supp figures final - nature research...the density plots show the distribution of average log2...
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![Page 1: Supp Figures final - Nature Research...The density plots show the distribution of average log2 ratios (calculated in regions covering -700 to +200bp relative to the TSS) for the …](https://reader033.vdocuments.us/reader033/viewer/2022042116/5e935c39bc863c491e1183f4/html5/thumbnails/1.jpg)
A MeDIP from 2ug DNA
MeDIP-WGA from 150ng DNA
60
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140In
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t enr
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Dpe
p3B
rdt
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inTu
ba3
Pap
olb
Dpp
a4O
ct4
LN11
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ICR
Tbx1
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ido1
Ube
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aIG
d
MeD
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Test
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p3B
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apol
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ppa4
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MeDIP-WGA1 C MeDIP WGA
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pooled MeDIPs
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Calr4
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C MeDIP-WGA
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Sycp15m
C (l
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eDIP
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t)
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pooled MeDIPs
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D MeDIP-WGA
pooled MeDIPs
log2 ratioMeDIP/input
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pooled MeDIPs
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Supplementary Figure 1. Validation of DNA methylation profiles obtained with MeDIP-WGA on low amounts ofDNA. A) MeDIP was performed with 2μg sonicated E9.5 genomic DNA (left), and with 150ng sonicated E9.5 genomic DNAfollowed by whole genome amplification (WGA, right). The graphs show the enrichments in the MeDIP vs input fraction ofmethylated sequences (gene promoters Dpep3-Oct4, intergenic region LN11, H19 ICR) over unmethylated CpG islandpromoters (Tbx15-Ube2f) and regions with no CpG (IGa, IGd). Values are normalized to IGd whose ratio is set to 1. Errorbars represent standard deviations from three experiments. B) MeDIP-WGA samples prepared from 150ng DNA from E9.5embryos were hybridized to Nimblegen HD2 arrays representing 12kb around gene promoters. In parallel, we hybridized 25pooled unamplified MeDIPs, each prepared from 2μg DNA from E9.5 embryos, on Nimblegen 385K arrays representing2 5kb around gene promoters (pooled MeDIPs could not be hybridized onto HD2 arrays because of limiting DNA2.5kb around gene promoters (pooled MeDIPs could not be hybridized onto HD2 arrays because of limiting DNAquantities). The graphs show smoothed MeDIP/Input ratios of individual oligos in three regions, which illustrates that MeDIPprofiles obtained from 150ng DNA are very accurate. Genes are shown below the graphs as grey boxes, and transcriptionstart sites as grey arrows. Red bars represent the position of CpGs. C) In this example, MeDIP enrichments are partiallylost after WGA in a discrete CpG-rich region overlapping the gene start site. D) To test the effect of WGA at the genomescale, we averaged log2 ratios obtained with MeDIP-WGA on HD2 arrays and pooled MeDIPs on 385k arrays in regionscovering -400 to +400bp relative to all transcription start sites. The heat map shows the average log2 ratio for all genes onchromosome 14 (n= 704). Genes are ranked according to the average log2 ratio measured with pooled MeDIPs.
