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Methylation of NMDA receptor NR2B gene as a function of age in the mouse brain
Neuroscience Letters 380 (2005) 223–228
Methylation of NMDA receptor NR2B gene as a function of age in the mouse brain C.R. Marutha Ravindran, Maharaj K. Ticku ∗ Department of Pharmacology, The University of Texas Health Science Center, San Antonio, TX 78229-3900, USA Received 30 November 2004; received in revised form 13 January 2005; accepted 14 January 2005
Abstract We have previously reported that there is an up-regulation of the NR2B gene expression in the adult cortex and cultured fetal cortical neurons of mice following chronic ethanol treatment due to demethylation of cytosine residues in the NR2B gene CpG island. In the present study, we investigated the methylation pattern of the NR2B CpG island as a function of the mouse age by digesting the cortex genomic DNA with HpaII enzyme, amplifying the interested regions by performing PCR and detecting the methylated regions by Southern hybridization so as to determine whether age affects the methylation process. We observed demethylation of various regions of NR2B gene (5227–5567), (5647–6003), (6091–6445), (6424–7024) of adult mouse cortex. Our results indicate that methylation of NR2B gene in the mouse brain is age-dependent phenomenon. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: NR2B gene; CpG; Demethylation; HpaII; PCR; Southern hybridization
N-Methyl-d-aspartate receptor (NMDAR) belongs to the subclass of ionotropic glutamate receptors and is widely distributed in the vertebrate brain. NMDAR mediates excitatory neurotransmission, synaptic plasticity and memory formation in the brain [6,24]. Three families of NMDA receptor subunits have been identified in the rat by molecular cloning: one NMDAR1 (NR1), four different NMDAR2 (NR2A-D) and one NMDAR3A (NR3A) [4,20,27,30]. The NR3 subunit was encoded on a single gene, whereas the NR2 subunit class (A–D) was encoded on four separate genes. The NR1 class is encoded on a single gene and molecularly diverse forms are created by alternative RNA splicing of three exons, there by generating as many as eight NR1 splice variants. The NR1 subunit, in association with the NR2 subunit, leads to the formation of highly active receptor channels [13,27]. However, unlike NR1, which is expressed constitutively, NR2B expression is highly regulated during development of the brain.
∗ Corresponding author. Tel.: +1 210 567 4255/4268; fax: +1 210 567 4226. E-mail address: [email protected] (M.K. Ticku).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.01.042
First discovered over 50 years ago in calf thymus [10], methylation of DNA occurs mostly at the 5 position of cytosine in higher eukaryotic cells. This plays an important role in the regulation of gene expression [2,5]. When DNA was analyzed for CG dinucleotide pairs, regions with higher proportions of CG sequences than would be expected were found to exist [26]. Such CG-rich regions are now referred as CpG islands, which constitute between 1 and 2% of the total genome and are associated with the promoters of ∼50% of all mammalian genes [1]. They are regions of ∼1 kb that differ from the rest of the genome by being G + C rich (65%) [2,3]. Our interest in this study stems from our previous observation that chronic ethanol treatment causes up-regulation of the NR2B gene [7–9,11,12,15]. In order to understand the molecular mechanisms underlying this up-regulation, we studied methylation in the CpG island following chronic ethanol treatment of cultured cortical neurons and observed that demethylation may be responsible for the changes in the expression of NR2B gene [23]. It has been reported that the global levels of DNA methylation tend to decrease with age in mouse [25,30]. In the present study, we investigated
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whether the methylation process in the NR2B gene is an agedependent phenomenon. Mice of different age groups (strain C57BL/6) such as 0, 15-, 30-, 45- and 60-day-old were purchased from Harlan (Indianapolis, IN). We consider 0 to 30 days old mice as younger ones and 45 to 60 days old mice as the adult group. They were used in accordance with institutional guidelines and procedures approved by the Animal Welfare Committee. Briefly, cerebral hemispheres were dissected from six mice of each age group separately and homogenized in an ice-cold lysis buffer (15 mM Tris–HCl pH 7.6, 60 mM KCl, 15 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and 0.5 mM spermidine) in the presence of 0.5% NP-40 which was added at the time of lysis. These samples were centrifuged (1000 × g, 10 min, 4 ◦ C) so as to extract the nuclei. These nuclei were suspended in a DNA extraction buffer (10 mM Tris–HCl pH 8.0, 10 mM EDTA pH 8.0, 0.5% SDS and fresh proteinase K 100 g/ml), and the DNA was isolated by standard procedures [29]. DNA (15 g) was digested overnight at 37 ◦ C with methyl-sensitive restriction endonucleases such as HpaII or MspI which is not sensitive to methylation. The regions of interest were amplified by PCR using primers 0.2 M (Table 1), dNTP 0.2 mM, 750 ng of template and the PCR conditions were as follows: 94 ◦ C for 4 min, 39 cycles of 93 ◦ C for 30 s, 50 ◦ C, 57 ◦ C, 63 ◦ C or 70 ◦ C for 30 s, 72 ◦ C for 2 min and 72 ◦ C for 8 min. Restriction fragments were separated by electrophoresis in 1.2% agarose gel using 1× TBE buffer and subsequently blotted onto nylon membranes (Roche, IN). The cerebral hemispheres were dissected as detailed above and homogenized in ice-cold lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1% Triton-X 100, 10 mM NaF, 1 mg/ml bacitracin, 50 mM -glycerophosphate, 1 mM phenyl methyl sulfonyl fluoride, 1 mM N-ethyl maleimide, 1 mM Na3 VO4 , 10 g/ml leupeptin). These samples were centrifuged (5000 rpm, 10 min, 4 ◦ C) so as to remove the supernatant containing protein. Seventy-five micrograms of protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) [21] and transferred to polyvinylidene difluoride membrane followed by treatment with 5% milk in Tris buffered saline
