3D model of the folded yeast genome Zhijun Duan, Mirela Andronescu, 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: Presented by Hershel Safer in Ron Shamir’s group meeting on D model of folded yeast genome – Hershel SaferPage 19 March 2011

Outline Context Experimental technique & validation Tools & calculations Findings 3D model of folded yeast genome – Hershel SaferPage 29 March 2011

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) 3D model of folded yeast genome – Hershel SaferPage 39 March 2011

Outline Context Experimental technique & validation Tools & calculations Findings 3D model of folded yeast genome – Hershel SaferPage 49 March 2011

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. 3D model of folded yeast genome – Hershel SaferPage 59 March 2011

Experimental technique 3D model of folded yeast genome – Hershel SaferPage 69 March 2011

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 3D model of folded yeast genome – Hershel SaferPage 79 March 2011

Validation: Controls 1.Constructed four libraries using all combinations of RE1 (HindIII, EcoRI) and RE2 (MseI, MspI) 2.Constructed two independent sets of experimental libraries differing by DNA concentration 3.Constructed five control libraries, one with non-cross linked cells, and four with yeast genomic DNA and the different combinations of restriction enzymes 3D model of folded yeast genome – Hershel SaferPage 89 March 2011

Validation: Interaction vs. genomic distance Interaction frequency decreases with genomic distance in experimental but not control libraries. Graph considers only long-distance interactions, >20kb. 3D model of folded yeast genome – Hershel SaferPage 99 March 2011

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. 3D model of folded yeast genome – Hershel SaferPage 109 March 2011

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. 3D model of folded yeast genome – Hershel SaferPage 119 March 2011

Validation: Consistency between HindIII & EcoRI Libraries constructed with different restriction enzymes exhibit similar interactions, especially for intra-chromosomal interactions. 3D model of folded yeast genome – Hershel SaferPage 129 March 2011

Outline Context Experimental technique & validation Tools & calculations Findings 3D model of folded yeast genome – Hershel SaferPage 139 March 2011

Circos: Visualize data in a circular layout 3D model of folded yeast genome – Hershel SaferPage 149 March 2011

Circos for network visualization 3D model of folded yeast genome – Hershel SaferPage 159 March 2011

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. 3D model of folded yeast genome – Hershel SaferPage 169 March 2011 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

Optimization to place interacting pairs of loci Formulate problem as subject to various physical constraints 3D model of folded yeast genome – Hershel SaferPage 179 March 2011

Outline Context Experimental technique & validation Tools & calculations Findings 3D model of folded yeast genome – Hershel SaferPage 189 March 2011

Density of self-interactions Density of intra-chromosomal interaction does not vary much with chromosome size 3D model of folded yeast genome – Hershel SaferPage 199 March 2011

Ratios of self- to non-self interactions Ratio of intra-chromosomal to inter-chromosomal interactions is inversely correlated with chromosome length 3D model of folded yeast genome – Hershel SaferPage 209 March 2011

Interaction among pairs of chromosome Compare ratios of observed vs. expected interactions Interactions are more prevalent between smaller chromosomes 3D model of folded yeast genome – Hershel SaferPage 219 March 2011

Self-interactions between regions of similar size 3D model of folded yeast genome – Hershel SaferPage 229 March 2011

Self-interactions within local regions 3D model of folded yeast genome – Hershel SaferPage 239 March 2011

Self-interaction between telomeric ends Intra-chromosomal interaction between telomeric ends varied widely 3D model of folded yeast genome – Hershel SaferPage 249 March 2011

Chromosome XII Chromosome XII has a very different conformation from all the other chromosomes. 3D model of folded yeast genome – Hershel SaferPage 259 March 2011

Inter-chromosomal interactions: Centromeres Inter-chromosomal interactions are dominated by interactions between centromeres 3D model of folded yeast genome – Hershel SaferPage 269 March 2011

Enrichment of chromosomal features: tRNA HindIII sites adjacent to tRNA genes were significantly enriched for interactions with sites neighboring other tRNA genes 3D model of folded yeast genome – Hershel SaferPage 279 March 2011

Enrichment: Early origins of DNA replication Observed enrichment of sites near early (but not late) origins of DNA replication 3D model of folded yeast genome – Hershel SaferPage 289 March 2011

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 3D model of folded yeast genome – Hershel SaferPage 299 March 2011

Picture of yeast chromosome arms From Bystricky et al., “Chromosome looping in yeast,” J Cell Biology (2005), 168(3): D model of folded yeast genome – Hershel SaferPage 309 March 2011

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 3D model of folded yeast genome – Hershel SaferPage 319 March 2011

Interactions between chromosome pairs 3D model of folded yeast genome – Hershel SaferPage 329 March 2011

3D model of yeast genome 3D model of folded yeast genome – Hershel SaferPage 339 March 2011

Comparing yeast & human genomes 3D model of folded yeast genome – Hershel SaferPage 349 March 2011

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. 3D model of folded yeast genome – Hershel SaferPage 359 March 2011

References 3C method: Dekker et al., “Capturing chromosome conformation,” Science (2002), 295: C 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): D model of human genome: Lieberman-Aiden et al., “Comprehensive mapping of long-range interactions reveals folding principles of the human genome,” Science 2009, 326: D model of folded yeast genome – Hershel SaferPage 369 March 2011