1. RNA Transcription Pre-Initiation Complex (PIC) binds promoter PIC recruits RNA Polymerase II Pol2 transcription elongation complex (TEC) transcribes sequence 7-methyl guanosine cap Spliceosome ribozymes remove introns Polyadenylation factors recruit poly-A polymerase

2. Transcription regulation Initiation –Chromatin structure –Promoter elements –Enhancer elements Elongation Stability –miRNA

3. Conformational control of transcription Transcription initiation depends on –DNA accessibility –Affinity for Pol2 DNA structure –Supercoiling –Kinks –Nucleosomes Matrix association Intron Exon CoreEnhancer Promoter -4000-500-40-+5010000+ MAR Transcription Elongation Complex Nucleosome Core Particle Transcription Bubble

4. Chromatin remodeling Nucleosome remodeling –SWI/SNF, ISWI, CHD –Displace nucleosome relative to DNA –Displace histone proteins Steric regulation of transcription factor binding Histone modifications –Methylation –Acetylation

5. Histone code Histone tails have many lysines –Terminal amine H-bonds with PO 4 backbone –Subject to multiple methylation/acetylation –Interactions between nucleosomes –Interaction btw histone & DNA Histone acetyltransferase (HAT) –p300/CBP,P/CAF –Transcribed genes Histone deacetylase (HDAC) –Sin3, NuRD –Silenced genes

6. Histone structure Amino terminal of H3/H4 link adjacent nucleosomes Acetylation (-CO-CH 3 ) blocks this association HATs activate genes HDACs repress Epigenetic inheritance

7. Gene specific transcription Promoter proximal region –Reporter construct –Canonical response elements Remote enhancer/insulator elements –Clusters of regulatory elements –Chromatin loops of 50+kb Exon Intron CoreEnhancer Promoter -4000-500-40-+5010000+ MAR

8. Gene specific transcription factors TFs may be as much as 6% of the genome Specific regulation requires teamwork –Combinatorial control –Synergistic, greater than additive interaction Common features –Basic, DNA binding domain –Phosphate binding –Dimer/oligomer-ization Options –Major groove –Over the top

9. Characteristics of Gene-specific TFs Brivanlou & Darnell 2002 Transcriptional activators Constitutive Sp1 CCAT NF1 Conditional Developmental GATA HNF Pit1 MyoD Bicoid Hox Signal Dependent Steroid Receptor GR ER PR TR Internal Signals SREBP p53 ChoREB NFkB Surface Receptor Nuclear Resident ETS CREB SRF FOS-JUN Latent Cytoplasmic SMAD STAT NFAT

10. DNA binding Charged AAs –Positive (blue N) groups (lys, arg, his) –Negative (red O) groups (asp glu) –O+N (asn, gln) Protein  -helix 3-4 NTPs c-jun jun side-chains compliment nucleotide polarity to give sequence recognition

11. Jun binding Jun (leucine zipper): 1 mtakmettfy ddaLnasfLp sesgpygysn pkiLkqsmtL nLadpvgsLk phLraknsdL 61 LtspdvgLLk LaspeLerLi iqssnghitt tptptqflcp knvtdeqegf aegfvralae 121 lhsqntlpsv tsaaqpvnga gmvapavasv aggsgsggfs aslhseppvy anlsnfnpga 181 lssgggapsy gaaglafpaq pqqqqqpphh lpqqmpvqhp rlqalkeepq tvpempgetp 241 plspidmesq erikaerkrm rnriaaskcr krkleriarl eekvktlkaq nselastanm 301 lreqvaqlkq kvmnhvnsgc qlmltqqlqt f Jun DNA binding: kaerkrm rnriaask DNA Consensus: TCA Dimer palindrome: TGA X tca Other side chains interact with DNA PO 4 backbone

12. TF Mechanisms PIC interaction Mediator interaction Target histone modifiers GMSA shows increase in PIC assembly with CREB activation domain (CRG) but not CREB DNA binding domain alone (DBD) Felinski et al., 2001 CREB Activation domain DNA binding

13. MyoD “Master control switch” for myogenesis Associates with muscle specific promoters –Recruits P/CAF HAT –Trigger differentiation Recruits HDAC1 –Represses myogenesis Dual specificity chromatin-IP shows MyoD complexed with HDAC1 during growth and P/CAF during differentiation GrowthDifferentiation

14. Cooperativity Most promoters contain dozens of TF domains Most TFs capable of binding dozens of cofactors Hydrophobic protein-protein interaction Combinatorial/network control of transcription Protein-protein binding map for MyoD TFII Other TF HATs Other TF

15. Enhanceosome Combination of GTFs & GSEs Family of xscription factors produce specific molecular surface Highly nonlinear Interferon-beta –ATF+jun –IRF-1 –p50+p65 –HMG-1 –Only effective as combined group Kim & Maniatis (1997)

16. Mediator Helps recruit Pol2 to PIC GTF cofactor Integrates GTF & gene specific TFs CTD kinase Many subunits (30+) –Head –Middle –Tail Chadick & Austurias, 2005

17. Mediator GTF interactions –TBP –TFIIH Cdk activity targets Pol2 CTD –CDK8/CDK11 + cyclin D –Gene specific activation/repression Gene specific TFs may act through mediator to recruit/activate pol2 and through HDAC/HATs to recruit GTFs

18. Regulation of elongation TEC velocity Pause/arrest Negative Elongation Factor (NELF) –Binds DNA & blocks PolII progress –NELF-B = Cofactor of BRCA1 (COBRA1) Reversible Block –Binds PolII/DSIF complex –Released by P-TEFb phosphorylation of PolII –NELF-P-TEFb competition

19. P-TEFb releases pause Cyclin dependent kinase –Cdk9 –Cyclin T/K Phosphorylates DSIF Phosphorylates CTD –Stimulates elongation –Blocks NELF binding Pol II DSIF NELF P-TEFb PO 4 Transcription Blocked Transcription Allowed Yamaguchi et al., 1999 Cell 97:41

20. Chromatin remodeling Set1/PAF methylate histone, loosen structure Chd1 displaces H2A/B, allowing transcription Competition displaces Set1, downstream histones less methylated Turner 2003

21. miRNA Short, noncoding RNA Drosha forms hairpins Dicer form short dsRNA Steric translation block Slicer dsRNA degradation Drosha Dicer Slicer Mature slicer (Ago) with ssRNA

22. Regulatory Circuits Cascade Feedback/Regulatory Inhibition Combinatorial activation One or more external signals allow activation Product inhibits expression of promoter External signal

23. Modulation of TF activity Many TFs are constitutively expressed Secondary control mechanisms –Phosphorylation (phos-myoD binds HDAC) –Ligand/dimer binding (steroid) –Inhibitor dissociation (NF-kb) –Subcellular localization (MEF2 dephos) Some TF form cascades –“IEG”s –Delayed Early Genes –Cyclins/cdks

24. Example: c-Jun Activator Protein-1 component –Phosphorylation by JNK/ERK Ser 63, S73 (protein binding domain) Required for activity –Phosphorylation by GSK Thr231, Thr239, Ser243, Ser249 (DNA binding domain) Prevents DNA binding –Association with JNK Facilitates ubiquitination Binds TATA-binding protein-Associated Factor-1 Cell cycle regulation –CyclinD/cdk4 transcription after mitogens –Repression by GSK during starvation