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

Chromosome Territories

How are they formed?

How do cell growth signals affect them?

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?)

Results: What happens when cells are transferred to low serum?

% of chromosome signal detected/% DAPI

Green = series of fluorescent DNA probes for Chromosome 10Blue = DAPI (DNA stain)

Statistically significant difference

What is Required for Chromosome Re-Positioning?

Mehta et al. Genome Biology 2010, 11:R5

Jasplakinolide: inhibitor of nuclear myosin functionLatrunculin A: inhibitor of actin polymerization

Inhibitor was added to proliferating cells before (top panel) or after (bottom panel) transfer to low serum

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?

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

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.)

Another aspect of the nuclear environmentwhich all nuclear activities must cope with:

Higher-order chromosomal organization

Role of Histone H1

chromosome compaction

proper alignment ofchromosomeson mitotic spindle

inhibit binding ofchromatin remodelers

Fig 6-1. Lodish et al. 2013

What activities are occurring in this nuclear environment?

Major Activities in Interphase Nucleus

Transcription

mRNA Processing

DNA replication

DNA repair

Ribosome assembly

Chromatin exists in loops in vivo:How do we know this?

Different DNA probesEach probe specific forsequences 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

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

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)

Red arrow indicates location where unique C-terminaldomain 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

RNA polymerase II Pre-initiation Complex (PIC)

Fig. 7-19. Lodish 2013

Prior to transcription During transcription

What direction will polymerase move?

Conclusions

• All eukaryotic cellular processes involving DNA must navigate a chromatin template in a highly dynamic environment

• Conserved structure of RNA pol II

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

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?