having 0.1% Tween 20 and overnight incubation with primary antibody NR2B [16,17] at a concentration of 1:5000 (Chemicon). Then the membrane was washed several times and peroxidase coupled secondary antibody (anti-mouse IgG/anti-rabbit IgG; New England Biolabs) was added and incubated for 1 h. The membrane was washed and specific bands were visualized using NBT/BCIP kit (Sigma). The M1 clone (NCBI accession number AF033356, 18) has the same sequence as that of the genomic DNA. Using this clone, we have amplified a long fragment of 2239 bp in size, and employing this fragment as a template, we amplified the regions of interest by PCR utilizing the primers as described in Table 1. These regions were selected randomly based on the number of sites present for digestion by the enzyme HpaII. The amplified fragments of DNA were separated by electrophoresis as detailed earlier, and the products were cloned in TOPO vector using the TOPO cloning kit (Invitrogen, CA). The clones were identified by colony PCR; and the clones were inoculated in LB medium containing ampicillin 100 g/ml and incubated at 37 ◦ C overnight. After sufficient growth, the cultures were harvested and the plasmids were isolated using the Qiagen plasmid midi kit (Qiagen Inc, CA). Further, the plasmids were digested with EcoRI enzyme, and the restriction fragments were separated by electrophoresis in 1.2% agarose gel containing 1× TBE buffer. After electrophoresis, the cloned products of DNA was cut down under UV light using the sterile single use stainless surgical blades (Maersk Medical Ltd, Sheffield); the plasmids were extracted using the QIA quick gel extraction kit (50) (Qiagen Inc) protocol; and the probes were prepared for Southern blot analysis which was performed as described by Sambrook et al. [29]. Southern blots were hybridized with (␣32 P) dCTP-labeled probes (Amersham-Pharmacia Biotec) in Quick HybR hybridization solution (Stratagene) and further processed as recommended by the supplier. Blots were exposed on an X-ray film based on the intensity of the bands at −70 ◦ C. Initially we identified the methylated sites in the cerebral cortex of the newly born mice. The genomic map and the sequences of the NR2B gene in which we studied methyla-
Table 1 Primers used for amplification in PCR Enzyme used for digesting the genomic DNA
HpaII
Primer sequences
Annealing temperature used (◦ C)
Size (bp)
(1) 5 -GTG AGG GGG TGA TGG AGG GGG TTT GG-3 5 -GGC AAG TCA AGC GTT CGG CAG CAG GAG AGG-3 Sequences amplified from 5227 to 5567 (2) 5 -TTT TTG AGA AAG GAA GGT-3 5 -ATT TCT CCA AAC CCT CAA AAT T-3 Sequences amplified from 5647 to 6003 (3) 5 -TAT TTT GGA TGG TTT TTT ATT TTT-3 5 -GCT CAA TGG GTT CTG ATT GTG C-3 Sequences amplified from 6091 to 6445 (4) 5 -GCA CAA TCA GAA CCC ATT GAG C-3 5 -GCT TTA GTA GAG GAG TCT CTG TGT GG-3 Sequences amplified from 6424 to 7024
70
340
50
356
57
354
63
600
C.R.M. Ravindran, M.K. Ticku / Neuroscience Letters 380 (2005) 223–228
Fig. 1. Effect of methylation pattern of the NR2B gene CpG island as a function of age of the mice in the region (5227–5567). (a) Lane 1: 100 bp ladder; lane 2: PCR product of the newly born mice; lane 3: 15-day-old mice; lane 4: 30-day-old mice; lane 5: 45-day-old mice; lane 6: 60-dayold mice; and lane 7: 1 kb ladder. (b) Southern hybridization with (␣32 P) dCTP-labeled probes. The X-ray film was exposed for 30 min at −70 ◦ C. Lane 1: newly born mice; lane 2: 15-day-old mice; lane 3: 30-day-old mice; lane 4: 45-day-old mice; and lane 5: 60-day-old mice. (c) Percentage of methylation in the region (5227–5567) of different age groups of mice in the NR2B gene CpG islands. Percentage methylation was measured by scanning and comparing the intensity of each band. Each value is % mean ± S.E.M. of three individual experiments. The symbol (* ) significant and p < 0.05 as compared to the control (newly born mice).
tion of different age groups of mice are given in our previous reports [23]. We identified the multiple sites methylated in the NR2B gene for HpaII enzyme. We observed complete digestion of the genomic DNA using the HpaII enzyme. The genomic DNA isolated from the cerebral cortex of the newly born mice was digested with HpaII enzyme (3 units/g DNA) and various regions of the DNA were amplified using different sets of primers for NR2B gene by performing PCR (Table 1). Using PCR to study methylation we require a very small amount of genomic DNA than restriction digestion. In fact, PCR is inherently more sensitive than restriction digestion and therefore PCR will always be able to find some template to amplify. Hence, we studied methylation in this study by using PCR. Following PCR, we identified methylation in the region (5227–5567), which yielded the DNA fragments of 340 bp in size, and it was consistent with the anticipated region studied (Fig. 1a). These experiments indicated that the region (5227–5567) was methylated, and in this region two sites for digestion by the enzyme HpaII were present. Following identification of the methylated site in the above region, we amplified the remaining regions of the NR2B gene in the cortex. Region (5647–6003) yielded the DNA
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Fig. 2. Effect of methylation pattern of the NR2B gene CpG island as a function of age of the mice in the region (5647–6003). (a) Lane 1: 100 bp ladder; lane 2: PCR product of the newly born mice; lane 3: 15-day-old mice; lane 4: 30-day-old mice; lane 5: 45-day-old mice; and lane 6: 60-day-old mice. (b) Southern hybridization with (␣32 P) dCTP-labeled probes. The Xray film was exposed for 5 min at −70 ◦ C. Lane 1: newly born mice; lane 2: 15-day-old mice; lane 3: 30-day-old mice; lane 4: 45-day-old mice; and lane 5: 60-day-old mice. (c) Percentage of methylation in the region (5647–6003) of different age groups of mice in the NR2B gene CpG islands. Percentage methylation was measured by scanning and comparing the intensity of each band. Each value is % mean ± S.E.M. of three individual experiments. The symbol (* ) significant, p < 0.05 as compared to the control (newly born mice).
