HAL Id: hal-02786601
https://hal.inrae.fr/hal-02786601
Submitted on 5 Jun 2020
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Contrasted regulation of pericentromeric hetero chromatin in mouse ground naive and primed
pluripotent stem cells
Matteo Tosolini, Vincent Brochard, Pierre Adenot, Martine Chebrout, Grillo Giacomo, Violette Navia, Nathalie Beaujean Bobineau, Claire Francastel,
Amélie Bonnet-Garnier, Alice Jouneau
To cite this version:
Matteo Tosolini, Vincent Brochard, Pierre Adenot, Martine Chebrout, Grillo Giacomo, et al.. Con- trasted regulation of pericentromeric hetero chromatin in mouse ground naive and primed pluripotent stem cells. France-Japan Epigenetics Workshop 2017, Nov 2017, Paris, France. 2017. �hal-02786601�
Contrasted regulation of pericentromeric heterochromatin in mouse ground naive and primed pluripotent stem cells
Tosolini Matteo
1, Brochard Vincent
1, Adenot Pierre
1, Martine Chebrout
1, Grillo Giacomo
2, Navia Violette
1, Beaujean Nathalie
1,3, Francastel Claire
2, Bonnet-Garnier Amélie
1and Jouneau Alice
12i-ESC
EpiSC
In vitro conversion
E3.5 E6.5
+ LIF, iMEK, iGSK3 (2i)
laminin
+ ActivinA, + FGF2
fibronectin
Naïve
pluripotency
Primed pluripotency
Focus on constitutive heterochromatin:
- Epigentic constituents?
- Role in the epigenetic barrier ?
Model: Conversion of WT and mutant ESC into cEpiSC: Suv39hdn :double KO of
Suv39h enzymes that deposit H3K9me3 at PCH (Lehnertz et al, Current Biol 2003)
DnmtTKO :dnmt1-/-; dnmt3a-/-;
dnmt3b-/- (Tsumura et al, Genes to cell, 2006)
H3K27me3H3K9me3DAPI
2i-ESC EpiSC
Relative enrichment of H3K27me3 and H3K9me3 discriminate naïve ESC from primed EpiSC
ChIP QPCR on Major satellites
Digestion of genomic DNA with methyl- sensitive enzymes (HpyCH4IV and southern
blotting for Major satellites:
unmethylatedmethylated
Grey level intensity
MEF DnmtTKO-ESC 2i-ESC EpiSC
Probst, Almouzni et al. TIG 2011
The constitutive heterochromatin compartment:
- composed of telomeres, centromeres and peri- centromeric (PCH) regions
- PCH = Major satellites : 234bp, ~ 10.000 repeats (3%
of the mouse genome)
- Compacted and mainly silenced
- Usually enriched in H3K9me3 and DNA methylation
= repressive transcriptionnal environment
Figure 1
Figure 2
DNA methylation increases between 2i-ESCs and EpiSCs, along with Dnmt3s enzymes
H3K27me3 H3K9me3
meCpG CpG
MS ncRNA
234bp 234bp 234bp 234bp
SUV39H1
SUV39H1
DnmtTKO 2i-ESC
DnmtTKO cEpiSC 2i-ESC
EpiSC
234bp 234bp 234bp 234bp
SUV39H1 DNMT3B
DNMT3B
234bp 234bp 234bp 234bp
DNMT3B DNMT3B
Suv39hdn 2i-ESC
Suv39hdn cEpiSC
2i-ESC EpiSC
DNMT3B
DNMT3A
H3 total
2i-ESC EpiSC
1 13
DNMT3B
H3 total
1 9
Western-blot for DNMT3s
CONCLUSION :
Transition from naïve to primed state of pluripotency is characterized by a more repressive status of PCH, with a loss of H3K27me3 and a gain of H3K9me3 and 5meC.
Regulation of transcription at PCH in naïve (ground) 2i-ESCs is uncoupled with their epigenetic state, while it is tightly controlled by heterochromatic marks in primed pluripotent cells.
DNMT3b is present at chromocenters in 42% of 2i-ESC and strong but difuse in EpiSC
Higher methylation of major satellites in EpiSC compared to 2i-ESCs
Increasing level of methylation
Control of satellite transcription by epigenetic
marks ?
Suv39hdn ESC =
H3K9me3
at PCH DnmtTKO mutant =5meC
In ESC, gain of H3K27me3 enriched cells, but none in cEpiSC
Gain of DNA methylation in cEpiSC, in spite of the absence of H3K9me3
In ESC : gain of H3K27me3 enriched cells, but none in cEpiSC
RNA level of Major satellites transcripts (qRT-PCR)
WT 2i-ESC WT cEpiSC Suv39hdn 2i-ESC Suv39hdn cEpiSC TKO 2i-ESC TKO cEpiSC
In absence of either H3K9me3 or DNA methylation at PCH:
derepression of satellite transcription during transition from naive to primed cells
Expression relative to WT 2i-ESC
0.0 0.5 1.0 1.5 2.0 2.5
2i-ESC EpiSC
*
H3K27me3 /DAPI H3K9me3 /DAPI
*
*
*
*
Mitosis figures (telophase)
2i-ESC EpiSC
SUV39H1 DAPI
No enrichment at chromocenters
93% cells with enrichment at chromocenters
2i-ESC EpiSC
MEF DnmtTKO-ESC 2i-ESC cEpiSC 2i-ESC cEpiSC
WT Suv39hdn
Suv39hdn WT
Background
WORKING MODEL
0 1 2 3 4 5 6 7 8 9
%ChIP/input
2i-ESC EpiSC
H3K9me3 H3K27me3
Only 2i-ESC show H33K27me3 enriched chromocenters (in 36% of the cells) All EpiSC display H3K9me3 enriched
chromocenters (vs 30% of 2i-ESC)
