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Download Guest lecture 3130 2015 - Scheid Signalling Lab @ York University
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Action at a Distance • Bacterial and eukaryotic enhancers stimulate transcription even though located some distance from their promoters • Four hypotheses attempt to explain the ability of enhancers to act at a distance – Change in topology ie. supercoiling – Sliding – Looping – Facilitated tracking 12-1 Hypotheses of Enhancer Action Change in topology Sliding Looping Facilitated tracking 12-2 Complex Enhancers • Many genes can have more than one activator-binding site permitting them to respond to multiple stimuli • Each of the activators that bind at these sites must be able to interact with the preinitiation complex assembling at the promoter, likely by looping out any intervening DNA 12-5 Control Region of the Metallothionine Gene • The metallothionine gene product helps eukaryotes cope with heavy metal poisoning • Turned on by several different agents • Complex enhancers enable a gene to respond differently to different combinations of activators • This gives cells exquisitely fine control over their genes in different tissues, or at different times in a developing organism 12-6 After identifying individual regions in the 5’ UTR of the Endo16 gene that bind nuclear proteins, the isolated binding regions were fused to a reporter cassette and reintroduced into sea urchin. Expression was monitored and it was determined that some regions act alone and others in combination with each other. Very important for the fine control of gene expression required during development.. Architectural Transcription Factors Architectural transcription factors are those transcription factors whose sole or main purpose seems to be to change the shape of a DNA control region so that other proteins can interact successfully to stimulate transcription. Important when control regions are in very close proximity to one another. 12-9 • Short DNA acts as rigid rod • Long DNA behaves more like a string and can be easily manipulate Example of Architectural Transcription Factor: Control region of the human T-cell receptor alpha chain (TCR ) gene • Within 112 bp upstream of the start of transcription are 3 enhancer elements • These elements bind to: – Ets-1, LEF-1, CREB – LEF-1 alone cannot activate gene. Role? • bends DNA at the minor grove by 130deg 12-11 • Used synthetic DNA containing LEF-1 binding site in electrophoretic assay to show DNA bending • When the site was positioned in the middle of the synthetic DNA, movement through a gel was greatly retarded DNA Bending Aids Protein Binding • The activator LEF-1 binds to the minor groove of its DNA target through its HMG domain and induces strong bending of DNA • LEF-1 does not enhance transcription by itself • Bending it helps other activators bind and interact with activators and general transcription factors 12-13 Enhanceosome • An enhanceosome is a nucleoprotein complex containing a collection of activators bound to an enhancer in such a way that stimulates transcription • Ex. IFN-beta contains 8 binding sites which must all be occupied by activators. The other end of the simple/complex enhancer spectrum • only activated when a cell is under attack by a virus. 12-14 Dilema: Some enhancers act at great distances from their promoters. ie. Drosophila cut locus is 85kb from promoters. Enhancer is likely to come in proximity to other genes, how does the cell prevent inappropriate activation? Insulators • Insulators can shield genes from activation by enhancers (enhancer blocking activity) • Insulators can shield genes from repression by silencers (barrier activity). Prevent condensing • Insulators define regions between DNA domains. ie. Between enhancers and promoters 12-17 Mechanism of Insulator Activity • One mechanism which can be ruled out is that insulators induce the condensation of DNA upstream of their location. – If a gene were placed upstream of such an insulator, it would always be silenced – Experiments in Drosophila show that such genes can still be active and can be activated by their own enhancers. 12-18 Mechanism of Insulator Activity • Sliding model – Activator bound to an enhancer and stimulator slides along DNA from enhancer to promoter • Looping model – Two insulators flank an enhancer, when bound they interact with each other isolating enhancer 12-19 Mechanism of Insulator Activity • Sliding model – Activator bound to an enhancer and stimulator slides along DNA from enhancer to promoter • Looping model – Two insulators flank an enhancer, when bound they interact with each other isolating enhancer 12-20 • However…some insulators work as single copies. • ie. The Drosophila hairy-wing insulator - single insulator = some insulator activity - two insulators = no insulator activity - insulator-enhancer-insulator = increased insulator activity Model of Multiple Insulator Action Canceling of insulator