fragments of 356 bp in size (Fig. 2a), region (6091–6445) yielded the DNA fragments of 354 bp in size (Fig. 3a), and region (6424–7024) yielded the DNA fragments of 600 bp in size (Fig. 4a). These regions for amplification by PCR were selected randomly based on the number of sites present in the genomic DNA for digestion by the enzyme HpaII. The amplified regions were also matched with the regions studied in the present study. These results indicated that all the above regions were also methylated and in these regions 2 to 3 sites for digestion by the enzyme HpaII were present in the cerebral cortex of the mice. Further, to study the methylation in different age groups of the mice, we selected randomly 15-, 30-, 45- and 60-dayold mice, dissected the cerebral cortex, isolated and digested the genomic DNA with HpaII enzyme and as detailed earlier amplified the above regions using different sets of primers (Table 1). The region (5227–5567) yielded the DNA fragments of 340 bp in size, region (5647–6003) yielded 356 bp of DNA fragments, region (6091–6445) yielded 354 bp of
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Fig. 3. Effect of methylation pattern of the NR2B gene CpG island as a function of age of the mice in the region (6091–6445). (a) Lane 1: 100 bp ladder; lane 2: PCR product of the newly born mice; lane 3: 15-day-old mice; lane 4: 30-day-old mice; lane 5: 45-day-old mice; lane 6: 60-day-old mice; and lane 7: 1 kb ladder. (b) Southern hybridization with (␣32 P) dCTP-labeled probes. The X-ray film was exposed for 5 min at −70 ◦ C. Lane 1: newly born mice; lane 2: 15-day-old mice; lane 3: 30-day-old mice; lane 4: 45-day-old mice; and lane 5: 60-day-old mice. (c) Percentage of methylation in the region (6091–6445) of different age groups of mice in the NR2B gene CpG islands. Percentage methylation was measured by scanning and comparing the intensity of each band. Each value is % mean ± S.E.M. of three individual experiments. (* ) significant and p < 0.05 as compared to the control (newly born mice).
DNA fragments, and the region (6424–7024) yielded 600 bp of DNA fragments in all the age groups similar to the newly born mice (Figs. 1a, 2a, 3a and 4a). Interestingly, the increase in age reduced the intensity of the bands, which indicated the demethylation of the NR2B gene CpG islands in the cerebral cortex of adult mice in all the regions (Figs. 1a, 2a, 3a and 4a). To identify the intensity of the bands in the amplified regions, we used the probes prepared from the M1 clone by Southern hybridization. As expected we observed the presence of very thick bands in all the above regions of the younger groups of mice. Only in the adult age groups (45 and 60 days) of mice, we observed the presence of faint bands as revealed by Southern hybridization, which indicated demethylation of the CpG island (Figs. 1b, 2b, 3b and 4b). Furthermore, the % of methylation in all the age groups of mice was measured by scanning and comparing the intensity of each band. Percentage of methylation in the control (newly born mice) was considered as 100% and the variation
Fig. 4. Effect of methylation pattern of the NR2B gene CpG island as a function of age of the mice in the region (6424–7024). (a) Lane 1: 1 kb ladder; lane 2: PCR product of the newly born mice; lane 3: 15-day-old mice; lane 4: 30-day-old mice; lane 5: 45-day-old mice; lane 6: 60-dayold mice; and lane 7: 100 bp ladder. (b) Southern hybridization with (␣32 P) dCTP-labeled probes. The X-ray film was exposed for 5 min at −70 ◦ C. Lane 1: newly born mice; lane 2: 15-day-old mice; lane 3: 30-day-old mice; lane 4: 45-day-old mice; and lane 5: 60-day-old mice. (c) Percentage of methylation in the region (6424–7024) of different age groups of mice in the NR2B gene CpG islands. Percentage methylation was measured by scanning and comparing the intensity of each band. Each value is % mean ± S.E.M. of three individual experiments. The symbol (* ) significant and p < 0.05 as compared to the control (newly born mice).
in the intensity of the band in each blot was given as ±S.D. values. Then the intensity of the band in the control was compared with the intensity of the band in the 15-, 30-, 45and 60-day-old mice (Figs. 1c, 2c, 3c and 4c). Data were analyzed by Student’s t test. Differences were considered to be statistically significant when p < 0.05. Further to show that PCR amplification is constant, we digested the genomic DNA with the enzyme MspI which is not sensitive to methylation as a control and amplified the region (6424–7024) yielded 600 bp of DNA fragments in size similarly. Interestingly, in this case there was no change in the intensity of the band observed following amplification in various age groups of mice (Fig. 5).
