Download Control of gene expression in eukaryotes Transcriptional regulation

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Control of gene expression in eukaryotes
  Transcriptional regulation
  Developmental mutants in Drosophila
  Master genes
  Effector genes
  Relevance to disease, evolution
General principles from study of lac operon:
  Gene expression controlled by binding of proteins
to upstream regulatory elements
(promoter / operator)
  Each gene controlled by both activators and repressors
  Separate response elements for different proteins
  Mutations in regulatory proteins or in the DNA elements
can alter expression
  Do these principles apply to eukaryotes?
Important differences in gene expression in eukaryotes:
Prokaryotes:
Eukaryotes:
Transcription and mRNA
processing happen in
nucleus
Translation happens in
cytosol
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Eukaryotic DNA packaged into chromatin
Structure of the nucleosome
Histone
proteins
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Chromatin can be in an “open” state, allowing transcription
or in a “closed” transcriptionally silent state
Eukaryotic transcription
involves a complex of
“basal” transcription
factors that help recruit
RNA polymerase to the
promoter
(defined by TATA box)
RNA polymerase can
access open but not
closed chromatin
Once bound, RNA polymerase can transcribe through chromatin
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Chromatin and gene regulation
  Chromatin can be open or closed (active or silent)
  RNA polymerase can access and transcribe through open
chromatin
  Regulation of chromatin structure is one level of
transcriptional regulation in eukaryotes
  Also have specific activators and repressors as in
prokaryotes that regulate transcription
  These can influence chromatin structure and/or affect binding
of basal transcriptional machinery
While molecular mechanisms of transcriptional regulation
in eukaryotes differ somewhat from prokaryotes due to
presence of chromatin, the basic concepts are conserved:
Activators and repressors bind regulatory elements in the DNA
(enhancers and silencers) to control access or activity of basal
transcriptional machinery
Binding of these factors is:
  Specific
  Modular
- elements function independently and can be
combined to give different patterns of
expression in different genes
Activators and repressors bind enhancers
and silencers to regulate transcription
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Investigating the control of gene expression in eukaryotes
Homeotic mutations in Drosophila
Embryogenesis in
Drosophila
melanogaster
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Different segments
in embryo give rise
to different
structures in adult
Homeotic mutants
  Isolated by Ed Lewis in 1970’s
  Wanted to understand development and differentiation
- isolated mutants with transformations of
body structures, e.g., Antp: antennae -> legs
Bithorax mutants - third thoracic segment transformed
to fate of second thoracic segment (wings instead of halteres)
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Many different mutants isolated affecting segment or
structure identity
  Mapped to two regions of the genome
-  the antennapedia (ANTP) complex
-  the bithorax (BX) complex
  Genes are arranged on chromosomes in same
order as segments they specify
Hox genes define
segment identity
Hox genes are arranged
in linear order on
chromosome that
corresponds to order of
segments they specify
ANTP-C
BX-C
Some homeotic mutations in protein-coding genes
and others in regulatory elements
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Hox gene expression
defines segment
identity
Hox genes are arranged
in linear order on
chromosome that
corresponds to
expression domain in
embryo
ANTP-C
BX-C
Visualising Hox gene expression in the embryo
Visualising mRNA expression
In situ RNA hybridisation
  Tagged Antisense RNA probe binds mRNA
  Antibody used to bind to tag
  Enzyme fused to antibody - makes coloured substrate
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Visualising protein expression
  Primary antibody
binds protein X
  Secondary antibody
binds primary
  Enzymatic reaction
or fluorescent tag
Hox gene
expression
established by
cascade of
developmental
genes
Hox proteins
  Homeotic genes encode Hox proteins
- when cloned, found to look like… lac repressor!
=> bind DNA , act as transcription factors
  Control expression of many other genes
- they are called “master” regulatory genes
- control other Hox genes
- and downstream “effector” genes
  All contain homeodomain
- conserved DNA-binding domain
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Homeodomain proteins control expression
of many other genes
parasegment number
Hox genes specify
identity of
segments in which
they are expressed
Removal of Hox
genes leads to
transformation of
segments to more
anterior fates
Why?
Posteriorly expressed
Hox genes repress
more anteriorly
expressed ones
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Explanation of bithorax mutant
Bithorax:
- Ubx gene deleted
- Antp expression expands
posteriorly
- T3 -> T2
- halteres -> wings
Target genes: e.g., wingless
- necessary for wing formation
(mutant has no wings)
- activated by Antp, repressed by Ubx
Variation in targets of Ubx could be associated with
evolution of diverse wing types
Explanation of antennapedia mutant:
Antennapedia:
- deletion of silencer element
- Antp gene now expressed in antennal segment
(antennae -> legs)
- Antp represses homothorax gene
(necessary for antennal formation)
- Antp turns on distal-less
(necessary for limb formation)
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Hox genes are remarkably highly conserved
  Mammals have four copies of whole cluster
  Highly conserved at protein sequence level
  Some can substitute across species
(i.e., mouse gene can rescue fly mutant)
Mammals have four
copies of Hox gene
cluster, arranged on
different chromosomes
Order on chromosome
also corresponds to
expression domain in
embryo
Mutations in several human HOX genes associated
with congenital defects
e.g., HOXD13 mutations result in synpolydactyly
(multiple fused digits)
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Another example of a “master” regulatory gene:
  Eyeless: necessary for eye formation in flies
  Also encodes homeodomain protein
  Also conserved - called Pax6 in mammals
  Eyeless gene sufficient to drive eye formation in flies
if expressed elsewhere
  Even expression of Pax6 can drive formation of ectopic
eyes in fly!
eyeless mutants in Drosophila have reduced or
totally absent eyes
Wild-type
eyeless mutants
Pax6 mutants in mouse also lack eyes
Wild-type
Pax6 mutant
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Pax6 mutations in humans affect eye development,
leading to aniridia, lens defects and cataracts
Driving eyeless expression in other tissues leads to ectopic eyes
- Achieved by fusing ey protein-coding sequence to regulatory
elements of gene normally expressed in limbs
Driving mouse Pax6 expression also leads to ectopic eyes
- can still activate correct downstream targets in fly
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Eyeless (or Pax6) directly activates several target genes:
Optix
Eyes absent
Sine oculis
  Also transcription factors
  Mutations in any of these genes lead to loss of eyes
=> All those genes necessary for proper eye formation
  Only eyeless is sufficient to drive eye formation
(it is “master” regulator)
Implies eyes did NOT evolve multiple times but just once
eyes
no eyes
eyes
Hypothetical common ancestor of insects and mammals
must have had some photoreceptive organ specified by
eyeless/Pax6 homologue
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Summary
  Transcription in eukaryotes involves combined
effects of activators and repressors
  These bind specific regulatory elements
  Mutations in proteins or in regulatory elements can
alter expression
  Master genes specify segments or body structures by
regulating many downstream genes
  Remarkable conservation of these genes across species
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