprinting of genes, Sci. Am. 263 (1990) 52–60. [29] A. Smallwood, P.O. Esteve, S. Pradhan, M. Carey, Functional cooperation between HP1 and DNMT1 mediates gene silencing, Genes Dev. 21 (2007) 1169–1178. [30] O. Steward, Topographic organization of the projections from the entorhinal area to the hippocampal formation of the rat, J. Comp. Neurol. 167 (1976) 285–314. [31] M. Szyf, K. Avraham-Haetzni, A. Reifman, J. Shlomai, F. Kaplan, A. Oppenheim, A. Razin, DNA methylation pattern is determined by the intracellular level of the methylase, Proc. Natl. Acad. Sci. U.S.A. 81 (1984) 3278–3282. [32] J.E. Torres-Munoz, C. Van Waveren, M.G. Keegan, R.J. Bookman, C.K. Petito, Gene expression profiles in microdissected neurons from human hippocampal subregions, Brain Res. Mol. Brain Res. 127 (2004) 105–114. [33] A. Unterberger, S.D. Andrews, I.C. Weaver, M. Szyf, DNA methyltransferase 1 knockdown activates a replication stress checkpoint, Mol. Cell Biol. 26 (2006) 7575–7586. [34] E. Vire, C. Brenner, R. Deplus, L. Blanchon, M. Fraga, C. Didelot, L. Morey, A. Van Eynde, D. Bernard, J.M. Vanderwinden, M. Bollen, M. Esteller, L. Di Croce, Y. de Launoit, F. Fuks, The Polycomb group protein EZH2 directly controls DNA methylation, Nature 439 (2006) 871–874. [35] I.C. Weaver, N. Cervoni, F.A. Champagne, A.C. D’Alessio, S. Sharma, J.R. Seckl, S. Dymov, M. Szyf, M.J. Meaney, Epigenetic programming by maternal behavior, Nat. Neurosci. 7 (2004) 847–854. [36] I.C. Weaver, F.A. Champagne, S.E. Brown, S. Dymov, S. Sharma, M.J. Meaney, M. Szyf, Reversal of maternal programming of stress responses in adult offspring through methyl supplementation: altering epigenetic marking later in life, J. Neurosci. 25 (2005) 11045–11054. [37] I.C. Weaver, M.J. Meaney, M. Szyf, Maternal care effects on the hippocampal transcriptome and anxiety-mediated behaviors in the offspring that are reversible in adulthood, Proc. Natl. Acad. Sci. U.S.A. 103 (2006) 3480– 3485. [38] J.A. Yoder, N.S. Soman, G.L. Verdine, T.H. Bestor, DNA (cytosine-5)methyltransferases in mouse cells and tissues. Studies with a mechanism-based probe, J. Mol. Biol. 270 (1997) 385–395. [39] J.A. Yoder, C.P. Walsh, T.H. Bestor, Cytosine methylation and the ecology of intragenomic parasites, Trends Genet. 13 (1997) 335–340.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Regional-specific global cytosine methylation and DNA methyltransferase expression in the adult rat hippocampus Shelley E. Brown a , Ian C.G. Weaver b,1 , Michael J. Meaney b,∗,1 , Moshe Szyf a,∗∗,1 a b
Department of Pharmacology and Therapeutics, McGill University, 3655 Sir William Oslar Promenade, Montr´eal, Qu´ebec H3G 1Y6, Canada Douglas Hospital Research Center, 6875 LaSalle Blvd, Montr´eal, Qu´ebec H4H 1R3, Canada
a r t i c l e
i n f o
Article history: Received 9 December 2007 Received in revised form 30 April 2008 Accepted 7 May 2008 Keywords: DNMT-1 DNMT-3a/b DNA methylation Hippocampus Maternal care
a b s t r a c t Recent observations suggest that DNA methylation plays an important role in memory and long-term potentiation (LTP) in the hippocampus and is involved in programming the offspring epigenome in response to maternal care. Global DNA methylation is believed to be stable postnatally and to be similar across tissues in the adult mammal. It has also been a long held belief that DNA methyltransferases (DNMTs) play a very limited role in postmitotic tissues. Recent data suggests a more dynamic role for DNA methylation in the brain postnatally, therefore we examined the global state of methylation and the expression of the known DNMTs in the different regions of the hippocampus. We observed strikingly different levels of global methylation in the adult rat dentate gyrus (DG) and CA1 region in comparison with the CA2 and CA3 regions. mRNA levels of DNA methyltransferases exhibited similar regional specificity and were correlated with global DNA methylation levels. These regional differences in global methylation and expression of the DNA methylation machinery in the adult brain are consistent with the emerging hypothesis that DNA methylation may play a dynamic physiological role in the adult brain. Crown Copyright © 2008 Published by Elsevier Ireland Ltd. All rights reserved.