activity 12-23 Summary • Some insulators have both enhancer-blocking and barrier activities, but some have only one or the other • Insulators may do their job by working in pairs that bind proteins that can interact to form DNA loops that would isolate enhancers and silencers so they can no longer stimulate or repress promoters • Insulators may establish boundaries between DNA regions in a chromosome 12-24 12.6 Regulation of Transcription Factors • Several activators do not active transcription by contacting the basal transcription apparatus directly. Rely on: • Coactivators = no activator function on its own, but collaborates with one or more activators to stimulate the expression of a set of genes 12-25 Coactivator Activator 12.6 Regulation of Transcription Factors • Phosphorylation of activators can allow them to interact with coactivators that in turn stimulate transcription 12-27 Model for the Activation of a Nuclear Receptor-Activated Gene 12-28 12.6 Regulation of Transcription Factors • Sometimes genes are inactivated by the destruction of their activators • Ubiquitylation of transcription factors can mark them for – Destruction by proteolysis – Stimulation of activity • Sumoylation is the attachment of the polypeptide SUMO which can target for incorporation into compartments of the nucleus • Methylation and acetylation can modulate activity 12-29 Ubiquitylation • Normal function of ubiquitylation is to mark proteins for destruction by the proteasome. ie. Aprx 20% of all proteins are made incorrectly and need to be quickly disposed of • A fine balance may exist between coactivators and corepressors which have ubiquitylating ability - ie. A corepressor may mark a coactivator for destruction, tipping the scale towards repression 12-30 Activator Sumoylation (SUMO or small ubiquitin like modifier) • Sumoylation is the addition of one or more copies of the 101-amino acid polypeptide SUMO (Small Ubiquitin-Related Modifier) to lysine residues on a protein • Process is similar to ubiquitylation • Results quite different – sumoylated activators are targeted to a specific nuclear compartment that keeps them stable 12-32 Activator Acetylation • Nonhistone activators and repressors can be acetylated by HATs • HAT is the enzyme histone acetyltransferase which can act on nonhistone activators and repressors • Such acetylation can have either positive or negative effects - ex. p53 acetylation by coactivator p300 results in increased DNA binding 12-33 Signal Transduction Pathways • Signal transduction pathways begin with a signaling molecule interacting with a receptor on the cell surface • This interaction sends the signal into the cell and frequently leads to altered gene expression • Many signal transduction pathways rely on protein phosphorylation to pass the signal from one protein to another • This leads to signal amplification at each step 12-34 Three pathways that use CBP/p300 to mediate transcription activation 12-35 Ras and Raf Signal Transduction 12-36 Lecture key words - Cell cycle Transcription factors Phosphorylation Heterodimerization Immunohistochemistry Immunprecipitation Growth factors Apoptosis Colony formation Nuclear localization Consensus sequences Motifs CDCA7 | a case study in cellular regulation • Cell cycle control is the endgame of cellular regulation - critical balance between proliferation and apoptosis CANCER CDCA7 | a case study in cellular regulation • Cell cycle control is the endgame of cellular regulation - critical balance between proliferation and apoptosis CANCER • Modes: -phosphorylation, -subcellular localization - heterodimerization CDCA7 | What is known • Myc and E2F target gene with peak expression at 1-S member of cell division cycle-associated • G Novel familyoverexpressed in human tumors • gene Frequently • JPO2 binds Myc and promotes Myc dependent transformation • JPO2 and CDCA7 share cysteine rich C-term which may bind DNA • Not known if CDCA7 interacts with Myc Myc | Just the facts • Discovered in Burkitt’s lymphoma • patients Member of bHLH-LZ family of transcription • • • • • • factors Requires heterodimerization with Max to transactivate Regulates the expression of ~10-15% of genes Role in development, cell division, cell growth, metabolism, angiogenesis Early response gene induced by growth factors, levels peak at G0-G1 Driving force of cell cycle and malignant transformation Active in 70% of human cancers • ~100,000 cancer deaths per year in the US due to Growth Factors Receptor Tyrosine Kinase P P P P PIP3 PI3K PIP2 PDK1 P P AKT CDCA7P P 14-33 TOR ricto r 14-33 P 14-33 Cytoplasm AKT P 14-33 14-33 CDCA7P 14-33 AKT P CDCA7 Myc Myc Transcription Pro-apoptotic Genes ? Nucleus CDCA7 | a case study in cellular regulation • Cell cycle control is the endgame of cellular regulation - critical balance between proliferation and apoptosis CANCER • Modes: -phosphorylation -subcellular localization -heterodimerization CDCA7 | CONSERVATION AKT consensus site CDCA7 T163 >90% conserved