Nature Genetics: doi: 10.1038/ng.708
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A
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-7 -6 -5 -4 -3 -2 -1 0 1 2kb relative to TSS
3-8 -7 -6 -5 -4 -3 -2 -1 0 1 2kb relative to TSS
3-8 -7 -6 -5 -4 -3 -2 -1 0 1 2kb relative to TSS
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CpG count in promoter CpG count in promoterCpG count in promoter
E3.5 E6.5 EPB E9.52
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Supplementary Figure 2. Global distribution of promoter DNA methylation in early mouse embryos. A) Globaldistribution of MeDIP signals along tiled regions for the three promoter classes: high CpG promoters (HCPs), intermediate CpGpromoters (ICPs) and low CpG promoters (LCPs). The analysis was performed as described in Fig. 1C. B) To relate MeDIPenrichments to promoter CpG content, we averaged log2 ratios in regions covering -700 to +200bp relative to all TSS andcounted CpGs in the same regions plus 200bp on both sides. The graphs show the average log2 ratio relative to CpG count forall promoters. The red dots mark the median log2 ratio for each CpG count. Profiles in E6.5 epiblasts (EPB) and E9.5 embryos
5mC (average log2 ratio MeDIP/input)
5mC (average log2 ratio MeDIP/input)
5mC (average log2 ratio MeDIP/input)
are identical to the ones observed in human and mouse somatic cells: MeDIP enrichments peak at promoters with intermediateCpG content and are globally low for promoters with CpG count >40. C) The density plots show the distribution of average log2ratios (calculated in regions covering -700 to +200bp relative to the TSS) for the three promoter classes: HCPs, ICPs andLCPs. We observe an increased fraction of highly enriched promoters in E9.5 embryos and E6.5 EPB compared to E3.5blastocysts, mostly in the LCP/ICP classes. HCPs remain globally hypomethylated throughout development.
Nature Genetics: doi: 10.1038/ng.708
![Page 3: Supp Figures final - Nature Research...The density plots show the distribution of average log2 ratios (calculated in regions covering -700 to +200bp relative to the TSS) for the …](https://reader033.vdocuments.us/reader033/viewer/2022042116/5e935c39bc863c491e1183f4/html5/thumbnails/3.jpg)
E3.5
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t)
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ut)
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ut)
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/Inpu
Slc38a4
-1
0 pooledMeDIPs
0 1 2 kb
Nnat
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D
Snrpn
-1
0 pooledMeDIPs
-1
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1ratio
MeD
IP/In
put)
E3.5
E6.5 EPB
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-1
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15mC
(log
2 r
Igf2r
E9.5pooled
MeDIPs
0 1 2 kb
Supplementary Figure 3. DNA methylation profiles at known imprinted genes in early mouse embryos. The graphs showsmoothed MeDIP/Input ratios of individual oligos around known imprinted genes in E3.5 blastocysts, E6.5 epiblasts (EPB) andE9.5 embryos. For validation, we also show MeDIP profiles obtained with unamplified pooled MeDIPs at E9.5 on Nimblegen385K arrays. Genes are shown below the graphs as grey boxes, and transcription start sites as grey arrows. Red bars representthe position of CpGs. A) The Slc38a4 gene has been shown to carry a maternally methylated DMR in the 5’ portion of the gene.Our data indicate that methylation is present in E3.5 blastocysts and potentially inherited from gametes. B) The imprinted Nnatgene has been shown to carry a maternally methylated germline DMR spanning the transcription start site C) For the imprinted
gAir
gene has been shown to carry a maternally methylated germline DMR spanning the transcription start site. C) For the imprintedSnrpn gene, it has been shown that a germline DMR spans the first exon and intron but not the upstream promoter sequence,which is in agreement with our MeDIP profiles. D) At the Igf2r locus, the germline DMR is intronic and coincides with thepromoter of the non-coding RNA Air. In contrast, the Igf2r promoter has been shown to contain a secondary DMR that does notinherit methylation from gametes but gains progressive methylation during development, which is confirmed by our MeDIPprofiles.
