Noordermeer 2022

Genomic Geography
our genome in 3 dimensions and

what can go wrong
Daan Noordermeer
Chromatin Dynamics team
Institute for Integrative Biology of the Cell - University Paris-Saclay & CNRS
Gif-sur-Yvette
Stability and Evolution of Genomes – 26 September 2022
Setup
• Part 1: Our genome in 3 dimensions and its

link with normal and perturbed ~ 1hr
genome function
15 min break
• Part 2: TAD formation and loop extrusion

~ 1.15hr
measured with single-cell precision
using multi-contact Nano-C

Part 1:
Our genome in 3 dimensions

and its link with normal and
incorrect genome function

The size of the human genome
• Our genome consists of 2 sets of 23 chromosomes

What is on our chromosomes?
• ~ 60,000 genes / transcripts
• regulatory information for gene activity
• structural elements
– telomeres
– centromeres
– …
• ~ 45% repeated sequences


– telomeres
– centromeres
– …
Encoded in ~ 2 sets of
3 * 109 basepairs
• 4 different ‘letters’: A,C,G,T


– telomeres
– centromeres
– …
Encoded in ~ 2 sets of
3 * 109 basepairs
• 4 different ‘letters’: A,C,G,T ≈

• How long is the human genome?

– Combined length of 46 human
chromosomes in the cell?
?

2 meters
?

2 meters
– Bonus question:
how many kilometers of DNA in our body? ?

2 meters
– Bonus question:
how many kilometers of DNA in our body?
2 meters × ~ 30 * 1012 cells = 60 000 000 000 km

?
(to generate this DNA, your DNA polymerases went
back and forth between the sun and Neptune about 6 times…)

• Challenge: how to fit 2 meters of DNA in a cell nucleus?

2 meters
COMPACTION
Mammalian
cell nucleus:
10 µm diameter


2 meters
COMPACTION
Mammalian
cell nucleus:
10 µm diameter
• How to maintain correct genome function?

– Transcription of genes
– Regulation of gene activity
– Maintenance of DNA: replication and repair


2 meters
COMPACTION
Mammalian
cell nucleus:
10 µm diameter
• How to maintain correct genome function?

– Transcription
Particularly inofmammalian
genes cells:
– Regulationuse
Widespread of gene activity
of DNA compaction to expand
genome
– function!
Maintenance of DNA: replication and repair

DNA compaction
• First-order DNA organization

– Nucleosomes and histones
– ~ 10 fold compaction

DNA compaction

– Nucleosomes and histones
– ~ 10 fold compaction
– Functionalization:
Chemical modifications of histone tails have epigenetic functions

DNA compaction

• Higher-order DNA organization
– All levels of 3 dimensional (3D) genome organization beyond
nucleosomes

DNA compaction
• Higher-order DNA organization

– Largest-scale organization: heterochromatin vs euchromatin
– Functionalization: zoning of transcriptionally active (competent)
and inactive (incompetent) domains
Peripheral
heterochromatin
Nucleolus-associated
heterochromatin
Euchromatin
Human liver
nucleus
Akhtar & Gasser, Nat Rev Genet 2007

Today’s focus
• Genomic cartography of 3D genome organization
– Chromosome Conformation Capture and Hi-C

– Topologically Associating Domains (TADs)
• In normal cell function
• As cause for disease

Carthography of 3D genome organization:
Chromosome Conformation Capture
• Fixing 3D DNA structure

Carthography of 3D genome organization:
Chromosome Conformation Capture
• Fixing 3D DNA structure

Chromosome Conformation Capture:
cutting and pasting
• Fragmentation by enzymatic digestion

– Crosslinks keep 3D interacting fragments together

cutting and pasting
• Enzymatic ligation of crosslinked fragments

– Fragments ligated based on initial 3D interactions

central principle
Characterization of ligation junction
=
Identification of 2 cross-linked fragments
=
Identification of 1 3D DNA interaction

characterization of ligation junctions
• Current preferred approach: high-throughput sequencing

Ligation junction
1. Pair-wise
contacts Single-end and
paired-end Illumina


Ligation junction
1. Pair-wise
paired-end Illumina
2. Single-molecule Single-molecule
(Oxford Nanopore
multi-contact and PacBio)


