Transcript 401Lecture5sp2013post

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?
Interior
Periphery
% of chromosome signal detected/% DAPI
Statistically
significant
difference
Green = series of fluorescent DNA probes for Chromosome 10
Blue = DAPI (DNA stain)
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
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 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
What activities are occurring
in this nuclear environment?
Major Activities in Interphase Nucleus
Transcription
mRNA Processing
Ribosome assembly
DNA replication
DNA repair
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
Fig. 6-35 Lodish et al. 2013
DNA
What result would you expect if DNA exists in loops?
Would you expect loops to be present at all stages of cell cycle?
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)
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
RNA polymerase II Pre-initiation Complex (PIC)
Prior to transcription
Fig. 7-19. Lodish 2013
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
Take home point?
Immunofluorescence
Triple-label RNA Fluorescent
With RNA pol II antibodies 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
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?