(A) Chromatin and expression landscape at the Zdbf2 locus in ESCs (top) and D7 EpiLCs (bottom). In hypomethylated naïve ESCs, Zdbf2 initiates from the pLiz promoter while a ~ 25 kb block of H3K27me3 …
Source data for Figure 1.
(A) Imprinted regulation of Liz and Zdbf2 in vivo. The Liz promoter is DNA methylated and silent on the maternal allele. Conversely, the paternal allele is expressed, leading to de novo DNA …
(A) H3K27ac ChIP-qPCR at control loci. The Jarid2 enhancer (E Jarid2) is active in ESCs and EpiLCs, whereas E Fgf5 is a poised enhancer (Buecker et al., 2014). pPax5 is a negative control. Data …
(A) Hi-C data from mouse ESCs (Bonev et al., 2017) overlaid with 4C-seq data from ESCs and EpiLCs (this study). The Zdbf2 locus is located in an inter-TAD, and 4C-seq shows that local interactions …
(A) Model for enhancer regulation based on 4C-seq data. (B) Model for deletion of E1-3.. (C) RT-qPCR of Liz (left) and Zdbf2 (right) during EpiLC differentiation in WT and the ∆E1-3 mutant. The ∆E1-3…
Source data for Figure 2.
(A) Alleles generated by CRISPR/Cas9 mediated deletions of enhancer elements. (B) RT-qPCR of Liz (left) and Zdbf2 (right) during EpiLC differentiation in WT and the ∆E3 mutant. There is no effect on …
(A) H3K27ac ChIP-qPCR in WT and Eed-/- ESCs. H3K27ac levels are unaffected at pLiz and E1-3, but E4 and pZdbf2 become aberrantly activated in Eed-/- ESCs. Data shown as ±s.e.m. from two biological …
Source data for Figure 3.
Ratios between 4C-seq signals is indicated in between the samples. While pZdbf2 interacts with E1-4 at greater frequencies in ∆Liz EpiLCs compared to ∆ Liz ESCs, it is not to the same extent as WT …
(A) 4C-seq tracks from the CTCF_PS VP in WT ESCs and EpiLCs. Ratios between 4C-seq signals is indicated between the samples, and gene and CTCF binding tracks (Stadler et al., 2011) are below. The …
Source data for Figure 4.
(A) 4C-seq tracks from the CTCF_PS VP in WT and Eed-/- ESCs. Ratios between 4C-seq signals is indicated between the samples. The partition loop still persists in Eed-/- ESCs, even though Zdbf2 is …
(A) Alleles generated by CRISPR/Cas9-mediated deletions of CTCF_PS. (B) 4C-seq tracks for WT and ∆ ESCs. Top: pLiz VP exhibits less interactions with E1-3 in absence of CTCF partition (high-lighted …
(A) H3K27me3 ChIP-qPCR in ESCs (Left) and EpiLCs (Middle) in WT and the ∆ mutant. There is no significant effect on polycomb dynamics in ESCs. In EpiLCs, the ∆ mutant retains residual H3K27me3 …
Reagent type (species) or resource | Designation | Source or reference | Identifier | Additional information |
---|---|---|---|---|
Cell Line (M. musculus) | E14TG2a (WT) | ATCC | CRL-1821 | |
Cell Line (M. musculus) | E14TG2a_∆Liz | Bourc’his Lab | Greenberg et al., 2017 | |
Cell Line (M. musculus) | E14TG2a_∆E1 | Bourc’his Lab | Greenberg et al., 2017 | |
Cell Line (M. musculus) | E14TG2a_∆E1-3 | This study | CRISPR/Cas9 generated mutant, sgRNA oligos are listed in Supplementary file 1 | |
Cell Line (M. musculus) | E14TG2a_∆E4 | This study | CRISPR/Cas9 generated mutant, sgRNA oligos are listed in Supplementary file 1 | |
