Epigenetic mechanisms in early mammalian development

Maria Elena Torres-Padilla

Helmholtz Center Munich

@METorresPadilla

Changes in cell potency and fate in the pre-implantation mouse embryo

Implantation

E3.5

E0.5

E1.5

E5.5

Fertilisation

‘Totipotency’

Pluripotency-associated genes

E2.5

Differentiation factors

Zygote 2-cell 8-cell Blastocyst

Oocyte

Is the chromatin ‘configuration’ the basis of cell plasticity?

Zygote

2-cell

8-cell

Morula

Blastocyst

Plasticity

Totipotency

Pluripotency /

Differentiation

Nuclear organisation of early embryos differs to that in pluripotent and differentiated cells

DNA FISH

Probe

(MajSat)

(Also work from Almouzni, Beaujean and Reik labs)

Somatic cell

DAPI

DAPI

Zygote

Is the nucleus in the embryo functionally regionalised?

Lamin / MajSat

DNA FISH & Immunostaining

Non-injected

ZF-GFP

ZF-Emerin

Targeting pericentromeric chromatin to the nuclear periphery leads to heterochromatin derepression

DNA / MajSat

noninjected

ZF-GFP

ZF-Emerin

MajSat

Jachowicz*, Santenard* et al. Genes & Dev 2013

RNA FISH

DNA FISH

Probe

(MajSat)

RNA FISH

Probe

(MajSat)

3D-Position

Transcription

Is this a general property of the nuclear periphery?

When is nuclear regionalisation first established in mammals?

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Zygote

2-cell

MEF

Nuclear periphery is mostly devoid of electron dense regions in early embryos

DAPI

*

*

*

*

*Nucleolar-like bodies (rich in nucleophosmin)

*

*

*

*

DAPI

DAPI

André Eid

Mapping LADs in the early mammalian embryo using DamID

Timely control of Dam activity (Auxin degron)

• All embryos develop
• Optimised concentration of mRNA
• Optimised auxin concentration
• Reciprocal genetic crosses

6-8 h stabilization of protein

zygote

2-cell

8-cell

Blastocyst

mRNA for AID-Dam.LaminB1 or AID-Dam

Collaboration with Jop Kind

X

Bl6

Cast

LADs appear as early as the zygote stage but display ‘atypical’ features at the 2-cell stage

A portion of the genome rearranges into LADs and iLADs during development

Gene expression

Validated using 3D-DNA FISH

LAD formation precedes TAD consolidation

(HiC data reanalysed, Wei Xie lab)

Borsos*, Perricone* et al. Nature 2019

How are LADs established?

‘Removal’ of H3K9me3 does not affect LAD formation

Lamin B1 DamID

Immunostaining in zygotes

LAD formation is prevented upon Kdm5b expression only in the paternal genome

Lamin B1 DamID

Immunostaining in zygotes

Is there a role of the architectural components of the Lamina?

Screening to investigate the mechanisms regulating LAD establishment in mouse embryos

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DamID

AID-LaminB1-Dam

TIR

Candidate Pools

Transcription factors

HDACs

ONM

INM

Lamina

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Chromatin

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Mrinmoy Pal

Disrupting structural nuclear lamina components does not seem to affect global LAD formation

Mrinmoy Pal

Lamin B1 DamID

How is chromatin so distinctive at the beginning of development?

Grewal & Elgin; Nature 2007

Mammalian heterochromatin comprises mainly repetitive elements

Genomic abundance

(Retrotransposons)

39.6%

Pericentromeric repeats and retrotransposons are transcribed after fertilisation

RNA-FISH/DAPI

zygote

2-cell

8-cell

IAP

Line L1 (Tf)

Major Satellite

RNA FISH

RNA-FISH/DAPI

RNA-FISH/DAPI

Santenard et al. 2010

Jachowicz et al., 2013

Fadloun et al., 2013

(also work from B. Knowles)

LTR

~10%

non-LTR

~20%

Satellite DNA

~2%

The H3K9me3-specific methyltransferase Suv39h1 is not expressed in early embryonic development

Immunostaining

Single cell mRNA

Can we impose ‘somatic’ heterochromatin through gain-of-function experiments in mouse embryos ?

Ectopic expression of Suv39h1

Adam Burton, Luis Altamirano

H3K9me3 is acquired at expressed genes in embryos

Suv39h1

mRNA

Cut&Run H3K9me3

Low

Medium

High

Not-expressed

Maternal

Expression level in wild-type 2-cell embryos

Control

(non-injected)

Suv39h1wt

-3 TSS TES +3 -3 TSS TES +3

De novo acquisition of H3K9me3 does not affect gene expression

Suv39h1.HA

mRNA

RNAseq

Suv39h2 but not Suv39h1 is expressed in early embryonic development

Burton et al. Nature Cell Biol 2020

Conclusions

• LAD formation precedes TAD consolidation

• Embryonic heterochromatin is characterised by non-repressive

H3K9me3

• Kdm5b expression prevents paternal LAD formation in zygotes,

suggesting parent-specific mechanisms

• Non-inheritance of nuclear organisation

• LADs emerge de novo after fertilisation in the zygote

• Early mammalian development is characterised by atypical

heterochromatin with robust transcriptional activation of transposons

• Global H3K9me3 disruption does not majorly affect LADs in embryos

Imaging Core @ IES

Andreas Ettinger

Acknowledgments

Ken-ichiro Abe

Adam Burton

Antoine Canat

Margarita Céspedes

Yung-Li Chen

Marion Genet

Manuel Guthmann

Clara Hermant

Tsunetoshi Nakatani

Marlies Oomen

Mrinmoy Pal

Federico Pecori

Tamas Schauer

Institute of Epigenetics & Stem Cells

Former members

Luis Altamirano-Pacheco

Máté Borsos

Joanna Jachowicz

Elias Ruiz-Morales

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Scientific Coordinator

Amelie Kraus

MPI Freiburg

Thomas Jenuwein

@METorresPadilla

Hubrecht Institute

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Jop Kind & Sara Perricone