Supplementary Table 1. Primers used for qPCR
Transcript Forward
Dusp1
Fkbp5
Hprt
5’- gagctgtgcagcaaacagtc-3’
5’-gggtgtacgccaacatgttc-3’
5’- acctctcgaagtgttggatacagg-3’
Sgk1
Crh
YY1
5’-cttctggactaatgatgatgcagc-3’
5‘-gcgggctcacctaccaag-3‘
5’-gatgatgctccaagaacaatagc-3’
Reverse
5'-cttccgagaagcgtgatagg-3’
5’-gaggagggccgagttcatt -3’
5'-cttgcgctcatcttaggctttg-3’
5'-tgccctttccgatcactttc-3’
5-ttcttcacccatgcggatcag-3’
5'-tagctttgagctctcaacgaac-3’
Primer sequences for total GR mRNA or exon 1 GR transcript variants and Atp5j are listed elsewhere.
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Supplementary Table 2. Primers used for bisulfite sequencing
Region
CpG1–9
Forward
5’-ttttttttttggagaaggaggtt-3’
CpG5–13
5’-gattgtgtagtatttttggtttag-3’
CpG12–18
5’-tgaagttaaagagtatttttgtta-3’
CpG19–26
5’-ttagagatttttaagaggttaggt-3’
CpG27–34
5’-gtatttgtgggggagtaaaagttat-3’
CpG36–47
5’-gggtagtttaggggtaggttt-3’
CpG48–54
5’-gtagaggggtttttggaatttag-3’
CpG64–76
5’-ggggaggtttgaatttggtag-3’
CpG76–112
5’-agggtgggttttgttttgtaat-3’
CpG108–125
5’-gtagtttttttgttagagtgatatat-3’
CpG132–154
5’-gtggggaaaaaaggttaagaagt-3’
CpG157–191
5’-gttgtygatagtygttaataggttgtat-3’
CpG192–220
5’-gagttyggagttagtgtttggagtt-3’
CpG221–252
5’-gttaggggttttttttttttttatgga-3’
CpG253–278
5’-gggatttggtaaatttttgttagtt-3’
Supplementary Table 3. DNA oligomers used for EMSA
Reverse
5’- taaaaaatataaaaactacccaatatatc-3’
5’-ccatcttaataaaaaacaataac-3’
5’-acctaacctcttaaaaatctctaa-3’
5’-ataacttttactcccccacaaatac-3’
5’-gggtagtttaggggtaggttt-3’
5’-ctaaattccaaaaacccctctac-3’
5’-atatatacaaacctaaaaacaaattac-3’
5’-ccaccraaaaaaattacaaaacaa-3’
5’-acaaaaccrcaaaaataatcc-3’
5’-tttctttaatttctcttctccctaactcc-3’
5’-aacractatcracaactacaaaaac-3’
5’-actcraactccaaacactaac-3’
5’-ccataaaaaaaaaaaaaacccctaac-3’
5’-aactaacaaaaatttaccaaatc-3’
5’-ccaaaaaaaaaaaactaactatcctaac-3’
Name wt CpG3 sense
Sequence
5’-agcttcggctcctttgccaagatggcggccctggctcccatg-3’ wt CpG3 antisense
5’-gatccatgggagccagggccgccatcttggcaaaggagccga-3’ mut TpG2 sense
5’-agctttggctcctttgccaagatggcggccctggctcccatg-3’ mut TpG2 antisense
5’-gatccatgggagccagggccgccatcttggcaaaggagccaa-3’ mut TpG3 sense
5’-agcttcggctcctttgccaagatggtggccctggctcccatg-3’ mut TpG3 antisense
5’- gatccatgggagccagggccaccatcttggcaaaggagccga-3’ mut CpA3 sense
5’-agcttcggctcctttgccaagatggcagccctggctcccatg-3’ mut CpA3 antisense
5’- gatccatgggagccagggctgccatcttggcaaaggagccga-3’
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Supplementary Table 4. Primers used for ChIP-qPCR analysis
Gene/region
GR
GR
GR
CGI shore
CGI
Dusp1
Sgk1
–6 kb
GRE
Fkbp5 GRE
3
Forward
5’-tccttcttgaggtgtcaagcttc-3’ 2
5’-cttggcacttctgatcggag-3’
5’-acttcatctgtgtacaatggac-3’
5’-ctgggtccactttcccac-3
5’-gttcagctgtgcaatccaga-3’
5’-acctcctcacgtgttcttgg-3’
Supplementary Table 5. Antibodies
Reverse
5’-atgtaaaggctgcccaatgtg-3’
5’-aatggtgaccgtgtggcgtc-3’
5’-tacagaacttgatccaatgtcac-3’
5’-ggcctgagtgggtctcttct-3’
5’-agggtgttctgtgctcttcaa-3’
5’-ggtatgagggggtcaggaat-3’
Antibody
FLAG M2
YY1(CM1, CM2)
(CM1)
Application (dilution) Company
Immunoblot (1:500) Sigma-Aldrich
Immunoblot (1:1000)
ChIP (1:50) custom-made
Santa Cruz Biotechnology YY1 (H-414 X) Immunoblot (1:1000)
ChIP (1:150)
EMSA (1:10)
ChIP (1:100) GR (H300 X)
IHC (1:1000) pCAF (H-369 X) EMSA (1:10)
Santa Cruz Biotechnology
Santa Cruz Biotechnology
HDAC1
HDAC2
ChIP (1:25)
ChIP (1:100)
Abcam
Abcam
Catalog number
F3165 sc-1703 X sc-8992 X sc-8999 X ab46985-100 ab7029
Ehmt2
Kdm5a
Suz12
ChIP (1:100)
ChIP (1:100)
ChIP (1:100)
Abcam
Abcam
Diagenode ab40542 ab70892
CS-029-100
Ezh2
Avp (PS 41)
ChIP (1:100)
IHC (1:100)
Merck Millipore
H. Gainer 4
07-689
Crh
β-Actin
IHC (1:500)
Immunoblot (1:3000)
Bachem
Cell Signaling
T-5007
8457
Primary antibodies were visualized using the appropriate secondary antibodies conjugated to
Dylight 488, Dylight 594 (1:500, Diagenode, 715-485-150, 711-585-152), Alexa 647 (1:500,
Dianova, Hamburg, Germany, 706-605-148), and IgG-Peroxidase (1:2000, Sigma-Aldrich,
A6782, A0545).
