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Outline
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Molecular Cell Biology Assessment
Review from last lecture
Role of nucleoporins in transcription
Activators and Repressors
Epigenetic mechanisms of inheritance
Cell type specification
– Restriction of cell fates
– Embryonic stem cells
• Pluripotency
• Chromatin structure
On-line assessment
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Molecular and cellular biology test
Multiple true/false format
Multi-institution study
3 points for participation
Link will be emailed today
Complete by next Tuesday (6/4)
Conclusions from Pax6 Gene
• Different control regions can control
transcription of the same gene in
different cell types
• Different subsets of transcription factors
bind to control regions of the same
gene in different cell types
• Control regions can be located far from
transcription start sites
From Last Lecture:
Techniques to Characterize CisActing Regulatory Regions
• Transgenic mice
– Create DNA constructs containing potential cis-acting
sequences + reporter gene and introduce into embryonic
stem cells
• Reporter Gene Assay (fig. 7-25)
– Cell culture approach
• Linker Scanning Mutation Analysis (fig. 7-21)
– In vivo or in cell culture
• DNase I Footprinting (fig. 7-23)
• Comparative genomics
Purple = in text book readings
From Last Lecture:
Linker-scanning Mutation of a Gene
Regulatory Region
Reporter gene activity
What can you tell from
comparing mutants
1, 2, 3 & 4?
Fig. 7-21
Review at home:
Techniques to Identify Regulatory Elements
Comparison of Eukaryotic Control Regions
Structure of a Transcription Factor
What do you notice when you compare (a) and (c)?
What do you notice when you compare (a) and (b)?
Fig. 7-22. Lodish
Example of a Typical Mammalian Enhancer
Enhanceosome on
the β-interferon
enhancer
What three things
determine whether a
given protein is
found associated
with the enhancer?
• WT1 represses EGR-1
• SRF/TCF and AP1 are transcription activators
• Based on this schematic, how is the mechanism of
action different for the eukaryotic WT1 (Wilms Tumor)
repressor compared to prokaryotic repressors (e.g. lac i)?
Summary
• Activators promote transcription and are
modular proteins composed of a DNA binding
domain and an activation domain
• Repressors inhibit transcription and are
modular proteins composed of a DNA binding
domain and a repressor domain
• Both repressor and activators recruit other
proteins to affect gene expression
• A cell must produce the specific set of
activators required for transcription of a
particular gene in order to express that gene
Can Proteins Serve Both a Regulatory
and a Structural Function in the Cell?
* = Dynamic
D’Angelo et al. 2009
Not discussed – you are not responsible for this information
A. Chromatin IP experiment identifying binding sites for Nup98
and lamin
What do you notice about the binding sites for Nup 98
and Lamin on this chromosome?
What does this suggest about the location of Nup98 in
the cell?
Capelson et al., Cell 140: 372 (2010)
Not discussed – you are not responsible for this information
What does the
expression data tell us
about the genes that
Nup98 associates
with?
What do these data
suggest about the
role(s) of Nup98?
What else are dynamic nucleoporins doing?
B. Expression levels of Nup98-associated genes
Capelson et al., Cell 140: 372 (2010)
Overview of Eukaryotic Transcription Control
At home: consider possible mechanisms by which eukaryotes
can regulate transcription which are not possible in prokaryotes
How are established
expression patterns inherited?
Cell Memory
Epigenetic mechanisms of inheritance
– DNA methylation (DNA methyltransferases)
– Histone modifications (e.g. methylation)
– noncoding RNAs
Memory of Winter
PRC2/VRN2=
Polycomb
Group Complex
(PcG)
Novel mechanisms for Gene Repression:
Epigenetic Silencing of the Flowering
Gene FLC by a Noncoding RNA
Luciferase activity
COLDAIR transcription is induced by cold
COLDAIR noncoding RNA binds Polycomb group proteins
FLC locus is methylated on histone H3
Silencing
Science 331: 76-79 (2011)
Cascade of Gene Expression Required
for Vernalization-Induced Silencing
Science 331: 76-79 (2011)
Strategy for Epigenetic Inheritance of
Histone Modifications
Reader/Writer Hypothesis
How is Histone H3 methylation
Maintained during DNA replication
What two properties
Must the H3K9 HMT
have?
Fig. 7-46. Lodish et al. 2013
Model for Repression by Polycomb
Complexes:
Establishing Gene Repression
Fig. 7-47
PRC2 contains a histone
methyl transferase (writer)
Where have we seen PRC2 before?
What is the role of the repressor?
Model for Repression by Polycomb Complexes:
Maintaining gene Repression
PRC1(the reader)
contains a protein
(Pc) that binds
H3K27Me
Not shown: PRC2
(the writer) can bind
PRC1 to maintain
the methyl mark after
DNA replication
Do plants have PRC1 complexes?
Fig. 7-47
Conclusions
• Changes in chromatin structure are necessary before
transcription can occur at most genes
• Activators can direct histone acetylation and
methylation to specific genes
• Repressors can direct histone deacetylation, histone
methylation and DNA methylation to specific genes
• Repressors and microRNAs can recruit polycomb
group complexes to promote heterochromatin
formation
• Epigenetic marks can be inherited by daughter cells