Expression of <i>Suz12</i> and <i>Tet</i> genes correlated with the presence of methylcytosine (5mC) and hydroxymethylcytosine (5hmC) at the <i>Avp</i> locus during hypothalamic-like differentiation.

<p>A, <i>Suz12</i> gene expression rapidly decreased following 4 days of Lif withdrawal. B, Similarly, <i>Tet1</i> and <i>Tet2</i>, but not <i>Tet3</i>, gene expression rapidly declined following 4 days of Lif withdrawal. C, MeDIP showed high hydroxymethylation (5hmC) at the <i>Avp</i> gene body and downstream enhancer which strongly decreased upon Lif-withdrawal. Application of cyclopamine evoked a further decline at the enhancer region. D, Conversely, methylation (5mC) at the <i>Avp</i> enhancer, but not at the gene body, increased as evidenced by MeDIP. Bars represent standard deviations from the mean (sem), *, <i>P</i><0.05; from3 independent experiments performed in duplicate.</p

Mecp2 occupancy at the <i>Avp</i> enhancer coupled with repressive chromatin marks and repressor complexes.

<p>A, Chromatin samples from the PVN were subjected to seqChIP. The first ChIP was done with antibodies against H3Ac (panacetylated-histone H3), an active mark, and H3K9me2 (dimethyl-histone H3, Lys-9), a repressive mark. The second round ChIP was carried out with anti-Mecp2. Hereby, Mecp2 was preferentially, with a distinct trend toward significance, contained in the chromatin fraction associated with the transcriptionally inactive <i>Avp</i> enhancer. B, PVN tissues from Mecp2 null male (-/y) mice showed increased H3Ac and decreased H3K9me2 chromatin marks at the <i>Avp</i> enhancer compatible with a repressor function of Mecp2. However, due to the knockout of <i>Mecp2</i>, the anti-Mecp2 antibody did not enrich for either chromatin fraction attesting to its specificity. C, Sequential ChIP experiments showed that Mecp2 occupancy at the ELS-responsive enhancer associated with the chromatin fraction containing Hdacs and Dnmts. Chromatin samples from hypothalamus were immunoprecipitated in the first ChIP experiment with antibodies against Hdac1, Hdac2, Dnmt1, Dnmt3a and Dnmt3b. Following on two-thirds of the product of the primary ChIP were precipitated in the second ChIP with anti-C-terminal Mecp2; the remaining sample was used for the analysis of the primary ChIP. DNA recovered from both ChIP steps was analyzed by qPCR for the presence of the <i>Avp</i> enhancer. Bars represent standard deviations from the mean (sem), *, <i>P</i><0.05; #, <i>P</i><0.07 from 5 independent experiments performed.</p

Hypothalamic-like differentiation displaced repressive histone marks and Suz12 occupancy at the ELS-responsive <i>Avp</i> enhancer.

<p>A, Schematic diagram of the <i>Avp</i> locus. Exons are shown as boxes and numbered with coding parts shadowed. Amplified DNA segments depicted above. Arrow marks translational start codon. ChIP analysis was done for the promoter, gene body and downstream enhancer regions. The strong-CpG island at the gene body and the intermediate-CpG island harboring the ELS-responsive enhancer are shown beneath by black and grey bars, respectively. B–D, Chromatin samples from undifferentiated (+Lif), Lif-withdrawn (-Lif) and further cyclopamine treated EB5 cells were immunoprecipitated with antibodies against activated RNA polymerase II, Suz12, H3K27me3 (trimethylation-histone H3, Lys27), H3K9me2 (dimethylation-histone H3, Lys 9), and H4Ac (pan-acetylated-histone H4). Recovered DNA was analyzed by qPCR for the presence of the <i>Avp</i> promoter (B), gene body (C) or enhancer (D). Bars represent standard deviations from the mean (sem),*, <i>P</i><0.05; from 4 independent experiments.</p

Hypothalamic-like differentiation triggered ELS-responsive DNA methylation at the <i>Avp</i> enhancer.

<p>A, ELS-responsive DNA methylation rapidly evolved following 4 days of Lif withdrawal and was maintained during subsequent cyclopamine treatment as evidenced by bisulfite sequencing of 25 clones. Shaded hemispheres represent percentage of methylation at each ELS-responsive CpG residue. Results are representative of three independent experiments. B, Comparison of ELS-responsive DNA methylation at the Avp enhancer in the SON, PVN and following hypothalamic-like differentiation of EB5 cells. C, Overall CpG methylation at CpG island 3 (CGI3) comprising the ELS-responsive <i>Avp</i> enhancer in the developing mouse hypothalamus. Postnatal days (PND) are indicated. D, Avp mRNA expression as evidenced by qRT-PCR analysis in the developing mouse dorsal hypothalamus. Bars represent standard deviations from the mean (sem), from 4–5 independent experiments.</p

Hypothalamic-like differentiation of the embryonic stem cell line EB5.