Nature Genetics: doi: 10.1038/ng.708
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AE3.5
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/Inpu
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0pooled
MeDIPs
Gdf3 Dppa3 (Stella)
D
0 1 2 kb
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‐ + ‐ + ‐ +Taqα1
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**
Dppa3 (3)
Zfp42 (2)
mC
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eDIP
/Inpu
t)
mC
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eDIP
/Inpu
t)
-1
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MeDIPs
Zfp42 (Rex1)
0 1 2 kb
E9.5pooled
MeDIPs
Elf5
-1
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1*(3)
0 1 2 kb
5m5m
Supplementary Figure 4. DNA methylation profiles at pluripotency and trophoblast genes in early mouse embryos. A)The graphs show smoothed MeDIP/Input ratios of individual oligos in E3.5 blastocysts, E6.5 epiblasts (EPB) and E9.5 embryos.Pluripotency-associated genes Gdf3 and Dppa3 (also known as Stella) are in a gene cluster on chromosome 6 and are de novomethylated in E9.5 embryos. For validation, we also show MeDIP profiles obtained with unamplified pooled MeDIPs at E9.5 onNimblegen 385K arrays. Genes are shown below the graphs as grey boxes and transcription start sites as grey arrows. Redbars represent the position of CpGs. B) The pluripotency gene Zfp42 (also known as Rex1) is de novo methylated in E9.5embryos. C) COBRA in the promoter of pluripotency genes Zfp42 and Dppa3 confirm that they are de novo methylated at thetransition from the epiblast to E9.5 embryos. The number of TaqαI sites in the PCR product is indicated in parenthesis. Thestars mark restriction fragments representing end products of the digestion. D) Methylation in the promoter region of theg p g p g ) y p gtrophoblast-specific gene Elf5 is detected in E6.5 epiblasts and E9.5 embryos.
Nature Genetics: doi: 10.1038/ng.708
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E3.5
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og2
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IP/In
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IP/In
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C (l
o
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Syce1
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og2
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MeDIPs
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Brdt5m
C-1E9.5
pooledMeDIPs
-101
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E9.5
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C (l
og2
ratio
MeD
IP/In
put)
Supplementary Figure 5. DNA methylation profiles at germline-specific genes de novo methylated during earlydevelopment. The graphs show smoothed MeDIP/Input ratios of individual oligos in E3.5 blastocysts, E6.5 epiblasts (EPB) and
-1
0
1 E9.5pooled
MeDIPs
Asz1
5mC
NM_175017
-1
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MeDIPs
5mC
development. The graphs show smoothed MeDIP/Input ratios of individual oligos in E3.5 blastocysts, E6.5 epiblasts (EPB) andE9.5 embryos for seven genes specific of germ cells, including one gene expressed in oocytes (NM_175017). For validation, wealso show MeDIP profiles obtained with unamplified pooled MeDIPs at E9.5 on Nimblegen 385K arrays. Genes are shown belowthe graphs as grey boxes and transcription start sites as grey arrows. Red bars represent the position of CpGs. Note that theMei1 and Syce1 gene promoters are not tiled on the Nimblegen 385K arrays. These profiles show a consistent wave of de novomethylation at germline genes during implantation, at the transition from E3.5 blastocysts to E6.5 epiblasts. We performedvalidations by COBRA and HpaII digestion for several of these genes (Fig. 2, Supplementary Fig. 6).
Nature Genetics: doi: 10.1038/ng.708
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A
C CGG
CCGGGGCC
CCGGGGCC
GGCCCCGG
CCGGGGCC
GGC C
CGGC
CGGC
CGGC
CGGC
CGGC
CGGC
HpaII
MspI
% resistant
B
CGGC
CGGC
0-7
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% resistant to digestion
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HpaII MspI
Supplementary Figure 6. Validation of DNA methylation by HpaII digestion. A) Genomic DNA is digested with themethylation-sensitive enzyme HpaII, which does not cut when the CG in its recognition site (CCGG) is methylated (blackdots). As a control, genomic DNA is also digested with the methylation-insensitive isoschizomer MspI. Digested andundigested control DNA are then used as templates for qPCR with primers (black arrows) spanning the site of interest. Wecalculate the percentage of alleles resistant to HpaII digestion, which reflects the level of methylation of the CCGG site inthe PCR fragment. B) The cartoon on the left shows the level of methylation for HpaII sites in the promoter of severalgermline-specific genes. Methylation was measured in blastocysts (E3.5), epiblast (EPB) and extraembryonic ectoderm(EE) from E6.5 embryos, adult liver and sperm. The percent of undigested alleles is given in the top left corner of thesquare This confirms that Tuba3 and Dpep3 promoters are methylated on around half the alleles in blastocysts whereassquare. This confirms that Tuba3 and Dpep3 promoters are methylated on around half the alleles in blastocysts, whereasothers (Brdt, Dazl, Prss21) are not. Smc1b is a control germline-specific gene not hypermethylated in somatic tissues. Thepromoter of the Oct4 gene, which is hypermethylated in extraembryonic lineages and differentiated tissues, is used as acontrol. Almost complete digestion is observed with MspI in all samples (right), which shows that the experimentalconditions allow full enzymatic digestion.
