Structure meets function:Chromosome folding in mammals

A primer on chromosome conformation capture, topologically associating domains, enhancer-promoter communication

Luca Giorgetti May 25. 2016FMI Baselluca.giorgetti@fmi.ch

Transcription Regulation And Gene Expression in Eukaryotes

FS 2016 Graduate Course G2P Matthias and RG Clerc

Pharmazentrum Hörsaal 2 16h15-18h00

promoter-proximalregulatory sequences

1. Proximal cis-regulatory elements

promoter

Transcriptional control in mammals

Essential for the correct regulation of genes during development, ensure tissue- and developmental stage specificity

enhancer 1 enhancer 2

1 kb - 1 Mb

promoter-proximalregulatory sequences

promoter

1. Proximal cis-regulatory elements2. Distal cis-regulatory elements (enhancers)

Transcriptional control in mammals

Spitz and Furlong, Nat. Rev. Gen. 2012

A paradigmatic case: o Shh expression in the posterior limb bud of E10.5 mice depends on an enhancer located 1 Mb awayo Enhancer-promoter proximity correlates with transcriptional state

Amano et al, Dev. Cell 2009

Long-range transcriptional control: Shh and the MFCS1 enhancer

Dominant model: Physical interactions with promoters are required to exert an enhancer’s regulatory functions

de Laat and Duboule, Nature 2013Schwarzer and Spitz, Curr. Op. Gen. Dev. 2014

Long-range regulation involves 3D interactions

A central question in molecular biology:How do enhancer/promoter interactions occur?

?

Long-range regulation involves 3D interactions

PCRs with candidate primers

Dekker et al, Science 2006

Covalent bond

Chromosome conformation capture (3C)

Job Dekker

PCRs with candidate primers

Dekker et al, Science 2006

Covalent bond

Chromosome conformation capture (3C)

A population-averaged biochemical techniqueMillions of cells per experiment

Job Dekker

Brain (does not express β-globin)Liver (expresses β-globin)

Tolhuis et al., Nat. Gen. 2002

Locus control region(LCR) β-globin gene

50 kb

Enhancer-promoter contacts are more frequent when the gene is transcribed

3C relies on PCR to detect interaction frequencies.

1. Need an a priori hypothesis on which regions are supposed to interact

2. Limited number of interactions that one can hope to detect (“one vs. few” approach)

Beyond 3C: overcoming technical limitations

De Santa et al, PLoS Biol. 2010

Enrich for the interactions of favorite locus with all others

(“one vs. all” approach)

High-throughput sequencing

Simonis et al, Nat. Gen. 2006

Beyond 3C: Circularized chromosome conformation capture (4C)

Wouter de Laat

Brain (does not express β-globin)Liver (expresses β-globin)

van de Werken et al., Nat. Meth. 2012

Brain

Liver

4C

3C

… 10 years later:

An impressive increasein resolution

50 kb

4C: increased resolution

Andrey et al, Science 2013

Hoxd13(inactive)

Viewpoint:

Hoxd9(active)

Hoxd13 Hoxd9500 kb

Interactions between enhancers and promoters occur within interaction “modules”

Ligation-mediatedamplification with manyprimers

High-throughput sequencing

Dostie et al, Gen. Res. 2006

Beyond 4C: 5C and Hi-C

Biotinylateligationjunctions and retrieve with streptavidin

“all vs. all”

High-throughput sequencing

“many vs. many”

Lieberman-Aiden, van Berkum et al, Science 2009

Job Dekker

5C Hi-C

a b

Linear genomic sequence

Contact map

b

aFraction of cells where fragments

a and b physically interact

low high

5C and Hi-C: Interaction maps

microfeeder.magnet.fsu.edu

The chromatin fiber is a polymer

Polymer physics and chromosome conformation capture

microfeeder.magnet.fsu.edu

The chromatin fiber is a polymer

Polymer physics is a well-established discipline that allows describing complex polymers (e.g. chromatin) in simplified but realistic terms

Tark-Dame and Hermann, J. Cell Sci. 2011

Polymer physics and chromosome conformation capture

Polymer physics predicts that in a polymer with uniform (or no) internal interactions, the contact probability between two loci scales like an inverse power law of their distance along the polymer

Any deviation from this behavior is a ‘surprising’ finding, indicative of non-random folding

Polymer coordinate

i j

Poly

mer

coor

dina

te

j

ipij

𝑗𝑗 − 𝑖𝑖

pij

Polymer physics and chromosome conformation capture

TADs: Sub-megabase regions of the genome, where genomic elements interact preferentially with each other

Nora et al, Nature 2012

500 kb1 Mb

The chromatin fiber is partitioned into topologically associating domains (TADs)

But what determines TAD boundaries?

