BiolChem 473

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Biol/Chem 473
Schulze lecture 2 Eukaryotic gene structure
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Prokaryotic vs. eukaryotic gene organization
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Eukaryotic genes have introns and exons
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Eukaryotic transcription is complex
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Eukaryotic RNA polymerases
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Eukaryotic RNA polymerases
but also MANY additional subunits.
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Eukaryotic RNA polymerases
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Eukaryotic RNA polymerases
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RNA polymerase II
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RNA polymerase II

• Has to transcribe a GREAT diversity of genes,
  that all have specific developmental and spatial
  expression profiles.
• RNA polymerase II has MANY subunits.

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Using reporter genes to dissect regulatory
sequences
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Using reporter genes to dissect regulatory
sequences
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Using reporter genes to dissect regulatory
sequences
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Eukaryotic promoters consist of two components
1 core promoter

• RNA pol II and the general transcription factors
  bind to the core elements in and around the
  transcription start site.
• This allows for a basal level of transcription.

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Eukaryotic promoters consist of two components
1 core promoter

• RNA pol II and the general transcription factors
  bind to the core elements in and around the
  transcription start site.
• This allows for a basal level of transcription.

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Eukaryotic promoters consist of two components
1 core promoter

• Not all promoters look alike in eukaryotes.
• Some genes have internal promoters.
• Some genes have no obvious promoters at all (even
  though they are expressed).

Kutach and Kadonaga (2000) Mol. Cell Biol.
20(13) 4754
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Eukaryotic transcription is complex
Within 100-200bp from transcription start
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Eukaryotic transcription is complex
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The first enhancer discovered SV40
fibroblasts
Reticulocyte control
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• And they are promiscuous they will influence any
  gene they have access to.

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How does an enhancer function?

• The enhancer appears to function to bring
  proteins into the vicinity of the promoter

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How does an enhancer function?

• The enhancer appears to function to bring
  proteins into the vicinity of the promoter

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How does an enhancer function?
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Specialized transcription factors
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Transcription factors an overview

• Can repress or activate transcription
• Activators bind enhancers repressors bind
  silencers
• Tend to be modularized
• Have distinctive DNA binding sites
• Helix-turn-helix, Zn finger, Leucine zipper,
  homeodomain,
• Can multimerize
• Can work combinatorially

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Transcription factors can work combinatorially
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Complexity of eukaryotic transcription
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Complexity of eukaryotic transcription
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Houston, we have a problem
Recall this loop can be enormous
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Houston, we have a problem
It almost certainly contains other genes
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Houston, we have a problem
But recall that enhancers are promiscuous
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• And they are promiscuous they will influence any
  gene they have access to.

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• If there is nothing blocking its way, an enhancer
  can activate any gene.

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Problem enhancers tend to be promiscuous!

• Enhancers located a great distance from their
  target genes have the potential to activate
  intervening non target genes
• How is the action of enhancers restricted to
  prevent promiscuous activation?

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Solution Insulators!

• DNA sequence elements that bind proteins with
  enhancer-blocking and/or chromatin barrier
  activity
• Insulator proteins have been extensively studied
  in flies (less so in humans)

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Proteins that function with insulators
flies
mammals
yeast
A number of these proteins (most?) can
self-associate
Geyer and Clark 2002
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Sequences associated with insulator function
Geyer and Clark 2002
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Insulators as enhancer-blockers
Insulators can disrupt enhancer-promoter
communication only when placed between them
Placement of insulators can create autonomous
domains of gene activity
Gerasimova and Corces 2001
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Insulators can organize the genome into
transcriptionally autonomous domains
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Insulators can organize the genome into
transcriptionally autonomous domains
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Overlay transcriptional and chromosomal
territories
Giant loop with active genes extending from
chromosomal territory
Active genes (white) congregate where
transcription factories (polymerases, splicing
factors etc) are enriched
Active genes (white) move away from het (yellow),
and back again when silent (black)
Different chromatin densities within a chromosome
territory
Gene poor, late replicating domains within
chromosome territories localize closer to the
nuclear periphery
Cremer Cremer (2001) Nat. Rev. Genet. 2(292)