Dynamic enhancer partitioning instructs activation of a growth-related gene during exit from naïve pluripotency

  1. Maxim Greenberg  Is a corresponding author
  2. Aurélie Teissandier
  3. Marius Walter
  4. Daan Noordermeer
  5. Deborah Bourc'his  Is a corresponding author
  1. PSL Research University, INSERM, CNRS, France
  2. Université Paris Sud, Université Paris-Saclay, CEA, CNRS, France
6 figures, 1 table and 2 additional files

Figures

Figure 1 with 3 supplements
The Zdbf2 locus exhibits dynamic enhancer activity during differentiation.

(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 …

https://doi.org/10.7554/eLife.44057.002
Figure 1—figure supplement 1
Zdbf2 expression dynamics during differentiation.

(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 …

https://doi.org/10.7554/eLife.44057.003
Figure 1—figure supplement 2
Chromatin landscape of the Zdbf2 locus in cellula and in vivo.

(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 …

https://doi.org/10.7554/eLife.44057.004
Figure 1—figure supplement 3
Zdbf2 locus interactions restricted within inter-TAD region.

(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 …

https://doi.org/10.7554/eLife.44057.005
Figure 2 with 1 supplement
Genetic deletions of enhancer elements impact pLiz/pZdbf2 regulation.

(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

https://doi.org/10.7554/eLife.44057.007
Figure 2—figure supplement 1
E3 deletion has minimal effect on locus regulation, E4 is devoid of PRC2 signature.

(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 …

https://doi.org/10.7554/eLife.44057.008
Figure 3 with 1 supplement
Polycomb regulates E4, but plays minor role in chromosome conformation.

(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 …

https://doi.org/10.7554/eLife.44057.010
Figure 3—figure supplement 1
Liz exerts minimal effect on chromosome conformation at the Zdbf2 locus 4C-seq tracks from the pZdbf2 VP in ∆Liz ESCs and EpiLCs and WT EpiLCs.

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 …

https://doi.org/10.7554/eLife.44057.011
Figure 4 with 3 supplements
The Zdbf2 locus is partitioned by CTCF, which instructs expression dynamics.

(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 …

https://doi.org/10.7554/eLife.44057.013
Figure 4—figure supplement 1
CTCF dynamics maintained in polycomb, DNA methylation, and Liz mutant background.

(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 …

https://doi.org/10.7554/eLife.44057.014
Figure 4—figure supplement 2
CTCF_PS deletion impacts locus regulation.

(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 …

https://doi.org/10.7554/eLife.44057.015
Figure 4—figure supplement 3
Polycomb and 3D organization both impact Zdbf2 activation.

(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 …

https://doi.org/10.7554/eLife.44057.016
Author response image 1
Author response image 2

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifierAdditional
information
Cell Line
(M. musculus)
E14TG2a (WT)ATCCCRL-1821
Cell Line (M. musculus)E14TG2a_∆LizBourc’his LabGreenberg et al., 2017
Cell Line (M. musculus)E14TG2a_∆E1Bourc’his LabGreenberg et al., 2017
Cell Line (M. musculus)E14TG2a_∆E1-3This studyCRISPR/Cas9 generated mutant, sgRNA oligos are listed in Supplementary file 1
Cell Line (M. musculus)E14TG2a_∆E4This studyCRISPR/Cas9 generated mutant, sgRNA oligos are listed in Supplementary file 1
Cell Line (M. musculus)E14TG2a_CTCF-AID-eGFP, Tir1Gift from E NoraNora et al., 2017
Cell Line (M. musculus)J1 (WT)ATCCSCRC-1010
Cell Line (M. musculus)J1_Dnmt tKOGift from M OkanoTsumura et al., 2006
Cell Line (M. musculus)J1_Eed-/-This studyCRISPR/Cas9 generated mutant, sgRNA oligos are listed inSupplementary file 1
Cell Line (M. musculus)J1_∆CTCF_PSThis studyCRISPR/Cas9 generated mutant, sgRNA oligos are listed in Supplementary file 1
Cell Line (M. musculus)J1_Eed-/-; ∆CTCF_PSThis studyCRISPR/Cas9 generated mutant, sgRNA oligos are listed in Supplementary file 1
Cell Line (M. musculus)J1 Clone 36 (WT)Gift from A WutzWutz and Jaenisch, 2000
Cell Line (M. musculus)J1 Clone 36_Eed-/-Gift from A WutzSchoeftner et al., 2006
AntibodyAnti-H3K27me2, mouse monoclonalActive Motif61435(1:5000)
AntibodyAnti-H3K27me3, rabbit monoclonalCell Signaling TechnologyC36B11(1:5000)
AntibodyAnti-H3K27ac,
rabbit polyclonal
Active Motif39133(1:5000)
AntibodyAnti-CTCF, rabbit polyclonalMillipore07–729(1:5000)
AntibodyAnti-GFP, mix of two mouse monoclonalRoche11814460001(1:2000)
AntibodyAnti-PCNA, mouse monoclonalDakoM0879(1:5000)
AntibodyAnti-EED, rabbit polyclonalOtherGift from R Margueron (1:5000)
AntibodyAnti-Lamin B1, rabbit polyclonalAbcamAb16048(1:5000)
AntibodyAnti-H3, rabbit polyclonalAbcamAb1791(1:10000)
Chemical Compound, DrugL-Absorbic AcidSigmaA4544
Chemical
Compound, Drug
Gsk3 inhibitorOtherCT-99021Gift from E Heard
Chemical Compound, DrugMEK inhibitorOtherPD0325901Gift from E Heard
Chemical Compound, DrugFGF2R and D Systems233-FB-025/CF
Chemical Compound, DrugActivin AR and D Systems338-AC-050/CF
Chemical Compound, DrugEZH2 InhibitorTocris BioscienceUNC 1999
Chemical Compound, DrugEZH2 Inhibitor Negative ControlTocris BioscienceUNC 2400
Chemical Compound, DrugIndole-3-acetic acid sodium salt (auxin analog)SigmaI5148-2G
Recombinant DNA ReagentpX459Addgene62988
Commercial Assay or KitEpiTect Bisulfite KitQiagen59104
Software, AlgorithmBWA v0.7.5aLi and Durbin, 2009
Software, AlgorithmPicard v1.130Broad Institute
Software, AlgorithmHOMER v4.7Heinz et al., 2010
Software, AlgorithmFASTX-Toolkit v0.0.13Greg Hannon Lab
Software, AlgorithmCutadaptMartin, 2011
Software, AlgorithmBismark v0.12.5Krueger and Andrews, 2011
Software, AlgorithmBowtie2 v2.1.0Langmead and Salzberg, 2012
Software, AlgorithmSTAR v2.5.0aDobin et al., 2013
Software, AlgorithmTrim Galore v0.4.0Babraham Institute
Software, AlgorithmFourCSeq v1.12.0Klein et al., 2015
Software, AlgorithmHTSeq v0.9.1Anders et al., 2015

Additional files

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