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Collegepart B.Burgering Deel 2
 

Collegepart B.Burgering Deel 2

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  • 1503 significant genes (1.7 fold) p<0.05
  • 1503 significant genes (1.7 fold) p<0.05
  • These results show that we can use microarrays to identify proteins that are known to physically interact. But these examples are all RING RING proteins, between putative E3 ligases. So to see if we can also see functional interactions with other enzymes like DUBS or E2’s, I have to go back to the bigger cluster.

Collegepart B.Burgering Deel 2 Collegepart B.Burgering Deel 2 Presentation Transcript

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  • DNA microarrays Microarray = raster van stipjes Elk stipje bevat DNA moleculen van een bepaald gen Elk stipje vertegenwordigd ander gen Intensiteit stipje = mRNA hoeveelheid (expressie niveau)
  • DNA microarray Representative cDNA mixture) T T C C C C G A A T G G C A T A C Een stip (gen A),na hybridisatie, fluorescentie op stip gen A = hoeveelheid cDNA = expressie (mRNA) van gen A mRNA cDNA Fluorescent label added G A A T T C G C G A T C G C A C T G A A T T C G C G A T C G C A C T G A A T T C G C G A T C G C A C T G A A T T C G C G A T C G C A C T C T T A A G C G C T A G C G T G A C T T A A G C G C T A G C G T G A A A G T C C C T G T T C G C T C C T T C C C C G A A T G G C A T A C T T C C C C G A A T G G C A T A C G A A T T C G C G A T C G C A C T C T T A A G C G C T A G C G T G A C T T A A G C G C T A G C G T G A C T T A A G C G C T A G C G T G A C T T A A G C G C T A G C G T G A
  • No clustering Clustering on experiments
  • Clustering on Genes & experiments Clustering on genes
  • Ubiquitin and ubiquitin-like pathways ubp15 yol138c ufd4 D 3 3 ste5 pex4 pex2 3 2 3 3 3 2 D 3 3 3 2 3 3 3 3 3 3 3 pex10 pex12 ubc7 ubp3 pep3 pep5 vps8 ubc4 slx5 slx8 rad18 rad5 not4 bre1 tom1 D = DUB 2 = E2 3 = E3 (putative)
  • ubp15 yol138c ufd4 D 3 3 ste5 pex4 pex2 3 2 3 3 3 2 D 3 3 3 2 3 3 3 3 3 3 3 pex10 pex12 ubc7 ubp3 pep3 pep5 vps8 ubc4 slx5 slx8 rad18 rad5 not4 bre1 tom1 D = DUB 2 = E2 3 = E3 (putative) Linking pathway components
  • slx8Δ slx5Δ pep3Δ rad18Δ rad5Δ pep5Δ rad6 rad18 rad5 pep3 pep5 slx5 slx8 Linking pathway components: Ring heterodimers have similar profiles
  • Regulation of transcription factors DNA binding
  • Binding to DNA
  • Binding to DNA The importance of hydrogen bridges
  • Voorkomen van verschillende DNA-bindings motieven (monomeren) Homeo-domein (b.v. hunchback, eve) POU-domein (b.v. Pit-1, Oct-1) Zink-vinger (b.v. nucleairehormoon receptoren)
  • Leucine-zipper (b.v. fos, jun) basic Helix-loop-Helix (b.v. MyoD, myc) Voorkomen van verschillende DNA-bindings motieven (dimeren)
  • Illustration: Jun, Fos (AP-1) form a complex with DNA FOS JUN FOS+JUN
  • Dimerizatie maakt regulatie via “combinatorial control” mogelijk Hetero-dimerizatie met een partner, die een andere DNA volgorde herkent Hetero-dimerizatie met een partner, die niet in staat is DNA te binden
  • Other determinants of binding to DNA: chromatin organization
  • Nucleosoom opbouw mbv histonen
  • The “histone code” hypothesis : the pattern of post-translational modifications occurring on the histone tails serves as binding sites for specific proteins.
    • Note that other chromatin modifying complexes include kinases, methylases and ubiquitin conjugating proteins.
    • Acetylation typically correlates with transcriptional activation while deacetylation correlates with repression.
  • Reading and writing the histone code
  • Writing the code: eg Histone Acetyl Transferases
      • Multiple families
      • Gene-specific or global activators of transcription