TADs are present everywhere in the genome and boundaries are conserved across spec

Dixon et al, Nature 2012

Nora et al., 2012

Dixon et al., 2012

TAD boundaries are possibly created by boundary elements (CTCF?)

Rao et al., Cell 2014

98% of ‘loops’ correspond to convergent CTCF binding sites

… but possibly also by CTCF-mediated ‘loops’ that organize sub-TAD structure

CTCF and cohesin : the main organizers of chromosome structure in mammals?

Merkenschlager and Nora, Ann Rev Genom Hum Genetics 2016

11-zinc-finger, sequence-specific DNA-binding proteinConserved in metazoaOnly known insulator protein in vertebratesLocalizes at virtually all loop anchors, but also at many more sites

Ring-like multiprotein that provides cohesion between sister chromatids Co-occupies thousands of CTCF sitesHas been implicated in the formation of CTCF-associated loops

Enhancer-promoter interactions occur within structural domains (e.g. TADs) most likely created by CTCF/cohesin

Long-range looping vs. Loops extrusion models

Fudenberg et al, bioRxiv 2015Sanborn et al PNAS 2015

Loop extrusionLong-range looping

How may CTCF and cohesin ‘create’ specific chromosomal structures?

De Wit et al., Mol. Cell 2015

I. All known enhancer-promoter interaction fall within the same TADs

Smallwood and Ren 2013

TADs act as regulatory microenvironments

II – Being in the same TAD favors transcriptional co-regulation

Tsix promoterinteractors

Xist promoterinteractors

FtxTsix Jpx Xpr

Xite

Rnf12Xist

Cnbp2

TsxChic

1Cdx4

Ppnx

Nap1L2

Slc16a2? ?

Linx

LeDily et al Genes & Dev. 2014

Nora et al, Nature 2012

Zhan et al, unpublished data

TADs act as regulatory microenvironments

Symmons et al. Genome Res 2014

III – enhancer activity correlates with TAD positions

TADs act as regulatory microenvironments

IV – Disrupting TAD boundaries leads to transcriptional mis-regulation

Nora et al, Nature 2012

Lupianez et al, Cell 2015

TADs act as regulatory microenvironments

Sexton et al, Cell 2012

1 Mb

TADs also exist in Drosophila

Crane et al, Nature 2015

TADs also exist in C.elegans

Le et al, 2013

Caulobacter1 Mb

TAD-like structures also exist in some bacteria

few bp

1 Mb

100 Mb

1000 bp

1. enhancer-promoter contacts, CTCF-mediated (?) loops

10 kb

100s of kb

Hierarchical folding of mammalian chromosomes

1. enhancer-promoter contacts, CTCF-mediated (?) loops2. TADs

10 kb

100s of kb

1 Mb

few bp

1 Mb

100 Mb

1000 bp

Hierarchical folding of mammalian chromosomes

Fraser et al, Mol. Systems Biol. 2015

Above the TAD level: TADs interact in meta-TAD hierarchical trees

Lieberman-Aiden et al, Science 2009

40 Mb

Active (“A) compartmentInactive (“B”) compartment

At even larger scale, A/B compartments can be detected

Mutually exclusive preferential contacts between active, gene rich and inactive, gene-poor regions

𝑗𝑗 − 𝑖𝑖

pij

The large-scale scaling behavior of contact probabilities in mammalian genomes is compatible with the idea that the physical state of chromosomes is a ‘crumpled globule’

𝛾𝛾 ≈ 1

Lieberman-Aiden 2009

The fractal globule hypothesis

Lieberman-Aiden 2009

The large-scale scaling behavior of contact probabilities in mammalian genomes is compatible with the idea that the physical state of chromosomes is a ‘crumpled globule’

The fractal globule hypothesis

However many other models can be found that predict the same scaling…

Zhan et al, Phys. Rev. E in revision

The large-scale scaling behavior of contact probabilities in mammalian genomes is compatible with the idea that the physical state of chromosomes is a ‘crumpled globule’

The fractal globule hypothesis

10 kb

100s of kb

1 Mb

10s of Mb

few bp

10 Mb

100 Mb

1000 bp

1. enhancer-promoter contacts, CTCF-mediated (?) loops2. TADs

3. A/B compartments

Hierarchical folding of mammalian chromosomes

• What is the cell-to-cell and temporal dynamics of chromosome folding, and enhancer-promoter communication in particular?

• How is it related to transcriptional dynamics, and cell-to-cell variability in transcription?

• How are TADs created and maintained? What is the molecular mechanism of CTCF/cohesinaction?

• Is transcription cause or effect of chromosomal structure (or both)?

• How do enhancers “use” the topological information encoded in TADs (and other structures) to target only a certain subset of enhancers?

• To which extent does enhancer-promoter communication rely on topological connectivity, rather than biochemical specificity?

Some outstanding open questions

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