Nature Genetics: doi: 10.1038/ng.708
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Tex12 Prss21
Blastocyst E3.5
Blastocyst E3.5
A B
EpiblastE6.5
EpiblastE6.5
Supplementary Figure 7. Bisulfite sequencing at germline-specific genes de novo methylated during earlyd l t W f d bi lfit i i th t i f li ifi T 12 (A) d
E9.5E9.5
development. We performed bisulfite sequencing in the promoter region of germline-specific genes Tex12 (A) andPrss21 (also known as Testisin, B). These results confirm the MeDIP profiles and show that these genes gain DNAmethylation during implantation in epiblast cells. Circles represent CpG dinucleotides either unmethylated (open) ormethylated (closed).
Nature Genetics: doi: 10.1038/ng.708
![Page 8: Supp Figures final - Nature Research...The density plots show the distribution of average log2 ratios (calculated in regions covering -700 to +200bp relative to the TSS) for the …](https://reader033.vdocuments.us/reader033/viewer/2022042116/5e935c39bc863c491e1183f4/html5/thumbnails/8.jpg)
AE3.5
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atio
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0 1 2 kb
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2 r
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2 ra E9.5
E9.5pooled
MeDIPs
Cryaa
BE3.5
E6.5EPB E9.5
‐ + ‐ + ‐ +Taqα1
***
Cplx4(3)
E6.5EE‐ +
Ad Retina‐ +
AdLens‐ +
AdKidney‐ +
Supplementary Figure 8. Promoter DNA methylation at eye genes is erased during eye development. A) The graphsshow smoothed MeDIP/Input ratios of individual oligos in E3.5 blastocysts, E6.5 epiblasts (EPB) and E9.5 embryos. Thepromoters of Cplx4, a gene expressed in retina, and Cryaa, a gene encoding an essential component of the lens, are denovo methylated during implantation. For validation, we also show MeDIP profiles obtained with unamplified pooled
Cryaa(3) **
*
y g p p p pMeDIPs at E9.5 on Nimblegen 385K arrays. Genes are shown below the graphs as grey boxes and transcription start sitesas grey arrows. Red bars represent the position of CpGs. B) Validation by COBRA confirms that Cplx4 and Cryaa are denovo methylated at E6.5 in the epiblast (EPB) and the extraembryonic ectoderm (EE). In adults (Ad), promoter methylationis erased in retina or lens, but maintained in other tissues such as kidney. The number of TaqαI sites in the PCR product isindicated in parenthesis. The stars mark restriction fragments representing end products of the digestion.
Nature Genetics: doi: 10.1038/ng.708
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1 E3.5
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(lo
Pcdha1Pcdha1a2
a3a4
a5a6
a7a8
a9a10
a11 a12
0 4 8 kb
Supplementary Figure 9. DNA methylation profiles at the protocadherin-alpha locus in early mouse embryos. This genelocus contains a constant C-terminal region and variable amino-terminal regions transcribed from multiple alternative promoters inthe nervous system. The graphs show smoothed MeDIP/Input ratios of individual oligos in E3.5 blastocysts, E6.5 epiblasts (EPB)and E9.5 embryos. For validation, we also show MeDIP profiles obtained with unamplified pooled MeDIPs at E9.5 on Nimblegen385K arrays. Genes are shown below the graphs as grey boxes and transcription start sites as grey arrows. Red bars representthe position of CpGs.