human monkey dog mouse chicken frog zebrafish 1 24 49 69 78 112 190 R X R X X T/S F/L R P R R R T F 261 363 AKT kinase 0.005% 371 T163 humCDCA7 zinc finger 261 361 CDCA7 is phosphorylated at t163 Phosphotase T163A WT T163A + CIP Radioactivity and mutational analysis Vector CDCA7+ CIP CDCA7 • Many ways to prove phosphorylation • Custom made antibody against phospho-T163 -FLAG T163A -P-T163 Vector CDCA7 is phosphorylated at t163 0 5 15 45 120 360 PDGF (min) Treatments w/ growth factors -P-T163 -FLAG Merge 1.0 2.2 3.6 4.7 4.0 3.9 Ratio P-T163/ Total CDCA7 T= 0’ T= 20’ T= 30’ Immunohistochemistry T= 40’ T= 50’ T= 60’ CDCA7 is phosphorylated at t163 by AKT Vector Inhibitors CDCA7 Akt inh VIII IP: -FLAG Blot: -P-T163 IP: -FLAG Blot: -FLAG CDCA7 | a case study in cellular regulation • Cell cycle control is the endgame of cellular regulation - critical balance between proliferation and apoptosis CANCER • Modes: -phosphorylation -subcellular localization -heterodimerization Where is cdca7 found? -Flag CDCA7 T163A CDCA7 DAPI CDCA7 | CONSERVATION >90% conserved human monkey dog mouse chicken frog zebrafish 24 49 69 78 112 1 190 261 363 371 T163 humCDCA7 NLS ? 157-186 NLS ? zinc finger 261 RRPRRRTFPGVASRRNPERRARPLTRSRSR How do we test for a nuclear localization signal? Isolate region in question and test its ability to target an innocuous protein to the nucleus 361 CDCA7 contains an NLS • Passive diffusion into nucleus <45 KDa SV40 SV40 KE 157-188 157-188 CDCA7 157-167 CDCA7 R176E 167-188 CDCA7 157-188 (T163A) R176/184E R171E R171/176E R184E 157-RRPRRRTFPGVASRRNPERRARPLTRSRSRIL-188 PHOSPHORYLATION ALTERS LOCALIZATION -CDCA7 Unstimulated PDGF PDGF + LY Nuclei Merge CDCA7 | a case study in cellular regulation • Cell cycle control is the endgame of cellular regulation - critical balance between proliferation and apoptosis CANCER • Modes: -phosphorylation -subcellular localization -heterodimerization CDCA7 | CONSERVATION >90% conserved human monkey dog mouse chicken frog zebrafish 24 49 69 78 112 1 190 261 363 371 T163 humCDCA7 NLS ? zinc finger NLS ? 157-186 261 361 RRPRRRTFPGVASRRNPERRARPLTRSRSR 14-3-3 consensus binding site R-[S/F/Y]-X-pS/T -X cdcA7 T163 Mekk2 T283 R G R R R K T T F F -P P P 14-3-3 | Just the facts • Large family of highly conserved, small, acidic polypeptides of 28-33 kDa • Seven different isoforms in humans, 14-3-3σ directly implicated in cancer • Binds to protein ligands at defined phosphoserine/threonine motif RSXpS/TXP • Over 200 known ligands • 14-3-3 regulates process relevant to cancer biology: cell-cycle progression, apoptosis and mitogenic signaling 14-3-3 | Modes of influence • 14-3-3 exists as a dimer and offers two binding sites for phospho-S/T motifs protein for: • Can function as adaptor a) two proteins that would otherwise not associate b) one protein with two 14-3-3 motifs = high affinity Adapted from Hermeking, 2005 • Affects change by: • Alteration of enzymatic activity – maintains RAF1 in inactive state • • • Alteration of DNA-binding activity – increases p53 Sequestration DNA-binding after DNA damage Altering protein-protein interactions Sequestration - BAD, FKHRL1, HDAC5 and CDC25C CDCA7 binds14-3-3 and is phospho dependent P165A X pT X P T163A F164A - R R161A R162A - R158A P159A R160A Vector Wildtype 14-3-3 consensus site S/F/Y Western blots Blot: -FLAG -P-T163 -14-3-3 14-3-3 alters CDCA7 localization -Flag DAPI CDCA7 T163A CDCA7 R161A CDCA7 R161A/ T163A CDCA7 Is 14-3-3 masking the NLS within the T163 region? CDCA7 | What is known • Myc and E2F target gene with peak expression at 1-S member of cell division cycle-associated • G Novel familyoverexpressed in human tumors • gene Frequently • JPO2 binds Myc and promotes Myc dependent transformation • JPO2 and CDCA7 share cysteine rich C-term which may bind DNA • Not known if CDCA7 interacts with Myc CDCA7 binds the transcription factor Myc D(170-370) CDCA7 D(153-370) CDCA7 D(230-370) CDCA7 D(260-370) CDCA7 T163A CDCA7 D(112-137) CDCA7 D(1-146) CDCA7 D(1-172) CDCA7 D(1-202) CDCA7 D(1-234) CDCA7 WT CDCA7 Co-immunoprecipitation CDCA7 WT T163A D(112-137) D(1-146) His-Myc Pulldown Blot: -FLAG D(1-172) D(1-202) D(1-234) D(260-370) D(230-370) D(170-370) D(153-370) Input Blot: -FLAG + + + + + + + - So how does cdca7 affect phenotype? APOPTOSIS PROLIFERATION 14-3-3/CDCA7 binding influence Mycinduced transformation Colony formation assay 14-3-3/CDCA7 binding influence Mycinduced apoptosis Rat1 Trypan blue exclusion Myc-Rat1 Sh1-Myc-Rat1 Growth Factors Receptor Tyrosine Kinase P P P P PIP3 PI3K PIP2 PDK1 P P AKT CDCA7P P 14-33 TOR ricto r 14-33 P 14-33 Cytoplasm AKT P 14-33 14-33 CDCA7P 14-33 AKT P CDCA7 Myc Myc Transcription Pro-apoptotic Genes ? Nucleus summary • CDCA7 is a novel target of AKT required for Mycapoptosis • dependent Phosphorylation of T163 inhibits CDCA7/Myc apoptosis by: • Promoting 14-3-3 binding • Disruption of Myc binding • Shuttling to the cytoplasm • Potential for medical intervention in Myc tumors where AKT is dysregulated