Announcements

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Announcements

• Exam this Wednesday my half is 40. Gerry
  Prodys half is 60.
• Exam regrade policy if you have a question about
  how I graded an answer, send me an email
  outlining exactly how you felt I went wrong, and
  submit the exam for regrade.
• You MUST (1) make this request in writing, and
  you MUST (2) submit the exam, both within 5
  business days of its return.
• PLEASE NOTE that I will regrade the entire exam
  your grade may go up or it may go down (more
  likely).

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Biol/Chem 473
Schulze lecture 4 Eukaryotic gene regulation
Early Drosophila development
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Eukaryotic transcription is complex
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Complexity of eukaryotic transcription
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Insulators may function to organize the genome
into transcriptionally autonomous domains
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Linking complex gene regulation to nuclear
organization
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The best model organism in the whole world!!!!
Drosophila melanogaster black bellied dew
lover fruit fly
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The early Drosophila embryo is a syncytium (a bag
of nuclei)
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(Maternal effect genes)
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Patterning genes classified by mutant effect on
segmentation
The hierarchical nature of these interactions can
be shown by genetics mutations in earlier levels
of the hierarchy severely disrupt later
patterning.
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Seminal publication and subsequent Nobel
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Maternal genes

• Required before the embryo starts transcribing
  its own genes
• Mother expresses these genes during oogenesis
  (mum packs a lunch)
• Localization of maternal gene products is the
  first step in the cascade

http//flymove.uni-muenster.de/Processes/Segmentat
ion/
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Earliest stages of development run on mRNAs and
proteins deposited by mum during oogenesis

• An efficient way to localize and concentrate
  proteins is by localization of mRNA in a
  translationally repressed state, followed by
  activation of translation when the mRNA reaches
  its destination.
• How are mRNAs localized?
• How are mRNAs repressed?
• How are localized mRNAs then, when required,
  de-repressed?

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Maternal effect Mum packs a lunch for the embryo
Anterior end
Posterior end
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Maternal effect Mum packs a lunch for the embryo

• The bicoid gene encodes a homeodomain
  transcription factor.
• The oskar gene encodes something we are not quite
  sure about.
• The gurken gene encodes TGFß homolog - EGF-R
  ligand, required for dorso / ventral patterning
  of the egg and embryo.
• The nanos gene encodes a translational repressor
  - zinc finger that targets Hunchback- and
  Bicoid-mRNAs to achieve posterior identity.

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Maternal effect Mum packs a lunch for the embryo

• Polarity of the oocyte cytoskeleton is essential
  for maternal factor localization (gurken plays a
  role here).
• Bicoid mRNA gets localized to anterior pole
  because another protein, called Staufen,
  recognizes a structure in the 3UTR of the bicoid
  message (and Staufen protein is itself localized
  by the polarity of the cytoskeleton.

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Maternal effect Mum packs a lunch for the embryo

• Bicoid mRNA gets localized to anterior pole
  because another protein, called Staufen,
  recognizes a structure in the 3UTR of the bicoid
  message (and Staufen protein is itself localized
  by the polarity of the cytoskeleton.

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Maternal effect Mum packs a lunch for the embryo
These dsRNA UTR sequences are required for
anterior localization of bicoid mRNA by Staufen
protein

• Bicoid mRNA gets localized to anterior pole
  because another protein, called Staufen,
  recognizes a structure in the 3UTR of the bicoid
  message (and Staufen protein is itself localized
  by the polarity of the cytoskeleton).

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Gap genes

• The gap genes are expressed by the zygote
  (activated in the syncytial blastoderm)
• Their expression patterns are regulated by the
  maternal gene products, and they regulate each
  other
• They divide the embryo into broad, unique domains
  containing different combinations of
  transcription factors

http//flymove.uni-muenster.de/Processes/Segmentat
ion/
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Pair-rule genes

• Pair-rule genes are expressed just before
  cellularization in patterns of 7 stripes (half
  the number of segments in a wild type larva)
• Some pair-rule genes respond to patterns already
  laid down by the maternal and gap genes, while
  others are regulated by other pair-rule genes
• Pair rule gene expression is very dynamic!
  (pretty pictures)

http//flymove.uni-muenster.de/Processes/Segmentat
ion/
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Segment polarity genes

• Segment polarity genes are expressed at the onset
  of gastrulation
• They are expressed in 14 segment bands, refining
  the patterns of all the previous genes in the
  hierarchy. Maternal, gap and pair-rule gene
  expression begins to fade away

http//flymove.uni-muenster.de/Processes/Segmentat
ion/
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Stripe boundaries are precisely defined
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Control regions upstream of pair-rule genes are
complex
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ASSIGNED PAPERTranscriptional regulation of a
pair-rule stripe in Drosophila
Small, S. et al., Genes Development (1991) 5
827-839
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Main players

• Even-skipped (eve) is a homeo-box containing
  transcription factor.
• Hunchback (hb) has multiple Zn fingers.
• Bicoid (bic) is a homeo-box containing
  transcription factor.
• Kruppel (Kr) contains a Zn finger.
• Giant (gt) contains a Leucine zipper.

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Wild type expression patterns of putative
regulators of eve stripe 2

• even-skipped protein is red
• hunchback protein is green
• overlap of expression is yellow

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Wild type expression patterns of putative
regulators of eve stripe 2

• even-skipped protein is red
• Kruppel protein is green
• overlap of expression is yellow

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Wild type expression patterns of putative
regulators of eve stripe 2

• even-skipped protein is green
• giant protein is red
• Overlap of expression is yellow

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Wild type expression patterns of putative
regulators of eve stripe 2
High levels of gt
High levels of Kr
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Wild type expression patterns of putative
regulators of eve stripe 2
High levels of gt
High levels of Kr
What predictions arise from this picture?
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eve-lacZ gene fusion
This is a transgene gets injected into the
embryo (P transformed embryos), integrates into
the genome and is expressed stably along with all
the other genes. BUT this transgene will express
lacZ in an even-skipped-stripe 2 dependent manner!
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Questions to ask about in vivo expression patterns

• How do we know the order in which patterning
  genes act in development?
• How were potential trans-acting regulators of
  stripe 2 identified?
• What would happen to the domain of expression of
  eve stripe 2 in giant or Kruppel mutant embryos?
• Do these vivo expression patterns indicate direct
  interaction between Kr and gt and the eve stripe
  2 regulatory region?