Fig. 5. Effect of methylation pattern of the NR2B gene CpG island as a function of age of the mice in the region (6424–7024) by digesting the genomic DNA with MspI. Lane 1: 100 bp ladder; lane 2: PCR product of the newly born mice; lane 3: 15-day-old mice; lane 4: 30-day-old mice; lane 5: 45-day-old mice; lane 6: 60-day-old mice; and lane 7: 1 kb ladder.
C.R.M. Ravindran, M.K. Ticku / Neuroscience Letters 380 (2005) 223–228
Fig. 6. Effect on the regulation of polypeptide levels of NR2B gene as a function of age in the mice. Seventy-five micrograms of protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane and Western Blotting was performed using the NBT/BCIP kit. Lane 1: protein of the newly born mice; lane 2: 15-day-old mice; lane 3: 30-day-old mice; lane 4: 45-day-old mice; lane 5: 60-day-old mice; lane 6: prestained SDS–PAGE standards, broad range.
To investigate these changes in the protein level, the isolated proteins (75 g) was separated and transferred to the polyvinylidene difluoride membrane. Initially, pilot experiment was performed by using various concentrations of antibody to detect the NR2B gene. The intensity of the band was increased from 1:10,000 to 1:5000 dilution without any significant increase in the background. Therefore, for further experiments, 1:5000 dilution of the antibody was used. Following Western Blotting we observed the up-regulation of the NR2B gene with increase in age especially in the adult age groups (45 and 60 days) of mice (Fig. 6). Our laboratory has reported that chronic ethanol treatment causes up-regulation in the levels of mRNA and polypeptides of NR1, NR2A and NR2B subunits in the cultured cortical neurons and in the rat brain regions [7–9,11,12,15,19]. Chronic ethanol treatment produced changes in NR1 at a post-transcriptional level and in the NR2 gene at the transcription level [19]. However, the molecular mechanisms involved for this increased gene expression is unknown. Generally CpG island-containing genes are frequently expressed in all tissues in a “housekeeping” fashion. Methylation at the 5 position of cytosine residues of CpG dinucleotides plays a major role in the regulation of gene expression [2,5]. It is already reported that the “CpG island” exists in the 5 part of the NR2B gene in mice and the M1 clone has the same sequence as that of the genomic DNA [18]. Also the CpG island exists in the other NMDA receptor subunits such as NR2A and NR2D [28,31]. Hypermethylation of promoter CpG islands is often associated with transcriptional silencing, whereas increased CpG island methylation downstream of transcription correlates with gene expression [14]. In our laboratory, treatment of cultured cortical neurons with 100 mM ethanol for 5 days caused a significant decrease in the neuron-restrictive silencer factor (NRSF) mRNA levels and an increase in the promoter activity (unpublished results). Further we have reported that decreased methylation could be responsible for an increase in expression of the NR2B gene following chronic ethanol treatment in cultured cortical neurons [19,23]. We have studied only the single site methylation of NR2B gene in our previous report [23]. In the present study, we have investigated the methylation of two or more sites for NR2B gene. HpaII is an isoschizomer of MspI enzyme. Unlike HpaII, MspI can cleave the sequence when the internal C residue in
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the sequence CCGG is methylated. In our experiments, only the external C was methylated. Moreover, the enzyme HpaII is methylation sensitive but MspI is not sensitive to methylation. We have observed an amplification of DNA following digestion with MspI enzyme in our previous study [23]. Similarly in the present study also we observed amplification of DNA in all the age groups of mice. Interestingly, there was no change in the intensity of the bands especially in the adult age groups (45 and 60 days) of mice since MspI is not sensitive to methylation and only the external C was methylated. Hypersensitivity to deoxyribonuclease I (DNase I) is commonly associated with transcriptional activation [22]. In view of this background, we studied methylation in different age groups of mice in the NR2B gene CpG islands. We studied methylation in the newly born mice initially since we expected the presence of an increase in methylation as the age goes up. As expected, we observed that the intensity of the bands were more in the newly born mice. But the intensity of the bands in 15 and 30 days old mice were less compared to the intensity of the bands in the newly born mice. Only in the adult mice (45 and 60 days) we observed the bands with lesser intensity, i.e., the demethylation process compared to the younger age groups of mice (Figs. 1a, 2a, 3a and 4a). There was a decrease in DNA methylation of 40% at 60 days old mice which indicated that the NR2B gene expression was more at this age compared to the other age groups of the mice. However, it is not known how the changes in demethylation process at various stages of brain development affect functional aspects of the NR2B gene in the brain. Moreover, it is unknown whether methyltransferase a methylating enzyme of cytosine residues on CpG dinucleotides is involved in the demethylation of NR2B gene observed in the present study. Similarly, there was up-regulation observed in the protein level of the NR2B gene with increase in age (new born to 60 days). However, up-regulation of NR2B expression and demethylation of the genomic DNA was more pronounced only in 45 and 60 days old mice. Thus, earlier stages of brain development may be more vulnerable to ethanol and similar agents due to higher levels of demethylation of the NR2B gene at the earlier stages of brain development. It is not known whether the demethylation of cytosines observed in our study is a global change (demethylation of cytosines occurs in both expressing and non-expressing tissues) or a gene specific change (demethylation of cytosines occurs only in the expressing tissues). However, it is reported that the global levels of DNA methylation in myeloma cells of mice tend to decrease with age [25,30]. However, demethylation of the NR2B gene CpG island observed in our current study in the mouse brain is age-dependent phenomenon. Acknowledgements We thank Dr. A.K. Mehta for his valuable help. This work was supported by funds from National Institute of Alcohol Abuse & Alcoholism grant #AA12297.