Nature Genetics: doi: 10.1038/ng.708
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-1
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eDIP
/Inpu
t)
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eDIP
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MeDIPs
Tssk2
5mC
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B
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eDIP
/Inpu
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eDIP
/Inpu
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y
E3.5
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E3.5
E6.5 EPB
-1
0
1
Cd4
-1
0
15mC
(log
2 ra
tio
-1
0
1
Rrh
-1
0
15mC
(log
2 ra
tio
0 1 2 kb
E9.5
E9.5pooled
MeDIPs
E9.5
E9.5pooled
MeDIPs
Supplementary Figure 10. DNA methylation profiles at genes methylated throughout early development. The graphs showsmoothed MeDIP/Input ratios of individual oligos in E3.5 blastocysts, E6.5 epiblasts (EPB) and E9.5 embryos for six testis-specific genes (A) and two somatic genes (B), all harboring a methylated region overlapping the TSS throughout early embryonicdevelopment. For validation, we also show MeDIP profiles obtained with unamplified pooled MeDIPs at E9.5 on Nimblegen 385Karrays. We performed validations by COBRA, bisulfite sequencing and HpaII digestion for several of these genes (Fig. 4,Supplementary Fig. 6 and Supplementary Fig. 11). Genes are shown below the graphs as grey boxes and transcription startsites as grey arrows. Red bars represent the position of CpGs.
Cd4 Rrh
Nature Genetics: doi: 10.1038/ng.708
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Spermatozoa E2.5Morula
E3.5 Blastocyst
E6.5Epiblast
Dpep3A
E9.5
Oocyte
BCkt2
CSpaca4
Spermatozoa
Oocyte
E2.5Morula
E3.5 Blastocyst
E6.5Epiblast
C t
E9.5
E3.5 Blastocyst BL6xJF1
SpacaSpermatozoa
Oocytepat
mat
ESpermatozoa
Cd4D
SpermatozoaRrh
p
Oocyte
E3.5 Blastocyst
E6.5 Epiblast E9.5
p
Oocyte
E3.5Blastocyst
E6.5Epiblast E9.5
E2.5Morula
Supplementary Figure 11. Bisulfite sequencing at promoters methylated in preimplantation embryos. A-B) Bisulfitesequencing in the promoter of testis-specific genes Dpep3 and Ckt2 reveals hypermethylation in oocytes but not spermatozoa,a mixture of methylated and unmethylated alleles in morulas and blastocysts, and full methylation in E6.5 epiblast and E9.5embryos. This suggests post-fertilization inheritance of DNA methylation from the oocyte, while paternal alleles are de novomethylated during implantation. C) Bisulfite sequencing in the promoter of testis-specific gene Spaca4. This gene is methylatedin both spermatozoa and oocytes, however hybrid BL6xJF1 embryos show that predominantly maternal alleles are DNAmethylated in E3 5 blastocysts D-E) Bisulfite sequencing in the promoter of eye gene Rrh and hematopoietic gene Cd4 Thesemethylated in E3.5 blastocysts. D E) Bisulfite sequencing in the promoter of eye gene Rrh and hematopoietic gene Cd4. Thesegenes are methylated in both spermatozoa and oocytes. Profiles in preimplantation embryos are consistent with one parentalallele inheriting DNA methylation, whereas all alleles become methylated after implantation. Circles represent CpGdinucleotides either unmethylated (open) or methylated (closed).