Cell Line (M. musculus) | E14TG2a_CTCF-AID-eGFP, Tir1 | Gift from E Nora | Nora et al., 2017 | |
Cell Line (M. musculus) | J1 (WT) | ATCC | SCRC-1010 | |
Cell Line (M. musculus) | J1_Dnmt tKO | Gift from M Okano | Tsumura et al., 2006 | |
Cell Line (M. musculus) | J1_Eed-/- | This study | CRISPR/Cas9 generated mutant, sgRNA oligos are listed inSupplementary file 1 | |
Cell Line (M. musculus) | J1_∆CTCF_PS | This study | CRISPR/Cas9 generated mutant, sgRNA oligos are listed in Supplementary file 1 | |
Cell Line (M. musculus) | J1_Eed-/-; ∆CTCF_PS | This study | CRISPR/Cas9 generated mutant, sgRNA oligos are listed in Supplementary file 1 | |
Cell Line (M. musculus) | J1 Clone 36 (WT) | Gift from A Wutz | Wutz and Jaenisch, 2000 | |
Cell Line (M. musculus) | J1 Clone 36_Eed-/- | Gift from A Wutz | Schoeftner et al., 2006 | |
Antibody | Anti-H3K27me2, mouse monoclonal | Active Motif | 61435 | (1:5000) |
Antibody | Anti-H3K27me3, rabbit monoclonal | Cell Signaling Technology | C36B11 | (1:5000) |
Antibody | Anti-H3K27ac, rabbit polyclonal | Active Motif | 39133 | (1:5000) |
Antibody | Anti-CTCF, rabbit polyclonal | Millipore | 07–729 | (1:5000) |
Antibody | Anti-GFP, mix of two mouse monoclonal | Roche | 11814460001 | (1:2000) |
Antibody | Anti-PCNA, mouse monoclonal | Dako | M0879 | (1:5000) |
Antibody | Anti-EED, rabbit polyclonal | Other | Gift from R Margueron (1:5000) | |
Antibody | Anti-Lamin B1, rabbit polyclonal | Abcam | Ab16048 | (1:5000) |
Antibody | Anti-H3, rabbit polyclonal | Abcam | Ab1791 | (1:10000) |
Chemical Compound, Drug | L-Absorbic Acid | Sigma | A4544 | |
Chemical Compound, Drug | Gsk3 inhibitor | Other | CT-99021 | Gift from E Heard |
Chemical Compound, Drug | MEK inhibitor | Other | PD0325901 | Gift from E Heard |
Chemical Compound, Drug | FGF2 | R and D Systems | 233-FB-025/CF | |
Chemical Compound, Drug | Activin A | R and D Systems | 338-AC-050/CF | |
Chemical Compound, Drug | EZH2 Inhibitor | Tocris Bioscience | UNC 1999 | |
Chemical Compound, Drug | EZH2 Inhibitor Negative Control | Tocris Bioscience | UNC 2400 | |
Chemical Compound, Drug | Indole-3-acetic acid sodium salt (auxin analog) | Sigma | I5148-2G | |
Recombinant DNA Reagent | pX459 | Addgene | 62988 | |
Commercial Assay or Kit | EpiTect Bisulfite Kit | Qiagen | 59104 | |
Software, Algorithm | BWA v0.7.5a | Li and Durbin, 2009 | ||
Software, Algorithm | Picard v1.130 | Broad Institute | ||
Software, Algorithm | HOMER v4.7 | Heinz et al., 2010 | ||
Software, Algorithm | FASTX-Toolkit v0.0.13 | Greg Hannon Lab | ||
Software, Algorithm | Cutadapt | Martin, 2011 | ||
Software, Algorithm | Bismark v0.12.5 | Krueger and Andrews, 2011 | ||
Software, Algorithm | Bowtie2 v2.1.0 | Langmead and Salzberg, 2012 | ||
Software, Algorithm | STAR v2.5.0a | Dobin et al., 2013 | ||
Software, Algorithm | Trim Galore v0.4.0 | Babraham Institute | ||
Software, Algorithm | FourCSeq v1.12.0 | Klein et al., 2015 | ||
Software, Algorithm | HTSeq v0.9.1 | Anders et al., 2015 |
List of oligonucleotides used in this study.