1. Outline Group Reading Quiz #2 on Thursday (covers week 5 & 6 readings Chromosome Territories Chromatin Organization –Histone H1 Mechanism of Transcription Activation –RNA pol II structure and function –Transcription Regulatory elements Identification & characterization in vivo Midterm Discussion

2. Chromosome Territories How are they formed? How do cell growth signals affect them?

3. Experiment Mouse cell line growing in media with serum (growth factors) Transfer cells to media with low serum (what does this do?) Monitor 3D-location of chromosomes in nucleus over time (what technique?)

4. Results: What happens when cells are transferred to low serum? Periphery Interior % of chromosome signal detected/% DAPI Green = series of fluorescent DNA probes for Chromosome 10 Blue = DAPI (DNA stain) Statistically significant difference

5. What is Required for Chromosome Re-Positioning? Mehta et al. Genome Biology 2010, 11:R5 Jasplakinolide: inhibitor of nuclear myosin function Latrunculin A: inhibitor of actin polymerization Inhibitor was added to proliferating cells before (top panel) or after (bottom panel) transfer to low serum

6. Major Conclusions Chromosomes are repositioned when cell growth signal are removed Repositioning may require myosin/actin machinery (similar to cell movement) Why are chromosomes repositioned?

7. Schematic summary of some of the processes and structures that influence the spatial organization of the genome Cope et al. Genome Biology 2010, 11:204

8. Remaining Questions About Nuclear Organization Take 2-3 minutes and think about what you have learned about nuclear organization over the last 2 weeks On index cards: 1) your name 2) one question you have about this topic (something that is unclear to you, something that you want to know more about etc.)

9. Another aspect of the nuclear environment which all nuclear activities must cope with: Higher-order chromosomal organization Role of Histone H1 chromosome compaction proper alignment of chromosomes on mitotic spindle inhibit binding of chromatin remodelers Fig 6-1. Lodish et al. 2013

10. What activities are occurring in this nuclear environment?

11. Major Activities in Interphase Nucleus Transcription mRNA Processing DNA replication DNA repair Ribosome assembly

12. Chromatin exists in loops in vivo: How do we know this? Different DNA probes Each probe specific for sequences separated by known distances in linear DNA Technique = FISH: Fluorescent In Situ Hybridization What result would you expect if DNA exists in loops? Would you expect loops to be present at all stages of cell cycle? Fig. 6-35 Lodish et al. 2013

13. Some regulatory proteins can bind DNA and cause loops to form, bringing distant genomic regions together Chromatin loops isolate genomic regions allowing coordination of gene activation or silencing Cope et al. Genome Biology 2010, 11:204

14. Overview of Transcription Transcription of a Gene Requires –Cis-acting signals on the DNA Promoters and Enhancers –Binding of many different proteins RNA polymerase II General transcription factors (e.g. TBP, TFIIB) Gene-specific transcription factors (e.g. HNF1, HNF4) Co-activators (HATs, chromatin remodeling complexes)

15. Red arrow indicates location where unique C-terminal domain extends from in RNA pol II Structures of RNA polymerase (Pol) II Fig. 7-10. Lodish 2013 What do you notice about the structures? What does this suggest about evolution of RNA pol II? YeastBacteria Yeast

16. RNA polymerase II Pre-initiation Complex (PIC) Fig. 7-19. Lodish 2013 Prior to transcriptionDuring transcription What direction will polymerase move?

18. Conclusions All eukaryotic cellular processes involving DNA must navigate a chromatin template in a highly dynamic environment Conserved structure of RNA pol II

19. Triple-label RNA Fluorescent In Situ Hybridization (FISH) Outlines indicate overlay of RNA Pol II staining Trfc, Hba and Hbb are genes that are located on 3 different chromosomes Take home point? Immunofluorescence With RNA pol II antibodies Cope et al. Genome Biology 2010, 11:204

20. On-Going Questions How do chromosomes get positioned into different chromosome territories? How do transcriptionally active regions on one chromosome associate with transcriptionally active regions on another chromosome? Are there subnuclear regions that promote transcription? Chromosomal translocations?