ARTICLES at the lysine-9(K9) residue of H3 and H4 histones is a well-estab ished marker of active chromatin/.29. Acetylation of the histone tails neutralizes the positively charged s,which disrupts his- tone binding to negatively charged DNA and thus promotes tran scription factor binding. We tested the hypothesis that the maternal effect on DNA methylation results in(i)increased histone acetyl- tion at the k9 residue of the H3 histone(s)associated with the exon 1, GR promoter and (ii) increased interaction between NGFI-A and the promoter sequence. We performed a chromatin immuno 123456789101112131415 precipitation(ChIP)analysis of histone H3-K9 acetylation and NGFI-A protein binding to the exon 1, GR promoter in the native chromatin environment in vivo Intact hippocampi from adult off- b avehicle: low LG/ABN spring of high- and low-LG-ABN mothers were crosslinked in vivo by paraformaldehyde perfusion. We then selectively immunopre cipitated protein-DNA complexes with either an acetylated H3-K9 histone primary antibody or an NGFI-A primary antibody. The protein-DNA complexes were uncrosslinked, and the precipitated genomic DNA was subjected to PCR amplification with primers pecific for the exon 1, GR promoter sequence. There were signifi cant Group effects for the association of both histone H3-K9 acety lation(t= 2.1, *P<0.001)and NGFI-A (t=3. 1, **P<00001)with 17 Gr These results indicated signifi- cantly greater histone H3-K9 acetylation association and threefold Figure 4 TSA effects on cytosine methylation. (a, b)Methylation analysis of greater binding of NGFI-A protein to the hippocampal exon 1,GR e the 17 CpG dinucleotides of the exon 1, GR promoter in hippocampi of promoter in the adult offspring of high- compared with low-LG- G ABN mothers (n= 5 animals/group).(a)Percentage of cytosine residues GR promoter involves DNA methylation, histone H3-K9 acetyla that were methylated(mean+ s.e. m )for the first 15 CpG dinucleotides tion and alterations in ngFi-a binding (P<0.05).(b)Percentage of methylated cytosines for the 5(site 16)and 3(site 17)CpG dinucleotides within the NgFl-a binding region (P<0.001;**P<0.003) Reversal of maternally mediated epigenetic marking These findings suggest that maternal care influences hippocampal GR expression, and thus HPA function in the offspring, through epigenetic alterations that regulate NGFl-A binding to the exon 1, promoter. A both the basal state of methylation and the first wave of de novo critical question is whether the impact of early experience is reversible methylation after birth occur similarly in both groups. Whereas it is and whether epigenetic programming is modifiable in adult, post generally accepted that DNA methylation patterns are formed prena- mitotic tissues? The generally accepted model is that the DNA methyla- o tally and that de novo methylation occurs early in development, there tion pattern is an irreversible reaction in adult post-mitotic cells is at least one documented example of postnatal de novo methylation However, recent data from in vitro experiments suggests that in certain of the Hoxa5 and Hoxb5 genes27 Because similar analyses are not doc- instances it is possible to induce replication-independent demethylation umented for other genes, it remains unknown whether changes in of ectopically methylated genes by increasing histone acetylation us ethylation are common around birth or whether they are unique to the histone deacetylase(HDAC) inhibitor trichostatin A(TSA)29,30 Cytosine methylation attracts methylated DNA binding proteins and The differences in the methylation status of the exon 1, GR pro- HDACs that prevent histone acetylation and thus transcription factor moter between the two groups developed between PI and P6, the binding29, 30. Activation of chromatin through HDAC inhibition might period when differences in the maternal behavior of high-and low- trigger DNA demethylation by increasing the accessibility of DNA to LG-ABN dams are apparent. 8. By P6, the NGFI-A response element demethylase activity 0. We tested the hypothesis that inhibition of 5 CpG dinucleotide (site 16) was effectively demethylated in the HDACs with TSA would result in increased K9 acetylation of H3-his- high-, but not in the low-LG-ABN group. The group difference in tones associated with the exon 1, GR promoter, DNA demethylation, CpG dinucleotide methylation remains consistent through to adult- NGFl-A binding and reversal of maternal programming of str hood (P90; Fig. le). These findings, together with those of the cross- responses in the adult offspring of low-LG-ABN mothers fostering study, suggest that the group difference in DNA We first used ChIP analysis to determine whether histone H3-K9 ethylation occurs as a function of a maternal behavior over the acetylation and NGFl-A protein binding to the exon 17 gr pro first week of life. The results of earlier studies indicate that the first moter is altered in the offspring of high-and low-LG-ABN mothers week of postnatal life is a 'critical period'for the effects of early through intracerebroventricular (i.c.v. ) infusion of the adult off- experience on hippocampal GR express pring with TSA (100 ng/ml)or vehicle. Statistical analysis revealed a significant Group x Treatment interaction effect for both the his- Maternal effects on chromatin structure and NGFI-A binding tone H3-K9 acetylation(F=4.93, P<0.05)and NGFI-A(F=8.9 The next question concerns the functional importance of such dif- P=0.01). Post-hoc analysis showed that for both assays, vehicle ferences in methylation. DNA methylation is associated with treated offspring of low-LG-ABN mothers showed significantly changes in chromatin activity states. Chromatin gates the accessi- ("P<0.01)less association than any other group. These results indi- bility of promoters to transcription factors. Histone acetylation cate greater histone H3-K9 acetylation association and more bind 850 VOLUME 7 NUMBER 8 AUGUST 2004 NATURE NEUROSCIENCEARTICLES both the basal state of methylation and the first wave of de novo methylation after birth occur similarly in both groups. Whereas it is