Nature Genetics: doi: 10.1038/ng.708
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Tex12 Sycp1
E9.5
BA
E9.5WT
E9.5Dnmt3b-/+
WT
E9.5Dnmt3b-/+
E9.5Dnmt3b-/-
(83%)
(61%)
(94%)
(79%)
E9.5Dnmt3b-/-
(61%)
(16%)
(15%)
DDpep3
E9.5WT
E9.5Dnmt3b-/+
(91%)
C Brdt
E9.5WT
E9 5
(94%)
E9.5Dnmt3b-/-
(90%)
(69%)
E9.5Dnmt3b-/+
E9.5Dnmt3b-/-
(85%)
(23%)
Supplementary Figure 12. Bisulfite sequencing at germline-specific genes in Dnmt3b knockout embryos. Weperformed bisulfite sequencing in the promoter region of germline-specific genes Tex12 (A), Sycp1 (B), Brdt (C) and Dpep3(D) in wild-type (WT) and Dnmt3b mutant E9.5 embryos. Tex12 and Sycp1 genes are slightly hypomethylated in Dnmt3b-/+embryos and show severe hypomethylation in Dnmt3b-/- embryos compared to wild-type embryos. Interestingly, Brdt andDpep3 only show partial or moderate hypomethylation in Dnmt3b-/- embryos, suggesting that DNMT3A cooperates withDNMT3B t th l t th t t Th t f t i th l ti i th lifi d f t i i iDNMT3B to methylate these targets. The percentage of cytosine methylation in the amplified fragment is given inparenthesis. Circles represent CpG dinucleotides either unmethylated (open) or methylated (closed).
Nature Genetics: doi: 10.1038/ng.708
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E3.5E6.5EPB E9.5
EScells
BE3.5
E6.5EPB E9.5
EScells NPs
A
10-1
log2 ratioMeDIP/Input
C
Gene promoters hypermethylated in ES cells
E3.5
38%
E6.5 EPB
69%
E9.5
85%
Supplementary Figure 13. Comparison of promoter DNA methylation in ES cells and early embryos. We usedpublished data measuring DNA methylation in differentiating ES cells derived from preimplantation blastocysts for regions
10-1
log2 ratioMeDIP/Inputlog2 ratio>0.3
log2 ratio<0.3
published data measuring DNA methylation in differentiating ES cells derived from preimplantation blastocysts for regionscovering -700 to +200bp relative to all gene start sites (Mohn F et al., 2008 Mol Cell 30:755-766). To compare with ourdata, we averaged oligo log2 ratios from early embryos in the same region (-700 to +200bp relative to the TSS) for allgenes that could be unambiguously mapped between both datasets. Promoters from all three promoter classes (LCPs,ICPs and HCPs) were considered. A) This first heatmap shows average promoter log2 ratios for 175 gene promoters denovo methylated during differentiation of ESCs into Pax6-positive neuronal progenitors (NPs). B) The heatmap showsaverage promoter log2 ratios for 675 gene promoters found to be hypermethylated in cultured ES cells. Most genesmethylated in ES cells are not methylated in blastocysts but gain DNA methylation in postimplantation embryos. C) Thegraphs show the fraction of gene promoters hypermethylated in ES cells that is also hypermethylated (average log2 ratio>0.3) in early embryonic stages.
Nature Genetics: doi: 10.1038/ng.708
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Supplementary Table 2. Primer sequences.