Ligation junction
Second part
1. Pair-wise of lecture
paired-end Illumina
2. Single-molecule Single-molecule
(Oxford Nanopore
multi-contact and PacBio)

• Gold-standard: Hi-C technology

– Aim: to characterize all ligation junctions present in the library
– Advantage: genome-wide cartography of all 3D contacts in the
genome

• Gold-standard: Hi-C technology

– Aim: to characterize all ligation junctions present in the library
– Advantage: genome-wide cartography of all 3D contacts in the
genome
– Disadvantage: requires massive amounts of sequencing and
computation
• Expensive
• Difficult to handle data sets (non-compressed ≈ 3000
Hi-C matrix at 1kb resolution = 18 Tb of data)
– Alternatives: cost- and data-efficient Chromosome
Conformation Capture derivatives to address specific questions
• Focus on genomic loci/regions, specifc TF-binding sites, …

Hi-C technology
• Illumina sequencing based

– Genome-wide cartography
– Ligation junction between all sites in the genome vs. all sites in
the genome
– Output: matrix of DNA interactions
Nearby interactions
along diagonal
Lieberman-Aiden,
Science 2009

Hi-C technology

the genome
Highest interaction frequency
Lowest interaction frequency
Nearby interactions
along diagonal
Lieberman-Aiden,
Science 2009

Hi-C technology

the genome
Lieberman-Aiden,
Science 2009

Largest-scale 3D genome organization
• Genome-wide Hi-C maps separate into mutually

exclusive domains
– Compartment A: gene rich, high gene activity
– Compartment B: gene poor, low gene activity
Enriched
contacts
Depleted
contacts
Lieberman-Aiden,
Science 2009


exclusive domains
‘B compartment’ Enriched
contacts
‘A compartment’
Depleted
contacts
Lieberman-Aiden,
Science 2009


exclusive domains
‘B compartment’
‘A compartment’ ?
Lieberman-Aiden,
Science 2009


exclusive domains
– Compartment A: gene rich, high gene activity à euchromatin
– Compartment B: gene poor, low gene activity à heterochromatin
‘B compartment’
‘A compartment’
Lieberman-Aiden,
Science 2009

Topologically Associating Domains (TADs)
• Mammalian genomes separate into insulated domains

– TAD definition: increased intra-domain interactions over outside
5 Mb Dixon et al, 2012 Nature

Nora et al, 2012 Nature





– Average ~ 1 Mb size (2000 – 3000 TADs in the genome)



– Average ~ 1 Mb size (2000 – 3000 TADs in the genome)
– Regionalize genomic functions:
• gene regulation, DNA replication, DNA repair, …


TADs as functional units of gene regulation
• Perturbed TAD structure can cause cancer and

embryonic defects
– Hand malformation due to different perturbations of the
same TAD
“short fingers” fusion of fingers “many fingers”
Lupianez et al, 2015 Cell

• The EPHA4 TAD

– Activity of EPHA4 gene is essential for hand development
– EPHA4 activity requires a distant enhancer element
located in the same TAD!
enhancer

• Genomic rearrangements cause hand malformations

– Duplications, inversions or deletions of small genomic regions
– All rearrangements modify one boundary of the TAD
– Activity of EPH4 gene not affected

• Healthy hand: surrounding genes have contacts in their

own TADs
4C-seq
(3D interactions
for one site in
the genome)

• Rearrangements position other genes with active EPHA4

enhancers within same domain
– F-syndrome: activation of WNT6 gene
border border
new TAD

• Rearrangements position other genes with active EPHA4

enhancers within same domain
– Polydactyly: activation of IHH gene
border border
new TAD

• TADs regionalize the function of active enhancers

– Perturbation and reorganization of TAD boundaries can cause:
• incorrect promoter-enhancer contacts
• incorrect gene activation

What are TAD boundaries?
• Most TAD boundaries contain CTCF binding sites

– CTCF: DNA binding factor
– CTCF is present at ~ 75% of TAD borders
Dixon et al, 2012 Nature

Phillips-Cremins & Corces, 2013 Mol Cell
Stability
Daan and Evolution of Genomes – 26 September 2022
Noordermeer
• Disruption of TAD borders can cause all kinds of diseases