Mammalian DNA is modified by addition of a methyl moiety to cytosines, which in most cases reside in cytosine guanosine (CG) dinucleotide sequences [15]. While about 80% of the CG dinucleotide sequences are methylated, the distribution of methylated cytosines in the DNA is cell- and tissue-specific [26]. The DNA methylation pattern is sculpted during development by the action of DNA methyltransferases (DNMTs) and demethylases to generate a tissue-specific pattern of methylation [4]. It was generally believed that although gene-specific differences in methylation are observed between different somatic tissues, the global level of methylation is similar across tissues. In critical regulatory regions DNA methylation silences the expression of genes and was therefore proposed as a mechanism for the differential expression of tissue-specific genes [26], parental imprinting [28], X inactivation [27] and silencing of repetitive and viral parasitic sequences [39]. These functions are stable over the life span, and since such models served as the basis for our understanding of DNA methylation this epigenetic process was considered to be immutable except in cases of pathology. Moreover, DNMTs were believed to maintain the fixed
∗ Corresponding author. Tel: +1 514 761 6131x3938; fax: +1 514 762 3043. ∗∗ Corresponding author. Tel.: +1 514 398 7107; fax: +1 514 398 6690. E-mail addresses: [email protected] (M.J. Meaney), [email protected] (M. Szyf). 1 McGill Program for the Study of Behaviour, Genes and Environment.
pattern of methylation in somatic tissues after birth during cell division. These basic concepts of DNA methylation are challenged by a number of observations. First, DNMTs are expressed in nondividing neuronal cells [14] suggesting that DNMTs are not simply involved in copying the DNA methylation pattern during cell division. Second, the state of DNA methylation of certain genes undergoes rapid and gene-specific changes during long-term potentiation [18] and following contextual fear conditioning in postmitotic neurons [21]. Third, the glucocorticoid receptor (GR) gene in the hippocampus of an adult rat is hypermethylated in response to exogenous infusion of methionine, suggesting that the DNA methylation machinery is active and responsive in the adult postmitotic brain [36]. These findings suggest a role for DNMTs in neuronal plasticity in the adult animal. Three functional DNMTs have been identified. DNMT1 has a 5–30-fold preference for hemi-methylated substrates [38] and has been designated as a maintenance methyltransferase that copies the pattern of methylation from the paternal strand to the nascent strand during cell division. DNMT3A and DNMT3B show equal preference for unmethylated and hemi-methylated DNA in vitro [24], and were proposed as de novo methyltransferases. However, several studies indicate that maintenance of the state of methylation of certain sequences requires the presence of de novo DNMTs [19]. The maintenance of DNA methylation involves most probably a combination of targeting factors [10], chromatin structure [12],
0304-3940/$ – see front matter. Crown Copyright © 2008 Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.05.028
50
S.E. Brown et al. / Neuroscience Letters 440 (2008) 49–53
and a combination of DNMTs [19]. Though DNMT1, DNMT3A and DNMT3B have distinct functions, they are essential for mammalian brain development [23]. In the following studies, we examined the level of global DNA methylation and expression of the DNA methylation machinery in the hippocampus, a brain region that shows considerable plasticity in adulthood. We previously suggested that maternal care in early life influences epigenetic programming and DNA methylation in the adult rat hippocampus [35]. We therefore compared global methylation across hippocampal cell fields between adult rats exposed to high or low licking and grooming (LG) maternal care in the first week of postnatal life, and compared the levels of global DNA methylation to the levels of DNMT expression. The animals used in all studies were derived from Long-Evans hooded rats born in our colony from animals originally obtained ´ from Charles River Canada (St. Constant, Quebec). The animals were mated with males drawn randomly from a breeding stock maintained in our colony. In cases where the offspring of high or low LG mothers were