Shopland-chromosome-org

Visualizing chromosome organization in the cell nucleus : 

Lindsay Shopland Institute for Molecular Biophysics Visualizing chromosome organization in the cell nucleus

Technology Wish List : 

Technology Wish List Detect specific chromosome sequences (in fixed cells) without disrupting nanostructure   Compare locations of 3 + different objects (genes, proteins) at the same time, with clear resolution of each object         Tag specific sequences in living cells without disrupting nanostructure or activity

100-year-old view of chromosomes : 

100-year-old view of chromosomes

Chromosomes during the cell cycle : 

Chromosomes during the cell cycle

Interphase chromosomes form “territories”, not rods : 

Interphase chromosomes form “territories”, not rods mitotic chromosomes interphase chromosomes

Probing chromosome structure in the nucleus : 

Probing chromosome structure in the nucleus Fluorescence in situ hybridization (FISH) dsDNA in fixed cell Labeled DNA probe denature hybridize * fluorescence imaging

Interphase chromosome structure : 

Interphase chromosome structure mitotic chromosomes interphase chromosomes Where is the important information? How does DNA fold up?

DNA folding: a long-standing mystery : 

DNA folding: a long-standing mystery (from Alberts et al, Molecular Biology of the Cell) 30 nm 800 nm “higher order” Most “higher-order” structures < resolution of light microscope Interphase nucleus Mitosis

Model:the “piebald” region of chromosome 14 : 

Model:the “piebald” region of chromosome 14 Mmu14 Low gene density - 20 genes/5 Mb Genes organized into discrete clusters separated by gene “deserts” (Peterson, et al., 2002) Patterned distribution of genes in DNA sequence

Bar-coding 5 Mb of chromosome : 

Gene Cluster Gene “Desert” Mouse chromosome 14: 5 Mb Bar-coding 5 Mb of chromosome NIH-3T3 Gene clusters Deserts NIH-3T3 fibroblast DNA

Predominant 3-D patterns in the nucleus : 

Predominant 3-D patterns in the nucleus 500 nm Thick (~ 400 nm) fiber and higher-order structures Frequent associations between gene clusters Gene sequence based Intermediate states 200 3-D reconstructions of NIH-3T3 chromosomes

Model of chromosome region folding and organization : 

Model of chromosome region folding and organization Thick (~ 400 nm) fiber and higher-order structures Genic sequence based – Sequence Matters Intermediate states - Dynamic Frequent associations between clusters – Function?

Modeling randomized chromosomes : 

Modeling randomized chromosomes 500nm

Summary : 

Summary Complex 3-D interactions between genes and deserts - Preservation, high resolution, multi-labels Associations between gene clusters – Co-regulation? Multi-labels, 3-D pattern analysis Sequence Matters – Cancer Genomics, high-throughput imaging Intermediate structures – Dynamics Living cells

Technology Wish List : 

Technology Wish List Detect specific chromosome sequences (in fixed cells) without disrupting nanostructure: Structural preservation in fixation, labeling, imaging b. Detecting sequences with sensitivity single copy, small (500 bp, ~20 KDa) c. Detecting sequences with high specificity d. Detecting a lot of chromosome sequence (5 Mb) in a cost-effective way

Technology Wish List : 

Technology Wish List 2. Compare locations of 3+ different objects (genes, proteins) Need distinct spectral signatures Clear identification of object “territories” when overlapping 3. Tag a specific sequence in living cells without disrupting nanostructure or activity DNA is inaccessible (double helix, bound by proteins) b. Nucleus is inaccessible

Acknowledgements : 

Acknowledgements Shopland Lab Megan McOsker Chris Lynch Kathy Snow Kate Thornton The Jackson Laboratory Tim O’Brien (Cornell Univ.) Carol Bult Kevin Peterson University of Heidelberg Christoph Cremer Gregor Kreth Nick Kepper Stefan Stein Johann von Hase