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Methylation of NMDA receptor NR2B gene as a function of age in the mouse brain C.R. Marutha Ravindran, Maharaj K. Ticku ∗ Department of Pharmacology, The University of Texas Health Science Center, San Antonio, TX 78229-3900, USA Received 30 November 2004; received in revised form 13 January 2005; accepted 14 January 2005
Abstract We have previously reported that there is an up-regulation of the NR2B gene expression in the adult cortex and cultured fetal cortical neurons of mice following chronic ethanol treatment due to demethylation of cytosine residues in the NR2B gene CpG island. In the present study, we investigated the methylation pattern of the NR2B CpG island as a function of the mouse age by digesting the cortex genomic DNA with HpaII enzyme, amplifying the interested regions by performing PCR and detecting the methylated regions by Southern hybridization so as to determine whether age affects the methylation process. We observed demethylation of various regions of NR2B gene (5227–5567), (5647–6003), (6091–6445), (6424–7024) of adult mouse cortex. Our results indicate that methylation of NR2B gene in the mouse brain is age-dependent phenomenon. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: NR2B gene; CpG; Demethylation; HpaII; PCR; Southern hybridization
N-Methyl-d-aspartate receptor (NMDAR) belongs to the subclass of ionotropic glutamate receptors and is widely distributed in the vertebrate brain. NMDAR mediates excitatory neurotransmission, synaptic plasticity and memory formation in the brain [6,24]. Three families of NMDA receptor subunits have been identified in the rat by molecular cloning: one NMDAR1 (NR1), four different NMDAR2 (NR2A-D) and one NMDAR3A (NR3A) [4,20,27,30]. The NR3 subunit was encoded on a single gene, whereas the NR2 subunit class (A–D) was encoded on four separate genes. The NR1 class is encoded on a single gene and molecularly diverse forms are created by alternative RNA splicing of three exons, there by generating as many as eight NR1 splice variants. The NR1 subunit, in association with the NR2 subunit, leads to the formation of highly active receptor channels [13,27]. However, unlike NR1, which is expressed constitutively, NR2B expression is highly regulated during development of the brain.
∗ Corresponding author. Tel.: +1 210 567 4255/4268; fax: +1 210 567 4226. E-mail address: [email protected] (M.K. Ticku).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.01.042
First discovered over 50 years ago in calf thymus [10], methylation of DNA occurs mostly at the 5 position of cytosine in higher eukaryotic cells. This plays an important role in the regulation of gene expression [2,5]. When DNA was analyzed for CG dinucleotide pairs, regions with higher proportions of CG sequences than would be expected were found to exist [26]. Such CG-rich regions are now referred as CpG islands, which constitute between 1 and 2% of the total genome and are associated with the promoters of ∼50% of all mammalian genes [1]. They are regions of ∼1 kb that differ from the rest of the genome by being G + C rich (65%) [2,3]. Our interest in this study stems from our previous observation that chronic ethanol treatment causes up-regulation of the NR2B gene [7–9,11,12,15]. In order to understand the molecular mechanisms underlying this up-regulation, we studied methylation in the CpG island following chronic ethanol treatment of cultured cortical neurons and observed that demethylation may be responsible for the changes in the expression of NR2B gene [23]. It has been reported that the global levels of DNA methylation tend to decrease with age in mouse [25,30]. In the present study, we investigated
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whether the methylation process in the NR2B gene is an agedependent phenomenon. Mice of different age groups (strain C57BL/6) such as 0, 15-, 30-, 45- and 60-day-old were purchased from Harlan (Indianapolis, IN). We consider 0 to 30 days old mice as younger ones and 45 to 60 days old mice as the adult group. They were used in accordance with institutional guidelines and procedures approved by the Animal Welfare Committee. Briefly, cerebral hemispheres were dissected from six mice of each age group separately and homogenized in an ice-cold lysis buffer (15 mM Tris–HCl pH 7.6, 60 mM KCl, 15 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and 0.5 mM spermidine) in the presence of 0.5% NP-40 which was added at the time of lysis. These samples were centrifuged (1000 × g, 10 min, 4 ◦ C) so as to extract the nuclei. These nuclei were suspended in a DNA extraction buffer (10 mM Tris–HCl pH 8.0, 10 mM EDTA pH 8.0, 0.5% SDS and fresh proteinase K 100 g/ml), and the DNA was isolated by standard procedures [29]. DNA (15 g) was digested overnight at 37 ◦ C with methyl-sensitive restriction endonucleases such as HpaII or MspI which is not sensitive to methylation. The regions of interest were amplified by PCR using primers 0.2 M (Table 1), dNTP 0.2 mM, 750 ng of template and the PCR conditions were as follows: 94 ◦ C for 4 min, 39 cycles of 93 ◦ C for 30 s, 50 ◦ C, 57 ◦ C, 63 ◦ C or 70 ◦ C for 30 s, 72 ◦ C for 2 min and 72 ◦ C for 8 min. Restriction fragments were separated by electrophoresis in 1.2% agarose gel using 1× TBE buffer and subsequently blotted onto nylon membranes (Roche, IN). The cerebral hemispheres were dissected as detailed above and homogenized in ice-cold lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1% Triton-X 100, 10 mM NaF, 1 mg/ml bacitracin, 50 mM -glycerophosphate, 1 mM phenyl methyl sulfonyl fluoride, 1 mM N-ethyl maleimide, 1 mM Na3 VO4 , 10 g/ml leupeptin). These samples were centrifuged (5000 rpm, 10 min, 4 ◦ C) so as to remove the supernatant containing protein. Seventy-five micrograms of protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) [21] and transferred to polyvinylidene difluoride membrane followed by treatment with 5% milk in Tris buffered saline