used in studies, no more than two animals per group were drawn from any single litter. At the time of weaning on day 22 of life, pups were housed in same-sex, same litter groups of 3–4 animals per cage until day 45 of life, and two animals per cage from this point until adulthood. All procedures were performed according to guidelines developed by the Canadian Council on Animal Care and protocol approved by the McGill University Animal Care Committee. Maternal behavior was scored using a version of the procedure previously described [5]. The frequency of maternal licking/grooming and arched-back nursing across a large number of mothers is normally and not bi-modally distributed [5]. To define the high and low LG mothers for the current study we observed the maternal behavior in a cohort of 32 mothers and devised the group mean and standard deviation (S.D.) for each behavior over the first 10 days of life. High LG mothers were defined as females for which the frequency of pup LG was greater than 1 standard deviation (S.D.) above the mean. Low LG mothers were defined as females for which the frequency of pup LG was greater than 1 S.D. below the mean. Whole brains were removed from adult (90-day-old) male offspring (n = 6 animals/group) by rapid decapitation less than 1 min following their removal from the home cage. Coronal sections (16 m) corresponding to stereotaxic levels from −2.30 to −3.80 mm from bregma [25] were thaw-mounted onto gelatinsubbed slides and temporarily stored within the cryostat (−20 ◦ C). The slides were vacuum-dried within a desiccator (4 ◦ C, 14 h) and stored at −80 ◦ C. The immunohistochemistry was performed as previously described [36]. Sections were stained with the anti-5mC antibody or the anti-NeuN antibody (Chemicon). Visualization of 5-mC and NeuN labeling was performed using a Zeiss Axioplan 2 Imaging fluorescence microscope, equipped with a high-resolution color digital camera and connected to a computer running Zeiss Axiovision 4.1 Software. The appropriate filter combination and a 63 plan-apochromatic oil-immersion objective was used to capture images over the entire area of the dentate gyrus (DG), and CA1, CA2 and CA3 hippocampal regions of Ammon’s horn. A minimum of three images were taken for each hippocampal region per slide, with four slides examined in total per animal. These images were converted to Tiff format and imported into an MCID Elite image analysis system for quantification. Individual neuronal nuclei were quantified for the intensity of 5-mC staining and the average of the intensity of the cells in each region was averaged. Approximately 20 nuclei were quantified in each field. By averaging the intensity per nucleus we avoid the confounding factor of differences in cell densities in the different brain regions. The analysis was performed by an observer blind to the experimental conditions.
Samples were analyzed in triplicate using total hippocampal RNA preparations from 12 different animals (n = 6 animals/ treatment). RNA extraction and reverse-transcription (RT) was performed as previously described [2]. Samples were taken every two cycles from 16 to 25 cycles to ensure the amplification was in the linear range. The primer sequences are: DNMT1 sense, 5 aacggaacactctctctcactca-3 ; anti-sense, 5 -tcactgtccgacttgctcctc-3 . DNMT3A sense, 5 -cagcgtcacacagaagcatatcc-3 ; anti-sense, 5 -ggtcctcactttgctgaacttgg-3 . DNMT3B sense, 5 -gaatttgagcagcccaggttg3 ; anti-sense, 5 -tgaagaagagccttcctgtgcc-3 . Neuronal-specific -III tubulin sense, 5 -tgcgtgtgtacaggtgaatgc-3 ; anti-sense, 5 aggctgcatagtcatttccaag-3 . Quantification of the bands was determined by densitometry using the NIH Image program. To calculate the final signal for each sample, the ROD value of the band for each sample for the gene of interest (dnmt1, dnmt3a, and dnmt3b) was divided by the ROD value the corresponding neuronal-specific -III tubulin band. To examine whether regional differences in DNA methylation exist in the hippocampus and whether these changes are affected by maternal care, dorsal–hippocampal coronal sections from the adult offspring of high and low LG dams were analyzed using antibodies specific for 5-mC to assess genomic methylation levels in neurons. The tissue sections were analyzed thoroughly over the entire area of the dentate gyrus, and CA1, CA2, and CA3 sub-fields of Ammon’s horn (Fig. 1A). We used this method for measuring global DNA methylation since this is the only method that allows assessment of the state of methylation in neurons without contamination by the surrounding glial cells. Representative confocal microscopy images of 5-mC staining within the different regions from each treatment group are shown in Fig. 1A (panels i–viii). Neuronal cells were differentiated from glial cells by using an antibody directed against NeuN, a neuronal marker (Fig. 1B). 