1. BS222 – Genome Science Lecture 5
3D genome organisation
Vladimir Teif

2. Module structure
Genomes, sequencing projects and genomic databases (VT) (Oct 9, 2018)
Sequencing technologies (VT) (Oct 11, 2018)
Genome architecture I: protein coding genes (VT) (Oct 16, 2018)
Genome architecture II: transcription regulation (VT) (Oct 18, 2018)
Genome architecture III: 3D chromatin organisation (VT) (Oct 23, 2018)
Epigenetics overview (PVW) (Oct 25, 2018)
DNA methylation and other DNA modifications (VT) (Oct 30, 2018)
NGS experiments and analysis I: the basics (VT) (Nov 1, 2018)
NGS experiments and analysis II: data integration (VT) (Nov 8, 2018).
Comparative genomics (JP, guest lecture) (Nov 13, 2018)
SNPs, CNVs, population genomics (LS, guest lecture) (Nov 15, 2018)
Histone modifications (PVW) (Nov 20, 2018)
Non-coding RNAs (PVW) (Nov 22, 2018)
Genome Stability (PVW) ) (Nov 27, 2018)
Transcriptomics (PVW) (Nov 29, 2018)
Year's best paper (PVW) (Dec 6, 2018)
Revision lecture (all lecturers; spring term)

3. Eukaryotes genomes are packed with nucleosomes in the chromatin

4. The history of the nucleosome
Olins & Olins (2013), Nature Rev. Mol Cell Biol., 4,

5. The history of the nucleosome
Olins & Olins (2013), Nature Rev. Mol Cell Biol., 4,

6. Nucleosome positioning affects gene expression
Image courtesy of Broad Communications
Shuleva et al., Nature Rev. Genet., 2014

7. The nucleosome
Nucleosome, with DNA in orange and histone proteins in blue.

8. The nucleosome Nucleosome histone proteins (left) and DNA (right)

9. The nucleosome

10. The nucleosome
Turner B.M. (2005) Nature Structural & Molecular Biology, 12,

11. The nucleosome

12. Nucleosome arrays in chromatin

13. Nucleosome arrays in chromatin
Nucleosome includes
147 DNA base pairs

14. The average distance between centres of neighbouring nucleosomes, called the nucleosome repeat length (NRL), determines chromatin fiber compaction
Routh et al. (2008), PNAS, 105, 8872–8877

15. NGS methods to map nucleosomesMM
NGS methods to map nucleosomes
MNase =
Micrococcal Nuclease (enzyme that cuts DNA, preferentially between nucleosomes)
Teif et al., Methods, 2012

16. Nucleosomes near transcription start
start site (TSS)
3’-end
of gene
Nucleosome occupancy
Jiang & Pugh, Nat Rev Genet., 2009

17. Why ordered nucleosome arrays?
Jiang & Pugh, Nat Rev Genet., 2009

18. Chromatin remodellers actively reposition nucleosomes
TSS
Zhang et al., Science, 2011

19. Chromatin remodellers have different nucleosome positioning rules
Yen et al., (2012) Cell 149, 1461–1473

20. Remodelers are recruited to specific sites and reposition specific nucleosomes, e.g. at TSS
Ho & Crabtree,
Nature, 2010

21. Multicellular organisms have evolved to attract nucleosomes to promoters
Tompitak et al. (2017), Biophys J. 112, 505–511

22. Nucleosome positioning changes during cell differentiation or activation
Human CD4+ T cells,
Teif and Rippe, Nucleic Acids Res., 2009
Schones et al., Cell, 2008

23. Next level of chromatin compaction: thousands to millions of DNA base pairs

24. CCCTC-binding factor (CTCF protein)
Co-discovered
by Prof Elena Klenova from our school
(Klenova et al.,
Mol Cell Biol, 1993)

25. CTCF proteins can form chromatin loops and organise genome in 3D

26. Example: CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2
Kurukuti et al. (2006), PNAS, 103, 10684–10689

27. CTCF-dependent looping
This gene is enclosed in the chromatin hub that contains several enhancers, and is active
This gene is isolated from all its enhancers and is inactive
Kurukuti et al. (2006) Proc Natl Acad Sci U S A. 103:

