DNA goes 3-dimensional: chromatin structure
Aleksandra Galitsyna
Invited lecture for SMTB 2020
Classical view on DNA
- DNA is a linear molecule of biopolymer that contains genetic information.
First proof that DNA is lineraly ordered
- Early Benzer experiments on E. coli and T4 bacteriophage:
Benzer, 1950-1960
https://slideplayer.com/slide/1400263/
Bacterial loan on the agar plate,
plaques are formed at the locations infected by the virus
First proof that DNA is lineraly ordered
- Co-transfection by two mutants:
Benzer, 1950-1960
https://slideplayer.com/slide/1400263/
Plaques are formed
(infection takes place, wild type)
Plaques are not formed
(no infection)
First proof that DNA is lineraly ordered
- Genetic map of bacteriophage
Benzer 1961
О - no infection, |- wild type
Mutations of phage,
ordered by similarity of patterns
DNA as double helix
- Watson, Crick and Franklin: X-ray crystallography (1953)
Pray Nature Education 2008
Levels of chromatin organisation
Sequence
Secondary structure
A-helix
B-helix
Z-helix
G-quadruplex
DNA hairpin
Chromatin is DNA-protein complex
DNA forms more complex structures upon the binding of proteins. For example, the binding of histones:
Alberts 2015 "Molecular Biology of the Cell" 6th edition
Chromatin is DNA-protein complex
Binding of transcription factors:
Robinson, 2016
Enhancers are regulators of gene expression
The promoter-enhancer protein complex is required for the activation of gene expression:
Promoter
How promoters and enhancers interact?
Enhancer can be located thousands of nucleotides away from its target promoter (gene start).
Hypothetical mechanisms of interactions:
Dean 2006
Regulatory networks of enhancers
Single gene might be regulated by multiple enhancers, and one enhancer might regulate several genes:
Regulatory networks of enhancers
Long-range regulation helps to generate different phenotypes of cells in multi-cellular organism:
Approaches to study long-range interactions and chromatin structure
Microscopy of the nucleus
Ros 2006 "Histology Atlas with Correlated Cell and Molecular Biology"
Two approaches:
microscopy and molecular biology
FISH-microscopy
Fluorescent in situ hybridization
FISH-microscopy
Bolzer et al., PLoS Biol. 2005
Speicher & Carter 2005 Nature
Fluorescent in situ hybridization
Chromosomes Conformation Capture (3C)
Formaldehyde crosslinking
DNA restriction
Ligation
DNA purification
DNA-DNA interactions library
3C: Dekker et al. 2002 Science
Hi-C: Chromosomes Conformation Capture + sequencing
Formaldehyde crosslinking
DNA restriction
Ligation
DNA purifiction
Sequencing
Mapping
Lieberman-Aiden et al. 2009 Science
DNA-DNA interactions map
Lieberman-Aiden et al. 2009 Science
Color is the frequency of interactions
DNA-DNA interactions map
Lieberman-Aiden et al. 2009 Science
Color is the frequency of interactions
The map of pairwise interactions of SMTB-2020 students
Met at summer school, study molecular biology together
Color: number of messages in Telegram
Chromosome territories
Bonev et al. 2016 Nature Reviews
Topologically associating domains (TADs)
Bonev et al. 2016 Nature Reviews
Compartments
Bonev et al. 2016 Nature Reviews
Loops
Architectural loops
Promoter-enhancer loops
Polycomb-loops
Bonev et al. 2016 Nature Reviews
Levels of chromatin organization
Loop extrusion is the major hypothesis of chromatin folding
MirnyLab Youtube channel
Simplified visualization of key components: DNA, extruder (cohesin) and barrier element (CTCF)
Loop extrusion model
Fudenberg et al. 2016 Cell Reports
cohesin is extruding factor
CTCF is a barrier element
Architectural protein CTCF
Rao et al. 2014 Cell
Ganji et al. 2018 Science
Ganji et al. 2018 Science
Direct proof of extrusion mechanism
Model of active chromatin folding
Mirny Lab Youtube channel
Chromatin is actively folded structure that is formed by extruding factors
Quiz: Reconstruction of 3D from Hi-C map
Loop
Quiz: Reconstruction of 3D from Hi-C map
Multiple loops
Quiz: Reconstruction of 3D from Hi-C map
TAD
More exotic structure...
hairpin
In vivo hairpins
Caulobacter crescentus
(bacteria)
In vivo hairpins
Caulobacter crescentus
Pseudoknot
Rare case
Clinical examples
Anania and Lupiáñez, 2020; Lupiáñez et al. 2015
Clinical examples
Anania and Lupiáñez, 2020; Lupiáñez et al. 2015
Clinical examples
Anania and Lupiáñez, 2020; Lupiáñez et al. 2015
Take-home message
Human: 2 meters of DNA inside of 10 um nucleus
100-stores building in a grain of rice