5-mC labeling intensity and background were consistent among slides. Individual neuronal nuclei were quantified for their 5-mC staining intensity to account for differences in cell number between the hippocampal regions. ANOVA results indicated a main effect of Region [F(3, 11) = 32.261, p < 0.001], but no significant effect of Group [high versus low LG mother; F(1, 21) = 4.092, p < 0.1] or Group × Region interaction effect [F(1, 3) = 2.094, p < 0.1]. Tukey’s HSD post hoc analysis revealed significantly greater 5-mC staining intensity within the DG (p < 0.009) and CA1 (p < 0.012) hippocampal regions in comparison to the CA2 and CA3 sub-fields of Ammon’s horn. Despite the trend for higher levels of methylation in the CA1 and DG of high LG animals, these differences were not statistically significant (p > 0.05). Although the staining intensity within the different regions was similar regardless of maternal care, differences in staining intensity between the different regions were observed in the adult hippocampus. Notably, the global methylation in the CA2 and CA3 regions was significantly lower than in the CA1 and DG. Our findings reveal a striking difference in global methylation between different regions across the hippocampus, which was not affected by early in life maternal care. The CA1 and DG regions differ in both cell density and cell type. We examined the spatial organization of the DNA methylation within the cells of these two hippocampal regions (Fig. 1C). While DNA methylation staining is distributed throughout the nucleus in the CA1-3 fields (Fig. 1C, upper panel, CA1 region shown), the nucleus of the DG granule cells shows focal 5-mC staining, forming punctate pockets of heterochromatin (Fig. 1C, lower panel). These differences in spatial organization of DNA methylation are surprising and suggest global differences in organization and packaging of the DNA between these two hippocampal regions. Since the changes in DNA methylation observed between CA1, DG, and CA2/3 regions were global, we reasoned that a global mechanism might be responsible for these differences. We therefore tested whether there are regional differences in expression of the
S.E. Brown et al. / Neuroscience Letters 440 (2008) 49–53
51
Fig. 1. Region-specific global DNA methylation and DNA methyltransferases mRNA in the rat hippocampus. (A) Confocal photomicrographs of representative 5-mC-positive neurons located within the CA1, CA2, CA3 hippocampal regions and dentate gyrus from adult high and low LG offspring (i–viii). Only large round nuclei corresponding to neuronal nuclei (indicated by arrows pointing downward) were included for analysis, partial or smaller nuclei (indicated by arrows pointing upward) were not included in the quantification. Scale bar, 20 M. Shown beneath is the mean ± S.E.M. number of 5-mC-positive nuclei counted from hippocampal samples (n = 6 animals/group with nine sections/animal). Tukey’s HSD post hoc analysis of CA1 versus CA2/3 (**p < 0.009), DG versus CA2/3 (*p < 0.012). (B) Confocal photomicrographs of representative NeuNpositive neurons located within the CA1, CA2, CA3 hippocampal regions and dentate gyrus from adult high LG offspring. (C) Confocal photomicrographs of representative 5-mC-positive neurons located within the CA1 and DG to illustrate the different organization of DNA methylation in these regions. The representative images are from high LG offspring, however no difference was observed between high and low LG offspring. 5-mC distribution in CA2 and CA3 regions were similar to CA1. (D) Mean ± S.E.M. DNA methyltransferases (DNMT-1, -3A and -3B) mRNA expression within the CA1, CA2–3, and DG. No difference was observed between high and low LG offspring. Student’s t-test analysis of CA1 versus CA2/3 (*p < 0.05), DG versus CA2/3 († p < 0.05), CA1 versus DG (§ p < 0.05), compared within groups.