PCR for COBRA
Oct4 AGAATTGAGGAGTGGTTTTAGAAATAATCCACCTAAAAACCCTTAAAAACTTAAC
Asz1 TAAGTTAAAGTTTTGGGAAAAAGTTAGGTTATTTCTAATAAAAATCCATCTCACCC
Sycp1 TGAGTTAGGTTTTTTAGAGGGGTTTASycp1 TGAGTTAGGTTTTTTAGAGGGGTTTAAAAAAAACTTTCCCAACATCCTC
Tex12 TAAGTTAGTTTGGAATTTTTTGGGTAGTAACTTACTCCAACTTCTCCACAATATAAA
Papolb TGTGGAAGATTTTTGTTTTTATTGATCCCTAAACCTAACTAAAACATCCTCTAA
Spo11 GAGAGGATTTGGTATAGAGGTAGGAAGTATCACCACCAAAAAACACTAAAAAAC
Piwil1 TATTTGTAGTGGGTTTTGGTATAGGGCACAAATAAACACAACTTAATTCCAAACCACAAATAAACACAACTTAATTCCAAAC
Dpep3 GAGTAGTTAGGGTGTAGGTTATTTATAGAGATCAAACACTAACACACATAAATCTAACC
Tssk2 TTTTATAGTGGGAATGAGGATAATGTTTCCACAAACCATAATATAAAAAATCACAC
Ckt2 GAGGTTTTTGTTTGTTATTGAGTGAAGTAGCCCTAAATATCATCTAAACTACACCTTCCT
Spaca4 TTTTATTTGGTAAGGTAAGGATGTTTGATACTAATCACAACCAACCTAAAAACAAC
Hdhd1a TGTTTTTTTAGGTGGTGGATTAATTTTHdhd1a TGTTTTTTTAGGTGGTGGATTAATTTTCCATTACAAACAAAACCAAAATTTATATTT
Cd4 GTAGGTAAAGATGAGAAGGGTTTATTGTAATTTTTCTACCTAAATCACCCAATACTA
Fyb AGGTAGGTGAGGTAGGATTTTTTAGAGAACTACCAAATAAATTTTCCCACTTTT
Rrh TTAGAGATATAGGGGAGTGAGATATAGTAACCTAAAAACCAAAACAAATAAAAACTC
Pou2af1 TGGGAAATTAGTTTTAGTTAAGAAGTTTGATACTTAAAACACAAAACCAACCAATTAATACTTAAAACACAAAACCAACCAATTA
Pscdbp TTAATTGGGTAGTAGATAGAGGTTTTTTATATCTCAAACCACAACAATCACATAA
Tlr6 TTTTAGATTTTTTTGTGGATAAAGGGTATCCCAAACTTAAAACTAAAATTAATCAAAC
Dazl GATTTTTGTTATTTTTTAGTTTTTTTAGGATAAAATTCTCTCAACTAACCTAACTTATTTCT
Adam2 TTTTGGGATGTTTAAGTTAATATGGAGAAATTACCAAAAAATCAAACAAAAAATT
Dppa3 TGATTAGGGTTGGTTTAGAATTTAGAGDppa3 TGATTAGGGTTGGTTTAGAATTTAGAGCCAATTACTAATAAAAAAATATTACCCCC
Zfp42 GGTTAATGGATTAAGGTAGTTGGTGATCATAACTAAACAAAAATCTTTACCCCAC
Cplx4 GGTTTTGGAGAATTGAAATTTTGTTAAAACCTAACATACACAAAACCCTTCTCT
Cryaa ATTTGGTTTGAGAGTTTTTGTTGTTTAGAAAAACCAATATAATTACATCTTACCTCAA
Crygd GGGGTTGGGAGTAGGGTTTATATAGTAAATCTCCCATAAACTTAAAATTTTCACCC CCC C C CC
Nature Genetics: doi: 10.1038/ng.708
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PCR for bisulfite sequencing
Sycp1 Same primers as for COBRA
Dazl Same primers as for COBRA
Tex12 Same primers as for COBRA
Dpep3 Same primers as for COBRA
Ckt2 Same primers as for COBRA
Cd4 Same primers as for COBRA
Rrh Same primers as for COBRA
Prss21 ATAAGGAGGAAGGTTATTAAAGAGAAGTAGTATCTACCTATCACTACCCCCAATCT
Piwil1 TTAGTTATTTATTTTAAGAATTTGGAAAAAACACCTACTAACAACCCCAACTAC
Tssk2 TTTTATAGTGGGAATGAGGATAATGTTTCCCAACTCCATAACAATATAAATAC
