Chromosome organization changes during cell differentiation involve structural, spatial, and temporal coordination. Studies using Hi-C and replication timing analysis at single-cell resolution show that topologically associating domains (TADs) reposition between A and B compartments in the nucleus, correlated with changes in lamina association and shifts in early versus late DNA replication. These changes gradually reprogram gene expression and facilitate lineage specification, such as X chromosome inactivation during differentiation. Coordinated alterations in 3D genome organization are thus a mechanism regulating gene expression and cell fate.

1. 03/10/19 1BIO401,GENOME BIOLOGY

2. 1. What are these changes? (Structural organization)
2. How do these changes occur? (Spatial organization)
3. How are these changes coordinated? (Temporal organization)
“Chromosome organization changes in a cell during differentiation”
03/10/19 2BIO401,GENOME BIOLOGY

3. Frasner et al, Microbiology and molecular biology reviews, 2015
Chromosomal changes during the Cell Cycle
During interphase During mitosis
Thread like structures Compact structures
CHROMATINS CHROMOSOMES
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4. DNA double helix
(2nm)
Nucleosome
(10nm)
Solenoid
(30nm)
250nm wide fibre
Chromatin
(700nm)
Chromosome
(1400nm)
DNA packaging in eukaryotes
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5. Replication foci:-
• Identified by FISH and Giemsa staining
• Each stretch of DNA replicated within 60 min
can be observed as bright spot in the nucleus,
which is called as replication foci.
• Remains stable as a unit after multiple cell
cycles
• Contains approximately 1Mb of DNA
• Also known as ‘1Mb chromatin domain’
R bands:-
• High GC content
• High transcription
activity
• Early replication
G bands:-
• Low GC content
• Low transcription
activity
• Late replication
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6. Nuclear
membrane
LAD
Nucleolus
Lamina Associated Domains (LADs)
• Interact with relatively stable part
of nucleus
• Interact with nuclear lamina
• Makes up around 40% of genome
• Boundaries are enriched in
transcriptional repressors and
architectural protein binding sites.
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7. Giemsa staining, FISH and electron microscopy gave 2-D view of genome
Solution comes in a way of a technique to view genome in 3D
Hi-C technique
Why to study packaging and organization of genome?
Gene regulation
Organization of chromatin Chromatin
morphogenesis
Genome
stability
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8. Crosslink and
isolate
Digestion and
biotin fill in
Ligation and
DNA isolation
Biotin removal and
size fractionation
Pulldown, Adapter
ligation and sequencing
Belton et.al, Methods. 2012 November
Hi-C technique
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9. Belton et.al, Methods. 2012 November03/10/19 9BIO401,GENOME BIOLOGY

10. What information does Hi-C give?
Mota-Gomez et al, Gene201903/10/19 10BIO401,GENOME BIOLOGY

11. TADs- Topologically Associated Domains
• Regarded as basic chromosomal units
• High ratio of chromosomal interactions
within the domain than outside the
domain
• Contains many chromatin loops.
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https://www.wikiwand.com/en/Topologically_associating_domain

12. A and B compartments
• TADs are divided into two spatially exclusive compartments in the
nucleus, A and B
Compartment A Compartment B
Gene rich Gene poor
High GC content Compact
Contains histone markers for active
transcription
Contains histone markers for gene
silencing
Majorly early replicating gene Majorly late replicating
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13. Story so far…
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14. Structural organisation
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15. 1. What are these changes? (Structural organization)
2. How do these changes occur? (Spatial organization)
3. How are these changes coordinated? (Temporal organization)
“Chromosome organization changes in a cell during differentiation”
03/10/19 15BIO401,GENOME BIOLOGY

16. Single Cell Replication Sequencing (scRepli-Seq)
• Previous methods give average image about replication time of thousands of
cells
• It is necessary to check whether these maps reflect the actual replication
profile of the single cell
• To address this, it is necessary to perform genomic analysis at single cell
level
Hiratani et al, Genes 2019
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17. Experimental overview of scRepli-Seq
Stain the cells with PI
Mid-S sorting gate
Next Generation Sequencing
(NGS)
Identification of early and late
replicating domain.
Hiratani et al, Genes 201903/10/19 17BIO401,GENOME BIOLOGY
Replication timing

18. 1. What are these changes? (Structural organization)
2. How do these changes occur? (Spatial organization)
3. How are these changes coordinated? (Temporal organization)
“Chromosome organization changes in a cell during differentiation”
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19. Experimental Setup
oct4- Stem cell marker
Nanog- Pluripotency marker
sox1- Neuroectodermal lineage marker
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20. 03/10/19
Hi-c Single cell RT profiles
by scRepli-Seq
DNA FISH
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21. Principle Component analysis (PC1)
PCA finds the principal components
of data.
Q. So what are principal components
then?
They are underlying structures in the
Data which show most variance or
Direction where Data is most spread
out.
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22. Comparison of RT and A/B compartments (Hi-C-PC1) during Differentiation
Early replicating genes are found in Compartment A while late replicating genes are found in Compartment B
primarily
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23. Compartment profiles of constitutively early and late genes
03/10/19
• >90% of regions that remained as
early replicating stayed in the A
compartment during differentiation
• A/B compartment switches are rare in
regions with constant RT.
• >80% of constitutively late regions
stayed in the B compartment
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24. Compartment changes are linked to sub-nuclear re-positioning
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Changes in RT, A/B compartments and subnuclear positioning are tightly coupled
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25. Sub-nuclear repositioning is a function of NL association
03/10/19
• Changes in A/B compartments correlated with changes in lamin B1 binding
• Repositioning toward or away from the nuclear periphery correlates with
increased or decreased NL association
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NL
0 2 4 6 8 10

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Compartment boundaries are newly formed near TAD boundaries
A/B compartment boundaries and early/late RT boundaries coincide with TAD boundaries
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27. Modes of A/B compartment changes
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Compartment changes takes place majorly by Boundary shifting and rarely by isolation
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28. A/B Compartment change during Reprogramming
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• Compartments change by boundary shifting and frequently effects single TADs
• Reprogramming and differentiation trajectories do not overlap
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29. 1. What are these changes? (Structural organization)
2. How do these changes occur? (Spatial organization)
3. How are these changes coordinated? (Temporal organization)
“Chromosome organization changes in a cell during differentiation”
03/10/19 29BIO401,GENOME BIOLOGY

30. 03/10/19
B to A compartment changes create a state for activation for required genes
• RT changes reflect compartment changes
• B to A changes preceding late to
early changes and transcriptional
upregulation are consistent with
compartment switching creating a
transcriptionally competent state for
activation of genes.
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31. 03/10/19
RT changes gradually but uniformly in differentiating cells
Differentiating cells
EpiSCs
RT changed gradually but uniformly within a differentiating population
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32. 03/10/19
RT changes during X chromosome inactivation
• RT changes from early to late during X chromosome inactivation.
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33. 03/10/19
mESC differentiation
Xist cloud formation precedes early to late changes in RT
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To summarise…
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Conclusion
• First study showing the spatiotemporal coordination of
genome organization within the nucleus.
• First insight into the mechanism by which gene
expression profile of cells change as they differentiate.
• Showed that regulation of gene expression occurs even
at an organizational level in 3D space.
• Showed that the cells have a way of sensing the spatial
changes occurring within the genome, as a response to
which they change the replication time.
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Applications of the study
• 3D chromatin organization of cancer cells can be checked to see B to A
transition within transformed cells.
• Better understanding of molecular mechanisms can be obtained by this
approach.
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37. Any Questions?
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