DNA microarrays contain small spots of DNA, with each spot representing a different gene. The intensity of each spot indicates the expression level of that gene's mRNA. Microarrays allow clustering of genes and experiments based on similar expression profiles, linking related pathway components. Post-translational modifications of histone tails, such as acetylation, provide binding sites that regulate chromatin structure and transcription.
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)
3. 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
15. 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
22. 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.
23. Marmorstein (2001) Nat. Rev. Mol. Cell. Biol. 2, 422. Multiplicity of non-enzymatic domains in histone modifying enzymes
24. “ Chromatin remodeling complexes” and “Chromatin modifying complexes” are important for transcriptional activation Chromatin modifying complex Chromatin remodeling complex
29. Signal transduction and transcription control why study this? omgeving mRNA mRNA export mRNA afbraak translatie signaal transductie
30. The PI3K/PKB/FOXO module PI3K PI-3P PTEN PI PKB FOXO cell membrane PDK1 nucleus 100% mutated in human cancer !
31. 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
32. 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
33. 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
34. De ‘vrije radicalen’ theorie van veroudering anti-veroudering O 2 . _ O 2 Schade aan DNA, RNA etc ATP energie repair
36. 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
37. 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
49. Signal transduction and transcription control omgeving Transcriptie apparaat mRNA mRNA export mRNA afbraak translatie questions signaal transductie
Editor's Notes
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.