Super-resolution imaging reveals principles of physical chromatin - - PowerPoint PPT Presentation

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Super-resolution imaging reveals principles of physical chromatin - - PowerPoint PPT Presentation

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! Super-resolution imaging reveals principles of physical chromatin folding in eukaryotes Frdric Bantignies Chromosome Conformation Symposium - Toulouse 04/12/2019 Inside cell nucleus, the genome is highly compacted and folded as a

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Frédéric Bantignies Chromosome Conformation Symposium - Toulouse 04/12/2019 ¡

!

Super-resolution imaging reveals principles of physical chromatin folding in eukaryotes

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Rosa ¡& ¡Shaw, ¡2013 ¡

̴ 10 µm ̴ 2 meters

  • f DNA

Inside cell nucleus, the genome is highly compacted and folded as a chromatin fiber

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Mar1-­‑Renom ¡and ¡Mirny, ¡PLoSComputa+onal ¡Biology ¡2011 ¡

The different level of genome organization

1953 ¡ 1997 ¡ 2005 ¡

  • ngoing ¡
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Lieberman ¡et ¡al, ¡2009 ¡(Hi-­‑C) ¡ Rao ¡et ¡al, ¡2014 ¡(in ¡situ ¡Hi-­‑C) ¡

Chromosome Conformation Capture (Hi-C)

Chroma1n ¡crosslink ¡ ¡ inside ¡nucleus ¡ Genera1on ¡of ¡contact ¡maps ¡ between ¡all ¡interac1ng ¡fragments ¡ Contact ¡density ¡map ¡

Mar1-­‑Renom ¡and ¡Mirny, ¡PLoSComputa+onal ¡Biology ¡2011 ¡

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Ø TADs represent genomic region of highly interacting chromatin with few interactions spanning their borders

Hi-C maps represent three main levels of genome folding

Adapted ¡from ¡Szabo, ¡Ban1gnies, ¡Cavalli, ¡Science ¡Advances ¡2019 ¡ and ¡Mota-­‑Gomez, ¡Lupianez, ¡Genes ¡2019 ¡ ¡

Topologically Associating Domains

Hi-C Single cell

?

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TADs are a conserved genomic feature with species specificities

Adapted from Szabo, Bantignies, Cavalli, Science Advances 2019

Fly Mammals

  • Median size: ~ ¡100 kb
  • Coincide well with the alternation of

repressed and active chromatin marks

  • Median size: ~ ¡900 kb
  • Presence of corner peaks (structural architectural loops)
  • Presence of Enhancer-Promoter loop (functional loops)

vs.

Sexton et al., Cell 2012 Nora et al., Nature 2012 Dixon et al., Nature 2012 Hou et al., Molecular Cell 2012

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45° rotation

TADs are considered as functional genomic units

  • Median size: ~ ¡100 kb
  • Coincide well with the alternation of

repressed and active chromatin marks (Sexton et al, 2012)

  • Median size: ~ ¡900 kb
  • Presence of corner peaks (structural architectural loops)
  • Presence of Enhancer-Promoter loop (functional loops)
  • Genes within TADs are co-regulated (Nora et al, 2012;

Zhan et al, 2017)

  • Enhancer/promoter contacts are restricted within TADs

(Symmons et al, 2014; Bonev et al, 2017)

  • Disruption of boundary leads to ectopic gene expression

(Lupianez et al, 2015; Hniz et al, 2016; Rodriguez- Carballo et al, 2017)

Fly Mammals

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45° rotation

TADs are considered as functional genomic units Fly Mammals

Whether TADs structure is compatible with their functional role ?

Indeed, they can represent the manifestation of average interactions from large cell populations and therefore we need to understand their structure before to claim that they represent functional domains

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We undertook a structural approach combining Hi-C / Oligopaint technology / super-resolution microscopy in Drosophila

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The Oligopaint 3D-FISH technology

Ø Represents a new generation of FISH probes entirely derived from synthetic DNA oligonucleotides Ø Production of ssDNA oligo pools able to recognize any portion of the genome in various organisms, from 10 kb to several Mb, avoiding repetitive sequences

Beliveau et al., Nature communications 2015; Beliveau et al., PNAS 2018 https://oligopaints.hms.harvard.edu

Chroma1n ¡fiber ¡

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Super-Resolution Microscopy (SRM)

Schermelleh, Heintzmann and Leonhardt, J.cell.Biol. 2010 100 nm 50-80 nm 30 nm Axial resolution (estimated)

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In Drosophila, TADs corresponds to the alternation of chromatin states

Ø Active chromatin: H3K4me3/H3K36me3/H3K27ac/gene dense/ubiquitously active Ø Repressed chromatin: H3K27me3/Polycomb proteins or Void chromatin/gene poor/specific activation during developmental programs

Adapted from Szabo, Bantignies, Cavalli, Science Advances 2019

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Oligopaint probe covering 3 Mb (~12 fluorescent oligos/kb)

3D-SIM super-resolution imaging reveals chromatin nano-structures or nanocompartments