DNMTs. To determine the levels of DNMTs in the different hippocampal regions, we examined the mRNA levels of dnmt-1, -3a and -3b. The hippocampus was dissected from the rat brain and further sectioned into the CA1, CA2/3, and the DG. Due to difficulty in cleanly separating the CA2 and CA3, and as these regions demonstrated similar levels and spatial organization of DNA methylation, these regions were dissected together. RNA was extracted and reverse transcribed using primers specific for the different isoforms of the dnmts. ANOVA results indicated a primary effect of Region [F(2, 5) = 8.76, p < 0.01] and a significant Region × Enzyme transcript interaction [F(2, 12) = 5.73, p < 0.05]. Thus our study reveals region-specific regulation of dnmt mRNA levels in the adult rat hippocampus. Post hoc analysis revealed that the levels of dnmt1 correlated with the overall levels of genomic DNA methylation, with
the highest levels of mRNA transcription observed in the CA1 and DG which are heavily methylated, while the lowest levels were detected in the CA2/3 (p < 0.05, CA2/3 versus CA1 or DG). Interestingly, the mRNA levels of dnmt3a and 3b displayed a complex relationship with dnmt1 mRNA levels and overall genomic DNA methylation. The mRNA levels of dnmt3a were highest in the DG, and lowest in the CA1 region, while the mRNA levels of dnmt3b displayed the opposite pattern, being highest in the CA1 and lowest in the DG (p < 0.05, CA1 versus DG). Interestingly, the level of dnmt3a in CA2/3 was comparable to the DG, suggesting DNMT3A may play a dominant role in regulating the levels of DNA methylation in this area of the hippocampus. Thus, the expression of dnmt mRNAs in the brain exhibits regional and isotypic specificity. The CA1 and DG regions, which are highly methylated relative to the CA2/3 region,
52
S.E. Brown et al. / Neuroscience Letters 440 (2008) 49–53
express two isotypes at higher levels than the CA2/3 region; dnmt1 and dnmt3b for CA1 and dnmt1 and dnmt3a for DG. Our study reveals region-specific variations in levels of global genomic DNA methylation and in expression of dnmt mRNAs across the dorsal hippocampus. The overall levels of DNA methylation were highest in the CA1 and dentate gyrus, and lowest in the CA2 and CA3 regions (Fig. 1A). However, the differences were not limited merely to the absolute levels of DNA methylation, but rather there were detectable differences in the spatial organization of the methylated DNA in the dentate gyrus as compared to cells in the Ammon’s horn (Fig. 1C). The idea that DNA methylation plays a role in nuclear organization and higher order chromatin architecture has been previously proposed based on data from cells with a knockout of DNA methylation enzymes [11]. Our data suggests that similar processes might be defining physiological and functional differences in the brain. Although we have previously shown that gene expression is significantly altered in the hippocampus of adult rats as a function of maternal care early in life [37], here we report the 5-mC staining intensity within the different sub-fields remained the same, regardless of maternal care. These findings remain consistent with our observations that suggest that alterations of cytosine methylation in the adult brain through maternal behavior are highly gene-specific and do not emerge as a function of a widespread alteration in the epigenome [36]. Additionally, most methylated CGs in the genome are found within repetititve sequences and not in promoter of genes, thus changes in methylation in the promoters of genes affected by maternal care may not affect the overall level of CG methylation. The differences in overall levels and spatial organization of methylated DNA reflect differences in cell type and physiological function. The DG is comprised of small tightly packed granule cells, whereas the CA1–3 contain larger and more widely separated pyramidal neurons and interneurons. Additionally, the dentate gyrus is a site of neurogenesis, even into adulthood [17]. Therefore the differences in spatial organization of DNA methylation between these different regions of the hippocampus might relate to differences in cell type and function. The CA1 differs from the CA2 and CA3 of the Ammon’s horn in their neuronal inputs, their role in learning and memory consolidation, and in their susceptibility to cytotoxicity. While the CA3 receives input from the dentate gyrus and layer II of the entorhinal cortex [30], the CA1 receives input not only from the CA3 through the trisynaptic circuit, but also from the subiculum. These differences in input may be associated with the differences in levels of DNA methylation. Synaptic transmission may cause changes in the epigenome; it was previously shown that MeCp2 binding to specific promoters may be affected through synaptic transmission [7], and