Conclusions (last lecture) Conserved structure of RNA pol II RNA pol II must navigate a chromatin template in a highly dynamic environment

Outline Mechanism of Transcription Activation RNA pol II structure and function Coordination of RNA processing Transcription Regulatory elements Identification & characterization in vivo Examples of mammalian transcriptional control regions Transcription Factors

Central Dogma Why aren’t tRNAs and rRNAs processed like mRNAs? What is different about how mRNAs are made? Fig. 1-9 Lodish 2013

Only RNA Polymerase II has a C-Terminal Domain CTD is Phosphorylated Prior to Transcription Initiation CTD Red arrow indicates location where unique C-terminal domain extends from in RNA pol II Fig. 7-10. Lodish 2013

The RNA Pol II C-Terminal Domain (CTD) facilitates capping, splicing and polyadenylation by recruitment of RNA processing factors. Hsin J , and Manley J L Genes Dev. 2012;26:2119-2137 ©2012 by Cold Spring Harbor Laboratory Press

5’ Capping Splicing 3’ Processing RNA Pol II CTD phosphorylation coordinates the transcription cycle with pre-mRNA processing and histone modification. 5’ Capping Splicing 3’ Processing Hirose Y , Ohkuma Y J Biochem 2007;141:601-608 © 2007 The Japanese Biochemical Society.

Drosophila chromosome stained with antibody to phosphorylated Pol II (Red) or unphosphorylated Pol II (Green) What do you notice? Fig. 7-13 Lodish 2013

Central Dogma Fig. 1-9 Lodish 2013

Alternative Splicing Simple Complex Which promoter is regulating mRNA2 transcription?

Pax6: Master regulator of eye development Ectopic Expression of Eyeless (PAX 6 homolog) in Drosophila results in aberrant formation of eye structures Antenna Lower leg Halder et al. Science 267: 1788 (1995)

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 PAX6: paired box and Homeobox DNA binding domain – highly conserved in mammals, flies and worms Mammals: required for retina, brain and pancreatic beta cells (Pax 6 +/-) = lack of irisLP = lens pit. Actually there are 3 different promoters utilized at different times during embryogenesis in diff tissues of the developing embryo Transcript in Lens and pancreas initiates at exon 0 Transcript in brain initiates at exon 1 Transcript in retina initiates at exon alpha (unique to vertebrates so invertebrates cannot restrict light by constricting retina in response to excessive light, must close eye) – may have been an evolutionary advantage in vertebrates. Aniridia (loss of iris) occurs in 1/60,000 individuals Fig. 7-6 Lodish et al. 2008

How can we study gene expression in vivo? One method: transgenic mouse Introduce “reporter gene” controlled by potential regulatory elements into a mouse transgenic mouse animation Distinct from reporter gene assay which is performed in cell culture

Answer Pax6 gene questions in your course manual What parts of the Pax6 gene contain regulatory elements? Upstream region and introns   What DNA constructs could have been used to create the transgenic mice shown in the bottom panels of Fig. 7-6? For the far left picture which shows staining in lens pit and pancreas: Could have used 8kb upstream of exon 0, directing expression of the beta gal gene (don’t need any other pax 6 sequences. For the embryo showing staining in the retina, could have used the intron between exons 4 and alpha (intron 5), directing expression of the beta gal gene (don’t need any other pax6 sequences. Take home point: these sequences are binding regulatory factors that are found only in certain tissues, and these are stimulating transcription in those tissues. Could this same information have been obtained from an in vitro cell culture experiment? Why or why not? Could get some of this info from different cell culture lines (like intestinal cells used in discussion section experiments) – but difficult to recreate cross-talk between tissues in vitro What experiments could you perform to identify important transcription control regions? How would you know where to start looking? Could take a comparative genomics approach and look at sequences that are highly conserved between mouse, human etc. or could look at individuals with mutant phenotypes (eyeless) and see where the mutations lie. Then could mutate these regions and look at effect on expression.

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 LP = lens pit. Actually there are 3 different promoters utilized at different times during embryogenesis in diff tissues of the developing embryo Transcript in Lens and pancreas initiates at exon 0 Transcript in brain initiates at exon 1 Transcript in retina initiates at exon alpha (unique to vertebrates (invertebrates cannot restrict light by constricting retina in response to excessive light, must close eye) – may have been an evolutionary advantage in vertebrates. (not present in worms, mollusks, insects) Fig. 7-6 Lodish et al. 2008

Conclusions Unique Pol II CTD provides a mechanism for coupling mRNA-specific processing with transcription Different enhancers/promoters can control transcription of the same gene in different cell types Different subsets of transcription factors bind to enhancers of the same gene in different cell types Enhancers can be located far from transcription start sites