having 0.1% Tween 20 and overnight incubation with primary antibody NR2B [16,17] at a concentration of 1:5000 (Chemicon). Then the membrane was washed several times and peroxidase coupled secondary antibody (anti-mouse IgG/anti-rabbit IgG; New England Biolabs) was added and incubated for 1 h. The membrane was washed and specific bands were visualized using NBT/BCIP kit (Sigma). The M1 clone (NCBI accession number AF033356, 18) has the same sequence as that of the genomic DNA. Using this clone, we have amplified a long fragment of 2239 bp in size, and employing this fragment as a template, we amplified the regions of interest by PCR utilizing the primers as described in Table 1. These regions were selected randomly based on the number of sites present for digestion by the enzyme HpaII. The amplified fragments of DNA were separated by electrophoresis as detailed earlier, and the products were cloned in TOPO vector using the TOPO cloning kit (Invitrogen, CA). The clones were identified by colony PCR; and the clones were inoculated in LB medium containing ampicillin 100 g/ml and incubated at 37 ◦ C overnight. After sufficient growth, the cultures were harvested and the plasmids were isolated using the Qiagen plasmid midi kit (Qiagen Inc, CA). Further, the plasmids were digested with EcoRI enzyme, and the restriction fragments were separated by electrophoresis in 1.2% agarose gel containing 1× TBE buffer. After electrophoresis, the cloned products of DNA was cut down under UV light using the sterile single use stainless surgical blades (Maersk Medical Ltd, Sheffield); the plasmids were extracted using the QIA quick gel extraction kit (50) (Qiagen Inc) protocol; and the probes were prepared for Southern blot analysis which was performed as described by Sambrook et al. [29]. Southern blots were hybridized with (␣32 P) dCTP-labeled probes (Amersham-Pharmacia Biotec) in Quick HybR hybridization solution (Stratagene) and further processed as recommended by the supplier. Blots were exposed on an X-ray film based on the intensity of the bands at −70 ◦ C. Initially we identified the methylated sites in the cerebral cortex of the newly born mice. The genomic map and the sequences of the NR2B gene in which we studied methyla-
Table 1 Primers used for amplification in PCR Enzyme used for digesting the genomic DNA
HpaII
Primer sequences
Annealing temperature used (◦ C)
Size (bp)
(1) 5 -GTG AGG GGG TGA TGG AGG GGG TTT GG-3 5 -GGC AAG TCA AGC GTT CGG CAG CAG GAG AGG-3 Sequences amplified from 5227 to 5567 (2) 5 -TTT TTG AGA AAG GAA GGT-3 5 -ATT TCT CCA AAC CCT CAA AAT T-3 Sequences amplified from 5647 to 6003 (3) 5 -TAT TTT GGA TGG TTT TTT ATT TTT-3 5 -GCT CAA TGG GTT CTG ATT GTG C-3 Sequences amplified from 6091 to 6445 (4) 5 -GCA CAA TCA GAA CCC ATT GAG C-3 5 -GCT TTA GTA GAG GAG TCT CTG TGT GG-3 Sequences amplified from 6424 to 7024
70
340
50
356
57
354
63
600
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Fig. 1. Effect of methylation pattern of the NR2B gene CpG island as a function of age of the mice in the region (5227–5567). (a) Lane 1: 100 bp ladder; lane 2: PCR product of the newly born mice; lane 3: 15-day-old mice; lane 4: 30-day-old mice; lane 5: 45-day-old mice; lane 6: 60-dayold mice; and lane 7: 1 kb ladder. (b) Southern hybridization with (␣32 P) dCTP-labeled probes. The X-ray film was exposed for 30 min at −70 ◦ C. Lane 1: newly born mice; lane 2: 15-day-old mice; lane 3: 30-day-old mice; lane 4: 45-day-old mice; and lane 5: 60-day-old mice. (c) Percentage of methylation in the region (5227–5567) of different age groups of mice in the NR2B gene CpG islands. Percentage methylation was measured by scanning and comparing the intensity of each band. Each value is % mean ± S.E.M. of three individual experiments. The symbol (* ) significant and p < 0.05 as compared to the control (newly born mice).
tion of different age groups of mice are given in our previous reports [23]. We identified the multiple sites methylated in the NR2B gene for HpaII enzyme. We observed complete digestion of the genomic DNA using the HpaII enzyme. The genomic DNA isolated from the cerebral cortex of the newly born mice was digested with HpaII enzyme (3 units/g DNA) and various regions of the DNA were amplified using different sets of primers for NR2B gene by performing PCR (Table 1). Using PCR to study methylation we require a very small amount of genomic DNA than restriction digestion. In fact, PCR is inherently more sensitive than restriction digestion and therefore PCR will always be able to find some template to amplify. Hence, we studied methylation in this study by using PCR. Following PCR, we identified methylation in the region (5227–5567), which yielded the DNA fragments of 340 bp in size, and it was consistent with the anticipated region studied (Fig. 1a). These experiments indicated that the region (5227–5567) was methylated, and in this region two sites for digestion by the enzyme HpaII were present. Following identification of the methylated site in the above region, we amplified the remaining regions of the NR2B gene in the cortex. Region (5647–6003) yielded the DNA
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Fig. 2. Effect of methylation pattern of the NR2B gene CpG island as a function of age of the mice in the region (5647–6003). (a) Lane 1: 100 bp ladder; lane 2: PCR product of the newly born mice; lane 3: 15-day-old mice; lane 4: 30-day-old mice; lane 5: 45-day-old mice; and lane 6: 60-day-old mice. (b) Southern hybridization with (␣32 P) dCTP-labeled probes. The Xray film was exposed for 5 min at −70 ◦ C. Lane 1: newly born mice; lane 2: 15-day-old mice; lane 3: 30-day-old mice; lane 4: 45-day-old mice; and lane 5: 60-day-old mice. (c) Percentage of methylation in the region (5647–6003) of different age groups of mice in the NR2B gene CpG islands. Percentage methylation was measured by scanning and comparing the intensity of each band. Each value is % mean ± S.E.M. of three individual experiments. The symbol (* ) significant, p < 0.05 as compared to the control (newly born mice).