– Developmental defects
• Disruption of TAD borders, internal reorganization of TADs (e.g.
Lupianez et al, 2015 Cell, Franke et al, 2016 Nature)
– Cancer
• Disruption of TAD borders (e.g, Katainen et al, 2015 Nat Genet, Flavahan et
al, 2016 Nature, Hnisz et al, 2016 Science)
– Reduced resistance to influenza (flu)
• SNP adding a new TAD border (Allen et al, 2017 Nat Medicine)

TADs as functional units of DNA repair:
hijacking of loop extrusion to facilitate repair
• DNA Double Strand Breaks (DSBs) pose great danger to

the cell and need to be repaired quick

• After DSB:
– Rapid spreading of repair proteins in large domains
– Rapid spreading of histone modifications in large domains
Article
a Viewpoint c 1.8 γH2AX
+DSB
Normalized read counts

1.5
4C–seq (–DSB) 0
2.5 Viewpoint
0 4C–seq
1.2
read counts
γH2AX
Normalized
–DSB
0 0
0.25 2.5 Viewpoint 4C–seq
53BP1
0 –DSB
0.15 MDC1 0
TAD
0 borders
0.6 Ub (+DSB/–DSB) Chr 1 88 90 92
~ 1.5 Mb
log2[ratio]
–0.1 DSB1 (AsiSI)

0.1 H1 (+DSB/–DSB)
0
d DSB1 chr 1: 89458599 600
)
SB
–0.2
1
D
(–
Chr 1 88 92 Arnould et al, 2021 Nature
i-C
DSB1 (AsiSI)
H
b 2.5
Stability and Evolution of Genomes – 26 September
Viewpoint 2022
Viewpoint
H2AX 00 0.5
ts
4C–seq
• After DSB:
– Rapid spreading of repair proteins in large domains
– Rapid spreading of histone modifications in large domains
Article
– Domain matches TADs that were present before the DSB
a Viewpoint c 1.8 γH2AX
+DSB
Normalized read counts

1.5
4C–seq (–DSB) 0
2.5 Viewpoint
0 4C–seq
1.2
read counts
γH2AX
Normalized
–DSB
0 0
0.25 2.5 Viewpoint 4C–seq
53BP1
0 –DSB
0.15 MDC1 0
TAD
0 borders
0.6 Ub (+DSB/–DSB) Chr 1 88 90 92
~ 1.5 Mb
log2[ratio]
–0.1 DSB1 (AsiSI)

0.1 H1 (+DSB/–DSB)
0
d DSB1 chr 1: 89458599 600
)
SB
–0.2
1
D
(–
Chr 1 88 92 Arnould et al, 2021 Nature
i-C
DSB1 (AsiSI)
H
b 2.5
Stability and Evolution of Genomes – 26 September
Viewpoint 2022
Viewpoint
H2AX 00 0.5
ts
4C–seq
• DSB repair machinery hitchhikes on the Cohesin loop

extrusion machinery
– One-sided loop extrusion by fixing Cohesin at DSB
– Allows rapid and directional spreading away from DSB
– Allows focussed spreading around DSB
Arnould et al, 2021 Nature

• Functionalization of DNA compaction

• Topologically Associating Domains (TADs)
– Separation of mammalian in 2000 – 3000 insulated domains
– Regionalize genomic functions
• gene regulation
• DNA repair
– Genetic structural variation can cause perturbations of TADs that
can result in gene activation without affecting genes or enhancers
• Chromosome Conformation Capture and Hi-C
– Characterization of ligation junctions to identify DNA interactions
– Genome-wide cartography of all 3D contacts in the genome

Part 1:

and its link with normal and
perturbed genome function
QUESTIONS?

