Outline
• Questions from last lecture?
• P. 40 questions on Pax6 gene
• Mechanism of Transcription Activation
– Transcription Regulatory elements
• Comparison between yeast and mammals
• Examples of mammalian transcriptional control regions
– Transcription Factors (activators and repressors)
• Mechanisms of Transcription repression
– Comparison between prok and euk repression
– Novel mechanism of repression by non-coding RNAs
Different Pax 6 enhancers are active in different tissues:
Results in different sites of transcription initiation and
alternative splicing in different tissues
Transcript a is detected in lense
and pancreas. Lacks exons 1 and α
Fig. 7-7 Lodish et al. 2008
Alternative Pax 6 promoters & enhancers are used to generate
different transcripts in different tissues
Transcript c:
detected in retina
lacks exons 0-4
contains exon α
exon α is unique to vertebrates
Fig. 7-7
Pax 6 Questions p. 40
1. What parts of the Pax6 gene contain regulatory
elements?
2. What DNA constructs could have been used to
generate the transgenic mice shown in the bottom
panels of Fig. 7-7?
3. Could this same information have been obtained
from an in vitro cell culture experiment? Why or why
not?
4. What experiments could you perform to identify
important transcription control regions? How would
you know where to start looking?
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
Readings
Fig 7.8 – example of how a SALLI gene
enhancer was identified in an intergenic
region 500 kb downstream of the SALL1
gene
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
Review at home:
Techniques to Identify Regulatory Elements
Comparison of Eukaryotic Control Regions
Structure of a Transcription Factor
Fig. 7-22. Lodish
Conclusions
• 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
Transcription Initiation Requires Binding of Many
Different Proteins to Enhancers and Promoters
What are 3 different functions of proteins that regulate
transcription by binding to specific DNA sequences?
1.
2.
3.
Transcription activators
Transcription repressors
General transcription factors e.g. TATA binding protein
At home: draw a picture of a eukaryotic promoter at the
time of transcription initiation (include all proteins expected
to be there)
Example of a Typical Mammalian Enhancer
Enhanceosome on
the β-interferon
enhancer
What determines
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)?
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)
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
• A cell must produce the specific set of activators
required for transcription of a particular gene in order
to express that gene
• Noncoding RNAs can recruit proteins that promote
silencing (e.g. histone methyl transferases)_