changes in MeCp2 levels can affect the levels of global DNA methylation [1]. The CA1 and CA3 regions differ in their roles in contextual memory. The CA3 region is important for formation of the context while the CA1 is involved in memory consolidation [8]. Furthermore, recent studies have found that increased DNA methylation is crucial for memory consolidation [21]. Together the differences in DNA methylation levels between the CA1 and CA3 could be related to the differences in function. Previous studies have found that the levels of DNA methylation are coordinated with the levels of DNA methyltransferases [20,31]. Indeed, we found that mRNA levels of the DNA methyltransferases dnmt1, dnmt3a, and dnmt3b correlated with the levels of DNA methylation (Fig. 1D). Levels of dnmt1 correlated most closely with the overall levels of DNA methylation, with highest expression in the dentate gyrus and CA1 cell fields. Levels of dnmt3a and 3b expression were highest in the dentate gyrus and the CA1, respectively. Interestingly, the pattern of expression of dnmt3a is similar to
the previously published pattern of expression of the methylatedDNA binding domain protein MeCp2, which shows high levels of expression in the DG in the adult hippocampus as compared to the Ammon’s horn [3]. No regional differences in expression have been found for the other MBD proteins, with the exception of MBD3, which shows higher levels of expression in the CA1–4 as compared to the dentate gyrus during embryonic development, but these differences are lost in adulthood [16]. A number of studies have examined differential mRNA expression between the CA1 and CA3 regions of the hippocampus [13,32]. Several genes were found to have higher levels of expression in the CA3 as compared to the CA1, including AMPA receptors GluR1 and 2, and cytoskeletal genes ACTB and ␥-actin [13]. However, without further analysis of the methylation pattern of the specific promoters of these genes it cannot be determined whether their expression is correlated to the global DNA methylation levels observed in this study. It was thought that the DNA methylation pattern was established during development and maintained through cell divisions by maintenance methyltransferases. According to this model there would be no need for DNA methyltransferases in non-dividing cells such as neurons. However it is apparent in this study as well as others that DNA methyltransferases are present in postmitotic neurons [9,14]. This raises the possibility that the DNA methylation pattern is less static and more dynamic than previously believed. Recent studies have found that DNA methyltransferases require targeting through other enzymes to methylate target promoters [29,34]. As well, the DNA methylation pattern in the brain is modulated through environmental signals [35]. Therefore it is possible that the DNA methylation pattern is undergoing constant remodeling and requires the presence of DNA methyltransferases to allow adaptation at the level of the DNA methylation pattern in response to environmental cues. In dividing cells, the expression of DNMT1 is crucial for cell survival, as in its absence cells undergo arrest in G1/S [22], a DNA damage response is triggered [33], and the cells undergo mitotic catastrophe [6]. Although the changes observed with the lack of DNMT1 pertain to dividing cells, there could be an as yet unidentified role for DNA methyltransferases independent of its catalytic activity in non-dividing cells. Acknowledgements The authors thank Alain Niveleau for providing the anti-5methylcytosine antibody, and Jacynthe Laliberte´ for her assistance with the confocal microscopy. SEB and ICGW are supported by fellowships from the CIHR. This study was funded by grants from the NCIC awarded to MS and from the HFSP, CIHR and NICHD awarded to MS and MJM. References [1] A. Brero, H.P. Easwaran, D. Nowak, I. Grunewald, T. Cremer, H. Leonhardt, M.C. Cardoso, Methyl CpG-binding proteins induce large-scale chromatin reorganization during terminal differentiation, J. Cell Biol. 169 (2005) 733– 743. [2] S.E. Brown, M. Szyf, Epigenetic programming of the ribosomal RNA promoter by MBD3, Mol. Cell Biol. (2007). [3] S. Cassel, M.O. Revel, C. Kelche, J. Zwiller, Expression of the methyl-CpG-binding protein MeCP2 in rat brain. An ontogenetic study, Neurobiol. Dis. 15 (2004) 206–211. [4] H. Cedar, A. Razin, DNA methylation and development, Biochim. Biophys. Acta 1049 (1990) 1–8. [5] F.A. Champagne, D.D. Francis, A. Mar, M.J. Meaney, Variations in maternal care in the rat as a mediating influence for the effects of environment on development, Physiol. Behav. 79 (2003) 359–371. [6] T. Chen, S. Hevi, F. Gay, N. Tsujimoto, T. He, B. Zhang, Y. Ueda, E. Li, Complete inactivation of DNMT1 leads to mitotic catastrophe in human cancer cells, Nat. Genet. 39 (2007) 391–396.