15 minutes break

Part 2:
TAD formation and loop extrusion
measured with single-cell
precision using multi-contact
Nano-C

Outline
• Linking 1D genome organization to 3D genome

architecture
– Characterization of CTCF binding at
boundaries of TADs
– Intersection of ChIP-seq vs Hi-C
Li-Hsin Sourav
• Mechanistic validations Chang Ghosh
– Multi-contact Nano-C

TADs and their boundaries
• Topologically Associating Domains (TADs)

– Insulated domains of ~1 Mb
in mammalian genomes
– Regionalize genomic functions
(gene regulation, DNA repair)
– Most TAD boundaries bind
the CTCF protein

How are TADs formed?
• Continuously ongoing Cohesin-mediate loop extrusion
1. Loading of Cohesin complex by its loading

factor Nipbl
2. Compaction by energy-consuming loop

extrusion
3. One-sided blocking at boundary

CTCF, RNA PolII, ...
4. Two-sided blocking creates

Sanborn et al, 2015 PNAS
an insulated TAD Fudenberg et al, 2016 Cell Rep
Vian et al, 2018 Cell
Chang et al, 2020 J Mol Biol

How are TADs formed?
• Continuously ongoing Cohesin-mediate loop extrusion
Normal cells Cohesin depletion
Sanborn et al, 2015 PNAS

Fudenberg et al, 2016 Cell Rep
Vian et al, 2018 Cell
Chang et al, 2020 J Mol Biol

Some more words on CTCF
• Essential protein
• Essential to create 3D architecture of mammalian chromosomes
• Binding enriched at TAD boundaries
– But majority of CTCF peaks are found far away from TAD boundaries!

• Binds a complex and non-symmetric DNA motif

• Binds a complex and non-symmetric DNA motif
• Blocks loop extrusion, but

more efficiently in one orientation

CTCF binding clusters at TAD boundaries to
create ‘transition zones’
• Does clustering buffer against dissociation from DNA or

otherwise inefficient loop extrusion blocking?
Phillips-Cremins & Corces, 2013 Mol Cell

Chang et al, 2020 JMB

Our aims
• Question 1: what distinguishes CTCF binding in

transition zones from elsewhere?
– Quantitatively confirm that CTCF clusters at TAD boundaries
• Question 2: determine how CTCF binding creates TAD
boundaries
– Quantitatively determine the impact of clustered CTCF binding
on loop extrusion
Chang L-H*, Ghosh S*, et al. BioRxiv https://doi.org/10.1101/2021.04.15.440007

Question 1: what distinguishes CTCF binding
in transition zones from elsewhere?
• Intersection of identified CBS with TAD boundaries in

mESCs
– Reanalysis of high-resolution Hi-C data (Bonev at al, 2017 Cell)
The large majority of

CBS are within TADs

1. CBS with higher signal are enriched at TAD boundaries

– The stronger the peak, the stronger the enrichment near TAD
boundaries
See also:
Madani Tonekaboni, 2019 Genome Res

1. CBS with higher signal are enriched at TAD boundaries

2. CBS close to other CTCF peaks are enriched at TAD
boundaries
See also:
Kentepozidou et al, 2020 Genome Biol

• 55% of CBS contain more than one CTCF binding motif
Example in 3 CBS:
See also:
Shu et al, 2011 NAR
Pugacheva et al, 2015 Genome Biol
Clarkson et al, 2015 NAR

• CBS motif number strongly scales with CBS ChIP-seq

peak signal
See also:
Shu et al, 2011 NAR
Pugacheva et al, 2015 Genome Biol
Clarkson et al, 2015 NAR

• CTCF binding at TAD boundaries

– Three-level enrichment of CBS binding
Nakahashi et al, Cell Rep 2013

Krietenstein et al, Mol Cell 2020
Huang et al, Nat Genet 2021
Oh et al, Cell 2021

• CTCF binding at TAD boundaries

– Three-level enrichment of CBS binding
Correct causality:
Strong TAD boundaries are
visible because of enrichment
of “strong” CBS, and not the
other way around
Nakahashi et al, Cell Rep 2013

Krietenstein et al, Mol Cell 2020
Huang et al, Nat Genet 2021
Oh et al, Cell 2021

3D genome organization with
single-cell precission
• Hi-C from pairwise contacts

– Averaged descriptions from Ligation junction
100,000s of cells
– No information how pairs of
contacts link to other contacts

3D genome organization with
single-cell precission
• Multi-contact reads identify contacts that occurred

simultenously in the same cell Ligation junction
– 3C combined with long-read
sequencing
– Analysis of large numbers of
“Multi-contact hubs” can
measure blocking of loop
extrusion

Development of multi-contact Nano-C
• Multiplexed multi-contact 3C
– Multiplexing of 10-15 viewpoints / experiment
– Hundreds to thousands of long reads / viewpoint / experiment