generally accepted that DNA methylation patterns are formed prena￾tally and that de novo methylation occurs early in development, there is at least one documented example of postnatal de novo methylation of the Hoxa5 and Hoxb5 genes27. Because similar analyses are not doc￾umented for other genes, it remains unknown whether changes in methylation are common around birth or whether they are unique to this GR promoter. The differences in the methylation status of the exon 17 GR pro￾moter between the two groups developed between P1 and P6, the period when differences in the maternal behavior of high- and low￾LG-ABN dams are apparent5,8. By P6, the NGFI-A response element 5′ CpG dinucleotide (site 16) was effectively ‘demethylated’ in the high-, but not in the low-LG-ABN group. The group difference in CpG dinucleotide methylation remains consistent through to adult￾hood (P90; Fig. 1e). These findings, together with those of the cross￾fostering study, suggest that the group difference in DNA methylation occurs as a function of a maternal behavior over the first week of life. The results of earlier studies indicate that the first week of postnatal life is a ‘critical period’ for the effects of early experience on hippocampal GR expression28. Maternal effects on chromatin structure and NGFI-A binding The next question concerns the functional importance of such dif￾ferences in methylation. DNA methylation is associated with changes in chromatin activity states18. Chromatin gates the accessi￾bility of promoters to transcription factors17. Histone acetylation at the lysine-9 (K9) residue of H3 and H4 histones is a well-estab￾lished marker of active chromatin17,29. Acetylation of the histone tails neutralizes the positively charged histones, which disrupts his￾tone binding to negatively charged DNA and thus promotes tran￾scription factor binding. We tested the hypothesis that the maternal effect on DNA methylation results in (i) increased histone acetyla￾tion at the K9 residue of the H3 histone(s) associated with the exon 17 GR promoter and (ii) increased interaction between NGFI-A and the promoter sequence. We performed a chromatin immuno￾precipitation (ChIP) analysis of histone H3-K9 acetylation and NGFI-A protein binding to the exon 17 GR promoter in the native chromatin environment in vivo. Intact hippocampi from adult off￾spring of high- and low-LG-ABN mothers were crosslinked in vivo by paraformaldehyde perfusion. We then selectively immunopre￾cipitated protein-DNA complexes with either an acetylated H3-K9 histone primary antibody or an NGFI-A primary antibody. The protein-DNA complexes were uncrosslinked, and the precipitated genomic DNA was subjected to PCR amplification with primers specific for the exon 17 GR promoter sequence. There were signifi￾cant Group effects for the association of both histone H3-K9 acety￾lation (t = 2.1, *P < 0.001) and NGFI-A (t = 3.1, **P < 0.0001) with the exon 17 GR promoter sequence. These results indicated signifi￾cantly greater histone H3-K9 acetylation association and threefold greater binding of NGFI-A protein to the hippocampal exon 17 GR promoter in the adult offspring of high- compared with low-LG￾ABN mothers (Fig. 2). Thus, maternal programming of the exon 17 GR promoter involves DNA methylation, histone H3-K9 acetyla￾tion and alterations in NGFI-A binding. Reversal of maternally mediated epigenetic marking These findings suggest that maternal care influences hippocampal GR expression, and thus HPA function in the offspring, through epigenetic alterations that regulate NGFI-A binding to the exon 17 promoter. A critical question is whether the impact of early experience is reversible and whether epigenetic programming is modifiable in adult, post￾mitotic tissues? The generally accepted model is that the DNA methyla￾tion pattern is an irreversible reaction in adult post-mitotic cells. However, recent data from in vitro experiments suggests that in certain instances it is possible to induce replication-independent demethylation of ectopically methylated genes by increasing histone acetylation using the histone deacetylase (HDAC) inhibitor trichostatin A (TSA)29,30. Cytosine methylation attracts methylated DNA binding proteins and HDACs that prevent histone acetylation and thus transcription factor binding29,30. Activation of chromatin through HDAC inhibition might trigger DNA demethylation by increasing the accessibility of DNA to demethylase activity30. We tested the hypothesis that inhibition of HDACs with TSA would result in increased K9 acetylation of H3-his￾tones associated with the exon 17 GR promoter, DNA demethylation, NGFI-A binding and reversal of maternal programming of stress responses in the adult offspring of low-LG-ABN mothers. We first used ChIP analysis to determine whether histone H3-K9 acetylation and NGFI-A protein binding to the exon 17 GR pro￾moter is altered in the offspring of high- and low-LG-ABN mothers through intracerebroventricular (i.c.v.) infusion of the adult off￾spring with TSA (100 ng/ml) or vehicle. Statistical analysis revealed a significant Group × Treatment interaction effect for both the his￾tone H3-K9 acetylation (F = 4.93, P < 0.05) and NGFI-A (F = 8.97, P = 0.01). Post-hoc analysis showed that for both assays, vehicle - treated offspring of low-LG-ABN mothers showed significantly (*P < 0.01) less association than any other group. These results indi￾cate greater histone H3-K9 acetylation association and more bind- 850 VOLUME 7 | NUMBER 8 | AUGUST 2004 NATURE NEUROSCIENCE Figure 4 TSA effects on cytosine methylation. (a,b) Methylation analysis of the 17 CpG dinucleotides of the exon 17 GR promoter in hippocampi of vehicle- and TSA-treated (100 ng/ml) adult offspring of high- and low-LG￾ABN mothers (n = 5 animals/group). (a) Percentage of cytosine residues that were methylated (mean ± s.e.m.) for the first 15 CpG dinucleotides (*P < 0.05). (b) Percentage of methylated cytosines for the 5′ (site 16) and 3′ (site 17) CpG dinucleotides within the NGFI-A binding region (*P < 0.001; **P < 0.003). © 2004 Nature Publishing Group http://www.nature.com/natureneuroscience