ARTICLES LOW-LG/ABN h-LG/ABN Figure 2 Chromatin immunoprecipitation analysis of the association between histone H3-K9 acetylation and NGFl-A binding to the exon 17 GR sequence in hippocampal tissue from adult offspring of high- and low-LG- ABN mothers(n= 4 animals/group).(a, b)Lanes were loaded with non- (middle) primary antibody immunoprecipitated (A), or non-immune IgG antibody immuno-precipitated (N) hippocampal extracts).(a)Representative Southern blot of the amplified exon l7 region from acetyl-histone H3-K9 immunoprecipitated hippocampal tissue(194 bp band )and B-actin(171 bp b band)control. (b)Representative Southern blot of the amplified exon 17 GR exon 1, ion of the GR from NGFI-A immunoprecipitated hippocampal tissue (194 bp band). DNA loading was controlled using primers specific for the ubiquitously expressed B-actin promoter-o region. Exon 1b estrogen receptor-a promoter region, which does not contain NGFl-A recogniti elements(493 bp), amplified from the same NGFI-A immunoprecipitated hippocampal tissue was run as a control for specificity and showed no signal. (c)Relative optical density(ROD; m )of exon 17 2061日 sequence amplified from acetyl-histone H3-K9 or NGFI-A immunoprecipitated hippocampal tissue of adult high-and low-LG-ABN offspring(n= 4 animals/group: *P<0.001; **P<00001) 203 to examine the methylation status of the cytosines within the exon 1 GR promoter during development( Fig. le). Statistical analysis of the Acetyl H3K9 data for the 5 CpG(site 16)revealed a highly significant effect of Group(F= 66.7, P<00001)and Age(F=21.1, P<00001)as well as a significant interaction effect(F= 13.7, P<00001). Tukey post-hoc analysis revealed that the Group effect on methylation status of the 5 CpG(site 16)was significant at P6, P21 and P90(P < 0.001),but 9 NGFI-A consensus sequence(Fig. Id, left panel). Thus, in the not at E20 or PI. Just before birth(embryonic day 20; E20)the entire low-LG-ABN offspring that were fostered to high-LG-aBN dams, region was unmethylated in both groups. Strikingly, one day after methylation of this 5 site within the exon 1, promoter was indistin- birth(postnatal day 1; Pl)the exon 1, gr promoter was de novo a guishable from that of the biological offspring of high-LG-ABN methylated in both groups. The 5 and 3 CpG sites of the exon 1,GR mothers. Likewise, the methylation of the same 5" CpG dinucleotide NGFI-A response element in the offspring of both high- and low- in the biological offspring of high-LG-ABN mothers reared by low- LG-ABN mothers, which exhibit differential methylation later in life, There was no effect or mparable to that of low-LG-ABN offspring. were de novo methylated to the same extent. These data show that Lg-abn dams was co cross-fostering at the cytosine within the 3 CpG dinucleotide(site 17; Fig. ld) 0. These findings suggest that variations in maternal care directly alter TSA TSA 100 ng/ml Vehicle 100 ng/ml the methylation status of the exon 1, promoter of the GR gene. Thus Maternal care we have demonstrated that a DNA methylation pattern can be estab lished through a behavioral mode of programming without germ line ANIANIANI A N transmission In parental imprinting, a well-established paradigm of inheritance of an epigenomic mark, the paternally and maternally (acetyl-H3K9 IP) inherited alleles are differentially methylated. These methylation pat terns are defined during maturation of spermatocytes and oocytes, (acetyl-H3K9 IP) nd are transmitted to the offspring through the germ line- Timing of the maternal effect on DNA methylation -[--- The maternal care of high- and low-LG-ABN mothers differs only during the first week of life,. Thus, we wondered whether this period ER-a exon 1b to the Ing of the difference in NGFI-A DNA methylation in the offspring. We used sodium bisulfite mapping Figure 3 HDAC inhibitior (TSA) eliminates maternal effect on histone acetylation and NGFl-A binding (a)Chromatin immunoprecipitation nalysis of the association between histone H3-K9 acetylation and NGFI-A binding to the exon 17 GR promoter sequence in hippocampal tissue from vehicle- and TSA-treated (100 ng/ml) adult offspring of high- and low-LG- ABN mothers(n= 4 animals/group; lane labels as described in Fig. 2). b)Relative optical density(ROD; mean ts.e. m )of exon 17 sequence amplified from acetyl-histone H3-K9 or NGFl-A immunoprecipitate hippocampal tissue(P<0.05; **P<0.01) NATURE NEUROSCIENCE VOLUME 7 NUMBER 8 AUGUST 2004ARTICLES NGFI-A consensus sequence (Fig. 1d, left