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

Fig. 7-7 Lodish et al. 2008

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 lenseand pancreas. Lacks exons 1 and α

Fig. 7-7

Alternative Pax 6 promoters & enhancers are used to generatedifferent transcripts in different tissues

Transcript c:detected in retinalacks exons 0-4contains exon αexon α is unique to vertebrates

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

Fig. 7-21

Reporter gene activity

What can you tell from comparing mutants 1, 2, 3 & 4?

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

• 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 ElementsComparison of Eukaryotic Control RegionsStructure 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 ofaction 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

COLDAIR transcription is induced by coldCOLDAIR noncoding RNA binds Polycomb group proteinsFLC locus is methylated on histone H3 Silencing

Science 331: 76-79 (2011)

Luciferase activity

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)_