S.E. Brown et al. / Neuroscience Letters 440 (2008) 49–53 [7] W.G. Chen, Q. Chang, Y. Lin, A. Meissner, A.E. West, E.C. Griffith, R. Jaenisch, M.E. Greenberg, Derepression of BDNF transcription involves calcium-dependent phosphorylation of MeCP2, Science 302 (2003) 885–889. [8] S. Daumas, H. Halley, B. Frances, J.M. Lassalle, Encoding, consolidation, and retrieval of contextual memory: differential involvement of dorsal CA3 and CA1 hippocampal subregions, Learn. Mem. 12 (2005) 375–382. [9] J. Deng, M. Szyf, Downregulation of DNA (cytosine-5-)methyltransferase is a late event in NGF-induced PC12 cell differentiation, Brain Res. Mol. Brain Res. 71 (1999) 23–31. [10] L. Di Croce, V.A. Raker, M. Corsaro, F. Fazi, M. Fanelli, M. Faretta, F. Fuks, F. Lo Coco, T. Kouzarides, C. Nervi, S. Minucci, P.G. Pelicci, Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor, Science 295 (2002) 1079–1082. [11] J. Espada, E. Ballestar, R. Santoro, M.F. Fraga, A. Villar-Garea, A. Nemeth, L. LopezSerra, S. Ropero, A. Aranda, H. Orozco, V. Moreno, A. Juarranz, J.C. Stockert, G. Langst, I. Grummt, W. Bickmore, M. Esteller, Epigenetic disruption of ribosomal RNA genes and nucleolar architecture in DNA methyltransferase 1 (Dnmt1) deficient cells, Nucleic Acids Res. 35 (2007) 2191–2198. [12] R.J. Gibbons, T.L. McDowell, S. Raman, D.M. O’Rourke, D. Garrick, H. Ayyub, D.R. Higgs, Mutations in ATRX, encoding a SWI/SNF-like protein, cause diverse changes in the pattern of DNA methylation, Nat. Genet. 24 (2000) 368–371. [13] S.D. Ginsberg, S. Che, Expression profile analysis within the human hippocampus: comparison of CA1 and CA3 pyramidal neurons, J. Comp. Neurol. 487 (2005) 107–118. [14] K. Goto, M. Numata, J.I. Komura, T. Ono, T.H. Bestor, H. Kondo, Expression of DNA methyltransferase gene in mature and immature neurons as well as proliferating cells in mice, Differentiation 56 (1994) 39–44. [15] Y. Gruenbaum, R. Stein, H. Cedar, A. Razin, Methylation of CpG sequences in eukaryotic DNA, FEBS Lett. 124 (1981) 67–71. [16] B.P. Jung, G. Zhang, R. Nitsch, J. Trogadis, S. Nag, J.H. Eubanks, Differential expression of methyl CpG-binding domain containing factor MBD3 in the developing and adult rat brain, J. Neurobiol. 55 (2003) 220–232. [17] M.S. Kaplan, J.W. Hinds, Neurogenesis in the adult rat: electron microscopic analysis of light radioautographs, Science 197 (1977) 1092–1094. [18] J.M. Levenson, T.L. Roth, F.D. Lubin, C.A. Miller, I.C. Huang, P. Desai, L.M. Malone, J.D. Sweatt, Evidence that DNA (cytosine-5) methyltransferase regulates synaptic plasticity in the hippocampus, J. Biol. Chem. 281 (2006) 15763–15773. [19] G. Liang, M.F. Chan, Y. Tomigahara, Y.C. Tsai, F.A. Gonzales, E. Li, P.W. Laird, P.A. Jones, Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements, Mol. Cell Biol. 22 (2002) 480–491. [20] D. Lucifero, S. La Salle, D. Bourc’his, J. Martel, T.H. Bestor, J.M. Trasler, Coordinate regulation of DNA methyltransferase expression during oogenesis, BMC Dev. Biol. 7 (2007) 36. [21] C.A. Miller, J.D. Sweatt, Covalent modification of DNA regulates memory formation, Neuron 53 (2007) 857–869. [22] S. Milutinovic, Q. Zhuang, A. Niveleau, M. Szyf, Epigenomic stress response. Knockdown of DNA methyltransferase 1 triggers an intra-S-phase arrest of DNA replication and induction of stress response genes, J. Biol. Chem. 278 (2003) 14985–14995.
