3D model of the folded yeast genome

1. 3D model of the folded yeast genome • ZhijunDuan, MirelaAndronescu, Kevin Schutz, Sean McIlwain, Yoo Jung Kim, Choli Lee, Jay Shendure, Stanley Fields, C. Anthony Blau & William S. Noble, “A three-dimensional model of the yeast genome,” Nature 2010, 465:363-67 • Presented by Hershel Safer in Ron Shamir’s group meeting on 9.3.2011. 9 March 2011

2. Outline • Context • Experimental technique & validation • Tools & calculations • Findings 9 March 2011

3. Chromosome conformation enables interactions • Activity in the cell’s nucleus depends on physical interactions: DNA functional elements can only interact if they are physically close in 3D space. • In this work, physical interactions between chromosomal locations are identified and used to infer 3D conformations of chromosomes: • Folding of individual chromosomes: interactions between loci on the same chromosome (intra-chromosomal) • Relative locations of chromosomes: interactions between loci on different chromosomes (inter-chromosomal) 9 March 2011

4. Outline • Context • Experimental technique & validation • Tools & calculations • Findings 9 March 2011

5. Chromosome conformation capture on a chip (4C) • Chromosome conformation capture (3C) is an experimental technique for identifying DNA-DNA interactions. Loci of interest are selected in advance. • 3C on a chip (4C) does 3C on a genome-wide basis, so results are not biased by choice of specific loci. • In this work, the measurement done by chip in the 4C protocol is replaced by deep sequencing. 9 March 2011

6. Experimental technique 9 March 2011

7. Sequence data • Used Illumina paired-end sequencing with 20 bp reads. • Read pairs were mapped to S. cerevisiae genome using MAQ. • Kept reads with MAQ score ≥20 • Read locations had to be consistent w.r.t. position of corresponding RE1 (HindIII or EcoRI) recognition site 9 March 2011

8. Validation: Controls • Constructed four libraries using all combinations of RE1 (HindIII, EcoRI) and RE2 (MseI, MspI) • Constructed two independent sets of experimental libraries differing by DNA concentration • Constructed five control libraries, one with non-cross linked cells, and four with yeast genomic DNA and the different combinations of restriction enzymes 9 March 2011

9. Validation: Interaction vs. genomic distance • Interaction frequency decreases with genomic distance in experimental but not control libraries. Graph considers only long-distance interactions, >20kb. 9 March 2011

10. Validation: Biases related to restriction sites • Looked for bias from different efficiencies of restriction enzyme digestion or ligation. • Examined fraction of instances that each HindIII site had an intra-chromosomal interaction. • Strong correlation between independent experimental libraries, not with control. 9 March 2011

11. Validation: Reproducibility given DNA concentration • DNA concentration during proximity-based ligation has large effect on signal-to-noise ratio. • Interaction patterns are broadly similar for two independent experimental libraries. 9 March 2011

12. Validation: Consistency between HindIII & EcoRI • Libraries constructed with different restriction enzymes exhibit similar interactions, especially for intra-chromosomalinteractions. 9 March 2011

13. Outline • Context • Experimental technique & validation • Tools & calculations • Findings 9 March 2011

14. Circos: Visualize data in a circular layout • http://mkweb.bcgsc.ca/circos/ 9 March 2011

15. Circos for network visualization 9 March 2011

16. Converting interaction frequencies to 3D maps • Model each chromosome as a string of beads spaced at 10 kb. • Attempt to place beads so that each pair is at a distance that is inversely proportional to their interaction frequency. • Intra-chromosomal: • Divide chromosome into 5 kb bins. Find mean interaction frequency between each pair of bins. • Estimate 3D distance as a function of interaction frequency based on physical properties of polymers • Inter-chromosomal: Use same distance as an intra-chromosomal interaction with the same frequency 9 March 2011

17. Optimization to place interacting pairs of loci • Formulate problem as • subject to various physical constraints 9 March 2011

18. Outline • Context • Experimental technique & validation • Tools & calculations • Findings 9 March 2011

19. Density of self-interactions • Density of intra-chromosomal interaction does not vary much with chromosome size 9 March 2011

20. Ratios of self- to non-self interactions • Ratio of intra-chromosomal to inter-chromosomal interactions is inversely correlated with chromosome length 9 March 2011

21. Interaction among pairs of chromosome • Compare ratios of observed vs. expected interactions • Interactions are more prevalent between smaller chromosomes 9 March 2011

22. Self-interactions between regions of similar size 9 March 2011

23. Self-interactions within local regions 9 March 2011

24. Self-interaction between telomeric ends • Intra-chromosomal interaction between telomeric ends varied widely 9 March 2011

25. Chromosome XII • Chromosome XII has a very different conformation from all the other chromosomes. 9 March 2011

26. Inter-chromosomal interactions: Centromeres • Inter-chromosomal interactions are dominated by interactions between centromeres 9 March 2011

27. Enrichment of chromosomal features: tRNA • HindIII sites adjacent to tRNA genes were significantly enriched for interactions with sites neighboring other tRNA genes 9 March 2011

28. Enrichment: Early origins of DNA replication • Observed enrichment of sites near early (but not late) origins of DNA replication 9 March 2011

29. Findings consistent with Rabl configuration • These observations are consistent with the Rabl configuration of yeast chromosomes. • Chromosomes tethered by centromeres to one pole of nucleus • Telomeres extend outward toward nuclear membrane • Small chromosome arms crowded within all 32 arms & so make frequent inter-chromosomal contact • Distal regions of long arms are in relatively uncrowded regions & so make less contact 9 March 2011

30. Picture of yeast chromosome arms • From Bystricky et al., “Chromosome looping in yeast,” J Cell Biology (2005), 168(3):375-87 9 March 2011

31. Chromosome territories & arm flexibility • Enrichment for self-interaction compared to non-self • Enrichment decreased with increased distance from centromere • Yeast chromosome arms more flexible than in mammals 9 March 2011

32. Interactions between chromosome pairs 9 March 2011

33. 3D model of yeast genome 9 March 2011

34. Comparing yeast & human genomes 9 March 2011

35. A few notes • Lieberman-Aiden work on the human genome was at Mb resolution. This work is at kb resolution. • Map resolution is constrained by cost of deep sequencing. • Methods based on 3C detect chromatin interactions in a collection of cells. Findings should be confirmed in single cells using methods such as FISH. 9 March 2011

36. References • 3C method: Dekker et al., “Capturing chromosome conformation,” Science (2002), 295:1306-11 • 4C method: Simonis et al., “Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C),” Nature Genetics 2006, 38(11):1348-54 • 3D model of human genome: Lieberman-Aiden et al., “Comprehensive mapping of long-range interactions reveals folding principles of the human genome,” Science 2009, 326:289-93 9 March 2011