fragments of 356 bp in size (Fig. 2a), region (6091–6445) yielded the DNA fragments of 354 bp in size (Fig. 3a), and region (6424–7024) yielded the DNA fragments of 600 bp in size (Fig. 4a). These regions for amplification by PCR were selected randomly based on the number of sites present in the genomic DNA for digestion by the enzyme HpaII. The amplified regions were also matched with the regions studied in the present study. These results indicated that all the above regions were also methylated and in these regions 2 to 3 sites for digestion by the enzyme HpaII were present in the cerebral cortex of the mice. Further, to study the methylation in different age groups of the mice, we selected randomly 15-, 30-, 45- and 60-dayold mice, dissected the cerebral cortex, isolated and digested the genomic DNA with HpaII enzyme and as detailed earlier amplified the above regions using different sets of primers (Table 1). The region (5227–5567) yielded the DNA fragments of 340 bp in size, region (5647–6003) yielded 356 bp of DNA fragments, region (6091–6445) yielded 354 bp of
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Fig. 3. Effect of methylation pattern of the NR2B gene CpG island as a function of age of the mice in the region (6091–6445). (a) Lane 1: 100 bp ladder; lane 2: PCR product of the newly born mice; lane 3: 15-day-old mice; lane 4: 30-day-old mice; lane 5: 45-day-old mice; lane 6: 60-day-old mice; and lane 7: 1 kb ladder. (b) Southern hybridization with (␣32 P) dCTP-labeled probes. The X-ray film was exposed for 5 min at −70 ◦ C. Lane 1: newly born mice; lane 2: 15-day-old mice; lane 3: 30-day-old mice; lane 4: 45-day-old mice; and lane 5: 60-day-old mice. (c) Percentage of methylation in the region (6091–6445) of different age groups of mice in the NR2B gene CpG islands. Percentage methylation was measured by scanning and comparing the intensity of each band. Each value is % mean ± S.E.M. of three individual experiments. (* ) significant and p < 0.05 as compared to the control (newly born mice).
DNA fragments, and the region (6424–7024) yielded 600 bp of DNA fragments in all the age groups similar to the newly born mice (Figs. 1a, 2a, 3a and 4a). Interestingly, the increase in age reduced the intensity of the bands, which indicated the demethylation of the NR2B gene CpG islands in the cerebral cortex of adult mice in all the regions (Figs. 1a, 2a, 3a and 4a). To identify the intensity of the bands in the amplified regions, we used the probes prepared from the M1 clone by Southern hybridization. As expected we observed the presence of very thick bands in all the above regions of the younger groups of mice. Only in the adult age groups (45 and 60 days) of mice, we observed the presence of faint bands as revealed by Southern hybridization, which indicated demethylation of the CpG island (Figs. 1b, 2b, 3b and 4b). Furthermore, the % of methylation in all the age groups of mice was measured by scanning and comparing the intensity of each band. Percentage of methylation in the control (newly born mice) was considered as 100% and the variation
Fig. 4. Effect of methylation pattern of the NR2B gene CpG island as a function of age of the mice in the region (6424–7024). (a) Lane 1: 1 kb ladder; lane 2: PCR product of the newly born mice; lane 3: 15-day-old mice; lane 4: 30-day-old mice; lane 5: 45-day-old mice; lane 6: 60-dayold mice; and lane 7: 100 bp ladder. (b) Southern hybridization with (␣32 P) dCTP-labeled probes. The X-ray film was exposed for 5 min at −70 ◦ C. Lane 1: newly born mice; lane 2: 15-day-old mice; lane 3: 30-day-old mice; lane 4: 45-day-old mice; and lane 5: 60-day-old mice. (c) Percentage of methylation in the region (6424–7024) of different age groups of mice in the NR2B gene CpG islands. Percentage methylation was measured by scanning and comparing the intensity of each band. Each value is % mean ± S.E.M. of three individual experiments. The symbol (* ) significant and p < 0.05 as compared to the control (newly born mice).
in the intensity of the band in each blot was given as ±S.D. values. Then the intensity of the band in the control was compared with the intensity of the band in the 15-, 30-, 45and 60-day-old mice (Figs. 1c, 2c, 3c and 4c). Data were analyzed by Student’s t test. Differences were considered to be statistically significant when p < 0.05. Further to show that PCR amplification is constant, we digested the genomic DNA with the enzyme MspI which is not sensitive to methylation as a control and amplified the region (6424–7024) yielded 600 bp of DNA fragments in size similarly. Interestingly, in this case there was no change in the intensity of the band observed following amplification in various age groups of mice (Fig. 5).