53
[23] M. Okano, D.W. Bell, D.A. Haber, E. Li, DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development, Cell 99 (1999) 247–257. [24] M. Okano, S. Xie, E. Li, Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases, Nat. Genet. 19 (1998) 219– 220. [25] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, San Diego, 1996. [26] A. Razin, A.D. Riggs, DNA methylation and gene function, Science 210 (1980) 604–610. [27] A.D. Riggs, X inactivation, differentiation, and DNA methylation, Cytogenet. Cell Genet. 14 (1975) 9–25. [28] C. Sapienza, Parental imprinting of genes, Sci. Am. 263 (1990) 52–60. [29] A. Smallwood, P.O. Esteve, S. Pradhan, M. Carey, Functional cooperation between HP1 and DNMT1 mediates gene silencing, Genes Dev. 21 (2007) 1169–1178. [30] O. Steward, Topographic organization of the projections from the entorhinal area to the hippocampal formation of the rat, J. Comp. Neurol. 167 (1976) 285–314. [31] M. Szyf, K. Avraham-Haetzni, A. Reifman, J. Shlomai, F. Kaplan, A. Oppenheim, A. Razin, DNA methylation pattern is determined by the intracellular level of the methylase, Proc. Natl. Acad. Sci. U.S.A. 81 (1984) 3278–3282. [32] J.E. Torres-Munoz, C. Van Waveren, M.G. Keegan, R.J. Bookman, C.K. Petito, Gene expression profiles in microdissected neurons from human hippocampal subregions, Brain Res. Mol. Brain Res. 127 (2004) 105–114. [33] A. Unterberger, S.D. Andrews, I.C. Weaver, M. Szyf, DNA methyltransferase 1 knockdown activates a replication stress checkpoint, Mol. Cell Biol. 26 (2006) 7575–7586. [34] E. Vire, C. Brenner, R. Deplus, L. Blanchon, M. Fraga, C. Didelot, L. Morey, A. Van Eynde, D. Bernard, J.M. Vanderwinden, M. Bollen, M. Esteller, L. Di Croce, Y. de Launoit, F. Fuks, The Polycomb group protein EZH2 directly controls DNA methylation, Nature 439 (2006) 871–874. [35] I.C. Weaver, N. Cervoni, F.A. Champagne, A.C. D’Alessio, S. Sharma, J.R. Seckl, S. Dymov, M. Szyf, M.J. Meaney, Epigenetic programming by maternal behavior, Nat. Neurosci. 7 (2004) 847–854. [36] I.C. Weaver, F.A. Champagne, S.E. Brown, S. Dymov, S. Sharma, M.J. Meaney, M. Szyf, Reversal of maternal programming of stress responses in adult offspring through methyl supplementation: altering epigenetic marking later in life, J. Neurosci. 25 (2005) 11045–11054. [37] I.C. Weaver, M.J. Meaney, M. Szyf, Maternal care effects on the hippocampal transcriptome and anxiety-mediated behaviors in the offspring that are reversible in adulthood, Proc. Natl. Acad. Sci. U.S.A. 103 (2006) 3480– 3485. [38] J.A. Yoder, N.S. Soman, G.L. Verdine, T.H. Bestor, DNA (cytosine-5)methyltransferases in mouse cells and tissues. Studies with a mechanism-based probe, J. Mol. Biol. 270 (1997) 385–395. [39] J.A. Yoder, C.P. Walsh, T.H. Bestor, Cytosine methylation and the ecology of intragenomic parasites, Trends Genet. 13 (1997) 335–340.