Spaca4 AAGGATGTTTGAAGGATAATTTTATAAAAACTCTACTCTACCCCACTAAAAAAC
Brdt TGTTTGGAAAATAAAATATAGGAAAGTTTTCACCCTTTCAATTTACACTAAATAAAA
qPCR on MeDIP samples
Dpep3 Same primers as for PCR after HpaII digestion
Brdt Same primers as for PCR after HpaII digestion
TCACCCTTTCAATTTACACTAAATAAAA
Brdt Same primers as for PCR after HpaII digestion
Tuba3 Same primers as for PCR after HpaII digestion
Prss21 Same primers as for PCR after HpaII digestion
Tex12 TGTCTCTTCACGCCATTTCCAACGTGGCGAGTGTGAAAG
Papolb AGAACCGACGGCACACGCGCGAGAATTTCCTCCAATC
Dppa4 GGCCTCAAGACAACAGGAAGTCCAGCAGTCTCCATCTTGATCCAGCAGTCTCCATCTTGA
Oct4 ATCCGAGCAACTGGTTTGTGGACGTCTGGACAGGACAACC
LN11 TGGCAGGCAGCTTGGTAGTCAATGGCTGGGACAGAGCAGC
H19 ICR GCATGGTCCTCAAATTCTGCAGCATCTGAACGCCCCAATTA
Tbx15 TCCCCCTTCTCTTGTGTCAGCGGAAGCAAGTCTCAGATCC
Dido1 CAGAGCTGGGTCCACACCATACDido1 CAGAGCTGGGTCCACACCATACGTTCTCAGGGACATGCACGAAG
Ube2f CAGCTGCAAACCGGTCGGATTCGGTCACCGAAGCGCAAGCATC
IGa TGGTTGGCATTTTATCCCTAGAACGCAACATGGCAACTGGAAACA
IGd CCCTCTGGCCCTGAATTTATCACCCAGCAATGCTTCAGT
Nature Genetics: doi: 10.1038/ng.708
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PCR after HpaII digestion
Oct4 ATCCGAGCAACTGGTTTGTGOct4 ATCCGAGCAACTGGTTTGTGGACGTCTGGACAGGACAACC
Dpep3 GCAGGTTACCCACAGAGACGGTGACCAAGACTGAGCACCA
Brdt GCAGACCCTGGCCAATCAGTGGCCGCCAGCTCTCGC
Prss21 CAAGACGTTGGTGCCACTGCACTGCCCCCAGTCTCAC
Dazl ATTTACAAAATGCCCGCAGAAACCTTCTCAATGTGGCTGAA
Reverse transcription qPCR
AACCTTCTCAATGTGGCTGAATuba3 GTTCCTCACCACTGGTCCTC
CCAGCACTGCAACTTGAGCSmc1b GACTGGCGCGGGGGAAG
CAGCGGAGCAGGGATCCAA
Reverse transcription qPCR
Brdt TCCTGTGGATGCTGATGCTTTGGAAACGTCCCAACGGTCCTCTTC
Dazl TTGGACATTATGCATTGTGACAGCAGAGATGTCCCTGGCAGGTCAGATTG
Sycp1 AGGACCGTTGGACAACGATTGCCCTTGGTAAAGTTTGGCTCTCTTGG
Tex12 GAGATAGATGGACTCTTCAAAGAAGCCTTCTCTAGCAAGAAGAACACAGCAGTAAC
Tuba3 GCTGAGCAATACCACGGCCATCCTCCCCTTCTTCTGCCTCTGC
Dppa3 ATGCCGCACAGCAGATGTGAAAGTATTCCTCAGCCCTGGGCCTC
Zfp42 AAGCCAGAGGGCGGTGTGTACCCTTCGAACGTGCACTGATACG
Pou2af1 TCCAGCCTGCAGTATCAACCTCTGGTTGAGCTCGTACGTGTTGC
Tlr6 AGAGGAACTTTGTCCCTGGCAAGAGAATGGGTTCCAGCAAGATGAGG
Gapdh CTACAGCAACAGGGTGGTGGACGGGTGCAGCGAACTTTATTGATGG
Rpl13A GGTCCTCAAGACCAACGGACTCGTGCGCTGTCAGCTCTCTAATG
Beta-actin GCGGACTGTTACTGAGCTGCGTGTTTGCTCCAACCAACTGCTGTC
Nature Genetics: doi: 10.1038/ng.708