3DGenomics for genome engineering Marc A. Marti-Renom Structural - PowerPoint PPT Presentation

3DGenomics for genome engineering Marc A. Marti-Renom Structural Genomics Group (ICREA, CNAG-CRG) http://marciuslab.org http://3DGenomes.org http://cnag.crg.eu

Resolution Gap Marti-Renom, M. A. & Mirny, L. A. PLoS Comput Biol 7, e1002125 (2011) Knowledge IDM INM DNA length 10 10 10 10 nt Volume 10 10 10 10 10 μ m Time 10 10 10 10 10 10 10 10 s Resolution 10 10 10 μ

Hybrid Method Baù, D. & Marti-Renom, M. A. Methods 58, 300—306 (2012). Experiments A Chr.18 -Pg B C D Computation

Chromosome Conformation Capture Dekker, J., Rippe, K., Dekker, M., & Kleckner, N. (2002). Science, 295(5558), 1306—1311. Lieberman-Aiden, E., et al. (2009). Science, 326(5950), 289—293.

Chromosome Conformation Capture CROSSLINK Protein Protein CUTTING Endonuclease digestion Sonication Biotin dCTP fill in Immunoprecipitation Immunoprecipitation LIGATION biotinilated linkers B B B B B B REVERSE B B CROSSLINKS B B B B DETECTION Multiplexed Digestion with Sonicate Mmel digestion amplification four base cutter Pull down Ligation B B -Pg PCR with PCR with PCR with Pull down speci fj c primers universal primers speci fj c primers Inverse PCR B B Contact B B B library B COMPUTATIONAL ANALYSIS Chromatin-associated factors Gene 3C 5C 4C Hi-C ChIP-loop ChIA-PET

Restraint-based Modeling Baù, D. & Marti-Renom, M. A. Methods 58, 300—306 (2012). Chromosome structure determination 3C-based data Biomolecular structure determination 2D-NOESY data

http://3DGenomes.org FastQ files to Maps -Pg Map analysis i+1 i i+2 Model building i+n Model analysis

previous applications... Baù, D. et al. Nat Struct Mol Biol (2011) Umbarger, M. A. et al. Mol Cell (2011) Le Dily, F. et al. Genes & Dev (2014) Trussart M. et al. Nature Communication (2017) Cattoni et al. Nature Communication (2017) Stadhouders R. et al. Nature Genetics (2017) in press Job Dekker George M. Church Lucy Shapiro

The 3D architecture of Caulobacter Crescentus Nierman W C et al. PNAS 2001 98 :4136-4141 4,016,942 bp & 3,767 genes

The 3D architecture of Caulobacter Crescentus Ori Ter Ori Ori 3 x 10 0 0.0 Origin 2.53 x 10 0 0.5 Minus Probe Genome Position (mbp) 2.06 x 10 0 1.1 5C interaction Z-scores 1.59 x 10 0 1.7 = + Strand Ter = - Strand 1.12 x 10 0 2.1 6.56 x 10 -1 2.5 1.88 x 10 -1 3.0 Terminus 3.5 -2.81 x 10 -1 Ori -7.5 x 10 -1 4.0 0.0 0.5 1.1 1.6 2.1 2.5 3.1 3.6 4.0 Plus Probe Genome Position (mbp) 169 5C primers on + strand ~13Kb 170 5C primers on – strand 28,730 chromatin interactions

5C interaction matrix ELLIPSOID for Caulobacter cresentus Ori Ter Ori Ori 0 3 x 10 0.0 0 2.53 x 10 0.5 Ori Ter 0 Minus Probe Genome Position (mbp) 2.06 x 10 1.1 3 5C interaction Z-scores 0 2.5 1.59 x 10 1.7 2 Ter Contact Frequency 1.5 0 2.1 1.12 x 10 1 0.5 -1 2.5 6.56 x 10 0 −0. 5 -1 3.0 1.88 x 10 −1 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 Genome Position (mbp) 3.5 -1 -2.81 x 10 Ori -1 4.0 -7.5 x 10 0.0 0.5 1.1 1.6 2.1 2.5 3.1 3.6 4.0 Plus Probe Genome Position (mbp)

3D model building with the 5C + TADbit 0 3 x 10 0.0 339 mers 0 2.53 x 10 0.5 Minus Probe Genome Position (mbp) 0 2.06 x 10 1.1 5C interaction Z-scores 0 1.59 x 10 1.7 0 2.1 1.12 x 10 2.5 -1 6.56 x 10 -1 3.0 1.88 x 10 3.5 -1 -2.81 x 10 4.0 -1 -7.5 x 10 0.0 0.5 1.1 1.6 2.1 2.5 3.1 3.6 4.0 Plus Probe Genome Position (mbp)

Genome organization in Caulobacter crescentus Arms are helical dif site 47±17Kb from Ter parS sites 25±17Kb from Ori Resolution of Centromer-like chromosomes

Moving the parS sites 400 Kb away from Ori ParB PopZ parS parS ? Wild-type ET166

Moving the parS sites results in whole genome rotation! Wild-type 0 3 x 10 0.0 0 2.53 x 10 0.5 0 Minus Probe Genome Position (mbp) 2.06 x 10 1.1 5C interaction Z-scores 0 1.59 x 10 1.7 ParS sites 0 2.1 1.12 x 10 ET166 -1 2.5 6.56 x 10 -1 3.0 1.88 x 10 3.5 -1 -2.81 x 10 -1 4.0 -7.5 x 10 500 nm 0.0 0.5 1.1 1.6 2.1 2.5 3.1 3.6 4.0 Plus Probe Genome Position (mbp) Arms are STILL helical

From Sequence to (Structure) to Function dense Technology ? Genome Modification dense ParS Function!

Structure alteration and disease three examples from the Mundlos (2) and Young (1) Labs…

Chromosome Conformation Capture Dekker, J., Rippe, K., Dekker, M., & Kleckner, N. (2002). Science, 295(5558), 1306—1311. Lieberman-Aiden, E., et al. (2009). Science, 326(5950), 289—293. Compartments A compartments 20 Mb B compartments Interaction preference TADs 2 Mb

Structure alteration and disease Lupiáñez, et al. (2015). Cell, 1—15.

Structural alteration and disease Franke, M., et al. (2016). Nature, 1—15. - Intra-TAD e g .5 B B KCNJ2 B SOX9 B B B B B KCNJ2 SOX9 B B B B B B B B B B B e Cen. Tel. Sex reversal d No phenotype D Cooks syndrome d o Inter-TAD Inter-TAD 2 Neo-TAD with duplicated gene Neo-TAD without gene y KCNJ2 SOX9 B KCNJ2 B KCNJ2 B SOX9 B a B B B B B B B B B B B B B B B B B B B B B B B B e Centromeric Telomeric Centromeric Telomeric e e g

Structural alteration and disease Hnisz, D. et al. (2016). Science. 25;351(6280):1454-1458

Future Pilot? http://www.4dnucleome.eu

David Castillo Yasmina Cuartero Irene Farabella Silvia Galan Mike Goodstadt Francesca Mugianesi Julen Mendieta Juan Rodriguez François Serra Paula Soler Aleksandra Sparavier Yannick Spill Marco di Stefano Caulobacter 3D Genome in collaboration with Job Dekker, Jorge Church and Luci Shapiro http://marciuslab.org http://3DGenomes.org http://cnag.crg.eu