• Development of multi-contact Nano-C assay

– Computational challenge: determine 3D contacts from
error-rich RNA reads
• (RNA) mappers do not like when reads have multiple hits anywhere
in the genome
Selected Viewpoint
Nano-C read

• Development of multi-contact Nano-C assay

– Computational challenge: determine 3D contacts from
error-rich RNA reads
• (RNA) mappers do not like when reads have multiple hits anywhere
in the genome
Li & Durbin, Bioinformatics 2009

How do individual CTCF binding sites
contribute to blocking of loop extrusion?
• Nano-C TAD-walks to visualize multi-contact hubs

– One TAD-walk represents a multi-contact hub from within a
single cell
TAD-walk:
(here: 4-way
multi-contact hub) Contact 1 Viewpoint Contact 2 Contact 3

• Nano-C TAD-walks to visualize multi-contact hubs

– One TAD-walk represents a multi-contact hub from within a
single cell
– Aim: quantify loop extrusion read-through and boundary strength
Multi-contact hub
obeys boundary
Multi-contact hub
crosses boundary

• Transition zone with clustered CTCF binding

Boundary shown today:

4 CTCF binding sites within ~50 kb
3 viewpoints surrounding CTCF sites
Approximate transition zone
1 2 3

What can Nano-C tell us (and what not?)
• Comparison between cells with (WT) and without

(Rad21-AID) loop extrusion
Rad21-AID mESCs:
Elzo de Wit




– Many multi-contacts hubs represent individual contacts
WT (VP1)


WT (VP1)
Rad21-AID (VP1)


– Loop extrusion and boundaries polarize direction of multi-contact
hubs
WT (VP1)
Rad21-AID (VP1)


– Loop extrusion and boundaries polarize direction of multi-contact
hubs

Loop extrusion promotes long range contacts
within the TAD
• Comparison between WT and Rad21-AID cells

– Comprehensive scanning of DNA within TAD

Boundary shown today:

4 CTCF binding sites within ~50 kb
3 viewpoints surrounding CTCF sites
Approximate transition zone
1 2 3

• Nano-C TAD-walks to visualize multi-contacts
Most multi-contacts
strictly obey boundary
Few multi-contacts
in mixed configuration
Sub-population of multi-contacts
that all cross the boundary



– Significant enrichment of multi-contacts that obey or cross the boundary


– Multi-contacts that all obey or all cross the boundary represent the
two scenarios of loop extrusion: blocking and read through

How do individual CBS contribute to blocking
of loop extrusion?
• Nano-C indicates that all CBS individually but

incompletely contribute to blocking of loop extrusion
– Detection of considerable read through of loop extrusion as well
– Read through of loop extrusion decreases with increasing CBS
numbers
• Validation of CBS contribution to blocking of loop

extrusion 1 2 3
– Global CTCF depletion

• CTCF-AID mESCs
– Deletion of single CBS
within transition zone
• Genome-editing CTCF-AID mESCs:
Nora et al, Cell 2017

Impact of perturbed CTCF binding
• Perturbation of CTCF • Perturbation of CTCF

binding to a single site binding to all sites
– CRISPR-Cas9 mediated – Rapid and inducible
deletion of a single site Auxin-mediated degradation
of CTCF protein
Auxin
Nishimura, Nat Methods 2009

Do individual CTCF peaks create stepwise TAD
insulation?
• CTCF perturbations increase loop extrusion read-through

– Multi-contacts that all cross the boundary further enriched
– Mild effect, particularly when only one CBS is removed
1 2 3

Impact of perturbed CTCF binding
• More contacts crossing the boundary = less blocking of

Cohesin loop extrusion
– Does the TAD boundary become more compacted when CTCF
binding is perturbed?
• Oligopaint high-resolution microscopy
Reduction of distances relative
to non-perturbed cells:
Jennifer Luppino &

Eric Joyce

TAD formation and loop extrusion measured with
single-cell precision using multi-contact Nano-C
• Three-level clustering of CTCF sites in extended

transition zones around TAD boundaries
– Non-standard intersection of ChIP-seq and Hi-C data
• Individual CTCF sites additively contribute to the
structure and insulating function of TAD boundaries
– Multi-contact 3C
– Validations using cells with perturbed CTCF binding

• Traffic light model for loop extrusion blocking

– Incomplete blocking at multiple sites
creates increased Cohesin density
• Causes for loop extrusion

read through?
– Incomplete blocking / CTCF
binding orientation?
– CTCF dissociation?
– Competition for binding of
Cohesin with other factors?

