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Journal Club 16 th of January 2001 Marius van den Beek

Journal Club 16 th of January 2001 Marius van den Beek. Nature 480,391 – 395 ( 15 December 2011 ) doi :10.1038/nature10492. S. Pombe RNAi -directed heterochromatin assembly at the Centromere.

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Journal Club 16 th of January 2001 Marius van den Beek

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  1. Journal Club 16th of January 2001 Marius van den Beek Nature 480,391–395 (15 December 2011) doi:10.1038/nature10492

  2. S. PombeRNAi-directed heterochromatin assembly at the Centromere Lejeune, E., Bayne, E.H. & Allshire, R.C., 2011. On the Connection between RNAi and Heterochromatin at Centromeres. Cold Spring Harbor Symposia on Quantitative Biology

  3. RNAi is required for proper distribution of H3K9meand PEV wm4 H3K9m2

  4. Ago2 required for looping interactions at Abd-B locus in Drosophila RNAi-independent role for Argonaute2 in CTCF/CP190 chromatin insulator function, 2011, Genes & Dev. Moshkovich N, Nisha P, Boyle PJ, Thompson BA, Dale RK, Lei EP

  5. Results – nuclear RNAi

  6. Results – Ago2, Dcr-2 bind to euchromaticinterbands in 3rd instar larvae Zoom in

  7. Results: Chromatin binding of Dcr2 requires AGO2 but not vice versa

  8. Results: Dcr-2 and Ago2 bind to Heatshock-loci (87A, 87C) 87A 87B

  9. Results: Loss of Dcr-2 or Ago2 results in upregulation of Heat-shock transcripts (in non-heatshocked S2 cells) NON-HEATSHOCK HEATSHOCK

  10. Results: Upregulation in Dcr-2/Ago2 mutant result of defective PolII-pausing.Defective pausing correlates with decreased NELF-E binding at the TSS non-hs non-hs

  11. Results: Defective pausing is not limited to heat-shock loci qRT-PCR

  12. PolIIredestribution after Heatshock is disrupted in RNAi mutants During heatshock elongating PolII (Ser-2-phosphorylated) localizes from interbands to heat-shock loci PolII localization to Heat-shock loci is still intact in Ago2-414, but PolII still present at interbands PolIIredestribution requires catalytically active RNAi pathway

  13. Results: Dcr-2 and Ago2 bind to PolII/NELF-E Ago2-binding requires Dcr-2 … and probably a catalytically active RNAi pathway

  14. Results: Ago2-IP highly enriched for antisense smallRNA AGO2-IP Small RNAs mapping to heat-shock promoters strongly enriched after heat-shock ?? Antisense enrichment Ago2/control Very pronounced during heatshock ??

  15. Summary • nuclear, chromatin-bound RNAi • Dcr-2 and Ago2 bound to regions of active transcription • Dcr-2 and Ago2 required for redistribution of PolII transcription during heat-shock and pausing of heat-shock gene transcription • Antisense small RNA enriched at heat-shock loci, particularly during heat-shock Open Questions How are Dcr-2, Ago2 interacting with PolII/NELF-E ? What is the role of antisense siRNAsduring/before/after heat-shock? Trigger or consequence? Why is the relocation of elongating PolIIdefective in RNAi mutants? What is the contribution of RNAi-mediated gene-pausing on the genome-scale? How are the H3K9me marks set, considering that Dicer-2, Ago2 bind the chromocenter?

  16. Background RNAi <-> Transcriptional regulation • Well-known role in post-transcriptional regulation, anti-viral defense and transposon defense • Transcriptional regulation debatable in higher eukaryotes • RNAi required for transmission of epigenetic marks in S.pombe cell cycle  next slide • In Drosophila, disruption of RNAi leads to H3K9me2 re-distribution away from the chromo-center  2 slides after

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