28. CTCF-dependent looping may be different in different cells  different gene expression
Aberrant CTCF binding is linked to:
Beckwith–Wiedemann syndrome;
Silver–Russell syndrome;
Cancer (different types).
De Almeida et al., Blood, 2012

29. A bridge is not enough to separate domains, we also need rings (see lecturer’s demonstration)
Sofueva and Hadjur, Briefings in Functional Genomics, 3, (2012)

30. Cohesin is a ring-shaped protein complex that
“extrudes” CTCF-dependent chromatin loops
Singh and Gerton, Current Opinion in Cell Biology, 37, 9-11

31. Cohesin is a ring-shaped protein complex that
“extrudes” CTCF-dependent chromatin loops
Hansen et al., eLife 6, e25776 (2017)

32. Cohesin rings also connect different chromosomes during metaphase
Sofueva and Hadjur, Briefings in Functional Genomics, 3, (2012)

33. How CTCF and cohesin fold genome
in domains
Barrington et al., Chromosome Res, 25, 51–60 (2017)

34. Watch this video (at home)
Which aspects of 3D genome are still not understood by scientists?

35. NGS methods to map 3D genome: Chromatin Conformation Capture, Hi-C
Rao et al., Nature 159, 1665–1680 (2014)

36. Hi-C: NGS method to map 3D genome
Main features of the Hi-C matrix:
The diagonal (self-contacts)
Plaid-like pattern (A/B domains)
(A: active, B: inactive)
Squares along diagonal (TADs)
(TAD = Topologically Associated Domain)
“Spots” off-diagonal (Loops)
Rao et al., Nature 159, 1665–1680 (2014)

37. Hi-C: NGS method to map 3D genome
Main features of the Hi-C matrix:
The diagonal (self-contacts)
Plaid-like pattern (A/B domains)
(A: active, B: inactive)
Squares along diagonal (TADs)
(TAD = Topologically Associated Domain)
“Spots” off-diagonal (Loops)
Rao et al., Nature 159, 1665–1680 (2014)

38. Topologically associated domains (TADs) change during the cell differentiation
Aranda et al. (2015), Science Advances, 1, e

39. LARGE SCALE CHROMATIN ORGANIZATION
Cavalli and Misteli, Nature Structural & Molecular Biology 20(3):290-9, 2013

40. Heterochromatin vs euchromatin

41. Gene expression is affected by 3D location

42. Gene expression is affected by 3D location
(Discovered in Drosophila, 1930)

43. Effects of 3D gene location in human diseases
Kleinjan and van Heyningen (1998) Hum Mol Genet, 7(10), 1611–1618

44. Chromosome-wide effects
Chromosome 18 is gene-poor, chromosome 19 is gene-rich
Gene-rich domains congregate at the centre of the nucleus
Translocations result in genes moving out of their ‘normal’
nuclear environment

45. Genes (red) are seen to “loop out” from chromosome
This would enable distant regions to interact

46. Genes located close to the nuclear membrane (lamina) tend to be silenced
Lamina-associated domains (“LADs”)
Dillon N. (2008) Dev. Cell 15(2), 182–186

47. Heterochromatin
Repressed genes
Heterochromatin
Repressed genes
Luperchio et al., Curr Opin Gene Dev, 25, (2014)

48. The 4D Nucleome project 4D = space (3D) + time Nucleome =
genome + nuclear proteome, RNA, etc
(the whole nucleus)
Aims to decipher:
the principles of nuclear organization in space & time;
the role nuclear organization plays in gene expression and cellular function;
how changes in nuclear organization affect normal development as well as various diseases

49. NUCLEOSOME, NUCLEOSOME REPEAT LENGTH (NRL)
Take home message
3D genome organisation modulates gene expression in different cell states
MUST KNOW:
Chromatin, lamina, nucleome
NUCLEOSOME, NUCLEOSOME REPEAT LENGTH (NRL)
NGS methods to map nucleosomes
NGS METHODS TO MAP 3D GENOME CONTACTS
Loops, subcompartments, A/B compartments
Topologically Associated Domains (TADs)
Lamina Associated Domains (LADs)
CTCF, Cohesin, loop extrusion