• Implications for TAD function

1. Changes to the DNA sequence over extended genomic regions
can influence TAD structure and function
• DNA regulation, DNA repair, etc.
• Disease (cancer), embryonic defects
2. Changes to individual CTCF binding sites may be buffered by
other CTCF sites within the boundary
• More moderate changes to regulatory influence of TADs

Acknowledgements
• Applied Mathematics, Computational

Biology and Predictive Medicine Group
IBENS – ENS, Paris
Andrea Papale
David Holcman
• Department of Genetics and Epigenetics

• Chromatin Dynamics Group
Institute
Li-Hsin Chang Other members: University of Pennsylvania School of Medicine,
Sourav Ghosh Sébastien Bloyer Philadelphia, USA
Mélanie Miranda Joanne Edouard
Jennifer Luppino
Julie Segueni Benoit Moindrot
Eric Joyce
François Charon

Part 2:
TAD formation and loop extrusion

measured with single-cell precision
using multi-contact Nano-C
QUESTIONS?

Noordermeer 2022

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Genomic Geography

our genome in 3 dimensions and

• Part 1: Our genome in 3 dimensions and its

• Part 2: TAD formation and loop extrusion

Stability and Evolution of Genomes – 26 September 2022

Our genome in 3 dimensions

Stability and Evolution of Genomes – 26 September 2022

• Our genome consists of 2 sets of 23 chromosomes

Stability and Evolution of Genomes – 26 September 2022

• Our genome consists of 2 sets of 23 chromosomes

Stability and Evolution of Genomes – 26 September 2022

• Our genome consists of 2 sets of 23 chromosomes

Stability and Evolution of Genomes – 26 September 2022

• How long is the human genome?

• How long is the human genome?

• How long is the human genome?

• How long is the human genome?

2 meters × ~ 30 * 1012 cells = 60 000 000 000 km

Stability and Evolution of Genomes – 26 September 2022

• Challenge: how to fit 2 meters of DNA in a cell nucleus?

Stability and Evolution of Genomes – 26 September 2022

• Challenge: how to fit 2 meters of DNA in a cell nucleus?

• How to maintain correct genome function?

Stability and Evolution of Genomes – 26 September 2022

• Challenge: how to fit 2 meters of DNA in a cell nucleus?

• How to maintain correct genome function?

Stability and Evolution of Genomes – 26 September 2022

• First-order DNA organization

Stability and Evolution of Genomes – 26 September 2022

• First-order DNA organization

Stability and Evolution of Genomes – 26 September 2022

• First-order DNA organization

Stability and Evolution of Genomes – 26 September 2022

• Higher-order DNA organization

Akhtar & Gasser, Nat Rev Genet 2007

Stability and Evolution of Genomes – 26 September 2022

• Genomic cartography of 3D genome organization

– Chromosome Conformation Capture and Hi-C

Stability and Evolution of Genomes – 26 September 2022

• Fixing 3D DNA structure

Stability and Evolution of Genomes – 26 September 2022

• Fixing 3D DNA structure

Stability and Evolution of Genomes – 26 September 2022

• Fragmentation by enzymatic digestion

Stability and Evolution of Genomes – 26 September 2022

• Enzymatic ligation of crosslinked fragments

Stability and Evolution of Genomes – 26 September 2022

Characterization of ligation junction

Stability and Evolution of Genomes – 26 September 2022

• Current preferred approach: high-throughput sequencing

Stability and Evolution of Genomes – 26 September 2022

• Current preferred approach: high-throughput sequencing

Stability and Evolution of Genomes – 26 September 2022

• Current preferred approach: high-throughput sequencing

Stability and Evolution of Genomes – 26 September 2022

• Gold-standard: Hi-C technology

Stability and Evolution of Genomes – 26 September 2022

• Gold-standard: Hi-C technology

Stability and Evolution of Genomes – 26 September 2022

• Illumina sequencing based

Stability and Evolution of Genomes – 26 September 2022

• Illumina sequencing based

Highest interaction frequency