Fig. 5. Effect of methylation pattern of the NR2B gene CpG island as a function of age of the mice in the region (6424–7024) by digesting the genomic DNA with MspI. Lane 1: 100 bp ladder; lane 2: PCR product of the newly born mice; lane 3: 15-day-old mice; lane 4: 30-day-old mice; lane 5: 45-day-old mice; lane 6: 60-day-old mice; and lane 7: 1 kb ladder.
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Fig. 6. Effect on the regulation of polypeptide levels of NR2B gene as a function of age in the mice. Seventy-five micrograms of protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane and Western Blotting was performed using the NBT/BCIP kit. Lane 1: protein of the newly born mice; lane 2: 15-day-old mice; lane 3: 30-day-old mice; lane 4: 45-day-old mice; lane 5: 60-day-old mice; lane 6: prestained SDS–PAGE standards, broad range.
To investigate these changes in the protein level, the isolated proteins (75 g) was separated and transferred to the polyvinylidene difluoride membrane. Initially, pilot experiment was performed by using various concentrations of antibody to detect the NR2B gene. The intensity of the band was increased from 1:10,000 to 1:5000 dilution without any significant increase in the background. Therefore, for further experiments, 1:5000 dilution of the antibody was used. Following Western Blotting we observed the up-regulation of the NR2B gene with increase in age especially in the adult age groups (45 and 60 days) of mice (Fig. 6). Our laboratory has reported that chronic ethanol treatment causes up-regulation in the levels of mRNA and polypeptides of NR1, NR2A and NR2B subunits in the cultured cortical neurons and in the rat brain regions [7–9,11,12,15,19]. Chronic ethanol treatment produced changes in NR1 at a post-transcriptional level and in the NR2 gene at the transcription level [19]. However, the molecular mechanisms involved for this increased gene expression is unknown. Generally CpG island-containing genes are frequently expressed in all tissues in a “housekeeping” fashion. Methylation at the 5 position of cytosine residues of CpG dinucleotides plays a major role in the regulation of gene expression [2,5]. It is already reported that the “CpG island” exists in the 5 part of the NR2B gene in mice and the M1 clone has the same sequence as that of the genomic DNA [18]. Also the CpG island exists in the other NMDA receptor subunits such as NR2A and NR2D [28,31]. Hypermethylation of promoter CpG islands is often associated with transcriptional silencing, whereas increased CpG island methylation downstream of transcription correlates with gene expression [14]. In our laboratory, treatment of cultured cortical neurons with 100 mM ethanol for 5 days caused a significant decrease in the neuron-restrictive silencer factor (NRSF) mRNA levels and an increase in the promoter activity (unpublished results). Further we have reported that decreased methylation could be responsible for an increase in expression of the NR2B gene following chronic ethanol treatment in cultured cortical neurons [19,23]. We have studied only the single site methylation of NR2B gene in our previous report [23]. In the present study, we have investigated the methylation of two or more sites for NR2B gene. HpaII is an isoschizomer of MspI enzyme. Unlike HpaII, MspI can cleave the sequence when the internal C residue in
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the sequence CCGG is methylated. In our experiments, only the external C was methylated. Moreover, the enzyme HpaII is methylation sensitive but MspI is not sensitive to methylation. We have observed an amplification of DNA following digestion with MspI enzyme in our previous study [23]. Similarly in the present study also we observed amplification of DNA in all the age groups of mice. Interestingly, there was no change in the intensity of the bands especially in the adult age groups (45 and 60 days) of mice since MspI is not sensitive to methylation and only the external C was methylated. Hypersensitivity to deoxyribonuclease I (DNase I) is commonly associated with transcriptional activation [22]. In view of this background, we studied methylation in different age groups of mice in the NR2B gene CpG islands. We studied methylation in the newly born mice initially since we expected the presence of an increase in methylation as the age goes up. As expected, we observed that the intensity of the bands were more in the newly born mice. But the intensity of the bands in 15 and 30 days old mice were less compared to the intensity of the bands in the newly born mice. Only in the adult mice (45 and 60 days) we observed the bands with lesser intensity, i.e., the demethylation process compared to the younger age groups of mice (Figs. 1a, 2a, 3a and 4a). There was a decrease in DNA methylation of 40% at 60 days old mice which indicated that the NR2B gene expression was more at this age compared to the other age groups of the mice. However, it is not known how the changes in demethylation process at various stages of brain development affect functional aspects of the NR2B gene in the brain. Moreover, it is unknown whether methyltransferase a methylating enzyme of cytosine residues on CpG dinucleotides is involved in the demethylation of NR2B gene observed in the present study. Similarly, there was up-regulation observed in the protein level of the NR2B gene with increase in age (new born to 60 days). However, up-regulation of NR2B expression and demethylation of the genomic DNA was more pronounced only in 45 and 60 days old mice. Thus, earlier stages of brain development may be more vulnerable to ethanol and similar agents due to higher levels of demethylation of the NR2B gene at the earlier stages of brain development. It is not known whether the demethylation of cytosines observed in our study is a global change (demethylation of cytosines occurs in both expressing and non-expressing tissues) or a gene specific change (demethylation of cytosines occurs only in the expressing tissues). However, it is reported that the global levels of DNA methylation in myeloma cells of mice tend to decrease with age [25,30]. However, demethylation of the NR2B gene CpG island observed in our current study in the mouse brain is age-dependent phenomenon. Acknowledgements We thank Dr. A.K. Mehta for his valuable help. This work was supported by funds from National Institute of Alcohol Abuse & Alcoholism grant #AA12297.
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