<p>A, Pluripotency factors rapidly decreased in differentiating EB5 cells over 4 days following withdrawal of leukemia inhibitory factor (-Lif) as evidenced by qRT-PCR analysis. B, Markers for neuroectoderm and rostral hypothalamic development conversely increased during EB5 cell differentiation. C, qRT-PCR analysis of markers for dorsal-ventral hypothalamic patterning and PVN neurons demonstrated strong increases following 25 days of differentiation. D, PVN markers (<i>Brn2</i> and <i>Arnt2</i>) further increased following treatment with the sonic hedgehog (Shh) antagonist cyclopamine as evidenced by qRT-PCR. Application of the Shh agonist purmorphamine blocked this increase. Bars represent standard deviations from the mean (sem) from at least 3 independent experiments.</p

Figure 6

<p>A, DZNep treatment inhibited Suz12 occupancy and Mecp2 recruitment at the <i>Avp</i> enhancer during hypothalamic-like differentiation of EB5 cells. Chromatin samples from cells treated with DZNep during differentiation were immunoprecipitated with antibodies against Suz12, Mecp2 or H4Ac and recovered DNA analyzed by qPCR for the presence of the <i>Avp</i> enhancer. Cells treated with DZNep (DZ) in the undifferentiated state for 24 hrs (+Lif) showed strongly reduced Suz12 binding. Following 4 days of differentiation (DZ,-Lif 4 days), Mecp2 binding was decreased while H4Ac was increased. In contrast, following 3 days of differentiation an additional 24 hours application of DZNep (-Lif 4days, DZ) did not affect Mecp2 occupancy. B, DZNep treatment led to reduced methylation at the <i>Avp</i> enhancer. MeDIP evidenced reduced 5hmC at the downstream enhancer in response to increasing doses of DZNep, while methylation levels at the promoter and gene body regions did not change. Bars represent standard deviations from the mean (sem), *, <i>P</i><0.05; from 5 independent experiments performed.</p

Methylation at the CpG island shore region upregulates <i>Nr3c1</i> promoter activity after early-life stress

<div><p>Early-life stress (ELS) induces long-lasting changes in gene expression conferring an increased risk for the development of stress-related mental disorders. Glucocorticoid receptors (GR) mediate the negative feedback actions of glucocorticoids (GC) in the paraventricular nucleus (PVN) of the hypothalamus and anterior pituitary and therefore play a key role in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis and the endocrine response to stress. We here show that ELS programs the expression of the GR gene (<i>Nr3c1</i>) by site-specific hypermethylation at the CpG island (CGI) shore in hypothalamic neurons that produce corticotropin-releasing hormone (Crh), thus preventing Crh upregulation under conditions of chronic stress. CpGs mapping to the <i>Nr3c1</i> CGI shore region are dynamically regulated by ELS and underpin methylation-sensitive control of this region's insulation-like function via Ying Yang 1 (YY1) binding. Our results provide new insight into how a genomic element integrates experience-dependent epigenetic programming of the composite proximal <i>Nr3c1</i> promoter, and assigns an insulating role to the CGI shore.</p></div

Allele frequency in a NAB population (n = 165) for the SNP in the <i>Avp</i> signal peptide and the strictly linked promoter deletion.

<p>Allele frequency in a NAB population (n = 165) for the SNP in the <i>Avp</i> signal peptide and the strictly linked promoter deletion.</p

Alterations in behavior and vasopressin <i>(Avp)</i> expression.

<p>Phenotypic measures of HAB/NAB/LAB mice reflecting behavior (A) on the elevated plus-maze (EPM), in the (B) tail-suspension test (TST) and (C) forced swim test (FST) and corresponding vasopressin (<i>Avp</i>) expression patterns as measured by (D) <i>in situ</i> hybridization (ISH) in the paraventricular nucleus (PVN) in male mice and by (E) quantitative PCR (qPCR) in the PVN of male and female mice. Data are shown as means+SEM; * p<0.05; ** p<0.01; *** p<0.001; Mann-Whitney-U test.</p

Regulation of gene products of the vasopressin and oxytocin systems from the MPI24K platform-based microarray experiment of HAB vs. LAB mice.

<p>Values indicate the percentage of gene expression in LAB compared to HAB mice with HAB = 100%. Superscript numbers indicate the validation status: (1) significant gene expression differences confirmed by both quantitative PCR (qPCR) and <i>in situ</i> hybridization; (2) no differences in gene expression confirmed by receptor autoradiography; (3) no differences in gene expression confirmed by qPCR and <i>in situ</i> hybridization, respectively.</p