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Supplementary Figure 1.
GR mRNA expression in pituitary and hippocampal subfields. GR expression was detected in
6-week- and 3-month-old mice by qPCR in pituitary. Data are shown relative to the expression levels in the Ctrl group and normalized to Atpj5. Hippocampal GR mRNA was quantified in dentate gyrus (DG), CA1 and CA3 by ISH in 6-week- and 3-month-old mice
(two-way ANOVAs, no interaction or main effects of ELS and age in pituitary [n = 5-7] or within the hippocampal subfields [n = 4-14]). Data are means ± SEM.
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Supplementary Figure 2.
Methylation of GR promoter CGI in PVN of Ctrl and ELS mice starting at CpG10. CpG1 is defined at –4,732 base pairs (bp) relative to the ATG start codon. First exons mapping to
CpGs are shown as boxes above. Exon size is not scaled. Three human homologous YY1 binding sites are predicted in the mouse GR promoter. The distal YY1 site maps upstream of exon 1.3 at the CpG shore (not shown), whereas the middle (m) and proximal (p) YY1 site
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locate within exon 1.3. The mYY1 locates between CpG11 and CpG12. The pYY1 at CpG14 shows poor methylation in both groups. CpG residues mapping to the mouse homologous region upstream of exon 1
7
and the conserved NGFI-A binding site in rat
5
are marked by asterisks and double asterisks respectively (One-way MANOVAs for each amplicon, no ELS effect on site-specific CpG methylation). Data are means ± SEM; n = 5 mice per group.
Supplementary Figure 3.
Methylation of CpG 1–3 was analysed in tissues from 6 adult mice; one way ANOVA
(influential factor tissue) followed by modified LSD (Bonferroni) post hoc tests (*P < 0.001,
#P < 0.017).
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Supplementary Figure 4.
YY1 binding to DNA probe containing GR promoter CGI shore. ( A ) Representative EMSA of a DNA probe containing the predicted YY1 binding site at CpG3 of GR CGI shore. Shift of
32
P-labeled DNA probe by nuclear extract from LLC-PK1 cells transfected with pRK7-
FLAG-YY1 (lane 2). An increasing molar excess (5-, 50-, 500-fold) of unlabeled oligonucleotides harboring the consensus sequence of YY1 (yy1) competes with
32
P-labeled
DNA probes (lanes 3–5), whereas excess of oligonucleotides containing a mutated YY1 binding site (yy2) fails to compete (lanes 6–8). Specificity of YY1 shifting DNA probe was confirmed by adding an YY1-specific antibody (αYY1) (lane 9) and an unrelated pCAFspecific antibody (αpCAF) (lane 10). ( B ) YY1 binding to wt probe with CpG3 and to mutated probes (mut).
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Supplementary Figure 5.
Evaluation of custom-made (CM) YY1-specific antisera.
( A ) Representative immunoblot of protein extracts from LLC-PK1 cells transfected with either a FLAG-YY1 encoding plasmid (pRK7-FLAG-YY1, [+]) or filling DNA alone (mock,
[–]). Compared are the immunoreactivities of the FLAG-specific antibody (αFLAG), two polyclonal YY1-specific antisera raised in rabbits (CM1 and CM2) and a commercial antibody specific to YY1 (H-414 X). Red line of the marker represents 70 kDa. ( B ) ChIP of
YY1 in hypothalamic N44 cells at dYY1 binding site upstream of exon 1.3 (white box) at –
4.6 kb (relative to GR translation start codon). Comparison was made to a genomic region lacking canonical YY1 binding sites (–6.0 kb relative to GR translation start codon). Arrows indicate position of primer pairs; d, distal; m, middle; p, proximal.
Supplementary Figure 6.
Knock down of YY1 in mouse hypothalamic N6 cells using scramble (sc) and YY1 siRNA.
YY1 mRNA levels were quantified by qPCR and expression data are normalized to Atpj5 and relative to YY1 expression in sc siRNA transfected cells. (*P = 0.001 by one sample t-test; n
= 3 experiments in duplicate).
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Supplementary Figure 7.
Representative images of immunohistochemical triple-staining to detect GR, Crh and Avp on
PVN sections from Ctrl, ELS, CUS and ELSxCUS mice. Scale bar = 50 µm.
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Supplementary Figure 8.
Corticosterone (Cort) levels before and after stress (interactions between ELS, CUS and time;
P = 0.003 by three-way ANOVA; *P < 0.017,
#
P < 0.05 by univariate F-tests; n = 10-18).
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