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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)

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outline
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
slide2

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

slide3

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
Pax 6 Questions p. 40
  • What parts of the Pax6 gene contain regulatory elements?
  • What DNA constructs could have been used to generate the transgenic mice shown in the bottom panels of Fig. 7-7?
  • Could this same information have been obtained from an in vitro cell culture experiment? Why or why not?
  • 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
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
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 cis acting regulatory regions
Techniques to Characterize Cis-Acting 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

slide8
Review at home: Techniques to Identify Regulatory ElementsComparison of Eukaryotic Control RegionsStructure of a Transcription Factor

Fig. 7-22. Lodish

conclusions
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
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
Example of a Typical Mammalian Enhancer

Enhanceosome on the β-interferon enhancer

What determines whether a given protein is found associated with the enhancer?

slide12

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)?
slide13
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
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)_