      • Distinct substrate specificities for different families
      • Can acetylate non-histone proteins (transcription factors)
      • Weakens interaction of basic tails with negatively charged phosphate backbone of DNA.
      • Weakens interactions that occur between nucleosomes, thus promoting decondensation of the chromatin fiber.
      • Provide a marker for recognition by other proteins. For example, a conserved “bromo” domain found in SWI/SNF and other transcription factors recognizes this marker.
    How acetylation might contribute to activation
  • Non-enzymatic domains in Chromatin Modification proteins Bromodomain recognition of acetyl-lysine Marmorstein (2001) Nat. Rev. Mol. Cell. Biol. 2, 422. Dhalluin et al. (1999) Nature 399, 491.
  • Marmorstein (2001) Nat. Rev. Mol. Cell. Biol. 2, 422. Multiplicity of non-enzymatic domains in histone modifying enzymes
  • “ Chromatin remodeling complexes” and “Chromatin modifying complexes” are important for transcriptional activation Chromatin modifying complex Chromatin remodeling complex
  •  
  • TATA-binding protein (TBP) bepaalt de opbouw van het RNA polymerase II transcriptie complex
  • Opbouw van het transcriptie complex: stapsgewijze binding van algemene factoren en pol II
  • By Alberts
  • Signal transduction and transcription control why study this? omgeving mRNA mRNA export mRNA afbraak translatie signaal transductie
  • The PI3K/PKB/FOXO module PI3K PI-3P PTEN PI PKB FOXO cell membrane PDK1 nucleus 100% mutated in human cancer !
  • cell-type dependent regulation of proliferation and death by FOXOs G1: G1: 64% 73% 29% 49% Con FOXO4 Con + noco FOXO4 + noco NIH3T3/SAOS/U87/DLD cells 3% 58% Con FOXO3a death BaF3/Jurkat cells
  • Relocalization of FOXOs by PI3K/PKB signalling HA-FOXO4 Con insulin HA-FOXO4 gagPKB HA-FOXO4 HA-SASA gagPKB 14-3-3 FOXO FOXO PKB P
  • Signalling conserved through evolution C.elegans Mammalians Daf-2 FoxO PKB PI3K Ins/IGF-R Daf-18 Age-1 Akt-1 Daf-16 PTEN SOD-3 MnSOD “ regulation of lifespan” and disease = ageing
  • De ‘vrije radicalen’ theorie van veroudering anti-veroudering O 2 . _ O 2 Schade aan DNA, RNA etc ATP energie repair
  • FOXO-induced protection from oxidative stress Kops et al. Nature 2002
  • Multiple pathways regulate FOXO: Oxidative stress can relocate and activate FOXO +FCS-H2O2 +FCS +H2O2 +FCS +H2O2 FOXO4 localization Insulin PI3K PKB 14-3-3 FOXO FOXO ROS P
  • Regulation of FOXOs by oxidative stress O · Ral JNK mdm2 p300 FOXO4 Ubi Ubi Ubi Ac Ac P P sirt1 usp7 Ubi Ubi Ubi  -cat PRMT1/6 meth PIN1 +FCS-H2O2 +FCS +H2O2 +FCS +H2O2 FOXO4 localization
  • AND NOW……………………………………….. The transcription factor code How to decipher this ?
  • FOXO4 is ubiquitinated upon peroxide treatment
  • Ubiquitination comes in multiple flavors
  • FOXO4 is monoubiquitinated upon peroxide treatment
  • Monoubiquitination causes nuclear localisation … ubi FOXO4 FOXO4 localization
  • Armando, NCB 2006 (USP7)
    • - both on lysines
    • both after oxidative stress
    • competition?
    TSA: deacetylase inhibitor  increase in FOXO4 acetylation Interplay between ubiquitination and acetylation
  • p300 mediated acetylation of FoxO4 Linking phosphorylation and acetylation?
  •  -catenin/FOXO binding increased by cellular oxidative stress Reading the code: Cross-talk between TFs
  • Regulation of FOXO activity 14-3-3 FOXO PKB FOXO  -catenin nucleus FOXO insulin O 2 . _ FOXO GENES FOXO  -catenin P
  • Cross-talk between FOXO and TCF nucleus FOXO O 2 . _ FOXO GENES FOXO ? TCF  -catenin  -catenin  -catenin
  • FOXO inhibits TCF
  • Signal transduction and transcription control omgeving Transcriptie apparaat mRNA mRNA export mRNA afbraak translatie questions signaal transductie