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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

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Outline

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  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? Statistically significant difference % of chromosome signal detected/% DAPI Periphery Interior Green = series of fluorescent DNA probes for Chromosome 10 Blue = DAPI (DNA stain)

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

  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 environmentwhich 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 Ribosome assembly DNA replication DNA repair

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

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

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

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

  18. Take home point? Immunofluorescence With RNA pol II antibodies 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 Cope et al. Genome Biology 2010, 11:204

  19. 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?

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