panel). Thus, in the low-LG-ABN offspring that were fostered to high-LG-ABN dams, methylation of this 5′ site within the exon 17 promoter was indistin￾guishable from that of the biological offspring of high-LG-ABN mothers. Likewise, the methylation of the same 5′ CpG dinucleotide in the biological offspring of high-LG-ABN mothers reared by low￾LG-ABN dams was comparable to that of low-LG-ABN offspring. There was no effect of cross-fostering at the cytosine within the 3′ CpG dinucleotide (site 17; Fig. 1d). These findings suggest that variations in maternal care directly alter the methylation status of the exon 17 promoter of the GR gene. Thus we have demonstrated that a DNA methylation pattern can be estab￾lished through a behavioral mode of programming without germ line transmission. In parental imprinting, a well-established paradigm of inheritance of an epigenomic mark, the paternally and maternally inherited alleles are differentially methylated. These methylation pat￾terns are defined during maturation of spermatocytes and oocytes, and are transmitted to the offspring through the germ line26. Timing of the maternal effect on DNA methylation The maternal care of high- and low-LG-ABN mothers differs only during the first week of life7,8. Thus, we wondered whether this period corresponds to the timing for the appearance of the difference in DNA methylation in the offspring. We used sodium bisulfite mapping to examine the methylation status of the cytosines within the exon 17 GR promoter during development (Fig. 1e). Statistical analysis of the data for the 5′ CpG (site 16) revealed a highly significant effect of Group (F = 66.7, P < 0.0001) and Age (F = 21.1, P < 0.0001) as well as a significant interaction effect (F = 13.7, P < 0.0001). Tukey post-hoc analysis revealed that the Group effect on methylation status of the 5′ CpG (site 16) was significant at P6, P21 and P90 (P < 0.001), but not at E20 or P1. Just before birth (embryonic day 20; E20) the entire region was unmethylated in both groups. Strikingly, one day after birth (postnatal day 1; P1) the exon 17 GR promoter was de novo methylated in both groups. The 5′ and 3′ CpG sites of the exon 17 GR NGFI-A response element in the offspring of both high- and low￾LG-ABN mothers, which exhibit differential methylation later in life, were de novo methylated to the same extent. These data show that NATURE NEUROSCIENCE VOLUME 7 | NUMBER 8 | AUGUST 2004 849 Figure 2 Chromatin immunoprecipitation analysis of the association between histone H3-K9 acetylation and NGFI-A binding to the exon 17 GR sequence in hippocampal tissue from adult offspring of high- and low-LG￾ABN mothers (n = 4 animals/group). (a,b) Lanes were loaded with non￾immunoprecipitated input (I), acetylated histone H3-K9 (top) or NGFI-A (middle) primary antibody immunoprecipitated (A), or non-immune IgG antibody immuno-precipitated (N) hippocampal extracts). (a) Representative Southern blot of the amplified exon 17 region from acetyl-histone H3-K9 immunoprecipitated hippocampal tissue (194 bp band) and β-actin (171 bp band) control. (b) Representative Southern blot of the amplified exon 17 region of the GR from NGFI-A immunoprecipitated hippocampal tissue (194 bp band). DNA loading was controlled using primers specific for the ubiquitously expressed β-actin promoter-α region. Exon 1b estrogen receptor-α promoter region, which does not contain NGFI-A recognition elements (493 bp), amplified from the same NGFI-A immunoprecipitated hippocampal tissue was run as a control for specificity and showed no signal. (c) Relative optical density (ROD; mean ± s.e.m.) of exon 17 sequence amplified from acetyl-histone H3-K9 or NGFI-A immunoprecipitated hippocampal tissue of adult high- and low-LG-ABN offspring (n = 4 animals/group; *P < 0.001; **P < 0.0001). Figure 3 HDAC inhibitior (TSA) eliminates maternal effect on histone acetylation and NGFI-A binding. (a) Chromatin immunoprecipitation analysis of the association between histone H3-K9 acetylation and NGFI-A binding to the exon 17 GR promoter sequence in hippocampal tissue from vehicle- and TSA-treated (100 ng/ml) adult offspring of high- and low-LG￾ABN mothers (n = 4 animals/group; lane labels as described in Fig. 2). (b) Relative optical density (ROD; mean ± s.e.m.) of exon 17 sequence amplified from acetyl-histone H3-K9 or NGFI-A immunoprecipitated hippocampal tissue (*P < 0.05; **P < 0.01). © 2004 Nature Publishing Group http://www.nature.com/natureneuroscience