Lecture 2, Fall 2004

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Lecture 2, Fall 2004 Transcriptional regulation
Cis-acting elements and Trans-acting factors.
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Cis-acting regulatory elements

• Promoter - minimal region required for GTF and
  polymerase.
• Regulatory sequences
• UAS - upstream activating sequence, usually
  within a few hundred bases of the promoter, term
  usually used when discussing yeast.
• Enhancer - regulatory sequence that functions at
  a distance (even downstream or within the
  transcription unit) and orientation independent
  manner.
• Regulatory sequence - widely used generic term.

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Promoter
TATA box recognized by TBP. INR (initiator) and
DPE (downstream promoter element) recognized by
TAFs
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Many eucaryotic genes are controlled by
combinations of activators and repressors.
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Identifying regulatory elements.

• Systematically mutate DNA and connect to a
  convenient reporter gene.
• luciferase, chloramphenical acetyl transferase
  (CAT), ß-galactosidase.
• Transfect DNA back into a suitable eucaryotic
  cell.
• Measure reporter gene activity.

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• Introducing DNA back into cells
• Chemical CaCl2, DEAE dextran, Cationic lipids
• Transient transfection vs stable transformation.
• Transfection - DNA typically extrachromosomal,
  cells are only cultured for a short while, DNA is
  not packaged as normal chromatin.
• Transformation - Cells stably incorporate the DNA
  into their chromosomes, DNA of interest is linked
  to a drug selectable marker, may be a better
  model for studying chromatin-dependent regulation.

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• Caveats
• Cell lines are usually immortal so they carry
  mutations in cell cycle control.
• Response may be cell-type specific - specific
  factors or combination of factors may be absent.
• Chromatin structure may be abnormal.
• If DNA has randomly inserted into the genome,
  neighboring sequences can affect transcription.

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• Regulatory elements in animal cells
• Numerous elements spread over many kilobases of
  DNA.
• Regulatory elements in yeast
• Small number located within a few hundred bases
  of the transcription start.

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How can regulatory sequences function from far
away and from varied locations? Flexibility in
the location of the regulatory elements can occur
because of DNA looping. Persistent length of
DNA is the length of DNA to make a smooth 90o
bend and is 200 bp.
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The locations of closely spaced regulatory
elements are often highly constrained because the
stiffness of DNA demands spacing that allows the
proteins to interact.
A 10 bp insertion can be less deleterious than a
5 bp insertion because proteins remain on the
same side of the DNA helix.
5 bp
10 bp
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Transcriptional activators - trans-acting factors
that recognize DNA sequences and regulate
transcription.
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• Identification of the first pol II activator -
  the awesome power of yeast genetics
• Key genetic evidence mutation in yeast Gal4 gene
  resulted in reduced expression of multiple genes
  involved in galactose metabolism.
• Systematic mutagenesis of Gal4-dependent genes
  (target genes) identified a common sequence
  located at various distances upstream from the
  promoters - UASGal.
• Gal4 gene was isolated by transforming gal4
  mutant with a library of yeast DNA fragments and
  identifying fragments that complement the gal4
  mutation.
• Gal4 expressed in E. coli was shown to bind the
  UASGal.

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• Identification of transcriptional activators from
  mammals.
• Genetic screens are not tractable so
  identification has relied on biochemical
  fractionation of extracts.
• DNA affinity chromatography was the key
  breakthrough in purification - these proteins are
  often present in a few thousand molecules per
  cell.
• Assays for protein activity Gel shift, DNase I
  footprinting, transcriptional activation in
  vitro.
• Amino acid sequencing of small amounts of protein
  provides essential information leading to the
  cloning of the gene for the protein.
• Subsequent use of expression systems provides a
  way to produce large amounts of normal and mutant
  forms of the protein for structure/function
  studies.

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DNA affinity chromatography.
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Electrophoretic Mobility Shift Assay - EMSA,
gel-shift.
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• Many DNA-binding, transcriptional regulators have
  modular structures.
• Distinct DNA-binding domains and transcriptional
  regulatory domains (eg. activation domain).

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Structure/function analysis of Gal4.
Note loss of both activation and DNA binding.
Suggests activation can not occur without DNA
binding.
Note loss of activation, retention of DNA
binding. Indicates that DNA binding can occur
without activation domain.
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Domain swap experiment suggests that the only
function of the DNA binding domain is to target
the protein to the promoter region.
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Trans-activation assay is often used for
structure/function analysis of mammalian
activators.
Lodish 10-37
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Protein - DNA recognition.
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Most proteins recognize a specific sequence via
the major groove
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Examples of an amino acid side chain recognizing
the edge of a base in the major groove
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• Zinc finger protein.
• Some bind as monomers, some as dimers.
• Many have multiple fingers resulting in
  relatively large regions of sequence recognition.
• Base recognition is made by side chains
  projecting from an alpha helix.

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Homeotic selector gene
Well-known bacterial regulator.

• Helix turn helix protein.
• Most well-known prokaryotic regulators are helix
  turn helix proteins.
• Homeotic selector genes contain a homeodomain
  that is essentially a helix turn helix motif.
• All bind as dimers.
• Sequence recognition is by amino acid side chains
  projecting from an alpha helix.

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• Helix-loop-helix protein.
• Motif provides DNA recognition and dimerization -
  dont confuse with helix-turn-helix which only
  provide DNA recognition.

• Leucine zipper protein.
• Dimer held together by short coiled-coil
  containing leucines every 7 amino acids and a
  hydrophobic residue every 3rd or 4th amino acid.

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• Advantages of dimerization
• Formation of different heterodimers allows
  recognition of more sequences without a
  proportional increase in the number of
  polypeptides.
• Repression of binding can be achieved with
  partners that can dimerize but lack DNA binding
  domain.
• Ligands can cause allosteric changes that alter
  the location of the DNA reading heads.
• Post-translational modifications like
  phosphorylation and acetylation can promote or
  inhibit dimerization which in turn affects
  binding.

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Binding as homodimers or heterodimers increases
the repertoire of sites recognized.
Binding of ligand shifts the DNA binding domains
so they align with the major grooves.
Dimerization partners that lack the DNA binding
domain act as repressors. One of the first
examples was MyoD and Id. Id inhibits muscle
differentiation.