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Conventional Wide Field Oligopaint probe covering 3 Mb (~12 fluorescent oligos/kb)

3D-SIM super-resolution imaging reveals chromatin nano-structures or nanocompartments

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3D-SIM Conventional Wide Field Oligopaint probe covering 3 Mb (~12 fluorescent oligos/kb)

3D-SIM super-resolution imaging reveals chromatin nano-structures or nanocompartments

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3D-SIM Conventional Wide Field Oligopaint probe covering 3 Mb (~12 fluorescent oligos/kb)

3D-SIM super-resolution imaging reveals chromatin nano-structures or nanocompartments

1 µm

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Dual labeling of the chromatin fiber

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Repressed TAD

Local chromatin compaction reflects the chromatin state

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Local chromatin compaction reflects the chromatin state

Repressed TAD Active TAD

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**

Repressed TAD Active TAD

Local chromatin compaction reflects the chromatin state

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Investigating TAD structures in vivo

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1 2 3

Investigating TAD structures in vivo

Equidistant dot probes

1 µm

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1 2 3 Equidistant dot probes

Repressed TADs spatially confine the chromatin fiber

1 µm

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1 2 3 Equidistant dot probes

Repressed TADs spatially confine the chromatin fiber

1 µm

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Repressed TADs form discrete 3D chromosomal units

  • r nanocompartments
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Repressed TADs form discrete 3D chromosomal units

  • r nanocompartments

Oligopaint probes

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Repressed TADs form discrete 3D chromosomal units

  • r nanocompartments

Oligopaint probes

1 µm

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Repressed TADs form discrete 3D chromosomal units

  • r nanocompartments

Oligopaint probes

1 µm

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Repressed TADs form discrete 3D chromosomal units

  • r nanocompartments

Oligopaint probes

1 µm

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Repressed TADs form discrete 3D chromosomal units

  • r nanocompartments

Oligopaint probes

1 µm

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  • No contacts in ~70%
  • f the cells
  • Overlap fraction < 0.1

in ~85% of the cells TAD 1 + TAD 2

Repressed TADs form discrete 3D chromosomal units

  • r nanocompartments

Oligopaint probes

1 µm

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Polymer modeling of the chromatin fiber

Simulated contact probability map Compare with experimental Hi-C map Optimize interaction potentials

2 kb beads Interaction

Simulate ensemble of different configurations Self-avoiding and self-interacting polymer model of the region of interest

Adapted from Giorgetti et al, Cell 2014

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Polymer modeling is consistent with the physical TAD-based chromatin compartmentalization

Daniel Jost

Simulated ¡ Experimental ¡

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1 2 3 TAD 2 TAD 1

Daniel Jost

Polymer modeling is consistent with the physical TAD-based chromatin compartmentalization

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1 2 3 TAD 2 TAD 1

Daniel Jost

Polymer modeling is consistent with the physical TAD-based chromatin compartmentalization

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1 2 3 TAD 2 TAD 1

Daniel Jost

Polymer modeling is consistent with the physical TAD-based chromatin compartmentalization

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75 %

  • f the cells

What about shorter inter versus intra-TAD distances?

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75 %

  • f the cells

25 %

What about shorter inter versus intra-TAD distances?

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The relative TAD positioning can explain shorter inter versus intra-TAD distances

75 %

  • f the cells

25 %

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The relative TAD positioning can explain shorter inter versus intra-TAD distances

75 %

  • f the cells

25 %

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Szabo ¡et ¡al., ¡Science ¡Advances ¡2018 ¡ Ac1ve/decondensed ¡ ¡ chroma1n ¡ (gene ¡dense ¡region/ ¡ ubiquitously ¡expressed) ¡ Nanocompartments/ ¡ repressed ¡TADs ¡ (gene ¡poor ¡region/ ¡ Developmental ¡genes/ ¡ Tissue ¡specific ¡expression) ¡

Organization of the chromatin fiber in Drosophila interphase nuclei

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Giacomo Cavalli Quentin Szabo Thierry Cheutin Anne-Marie Martinez Bernd Schuettengruber Laurianne Fritsch Giorgio L. Papadopoulos Boyan Bonev Satish Sati Yuki Ogiyama Sandrine Denaud Vincent Loubière Ivana Jerkovic Axelle Donjon Alumni Virginie Roure Benjamin Leblanc Itys Comet Fillipo Ciabrelli Caroline Jacquier NOLLMANN lab

Centre de Biochimie Structurale CNRS Univ Montpellier

Marcelo Nollmann Diego Cattoni Julian Gurgo Daniel Jost

TIMCS-IMAG CNRS Univ Grenoble Alpes

Jia-Ming Chang

National Chengchi University

Tom Sexton

Institut de Génétique et de Biologie Moléculaire et Cellulaire CNRS INSERM Univ Strasbourg

BioCampus Montpellier Ressources Imagerie facility Julio Mateos Langerak

CAVALLI lab

BioCampus Drosophila facility Amos Tanay

Weizmann Institute Israël

Ting Wu

Harvard Medical School Boston