Collegepart B.Burgering Deel 2

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

Collegepart B.Burgering Deel 2

by BMW, Utrecht University on Nov 23, 2009

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Collegepart B.Burgering Deel 2 Webinar Transcript